WO2012023288A1 - Fam161a as a target gene for cancer therapy and diagnosis - Google Patents

Abstract

Objective methods for diagnosing cancer or a predisposition to developing cancer, particularly lung cancer is described herein. In one embodiment, the present invention provides a diagnostic method that utilizes the expression level of FAM161A as an index of cancer. The present invention further provides methods of assessing or determining the prognosis of a patient with lung cancer. The present invention further provides methods of screening for therapeutic substances useful in the treatment of cancer, e.g. lung cancer. The invention further provides methods of inhibiting the cancer cell growth and treating and/or preventing cancer. The invention also features double stranded molecules against FAM161A or CSNK2A2 gene, as well as vector encoding them and compositions containing them. The invention further provides a FAM161A polypeptide having a dominat-negative effect.

Description

FAM161A AS A TARGET GENE FOR CANCER THERAPY AND DIAGNOSIS

The present invention relates to the field of biological science, more specifically to the field of cancer research, cancer diagnosis and cancer therapy. In particular, the present invention relates to a novel biomarker and therapeutic target for lung cancer.

Priority
The present invention claims the benefit of US Provisional Application No. 61/375,447, filed on August 20, 2010 and Japanese Patent Application No. 2010-201361, filed on August 22, 2010, the entire contents of which are incorporated by reference herein.

Primary lung cancer is the leading cause of cancer death in most countries, and non-small lung cancer (NSCLC) accounts for about 80% of those cases (NPL1). An accurate molecular mechanism has yet to be determined, though many genetic alterations related to development and the progress of the lung cancer have been reported (NPL2). Nevertheless, a patient with advanced lung cancer frequently progresses to fatality despite improvements in surgical technique and the chemoradiotherapy (NPL1). Accordingly, developing a more complete understanding of the biology of lung cancer and more effective treatments to improve the survival of patients is of the utmost importance (NPL3).

Within the last two decades, some newly developed cytotoxic agents, such as paclitaxel, docetaxel, gemcitabine, and vinorelbine, have appeared to offer multiple choices for treatment of patients with advanced NSCLC. Unfortunately, such regimens show only a modest survival benefit as compared with conventional cisplatin-based therapies (NPL4-5). The concept of specific molecular targeting using therapeutic monoclonal antibodies and other small molecular agents has been applied to the development of a reformative cancer treatment strategy (NPL6). Many molecular targeting therapies have been investigated in phase II and phase III trials for the chemotherapy of advanced lung cancers; examples include tyrosine kinase inhibitors of epidermal growth factor such as gefitinib and erlotinib; tyrosine kinase inhibitors of vascular endothelial growth factor such as vandetanib, sorafenib, sunitinib; and monoclonal antibodies of epidermal growth factor or vascular endothelial growth factor such as bevacizumab and cetuximab (NPL6-10). However, these treatment programs suffer from the problem of high toxicity. Moreover, positive response remains very limited (NPL6-10).

Systematic analysis of expression levels of thousands of genes using a cDNA microarray technology has been shown to be an effective means to identify target molecules associated with carcinogenic pathways that can be candidates for development of novel therapeutics and diagnostics. To isolate potential molecular targets for diagnosis and/or treatment of NSCLC, genome-wide expression profiles of 101 lung cancer tissue samples were analyzed using a cDNA microarray containing 27,648 genes or expressed sequence tags, using tumor-cell populations purified by laser microdissection (PL1-2, NPL3, 11-12). To verify the biological and clinicopathologic significance of the respective gene products, a screening system that combines tumor tissue microarray analysis of clinical lung cancer materials with RNA interference technique has been established (NPL13-15). Through this systematic approach, a novel oncogene, Family with sequence similarity 161, member A (FAM161A) is herein identified. FAM161A is constructed 660 amino acids residues and encodes a cytoplasmic protein. At present, its molecular mechanism remains unknown.

[PL 1] WO 2004/031413
[PL 2] WO 2007/091328

[NPL 1] Ahmedin J. et al. CA Cancer J Clin 2007;57:43-66.
[NPL 2] Sozzi G. Eur J Cancer 2001;37 Suppl7:S63-73.
[NPL 3] Daigo Y. et al. Gen Thorac Cardiovasc Surg 2008;56:43-53.
[NPL 4] Kelly K. et al. J Clin Oncol 2001;19:3210-18.
[NPL 5] Schileer JH. et al. N Engl J Med 2002;346:92-8.
[NPL 6] Thatcher N. Lung Cancer 2007;57 Suppl 2:S18-23.
[NPL 7] Sandler A. N Engl J Med 2006;355:2542-50.
[NPL 8] Shephered FA. et al. N Engl J med 2005;353:123-32.
[NPL 9] Thatcher N. et al. Lancet 2005;366:1527-37.
[NPL 10] Cesare G. Et al. Oncologist 2007;12:191-200.
[NPL 11] Kikuchi T. et al. Oncogene 2003;22:2192-205.
[NPL 12] Kakiuchi S. et al. Mol Cancer Res 2003;1:485-99.
[NPL 13] Kato T. et al. Cancer Res 2005;65:5638-46.
[NPL 14] Hayama S. et al. Cancer Res 2007;67:4113-22.
[NPL 15]Taniwaki M. et al. Clin Cancer Res 2007;13:6624-31.

The present invention relates to FAM161A, and the roles it plays in carcinogenesis. As such, the present invention relates to novel compositions and methods for detecting, diagnosing, either or both of treating and preventing lung cancer, as well as methods of screening for candidate substances for cancer prevention and treatment.

Central to the present invention is the discovery that family with sequence similarity 161, member A (FAM161A) is expressed strongly in the majority of lung cancers. Furthermore, functional knockdown of endogenous FAM161A gene by siRNA in cancer cell lines resulted in suppression of cancer cell growth, suggesting its essential role in maintaining viability of cancer cells. Since it is only scarcely expressed in adult normal organs, FAM161A gene appears to be an appropriate and promising molecular target for a novel therapeutic approach with minimal adverse effect. Moreover, knockdown of endogenous FAM161A gene induced reduction of protein level of Casein kinase 2, alpha prime polypeptide (CSNK2A2), identified as FAM161A interacting protein, and exogenous FAM161A increased the protein level of CSNK2A2. Screening of the CSNK2A2 downstream protein indicates that CSNK2A2 activates the MAPK cascade; accordingly, FAM161A is presumed to play an important role in cancer cell growth through stabilization of CSNK2A2 and subsequent activation of MAPK cascade. Taken together, these results suggest FAM161A may be a promising molecular target for cancer therapy through inhibition of its expression or activity, or interaction with CSNK2A2.

Thus, it is an object the present invention is to provide a method of detecting or diagnosing lung cancer in a subject by determining an expression level of FAM161A in a subject derived biological sample. An increase in the expression level of the gene as compared to a normal control level of the gene indicates the presence of lung cancer in the subject or that the subject suffers from lung cancer.

It is another object of the present invention to provide methods of monitoring, assessing or predicting a prognosis of a subject with lung cancer, including the step of determining the expression level of the FAM161A gene in a cancerous tissue sample from the subject wherein an increase in expression of the FAM161A gene as compared to a selected control is indicative of a poor prognosis. In another aspect, the present invention provides a method of screening for a candidate substance for either or both of treating and preventing lung cancer. Such a substance would bind with the FAM161A polypeptide, reduce the expression of the FAM161A gene or a reporter gene surrogating the FAM161A gene, reduce the biological activity of the FAM161A or CSNK2A2 polypeptide, inhibit the binding between the FAM161A and the CSNK2A2 polypeptides, or inhibit the phosphorylation activity of the CSNK2A2 polypeptide.

The present invention also relates to the discovery that multiple tissue northern blot analysis identified FAM161A and CSNK2A2 expression only in testis and not in any other 22 normal tissues, and that double-stranded molecules composed of specific sequences (in particular, SEQ ID NOs: 11, 12, 13 and 14 ) are effective for inhibiting cellular growth of lung cancer cells. Accordingly, small interfering RNAs (siRNAs) targeting FAM161A and/or CSNK2A2 genes are provided by the present invention. These double-stranded molecules may be utilized in an isolated state or encoded in vectors and expressed from the vectors. Accordingly, it is an object of the present invention to provide such double stranded molecules as well as vectors and host cells expressing them.

It is a further object of the present invention to provide methods for inhibiting cell growth and treating lung cancer, by administering the double-stranded molecules or vectors of the present invention to a subject in need thereof. Such methods encompass administering to a subject in need thereof a composition composed of one or more of the double-stranded molecules or vectors of the present invention. Accordingly, the present invention encompasses compositions for treating a lung cancer, containing at least one of the double-stranded molecules or vectors of the present invention.

It is yet another of the present invention to provide a polypeptide containing a CSNK2A2-binding domain of a FAM161A polypeptide, such polypeptide inhibiting a interaction of the FAM161A with NFKBIL2 polypeptide. Preferably, the polypeptide of the present invention includes the amino acid sequence of SEQ ID NO: 32 or a functional equivalent thereof.

In summary, it is an object of the present invention to provide the following [1] to [44]
[1] A method of detecting or diagnosing cancer, or a predisposition for developing cancer in a subject, comprising determining an expression level of a FAM161A gene in a subject-derived biological sample, wherein an increase of the expression level in the subject-derived biological sample as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, wherein the expression level is determined by any one of method selected from the group consisting of:
(a) detecting an mRNA of a FAM161A gene;
(b) detecting a protein encoded by a FAM161A gene; and
(c) detecting a biological activity of a protein encoded by a FAM161A gene.
[2] The method of [1], wherein the FAM161A expression level is at least 10% greater than the normal control level.
[3] The method of [1], wherein the subject-derived biological sample is biopsy sample.
[4] A kit for detecting or diagnosing cancer or a predisposition therefor, which comprises a reagent selected from the group consisting of:
(a) a reagent for detecting an mRNA of a FAM161A gene;
(b) a reagent for detecting a protein encoded by a FAM161A gene; and
(c) a reagent for detecting a biological activity of a protein encoded by a FAM161A gene.
[5] The kit of [4], wherein the reagent comprises a probe or a primer set to the mRNA of the FAM161A gene, or an antibody against the protein encoded by the FAM161A gene.
[6] A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof;
(b) detecting the binding activity between the polypeptide or the functional equivalent and the test substance; and
(c) selecting the test substance that binds to the polypeptide or the functional equivalent.
[7] A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing a FAM161A gene;
(b) detecting an expression level of the FAM161A gene in the cell of the step (a); and
(c) selecting the test substance that reduces the expression level detected in the step (b) in comparison with the expression level of the FAM161A gene detected in the absence of the test substance.
[8] A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof;
(b) detecting a biological activity of the polypeptide or the functional equivalent of the step (a); and
(c) selecting the test substance that suppresses the biological activity detected in the step (b) in comparison with the biological activity detected in the absence of the test substance.
[9] The method of [8], wherein the biological activity is a cell proliferation enhancing activity or a binding activity to CSNK2A2 polypeptide.
[10] A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a candidate substance with a cell into which a vector, comprising the transcriptional regulatory region of FAM161A gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) selecting the test substance that reduces the expression or activity level of said reporter gene as compared to a control.
[11] A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a FAM161A polypeptide or a functional equivalent thereof with a CSNK2A2 polypeptide or a functional equivalent thereof in the presence of a test substance;
(b) detecting binding between the polypeptide(s) or the functional equivalent(s); and
(c) selecting the test substance that inhibits binding between the polypeptide(s) or the functional equivalent(s).
[12] The method of [11], wherein the functional equivalent of FAM161A polypeptide comprise a CSNK2A2-binding domain of the FAM161A polypeptide.
[13] The method of [12], wherein the CSNK2A2-binding domain of the FAM161A polypeptide comprise the position 342 to 363 of SEQ ID NO: 18.
[14] The method of [11], wherein the functional equivalent of CSNK2A2 polypeptide comprise a FAM161A-binding domain of the CSNK2A2 polypeptide.
[15] A method of screening for a candidate substance for either or both of treating and preventing cancer or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing FAM161A gene and CSNK2A2 gene;
(b) detecting the CSNK2A2 polypeptide level in the cell of step (a); and
(c) selecting the test substance that decreases the CSNK2A2 polypeptide level of step (b) in comparison with the CSNK2A2 polypeptide level detected in the absence of the test substance.
[16] A method of screening for a candidate substance for either or both of treating and preventing cancer or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a cell expressing CSNK2A2 and ERK1 and/or ERK2 polypeptide with a test substance;
(b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide; and
(c) selecting the test substance that reduces the phosphorylation level of the ERK1 and/or ERK2 polypeptide in comparison with the phosphorylation level detected in the absence of the test substance.
[17] A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a CSNK2A2 polypeptide or a functional equivalent thereof with an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof in the present of a test substance under a condition that allows phosphorylation of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
(b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
(c) comparing the phosphorylation level detected in the step (b) with the phosphorylation level detected in the absence of the test substance; and
(d) selecting the test substance that reduces the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent as a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth.
[18] The method of [17], wherein the functional equivalent of ERK1 or ERK2 polypeptide comprises a fragment of the ERK1 or ERK2 polypeptide having at least one serine or threonine phosphorylation site.
[19] A kit for measuring a phosphorylation activity of a CSNK2A2 polypeptide, wherein the kit comprises the following components (a) and (b):
(a) an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof; and
(b) a reagent for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent thereof.
[20] A kit for detecting for the ability of a test substance to reduce a phosphorylation level of an ERK1 and/or ERK2 polypeptide by a CSNK2A2 polypeptide, wherein the kit comprises the following components of (a) to (c):
(a) a CSNK2A2 polypeptide or a functional equivalent thereof;
(b) an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof; and
(c) a reagent for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent thereof.
[21] The kit of [19] or [20], wherein the functional equivalent of the ERK1 or ERK2 polypeptide comprises a fragment of the ERK1 or ERK2 polypeptide having at least one serine or threonine phosphorylation site.
[22] An isolated double-stranded molecule that, when introduced into a cell expressing FAM161A and/or CSNK2A2 gene, inhibits an expression of a FAM161A or CSNK2A2 gene as well as cell proliferation, said molecule comprising a sense strand and an antisense strand complementary thereto, said strands hybridized to each other to form the double-stranded molecule, the sense strand comprises the nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13 and 14.
[23] The double-stranded molecule of [22], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pairs in length.
[24] The double-stranded molecule of [22] or [23], which consists of a single polynucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[25] The double-stranded molecule of [24], which has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from a group consisting of SEQ ID NOs: 11, 12, 13 and 14, [B] is an intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is an antisense strand including a complementary sequence to the target sequence.
[26] A vector encoding the double-stranded molecule of any one of [22] to [25].
[27] Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to a target sequence of SEQ ID NO: 11, 12, 13 or 14 and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing FAM161A and/or CSNK2A2 gene, inhibits the cell proliferation.
[28] A method for either or both of treating and preventing cancer, wherein the method comprises the step of administering an pharmaceutically effective amount of at least one isolated double-stranded molecule against FAM161A or CSNK2A2 gene, or vector encoding the double-stranded molecule, wherein the double-stranded molecule that, when introduced into a cell expression FAM161A and/or CSNK2A2 gene, inhibits an expression of FAM161A or CSNK2A2 gene as well as cell proliferation.
[29] The method of [28], wherein the double-stranded molecule is that of any one of [22] to [25].
[30] The method of [28], wherein the vector is that of [26] or [27].
[31] A composition for either or both of treating and preventing cancer, wherein the composition comprises at least one isolated double-stranded molecule against FAM161A or CSNK2A2 gene, or vector encoding the double-stranded molecule, wherein the double-stranded molecule that, when introduced into a cell expression FAM161A and/or CSNK2A2 gene, inhibits an expression of FAM161A or CSNK2A2 gene as well as cell proliferation.
[32] The composition of [31], wherein the double-stranded molecule is that of any one of [22] to [25].
[33] The composition of [31], wherein the vector is that of [26] or [27].
[34] A method for monitoring, assessing or predicting a prognosis of a subject with cancer, wherein said method comprises a step of determining an expression level of the FAM161A gene in a subject-derived biological sample, wherein an increase of said expression level compared to a good prognosis control level of the FAM161A gene indicates a poor prognosis of said subject, wherein said expression level is determined by a method selected from the group consisting of:
(a) detecting an mRNA of the FAM161A gene;
(b) detecting a FAM161A polypeptide; and
(c) detecting a biological activity of a FAM161A polypeptide.
[35] A kit for use in monitoring, assessing or predicting a prognosis of a subject with cancer, wherein said kit comprises at least one reagent selected from the group consisting of:
(a) a reagent for detecting an mRNA of the FAM161A gene;
(b) a reagent for detecting a FAM161A protein; and
(c) a reagent for detecting a biological activity of a FAM161A protein.
[36] The kit of [35], wherein the reagent comprises an oligonucleotide that has a complementary sequence to a part of an mRNA of the FAM161A gene and specifically binds to said mRNA; or an antibody against the FAM161A protein.
[37] A polypeptide comprising a CSNK2A2-binding domain of a FAM161A polypeptide, wherein the polypeptide lacks a biological function of the FAM161A polypeptide, and wherein the biological function is a function to control a subcellular localization of a CSNK2A2 polypeptide.
[38] The polypeptide of [37], wherein the polypeptide is selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO:32;
b) a polypeptide that comprises an amino acid sequence of SEQ ID NO:32 in which one or more amino acids are substituted, deleted, inserted, and/or added; and
c) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:32.
[39] The polypeptide of [37] or [38], which is modified with a cell-membrane permeable substance.
[40] A polynucleotide encoding the polypeptide of [37] or [38].
[41] A vector encoding the polypeptide of [37] or [38].
[42] A method of either or both of treating and preventing cancer in a subject, wherein the method comprises the step of administering to the subject a pharmaceutically effective amount of the polypeptide of any one of [37] to [39] or the vector of [41].
[43] A composition for either or both of treating and preventing cancer, wherein the composition comprises a pharmaceutically effective amount of the polypeptide of any one of [37] to [39] or the vector of [41].
[44] The method of any one of [1] to [3], [6] to [18], [28] to [30], [34], the kit of any one of [4], [5], [35] and [36] or the composition of any one of [31] to [33] and [43], wherein the cancer is lung cancer.

It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention.

It will also be understood that both the foregoing summary of the present invention and the following detailed description are of exemplified embodiments, and not restrictive of the present invention or other alternate embodiments of the present invention. Other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.

Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and its preferred embodiments that follows:
Figure 1 demonstrates the expression of FAM161A in lung tumors and in normal tissues. Part A demonstrates the expression of FAM161A in 15 clinical lung cancers [lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCLC), and small-cell lung cancer (SCC)] and 15 lung cancer cell lines, examined by semiquantitative RT-PCR. Expression of beta-actin (ACTB) served as a quantity control. Part B demonstrates the expression of FAM161A in normal tissues, detected by Northern blot analysis. Part C depicts the subcellular localization of the FAM161A proteins on SBC-5 cells at interphase and mitotic phase examined by confocal microscopy. Part D demonstrates the expression of FAM161A in five normal human tissues as well as lung cancer tissues [lung adenocarcinoma (ADC), squamous cell carcinoma (SCC), large cell carcinoma (LCC), and small cell lung cancer (SCLC)], detected by immunohistochemical staining using the rabbit polyclonal anti-FAM161A antibody; counterstaining with hematoxylin (X 200). Part E demonstrates the subcellular localization of FAM161A in mammalian cells. With regard to specificity of the FAM161A antibody, there is downregulations of FAM161A protein in SBC-5 cells transfected with siRNAs against FAM161A compared to SBC-5 cells transfected with siRNAs against EGFP. Figure 2 demonstrates the expression of CSNK2A2 in lung tumors and in normal tissues.Part A demonstrates the expression of CSNK2A2 protein in 6 lung cancer cell lines and normal epithelial cells by western blot analysis. Part B demonstrates the subcellular localization of endogenous CSNK2A2 protein in lung cancer cells. CSNK2A2 is stained at the cytoplasm of the cell in LC319, but not in A549 cells. Part C demonstrates the expression of CSNK2A2 in normal tissues, detected by Northern blot analysis. Figure 3 demonstrates the inhibition of growth of NSCLC cells by siRNAs against FAM161A and CSNK2A2. Part A demonstrates the expression of FAM161A in response to siRNA treatment for FAM161A (si-FAM161A-#A or si-FAM161A-#B) or control siRNAs [si-enhanced green fluorescent protein (si-EGFP) or si-luciferase (si-LUC)] in SBC5 and LC319cells, analyzed by semiquantitative RT-PCR (top panels). MTT assays of the tumor cells transfected with si-FAM161As or control siRNAs (bottom panels). Part B demonstrates the expression of CSNK2A2 in response to siRNA treatment for CSNK2A2 (si-CSNK2A2-#2 or si-CSNK2A2-#3) or control siRNAs (si-EGFP or si-LUC) in LC319cells, analyzed by semiquantitative RT-PCR (top panels). MTT assays of the tumor cells transfected with si-CSNK2A2s or control siRNAs (bottom panels). Figure 4 demonstrates the results of flow cytometric analysis of the SBC5 and LC319 cells at 72 hours after transfection of the siRNAs for FAM161A (si-FAM161A-#B) and control siRNAs (si-EGFP). Transfection of si-FAM161A-#B resulted in subsequent increase of sub-G1fraction at 72 hours (bottom panels). Figure 5 demonstrates the interaction of FAM161A with CSNK2A2 in lung cancer cells. Part A demonstrates the co-localization of exogenous FAM161A and exogenous CSNK2A2 in COS-7 cells. Part B demonstrates the immunoprecipitation of exogenous FAM161A and CSNK2A2 from COS-7 cell (left panels), and immunoprecipitation of exogenous FAM161A and CSNK2A2 from SBC5 cell (middle, right panels) IP, immunoprecipitation. Figure 6 demonstrates the enhancement of cellular growth by FAM161A and CSNK2A2 introduction into mammalian cells. Part A demonstrates the transient expression of FAM161A and CSNK2A2 in COS-7 cells (left) and HEK293T cells (right) detected by western blot analysis. Part B demonstrates the cell viability evaluated by the MTT assay. Assays were done thrice and in triplicate wells. Figure 7 demonstrates the identification of a FAM161A-interacting protein CSNK2A2 that stabilized by FAM161A protein, and ERK1/2 phosphorylation by CSNK2A2 protein. Part A demonstrates the levels of exogenous CSNK2A2 proteins and endogenous CSNK2A2 transcripts and proteins, detected by western blot analysis and semiquantitative RT-PCR analysis in COS-7, HEK293T and SBC5 cells. Part B demonstrates the levels of endogenous CSNK2A2 and FAM161A gene and proteins, detected by western blot analysis in LC319 and SBC5 cells that were initially transfected with si-FAM161A. Part C demonstrates the phosphorylation of ERK1/2 by exogenous CSNK2A2 in COS-7 cell (left), and dephosphorylation of endogenous ERK1/2 protein in SBC5 and LC319 cells transfected with si-CSNK2A2 (right). IB, immunoblot. Part D demonstrates the cognate interaction of exogenous FAM161A with endogenous CSNK2A2 in LC319 cells by Immunoprecipitation experiments. Part E demonstrates the cognate interaction of exogenous FAM161A with endogenous CSNK2A2 in SBC-5 cells by Immunoprecipitation experiments. Figure 8 demonstrates the identification of CSNK2A2-interacting region in FAM161A and inhibition of growth of lung cancer cells by dominant-negative peptides of FAM161A. Part A is a schematic drawing of eight N-terminal Flag-tagged FAM161A partial proteins constructs lacking either or both of the terminal regions. Part B demonstrates the identification of the region in FAM161A that binds to CSNK2A2 by immunoprecipitation experiments using LC319 cells transfected with Flag-tagged FAM161A partial protein expression vector. Part C demonstrates the identification of the region in FAM161A that binds to CSNK2A2 by immunoprecipitation experiments using LC319 cells transfected with Flag-tagged FAM161A partial protein expression vector. The area where is crossover between FAM161A 294-373 construct and FAM161A 331-440 construct were indicated to be FAM161A-interacting region. Part D is a schematic drawing of three cell permeable peptides of FAM161A covering FAM161A 331-373 that corresponds to the CSNK2A2-interacting region in FAM161A. Part E demonstrates the reduction of the complex formation between exogenous FAM161A and endogenous CSNK2A2 proteins, detected by Immunoprecipitation assay in LC319 cells that were treated with the P2-FAM161A 342-363 peptides. Part F depicts the results of an MTT assay showing growth suppressive effect of P2-FAM161A 342-363 peptides that were introduced into LC319 cells that expressed both FAM161A and CSNK2A2 proteins. Part G depicts the results of an MTT assay showing no off-target effect of the P2-FAM161A 342-363 peptides on BEAS-2B cells that were not expressed FAM161A and CSNK2A2 mRNA. Part H demonstrates the expressions of FAM161A and CSNK2A2 mRNA in lung cancer LC319 cells and normal human bronchial epithelial cell line BEAS-2B cells, examined by RT-PCR analysis. Figure 9 demonstrates the association of FAM161A overexpression with poor prognosis for NSCLC patients. Part A provides representative examples for positive and negative FAM161A expression in lung ADC tissues and a normal lung tissue (original magnification, X 100). Part B demonstrates the Kaplan-Meier analysis of survival of patients with NSCLC (P = 0.0017, log-rank test).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

The disclosure of each publication, patent or patent application mentioned in this specification is specifically incorporated by reference herein in its entirety. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definition
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.

The terms "isolated" and "purified" used in relation with a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates that the substance is substantially free from at least one substance that can be included in the natural source. Thus, an isolated or purified polypeptide refers to a polypeptide that are substantially free of cellular material for example, carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, in some embodiments it is also substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In one embodiment, proteins including antibodies of the present invention are isolated or purified.

As used herein, the phrase "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or polynucleotides. In the context of the present invention, a biological sample may preferably contain a lung tissue or lung cells.

The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.

Amino acids can be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms "gene", "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleic acid residues and, unless otherwise specifically indicated are referred to by their commonly accepted single-letter codes. The terms apply to nucleic acid (nucleotide) polymers in which one or more nucleic acids are linked by ester bonding. The nucleic acid polymers may be composed of DNA, RNA or a combination thereof and encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.

In the context of the present invention, the phrase "FAM161A gene" encompasses polynucleotides that encode the human FAM161A or any of the functional equivalents of the human FAM161A gene. Likewise, the phrase "CSNK2A2 gene" encompasses polynucleotides that encode the human CSNK2A2 or any of the functional equivalents of the human CSNK2A2 gene. The phrase "ERK1 gene" encompasses polynucleotides that encode the human ERK1 or any of the functional equivalents of the human ERK1 gene. The phrase "ERK2 gene" encompasses polynucleotides that encode the human ERK2 or any of the functional equivalents of the human ERK2 gene.

The FAM161A gene, the CSNK2A2 gene, the ERK1 gene and the ERK2 gene can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.

Unless otherwise defined, the terms "cancer" refers to cancers over-expressing the FAM161A gene. Examples of cancers over-expressing FAM161A include, but are not limited to, lung cancer, including NSCLC and SCLC. Furthermore, NSCLC includes adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC).

To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels". While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.

To the extent that certain embodiments of the present invention encompass the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence, such methods may include any of the following steps: the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effective treatment and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. A treatment may also deemed "efficacious" if it leads to clinical benefit such as, reduction in expression of the FAM161A gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.

Genes and Polypeptides
The present invention is based in part on the discovery that the gene encoding FAM161A is over-expressed in cancer as compared to non-cancerous tissue and FAM161A interacts with CSNK2A. The present invention is further based on the discovery that CSNK2A activates MAPK cascade through the phosphorylation of ERK1 and/or ERK2.

The FAM161A (family with sequence similarity 161, member A) polypeptide is constructed about 660 amino acids residues and encodes a cytoplasmic protein. The CSNK2A2 (casein kinase 2, alpha prime polypeptide) polypeptide is one of protein kinase CK2 (casein kinase 2) subunits that are a ubiquitous and pleiotropic serine/threonine phosphotransferase which is highly conserved throughout eukaryotes. CK2 is well known protein complex and is considered the target of cancer therapy. The ERK1 polypeptide (extracellular signal-regulated kinases 1: also referred to as MAPK3) and the ERK2 polypeptide (extracellular signal-regulated kinases 2: also referred to as MAPK1) are members of the MAP kinase family. MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. The activation of MAP kinases requires their serine/threonine phosphorylation by upstream kinases. Molecular weights of ERK1 polypeptide and ERK2 polypeptide are 44kDa and 42kDa, respectively.

Nucleic acid and polypeptide sequences of the above mentioned genes of interest to the present invention include, but are not limited to, the following examples:
FAM161A: SEQ ID NO: 17 and 18;
CSNK2A2: SEQ ID NO: 19 and 20;
ERK1: SEQ ID NO: 21, 22, 23, 24, 25 and 26; and
ERK2: SEQ ID NO: 27, 28 and 29.

Additional sequence data is available via following accession numbers:
FAM161A: NM_032180.2;
CSNK2A2: NM_001896.2;
ERK1: NM_001109891, NM_001040056 and NM_002746.2; and
ERK2: NM_002745.4 and NM_138957.2.

The present invention contemplates "functional equivalents" and deems such to be "polypeptides" in context. Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to that of the original reference protein. Namely, any polypeptide that retains the biological ability of the original reference peptide may be used as such a functional equivalent or a functional fragment in the present invention. Such functional equivalents include those wherein one or more amino acids are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the protein. Alternatively, the polypeptide may be composed an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the respective protein, more preferably at least about 90% to 95% homology, even more preferably 96%, 97%, 98% or 99% homology. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the naturally occurring nucleotide sequence of the gene.

The present invention also contemplates functional equivalents of the FAM161A polypeptide, CSNK2A2 polypeptide, ERK1 polypeptide and ERK2 polypeptide. These polypeptides may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a functional equivalent to that of the original reference, it is within the scope of the present invention.

Examples of functional equivalents of FAM161A polypeptide include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the FAM161A protein. Also, functional equivalents of CSNK2A2 polypeptide include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the CSNK2A2 protein. Also, functional equivalents of ERK1 polypeptide or ERK2 polypeptide include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the ERK1 protein or ERK2 protein.

In other embodiments, functional equivalents of above polypeptides can be encoded by a polynucleotide that hybridizes under stringent conditions to the natural occurring nucleotide sequence of the FAM161A gene, CSNK2A2 gene, ERK1 gene or ERK2 gene.

The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing substances such as formamide. For selective or specific hybridization, a positive signal is at least two times of background, preferably 10 times of background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.

In the context of the present invention, the optimal condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the above human protein can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions are often preferably used. An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can routinely adjust these and other factors to arrive at the desired stringency.

It is generally known that a modification of one, two or more amino acid in a protein will not influence the function of the protein; in some cases, it may even enhance the desired function of the original protein. In fact, mutated or modified proteins (i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition) have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).

Accordingly, one of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence which alter a single amino acid or a small percentage of amino acids (i.e., less than 5%, more preferably less than 3%, even more preferably less than 1%) or those considered to be a "conservative modifications", wherein the alteration of a protein results in a protein with similar functions, are acceptable in the context of the instant invention. Thus, the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in an originally disclosed reference sequence.

So long as the activity the protein is maintained, the number of amino acid mutations or modifications is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence, more preferably less than 3%, even more preferably less than 1%. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 or 6 amino acids or less, and even more preferably 3 or 4 amino acids or less.

An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified polypeptides are included in the present protein. However, the present invention is not restricted thereto and includes non-conservative modifications, so long as the resulting modified peptide retains at least one biological activity of the original protein. In the context of the present invention, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

An example of a protein modified by addition of one or more amino acids residues is a fusion protein of the FAM161A, CSNK2A2, ERK1 or ERK2 protein. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the FAM161A, CSNK2A2, ERK1 or ERK2 gene with a DNA encoding another peptide or protein, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. The "other" component of the fusion protein is typically a small epitope composed of several to a dozen amino acids. There is no restriction as to the peptides or proteins fused to the FAM161A, CSNK2A2, ERK1 or ERK2 protein so long as the resulting fusion protein retains any one of the objective biological activities of the FAM161A, CSNK2A2, ERK1 or ERK2 proteins. Exemplary fusion proteins contemplated by the instant invention include fusions of the FAM161A, CSNK2A2, ERK1 or ERK2 protein and other small peptides or proteins such as FLAG (Hopp TP, et al., Biotechnology 6: 1204-10 (1988)), a polyhistidine (His-tag) such as 6xHis containing six His (histidine) residues or 10xHis containing 10 His residues, Influenza aggregate or agglutinin (HA), human c-myc fragment, Vesicular stomatitis virus glycoprotein (VSV-GP), p18HIV fragment, T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, and the like. Other examples of proteins that can be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such. Other examples of modified proteins contemplated by the present invention include polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

In addition to peptides and proteins, the present invention encompasses genes and polynucleotides that encode such functional equivalents and functional fragments of the protein. In addition to hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the protein, using a primer synthesized based on the sequence above information. Polynucleotides and polypeptides that are functionally equivalent to the human gene and protein, respectively, normally have a high homology to the originating nucleotide or amino acid sequence of. "High homology" typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 90% to 95% or higher, even more preferably 96% to 99% or higher. The homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".

Percent sequence identity and sequence similarity can be readily determined using conventional techniques such as the BLAST and BLAST 2.0 algorithms, which are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). Such algorithms involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.

The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).

A protein useful in the context of the present invention can have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has any one of the biological activities of the FAM161A protein (SEQ ID NO: 18), CSNK2A2 protein (SEQ ID NO: 20), ERK1 protein (SEQ ID NO:22, 24, 26) or ERK2 (SEQ ID NO: 29) it is useful in the present invention.

The present invention also encompasses partial peptides of the FAM161A protein, the CSNK2A2 protein, the ERK1 protein and ERK2 protein and their use in screening methods. A partial peptide having an amino acid sequence specific to the FAM161A protein, the CSNK2A2 protein, ERK1 protein or ERK2 protein is preferably composed of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.

A partial FAM161A peptide used for screenings in accordance with the present invention typically contains, at a minimum, at least one binding domain of FAM161A, more preferably CSNK2A2 binding region. Such partial peptides are also encompassed by the phrase "functional equivalent" of the FAM161A protein. In a similar fashion, a partial CSNK2A2 peptide suitable for use in connection with screenings of the present invention typically contains, at a minimum, at least one binding domain of CSNK2A2, more preferably the FAM161A binding region. Such partial peptides are also encompassed by the phrase "functional equivalent" of the CSNK2A2 protein.

The polypeptide or fragments used for the present method can be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
(1) Peptide Synthesis, Interscience, New York, 1966;
(2) The Proteins, Vol. 2, Academic Press, New York, 1976;
(3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
(4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
(5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
(6) WO99/67288; and
(7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, the protein can be obtained adopting any known genetic engineering methods for producing polypeptides (e.g., Morrison DA., et al., J Bacteriol. 1977 Oct;132(1):349-51; Clark-Curtiss JE & Curtiss R 3rd. Methods Enzymol. 1983;101:347-62). For example, first, a suitable vector including a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence including a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the FAM161A, CSNK2A2, ERK1 or ERK2 is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.

In addition to the protein of interest, the vector may also contain a promoter to induce protein expression. Any commonly used promoters can be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF- alpha promoter (Kim DW, et al. Gene. 1990 Jul 16;91(2):217-23), the CAG promoter (Niwa H, et al., Gene. 1991 Dec 15;108(2):193-9), the RSV LTR promoter (Cullen BR. Methods Enzymol. 1987;152:684-704), the SR alpha promoter (Takebe Y, et al., Mol Cell Biol. 1988 Jan;8(1):466-72), the CMV immediate early promoter (Seed B & Aruffo A. Proc Natl Acad Sci U S A. 1987 May;84(10):3365-9), the SV40 late promoter (Gheysen D & Fiers W. J Mol Appl Genet. 1982;1(5):385-94), the Adenovirus late promoter (Kaufman RJ, et al., Mol Cell Biol. 1989 Mar;9(3):946-58), the HSV TK promoter, and the like.

The introduction of the vector into host cells to express the FAM161A, CSNK2A2, ERK1 or ERK2 gene can be performed according to any methods, for example, the electroporation method (Chu G, et al., Nucleic Acids Res. 1987 Feb 11;15(3):1311-26), the calcium phosphate method (Chen C & Okayama H. Mol Cell Biol. 1987 Aug;7(8):2745-52), the DEAE dextran method (Lopata MA, et al., Nucleic Acids Res. 1984 Jul 25;12(14):5707-17; Sussman DJ & Milman G. Mol Cell Biol. 1984 Aug;4(8):1641-3), the Lipofectin method (Derijard B, et al., Cell. 1994 Mar 25;76(6):1025-37; Lamb BT, et al., Nat Genet. 1993 Sep;5(1):22-30; Rabindran SK, et al., Science. 1993 Jan 8;259(5092):230-4), and the like.
The proteins can also be produced in vitro by using an in vitro translation system.

Method of Detecting or Diagnosing Lung Cancer:
As demonstrated herein, the expression of FAM161A is significantly and specifically elevated in lung cancer cells (Fig. 1). Thus, the genes identified herein as well as their transcription and translation products find utility as diagnostic markers for lung cancer. Accordingly, by measuring the expression of FAM161A in a cell sample, lung cancer can be diagnosed. Specifically, the present invention provides a method for detecting, diagnosing and/or determining the presence of or a predisposition for developing lung cancer by determining the expression level of FAM161A in the subject. Lung cancers that can be diagnosed by the present method include NSCLC and SCLC. In the context of the present invention, "NSCLC" includes adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large-cell carcinoma (LCC).

According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from the disease. Alternatively, the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.

The present invention also provides a method for detecting or identifying cancer cells in a subject-derived lung tissue sample, said method including the step of determining the expression level of the FAM161A in the subject-derived tissue sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the subject-derived tissue sample.

Such result may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease. In other words, the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease. For example, according to the present invention, when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the FAM161A, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc. For example, some well-known diagnostic lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA. Namely, in this particular embodiment of the present invention, the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.

Particularly preferred embodiments of the present invention are set forth below as items [1] to [11]:
[1] A method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject, said method including the steps of:
(a) detecting the expression level of the FAM161A gene in a subject-derived biological sample; and
(b) correlating an increase in the expression level detected as compared to a normal control level of the gene to the presence of disease;
[2] The method of [1], wherein the expression level is at least 10% greater than the normal control level.
[3] The method of [1] or [2], wherein the expression level is detected by a method selected from among:
(a) detecting an mRNA of a FAM161A gene,
(b) detecting a protein encoded by a FAM161A gene, and
(c) detecting a biological activity of a protein encoded by a FAM161A gene;
[4] The method of [1], wherein the cancer is lung cancer;
[5] The method of [3] or [4], wherein the expression level is determined by detecting hybridization of a probe to the mRNA of the FAM161A gene;
[6] The method of [3] or [4], wherein the expression level is determined by detecting the binding of an antibody against the protein encoded by the FAM161A gene;
[7] The method of any one of [1] to [6], wherein the biological sample includes biopsy sample, sputum, blood, pleural effusion or urine;
[8] The method of any one of [1] to [7], wherein the subject-derived biological sample includes a lung epithelial cell;
[9] The method of [1], wherein the subject-derived biological sample includes a lung cancer cell;
[10] The method of [1], wherein the subject-derived biological sample includes a lung cancerous epithelial cell; and
[11] The method of [1], wherein the subject-derived biological sample includes a lung tissue or lung cells.

The method of diagnosing cancer is described in more detail below.
A subject to be diagnosed by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.

It is preferable to collect a biological sample from the subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it includes the objective transcription or translation product of FAM161A gene. Examples of suitable biological samples include, but are not limited to, bodily tissues which are desired for diagnosing or are suspicion of suffering from lung cancer, and fluids, such as biopsy, blood, sputum, pleural effusion and urine. Preferably, the biological sample contains a cell population including an epithelial cell, more preferably a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell may be purified from the obtained bodily tissues and fluids, and then used as the biological sample.

According to the present invention, the expression level of FAM161A gene in the subject-derived biological sample is determined and then correlated to a particular healthy or disease state by comparison to a control sample. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, FAM161A mRNA may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes (e.g., lung cancer specific genes) including FAM161A gene. Those skilled in the art can prepare such probes utilizing the known sequence information for the FAM161A gene (SEQ ID NO 17). For example, the cDNA of FAM161A may be used as the probes. If necessary, the probe may be labeled with a suitable label, such as dyes, fluorescent and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.

Alternatively, the transcription product of FAM161A gene may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primer pairs (SEQ ID NOs: 1 and 2 or 7 and 8) used in the Example may be employed for the detection by RT-PCR or Northern blot, but the present invention is not restricted thereto.

A probe or primer suitable for use in the context of the present method will hybridize under stringent, moderately stringent, or low stringent conditions to the mRNA of FAM161A gene. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Alternatively, diagnosis may involve detection of a translation product. For example, the quantity of FAM161A protein may be determined and correlated to a disease or normal state. The quantity of the translation products/proteins may be determined using, for example, immunoassay methods that use an antibody specifically recognizing the protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment retains the binding ability to FAM161A protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and fragments thereof.

Alternatively, the intensity of staining may be observed via immunohistochemical analysis using an antibody against FAM161A protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of FAM161A.

Alternatively, cell proliferation enhancing activity may be correlated to the FAM161A gene expression level. As discovered herein, inhibiting the expression of FAM161A gene leads to suppression of cell growth in lung cancer cells; as such, the FAM161A protein is presumed to promote cell proliferation. Thus, to determine the cell proliferation enhancing activity of FAM161A protein, a cell is first cultured in the presence of a biological sample. Then, by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability, the cell proliferation enhancing activity of the biological sample can be determined and the relative FAM161A expression correlated thereto.

In the context of the present invention, methods for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begin with a determination of FAM161A gene expression level. Once determined, using any of the aforementioned techniques, this value is as compared to a control level.

In the context of the present invention, the phrase "control level" refers to the expression level of a test gene detected in a control sample and encompasses both a normal control level and a cancer control level. The phrase "normal control level" refers to a level of gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer. A normal individual is one with no clinical symptom of lung cancer. A normal control level can be determined using a normal cell obtained from a non-cancerous tissue. A "normal control level" may also be the expression level of a test gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from lung cancer or esophageal cancer. On the other hand, the phrase "cancer control level" refers to an expression level of a test gene detected in the cancerous tissue or cell of an individual or population suffering from lung or esophageal cancer. An increase in the expression level of FAM161A detected in a subject-derived sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing lung cancer. In the context of the present invention, the subject-derived sample may be any tissues obtained from test subjects, e.g., patients known to have or suspected of having cancer. For example, tissues may include epithelial cells. More particularly, tissues may be epithelial cells collected from a suspected cancerous area. Alternatively, the expression level of FAM161A in a sample can be compared to a cancer control level of FAM161A gene. A similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer. When the expression levels of other cancer-related genes are also measured and compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.

The control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of FAM161A gene in samples from subjects whose disease state are known. Furthermore, the control level can be a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of FAM161A gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample. Moreover, it is preferred, to use the standard value of the expression levels of FAM161A gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.

To improve the accuracy of the diagnosis, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in lung cancer may also be determined, in addition to the expression level of the FAM 161A gene. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing lung cancer.

In the context of the present invention, gene expression levels are deemed to be "altered" or "increased" when the gene expression changes or increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level. Accordingly, the expression level of lung cancer marker genes including FAM161A gene in a biological sample can be considered to be increased if it increases from a control level of the corresponding lung cancer marker gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.

Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.

Such result may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease. In other words, the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease. For example, according to the present invention, when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the FAM161A gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc. For example, some well-known diagnostic lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA. Namely, in this particular embodiment of the present invention, the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.

To facilitate the afore-mentioned uses, the present invention provides diagnostic reagents for diagnosing cancer. Such reagent can be selected from among:
(a) a reagent for detecting mRNA of the FAM161A gene;
(b) a reagent for detecting the FAM161A protein; and
(c) a reagent for detecting the biological activity of the FAM161A protein.

Specifically, such reagent is an oligonucleotide that hybridizes to the FAM161A polynucleotide, or an antibody that binds to the FAM161A polypeptide.
In other words, the present invention also provides a kit for use in diagnosis or detection of cancer, wherein the kit includes a reagent that binds to a transcription or translation product of the FAM161A gene.

The findings of the present invention reveal that FAM161A is not only a useful diagnostic marker, but also suitable target for cancer therapy. Therefore, cancer treatment targeting FAM161A can be achieved by the present invention. In the present invention, the cancer treatment targeting FAM161A refers to suppression or inhibition of FAM161A activity and/or expression in the cancer cells. Any anti- FAM161A agents may be used for the cancer treatment targeting FAM161A. In the present agents may be used for the cancer treatment targeting FAM161A. In the present invention, the anti- FAM161A agents include following substance as active ingredient:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof.
Accordingly, in a preferred embodiment, the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of:
a) determining the expression level of FAM161A gene in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of FAM161Agene with a normal control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of FAM161A is increased as compared to the normal control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
Alternatively, such a method may include the steps of:
a) determining the expression level of FAM161A gene in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of FAM161A gene with a cancerous control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of FAM161A gene is similar or equivalent to the cancerous control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).

Method for Assessing the Prognosis of a Subject with Cancer
The present invention is based, in part, on the discovery that FAM161A (over)expression is significantly associated with poorer prognosis of subjects with cancer. Thus, the present invention provides a method for predicting, monitoring or assessing the prognosis of a subject with cancer, by determining the expression level of the FAM161A gene in a subject-derived biological sample, wherein an increase of said expression level as compared to a good prognosis control level of the FAM161A gene is indicative of a poor prognosis (poor survival).

Herein, the term "prognosis" refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.

The terms "assessing (or predicting) the prognosis" refer to predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the subject (e.g., malignancy, likelihood of curing cancer, estimated time of survival, and the like). For example, a determination of the expression level of FAM161A over time enables a predicting of an outcome for the subject (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).

In the context of the present invention, the phrase "assessing (or predicting) the prognosis" is intended to encompass predictions and likelihood analysis of cancer, progression, particularly cancer recurrence, metastatic spread and disease relapse. The present method for predicting or assessing prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria for example, disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.

Specifically, the present invention provides the following methods [1] to [8]:
[1] A method for predicting or assessing a prognosis of a subject with cancer, wherein the method includes steps of:
(a) determining an expression level of the FAM161A gene in a subject-derived biological sample;
(b) comparing the expression level determined in step (a) to a control level; and
(c) predicting the prognosis of the subject based on the comparison of (b);
[2] The method of [1], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis;
[3] The method of [1], wherein the control level is a poor prognosis control level and a similar expression level to the control level indicates poor prognosis;
[4] The method of [2], wherein the increase is at least 10% greater than said control level; and
[5] The method of any one of [1] to [4], wherein the expression level is determined by a method selected from among:
(a) detecting an mRNA of the FAM161A gene;
(b) detecting a FAM161A polypeptide; and
(c) detecting a biological activity of a FAM161A polypeptide;
[6] The method of any one of [1] to [5], wherein the subject-derived biological sample is a sample containing a cancerous tissue, or cancerous cells, or blood sample.
[7] The method of any one of [1] to [6], wherein the cancer is lung cancer; and
[8] The method of [7], wherein the lung cancer is NSCLC.
The method of predicting or assessing the prognosis of a subject with cancers will be described in more detail below.

The method of the present invention can applied to any cancer that overexpresses the FAM161A gene. Cancer is preferably lung cancer, more preferably NSCLC.
The subject-derived biological sample used for the method of the present invention can be any sample derived from the subject for predicting or assessing so long as transcription product or translation product of the FAM161A gene can be detected in the sample. For example, a subject-derived biological sample may be a bodily tissue sample or a bodily fluid sample. Examples of bodily fluid samples include sputum, blood, serum, plasma, pleural effusion, and so on. In preferred embodiments, a subject-derived biological sample is a tissue sample containing a cancerous area. For example, a lung cancer tissue sample is a preferred sample. In another preferred embodiments, a subject-derived biological sample is a subject-derived blood sample. Moreover, a subject-derived biological sample can be cells purified or obtained from a tissue. Subject-derived biological samples can be obtained from a patient at various time points, including before, during, and/or after a treatment.

According to the present invention, it was shown that the higher expression level of the FAM161A gene determined in a subject-derived biological sample, the poorer prognosis for post-treatment remission, recovery, and/or survival and the higher likelihood of poor clinical outcome. Thus, according to the present method, the "control level" used for comparison can be, for example, the expression level of the FAM161A gene determined before any kind of treatment in an individual or a population of individuals who showed good or positive prognosis, after the treatment, which herein is referred to as "good prognosis control level". Alternatively, the "control level" can be the expression level of the FAM161A gene determined before any kind of treatment in an individual or a population of individuals who showed poor or negative prognosis, after the treatment, which herein will be referred to as "poor prognosis control level". The "control level" may be a single expression pattern derived from a single reference population or from a plurality of expression patterns. Thus, the control level can be determined based on the expression level of the FAM161A gene determined before any kind of treatment in a subject with cancer, or a population of subjects whose prognosis are known. In some embodiments, the standard value of the expression levels of the FAM161Agene in a subject group with known prognosis is used. The standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.

As noted above, the control level can be determined at the same time with the test sample by using a sample(s) previously collected and stored before any kind of treatment from cancer subject(s) (control or control group) whose prognosis are known. Alternatively, the control level can be determined by a statistical method based on the results obtained by analyzing the expression level of the FAM161A gene in samples previously collected and stored from a control group. Furthermore, the control level can be a database of expression patterns from previously tested cells or subjects. Moreover, the expression level of the FAM161A gene determined in a subject-derived biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples. In some embodiments, a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample is used.

According to the present invention, a similarity between a measured or calculated expression level of the FAM161A gene and a level corresponding to a good prognosis control level indicates a more favorable patient prognosis. Likewise, an increase in the expression level as compared to the good prognosis control level indicates a less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. In the present invention, a good prognosis refers to a positive prognosis or favorable prognosis. On the other hand, a decrease in the expression level of the FAM161A gene in comparison as compared to a poor prognosis control level indicates a more favorable prognosis of the subject, with a similarity between the two indicating a less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. In the present invention, a poor prognosis refers to a negative prognosis or less favorable prognosis. In the context of the present invention, a cancer cell(s) obtained from a subject who showed good or poor prognosis of cancer after treatment is a preferable subject-derived biological sample for good or poor prognosis control level, respectively.

An expression level of the FAM161A gene in a subject-derived biological sample can be considered altered (i.e., increased or decreased) when the expression level differs from the control level by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.

The difference in the expression level between the test sample and the control level can be normalized to a control, e.g., housekeeping gene. For example, polynucleotides whose expression levels are known not to differ between the cancerous and non-cancerous cells, including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1, can be used to normalize the expression levels of the FAM161A gene.

The expression level of the FAM161A gene in a subject-derived sample can be determined by the methods described above in the section entitled "(2) Method for Diagnosing or Detecting Cancer".

Subjects to be predicted or assessed for the prognosis of cancer according to the method of the present invention can be a mammal including human, non-human primate, mouse, rat, dog, cat, horse, and cow.

Alternatively, according to the present invention, an intermediate result can also be provided in addition to other test results for assessing the prognosis of a subject with cancer. Such intermediate result can assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject and/or monitor the course of patient therapy. Additional information that can be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.

To facilitate the afore-mentioned utility, the present invention provides a reagent for assessing prognosis of cancer. Such reagent can be selected from among:
(a) a reagent for detecting mRNA of the FAM161A gene;
(b) a reagent for detecting the FAM161A polypeptide; and
(c) a reagent for detecting the biological activity of the FAM161A polypeptide.
Examples of such reagents include an oligonucleotide that hybridizes to the FAM161A polynucleotide, or an antibody that binds to the FAM161A polypeptide.

A Kit for Diagnosing Lung Cancer:
In addition to assessing the prognosis of cancer, and/or monitoring the efficacy of a cancer therapy, the present invention provides kit and methods for diagnosing lung cancer. Specifically, the kit includes at least one reagent for detecting the expression of the FAM161A gene in a subject-derived biological sample, which reagent may be selected from the group of:
(a) a reagent for detecting an mRNA of the FAM161A gene;
(b) a reagent for detecting a protein encoded by a FAM161A gene;
(c) a reagent for detecting a biological activity of a protein by a FAM161A gene; and
Suitable reagents for detecting mRNA of the FAM161A gene include nucleic acids that specifically bind to or identify the FAM161A mRNA, such as oligonucleotides that have a complementary sequence to a part of the FAM161A mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the FAM161A mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art. If needed, the reagent for detecting the FAM161A mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the FAM161A mRNA may be included in the kit.

A probe or primer of the present invention is typically a substantially purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bp of consecutive sense strand nucleotide sequence of a nucleic acid including a FAM161A sequence, or an anti sense strand nucleotide sequence of a nucleic acid including a FAM161A sequence, or of a naturally occurring mutant of these sequences. In particular, for example, in a preferred embodiment, an oligonucleotide having 5-50 bp in length can be used as a primer for amplifying the genes, to be detected. More preferably, mRNA or cDNA of a FAM161A gene can be detected with oligonucleotide probe or primer of a specific size, generally 15- 30 bp in length. In preferred embodiments, length of the oligonucleotide probe or primer can be selected from 15-25 bp. Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR). In these assays, probes or primers can also include tag or linker sequences. Further, probes or primers can be modified with detectable label or affinity ligand to be captured. Alternatively, in hybridization based detection procedures, a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).

On the other hand, suitable reagents for detecting the FAM161A protein include antibodies to the FAM161A protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used as the reagent, so long as the fragment retains the binding ability to the FAM161A protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and fragments thereof. Furthermore, the antibody may be labeled with signal generating molecules via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods may be employed for the present invention. Moreover, more than one reagent for detecting the FAM161A protein may be included in the kit.

Furthermore, the biological activity can be determined by, for example, measuring the cell proliferation enhancing activity due to the expressed FAM161A protein in the biological sample. For example, the cell may be cultured in the presence of a patient-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability or the cell proliferation enhancing activity of the biological sample can be determined. If needed, the reagent for detecting the FAM161A mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the biological activity of the FAM161A protein may be included in the kit.

The kit may contain more than one of the aforementioned reagents. Furthermore, the kit may include a solid matrix and reagent for binding a probe against the FAM161A gene or antibody against the FAM161A protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the FAM161A protein.
A kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, etc.) with instructions for use. These reagents and such may be retained in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.

According to an aspect of the present invention, the kit of the present invention for diagnosing cancer may further include either of positive or negative controls sample, or both. The positive control sample of the present invention may be established lung cancer cell lines. In a preferred embodiment, such clell lines are selected from the group consisting of:
lung adenocarcinoma (ADC) cell lines such as H1781, H1373, LC319, A549, PC14, and the like;
lung squamous cell carcinoma (SCC) cell lines such as SKMES1, H520, H1703, H2170, LU61,and the like;
small cell lung cancer (SCLC) cell lines such as SBC3, SBC5, DMS114, DMS273, and the like; and
large cell carcinoma (LCC) cell lines such as LX1 and the like.

Alternatively, the FAM161A positive samples may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s), including lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC), small cell lung cancer (SCLC), and/or large cell carcinoma (LCC). Alternatively, positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of an FAM161A mRNA or protein more than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a normal range and a cancerous range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include an FAM161A standard sample providing a cut-off value amount of an FAM161A mRNA or polypeptide. On the contrary, negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, or may be prepared by preparing a sample containing an FAM161A mRNA or protein less than cut-off value.

Likewise, the kit of the present invention for assessing the prognosis of cancer may further include either of a good prognosis control sample or a poor prognosis control sample, or both. As described in "Method for Assessing the Prognosis of a Subject of Cancer", a good control may be an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment. Meanwhile, a poor control may be an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment.

In a preferred embodiment, a good prognosis control sample may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed good or positive prognosis of lung cancer, after the treatment. In a preferred embodiment, such lung cancer tissue may be an NSCLC or SCLC tissue(s) obtained from a lung cancer patient(s). In a more preferred embodiment, such NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma (LCC) tissue(s).

Alternatively, a good prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of a FAM161A mRNA or protein less than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a good prognosis range and a poor prognosis range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include a FAM161A standard sample providing a cut-off value amount of a FAM161A mRNA or polypeptide.

On the contrary, a poor prognosis control sample may be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed poor or negative prognosis of lung cancer, after the treatment. In a preferred embodiment, such lung cancer tissue may be an NSCLC tissue(s) obtained from a lung cancer patient(s). In a more preferred embodiment, such NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma (LCC) tissue(s).

Alternatively, a poor prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of a FAM161A mRNA or protein more than the cut-off value.

As an embodiment of the present invention, when the reagent is a probe against the FAM161A mRNA, the reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one detection site. The measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid (probe). A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a strip separated from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of FAM161A mRNA present in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.

The kit of the present invention may further include positive and/or negative controls sample, and/or a FAM161A standard sample. The positive control sample of the present invention may be prepared by collecting FAM161A positive samples. Such CSNK2A2 positive samples may be obtained, for example, from established lung cancer cell lines, including lung adenocarcinoma cell (ADC) lines such as A427, NCI-H1781, A549, LC319 and the like; lung squamous cell carcinoma (SCC) cell lines such as NCI-H26, EBC-1, NCI-H520, NCI-H2170 and the like; and SCLC cell lines such as DMS114, DMS273, SBC-3, SBC-5, H196, H446 and the like. Alternatively, the FAM161A positive samples may be obtained from clinical lung cancer tissues, including lung adenocarcinoma tissues, lung squamous cell carcinoma tissues and SCLC tissues. Alternatively, positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of a FAM161A mRNA or protein more than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a normal range and a cancerous range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include a FAM161A standard sample containing a cut-off value amount of a FAM161A mRNA or polypeptide. On the contrary, negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, or may be prepared by preparing a sample containing a FAM161A mRNA or protein less than cut-off value.

Furthermore, the present invention also provides a reagent for detecting or diagnosing cancer. In preferred embodiment, such a reagent may include an oligonucleotide that hybridizes to the mRNA of the FAM161A gene, or an antibody against the protein encoded by the FAM161A gene.

Screening for an Anti-Cancer Substance
Through the present invention, it has been demonstrated that FAM161A is involved in cancer cell growth. Accordingly, substances that suppress an expression level of FAM161A gene and/or a biological activity of FAM161A polypeptide are expected to be useful for the treatment or prevention of cancer. Such substances can be screened using a FAM161A gene, polypeptides encoded by the gene, or transcriptional regulatory region of the gene. Thus, the present invention also provides a method of screening for a candidate substance for either or both of treating and preventing cancer using FAM161A gene, FAM161A polypeptide, or transcriptional regulatory region of the gene.

In the present invention, it has been further demonstrated that FAM161A polypeptide interacts with CSNK2A2 polypeptide. As demonstrated herein, FAM161A polypeptide activates MAPK cascade through the interaction with CSNK2A2 polypeptide, to induce cancer cell growth. Accordingly, substances that inhibit the interaction between FAM161A polypeptide and CSNK2A2 polypeptide are also expected to be useful for either or both of treating and preventing cancer. Such substances can be screened by identifying substances that inhibits the binding between FAM161A polypeptide and CSNK2A2 polypeptide. Thus, the present invention also provides a method of screening for a candidate substance for either or both of treating and preventing cancer by identifying a substance that inhibits the binding between FAM161A polypeptide and CSNK2A2 polypeptide.

The substances screened by the present screening method may be suitable candidate substances for either or both of treating and preventing cancer, and/or inhibiting cancer cell growth. In the present invention, the cancer is preferably characterized by an association with FAM161A overexpression. Accordingly, the screened substances may be preferably applied to the cancers correlated or associated with FAM161A overexpression. In the preferred embodiments, the cancers correlated or associated with FAM161A overexpression are lung cancer, including NSCLCs and SCLCs. NSCLCs include, lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) and lung large cell carcinoma (LCC).

In the context of the present invention, substances to be identified through the present screening methods include any compound or composition including several compounds. Furthermore, the test substance exposed to a cell or protein according to the screening methods of the present invention may be a single substance or a combination of substances. When a combination of substances is used in the methods, the substances may be contacted sequentially or simultaneously.

Any test substance, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide substances, synthetic micromolecular substances (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural substances can be used in the screening methods of the present invention. The test substance can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-substance" library method and (5) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of substances (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of substances may be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698; 5,403,484, and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Pat. Application 2002103360).

A substance or compound in which a part of the structure of the substance screened by any one of the present screening methods is converted by addition, deletion and/or replacement, is included in the substances obtained by the screening methods of the present invention.

Furthermore, when the screened test substance is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA is confirmed it's usefulness in preparing the test substance which is a candidate for treating or preventing cancer.

Test substances useful in the screenings described herein can also be antibodies that specifically bind to FAM161A or CSNK2A2 protein or partial peptides thereof that lack the biological activity of the original proteins in vivo.

Although the construction of test substance libraries is well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.

It is herein revealed that suppression of the expression level and/or biological activity of FAM161A lead to suppression of the growth of cancer cells. Therefore, when a substance suppresses the expression and/or activity of FAM161A, such suppression is indicative of a potential therapeutic effect in a subject. In the context of the present invention, a potential therapeutic effect refers to a clinical benefit with a reasonable expectation. Examples of such clinical benefit include but are not limited to;
(a) reduction in expression of the FAM161A gene,
(b) a decrease in size, prevalence, or metastatic potential of the cancer in the subject,
(c) preventing cancers from forming, or
(d) preventing or alleviating a clinical symptom of cancer.

(i) Molecular modeling:
Construction of test substance libraries is facilitated by knowledge of the molecular structure of substances known to have the properties sought, and/or the molecular structure of FAM161A or CSNK2A2 protein. One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.

Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new substances that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new substance will link to the target molecule and allow experimental manipulation of the structures of the substance and target molecule to perfect binding specificity. Prediction of what the molecule-substance interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.

An example of the molecular modeling system described generally above includes the CHARMM and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles have been published on the subject of computer modeling of drugs interactive with specific proteins, examples of which include Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect to a model receptor for nucleic acid components, Askew et al., J Am Chem Soc 1989, 111: 1082-90.

Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17: 266-78; Shoichet et al., Science 1993, 259: 1445-50.

Once a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors, or "test substances" may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly wherein the lung cancer.

(ii) Combinatorial chemical synthesis:
Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.

Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small substance libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology 1995 supplement; Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino substances, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

(iii) Other candidates:
Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 10⁶ -10⁸ chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples. Furka et al. (14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Patent 4,631,211) and Rutter et al. (US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target. Tuerk and Gold (Science. 249:505-510 (1990)) discloses SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers. In the SELEX method, a large library of nucleic acid molecules (e.g., 10¹⁵ different molecules) can be used for screening.

I. Screening for a Substance Binding to FAM161A Polypeptide :
In context of the present invention, over-expression of FAM161A was detected in lung cancer, in spite of no expression in normal organs (Fig. 1). Accordingly, using the FAM161A genes, proteins encoded by the genes, the present invention provides a method of screening for a substance that binds to FAM161A polypeptide. Due to the expression of FAM161A in lung cancer, a substance that binds to FAM161A polypeptide is expected to suppress the proliferation of lung cancer cells, and thus be useful for treating or preventing lung cancer. Therefore, the present invention also provides a method of screening for a substance that suppresses the proliferation of lung cancer cells, and a method of screening for a substance for treating or preventing lung cancer using the FAM161A polypeptide. One particular embodiment of this screening method includes the steps of:
(a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof ;
(b) detecting the binding activity between the FAM161A polypeptide or the functional equivalent and the test substance; and
(c) selecting the test substance that binds to the FAM161A polypeptide or the functional equivalent.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance or compound on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on either or both of the treatment and prevention lung cancer or the inhibition of cancer associated with over-expression of FAM161A, the method including steps of:
(a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof encoded;
(b) detecting the binding activity between the FAM161A polypeptide or the functional equivalent. and the test substance; and
(c) correlating the potential therapeutic effect of the test substance with the binding activity detected in the step (b), wherein the potential therapeutic effect is shown, when the test substance that binds to the FAM161A polypeptide or the functional equivalent as a candidate substance for treating or preventing lung cancer.

In the context of the present invention, the therapeutic effect may be correlated with the binding activity of the test substance to FAM161A polypeptide(s) or a functional equivalent thereof. For example, when the test substance binds to a FAM161A polypeptide or a functional equivalent thereof, the test substance may identified or selected as a candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not binds to FAM161A polypeptide or a functional fragment thereof, the test substance may identified as the substance having no significant therapeutic effect.

The method of the present invention is described in more detail below.
The FAM161A polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof. The polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides. In preferred embodiments, the polypeptide is isolated from cells expressing FAM161A or chemically synthesized to be contacted with a test substance in vitro.

As a method of screening for proteins, for example, that bind to the FAM161A polypeptide, many methods well known by a person skilled in the art can be used. Such a screening can be conducted using, for example, the immunoprecipitation method, specifically, in the following manner. The gene encoding the FAM161A polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.

The promoter to be used for the expression may be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141 (1982)), the EF-alpha promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa et al., Gene 108: 193 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)) the SR alpha promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), the CMV immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946 (1989)), the HSV TK promoter and so on.

The introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B., Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)) and so on.

The FAM161A polypeptide can be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors that can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available. A fusion protein prepared by introducing only small epitopes composed of several to a dozen amino acids so as not to change the property of the FAM161A polypeptide by the fusion is also provided herein. Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the FAM161A polypeptide (Experimental Medicine 13: 85-90 (1995)).

In the context of immunoprecipitation, an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent. The immune complex is composed of the FAM161A polypeptide, a polypeptide including the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the FAM161A polypeptide, besides using antibodies against the above epitopes, which antibodies can be prepared as described above. An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody. If the polypeptide encoded by FAM161A gene is prepared as a fusion protein with an epitope, such as GST, an immune complex can be formed in the same manner as in the use of the antibody against the FAM161A polypeptide, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the FAM161A polypeptide is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, ³⁵S-methionine or ³⁵S-cystein, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.

West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used as a method of screening for proteins binding to the FAM161A polypeptide using the polypeptide. In particular, a protein binding to the FAM161A polypeptide can be obtained by preparing a cDNA library from cultured cells expected to express a protein binding to the FAM161A polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled FAM161A polypeptide with the above filter, and detecting the plaques expressing proteins bound to the FAM161A polypeptide according to the label. The polypeptide of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the FAM161A, or a peptide or polypeptide (for example, GST) that is fused to the FAM161A polypeptide. Methods using radioisotope or fluorescence and such may be also used.

Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)").

In the two-hybrid system, a polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein. Examples of suitable reporter genes include, but are not limited to, the Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene. Therefore, in the present invention, in some embodiments, test substance is also contacted with FAM161A polypeptide in the cells expressing the polypeptide. Accordingly, a method for screening of the present invention also comprises a step for culturing cells expressing FAM161A polypeptide.

A substance binding to the FAM161A polypeptide can also be screened using affinity chromatography. For example, the polypeptide of the invention may be immobilized on a carrier of an affinity column, and a test substance, containing a protein capable of binding to the polypeptide of the invention, is applied to the column. A test substance herein may be, for example, cell extracts, cell lysates, etc. After loading the test substance, the column is washed, and substances bound to the polypeptide of the invention can be prepared. When the test substance is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.

A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention. When such a biosensor is used, the interaction between the polypeptide of the invention and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the invention and a test substance using a biosensor such as BIAcore.

The methods of screening for molecules that bind when the immobilized FAM161A polypeptide is exposed to synthetic chemical substances, or natural substance banks or a random phage peptide display library, and the methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9 (1996)) to isolate not only proteins but chemical substances that bind to the FAM161A protein (including agonist and antagonist) are well known to those skilled in the art.

In addition to the full length of FAM161A polypeptide, fragments of the polypeptides may be used for the present screening, so long as the fragment utilized retains at least one biological activity of the natural occurring FAM161A polypeptide. Examples of biological activities contemplated by the present invention include cell proliferation enhancing activity, a binding activity to CSNK2A2 polypeptide and so on.

FAM161A polypeptides or functional equivalent thereof may be further linked to other substances, so long as the polypeptides and fragments retain at least one of their biological activities. Useful substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.

FAM161A polypeptides or fragments used for the present method may be obtained from nature, as naturally occurring proteins via conventional purification methods, or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; and
7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, FAM161A polypeptides may be obtained through any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector including a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence including a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the FAM161A or CSNK2A2 polypeptide is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. A promoter may be used for the expression. Any commonly used promoters may be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SR alpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such. The introduction of the vector into host cells to express the LSD1 gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.

The FAM161A polypeptide may also be produced in vitro adopting and in vitro translation system.
The FAM161A polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.
Test substances screened by the present method as substances that bind to FAM161A polypeptide can be candidates substances that has the potential to treat or prevent cancers. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance for cancers. For example, these candidate substances may further examined their ability of suppressing cancer cell proliferation by being contacted with a cancer cell overexpressing FAM161A gene.

II. Screening for a Substance that Suppresses the Biological Activity of FAM161A:
In the context of the present invention, the FAM161A polypeptide is characterized as having the activity of promoting cell proliferation of lung cancer cells, and the binding to CSNK2A2 polypeptide. Using this biological activity as an index, the present invention provides a method for screening a candidate substance that suppresses the proliferation of lung cancer cells, and a method of screening for a candidate substance for either or both of treating and preventing lung cancer.

Thus, the present invention provides a method of screening for a candidate substance for either or both of treating and preventing cancer relating to FAM161A gene including the steps as follows:
(a) contacting a test substance with a FAM161A polypeptide or functional equivalent thereof;
(b) detecting the biological activity of the FAM161A polypeptide or the functional equivalent of step (a); and
(c) selecting the test substance that suppresses the biological activity of the FAM161A polypeptide or the functional equivalent as compared to the biological activity detected in the absence of the test substance.

According to the present invention, the therapeutic effect of the test substance on suppressing the activity to promote cell proliferation, or a candidate substance for treating or preventing cancer relating to FAM161A may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance for treating or preventing cancer relating to FAM161A, using the FAM161A polypeptide or functional equivalents thereof including the steps as follows:
(a) contacting a test substance with the FAM161A polypeptide or a functional equivalent thereof; and
(b) detecting the biological activity of the FAM161A polypeptide or the functional equivalent of step (a), and
(c) correlating the biological activity of (b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention provides methods for evaluating or estimating a therapeutic effect of a test substance in the context of the treatment, prevention or inhibition of a associated with over-expression of FAM161A (e.g., lung cancer), such methods including steps of:
(a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof;
(b) detecting the biological activity of the polypeptide or the functional equivalent of step (a); and
(c) correlating the potential therapeutic effect of the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the FAM161A polypeptide as compared to the biological activity of said polypeptide detected in the absence of the test substance.

In the context of present invention, the therapeutic effect may be correlated with the biological activity of a FAM161A polypeptide or a functional equivalent thereof. For example, when the test substance suppresses or inhibits the biological activity of a FAM161A polypeptide or a functional equivalent thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity of a FAM161A polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

As demonstrated herein, suppressing the expression of FAM161A gene reduces cell growth. Thus, by screening for a candidate substance that reduces the biological activity of FAM161A polypeptide, a candidate substance that has the potential to treat or prevent lung cancer can be identified. Potential of these candidate substances to treat or prevent lung cancer may be evaluated by second and/or further screening to identify therapeutic substance for lung cancer.

The method of the present invention will be described in more detail below.
Any polypeptides can be used for screening so long as they suppress a biological activity of a FAM161A polypeptide. Such biological activity includes cell proliferation enhancing activity and the binding activity to CSNK2A2 polypeptide.
For example, full length of human FAM161A protein can be used and polypeptides functionally equivalent to the protein can also be used. Such polypeptides may be expressed endogenously or exogenously by cells.

The substance isolated by this screening is a candidate for antagonists of the FAM161A polypeptide. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding FAM161A. Moreover, a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the FAM161A polypeptide with molecules (including DNAs and proteins).

When the biological activity to be detected in the present method is cell proliferation enhancing activity, it can be detected, for example, by preparing cells which express the FAM161A polypeptide, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring survival cells or the colony forming activity, for example, shown in Fig. 3. The substances that reduce the speed of proliferation of the cells expressed FAM161A are selected as candidate substance for treating or preventing lung cancer. In some embodiments, cells expressing FAM161A gene is isolated and cultured cells exogenously or endogenously expressing FAM161A gene in vitro.

More specifically, the method includes the step of:
(a) contacting a test substance with cells expressing FAM161A gene;
(b) measuring cell proliferation enhancing activity; and
(c) selecting the test substance that reduces the cell proliferation enhancing activity in the comparison with the cell proliferation enhancing activity in the absence of the test substance.
In preferable embodiments, the method of the present invention may further include the steps of:
(d) selecting the test substance that has no effect to the cells no or little expressing FAM161A gene.

According to the present invention, the therapeutic effect of the test substance on suppressing the activity to promote cell proliferation, or a candidate substance for treating or preventing cancer relating to FAM161A (e.g., lung cancer) may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance for treating or preventing cancer relating to FAM161A, using the FAM161A polypeptide or fragments thereof including the steps as follows:
(a) contacting a test substance with the FAM161A polypeptide or a functional equivalent thereof; and
(b) detecting the cell proliferation enhancing activity of the polypeptide or the functional equivalent of step (a), and
(c) correlating the cell proliferation enhancing activity of (b) with the therapeutic effect of the test substance.

In the context of present invention, the therapeutic effect may be correlated with the cell proliferation enhancing activity of a FAM161A polypeptide or a functional equivalent thereof. For example, when the test substance suppresses or inhibits the cell proliferation enhancing activity of a FAM161A polypeptide or a functional equivalent thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the cell proliferation enhancing activity of a FAM161A polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

Cells expressing FAM161A polypeptides include cells isolated cell. For example, cell lines established from lung cancer (e.g. SBC5 and LC319), and purified cells from clinical cancer tissues can be used for the present screening method. Such cells can be used for the above screening of the present invention so long as the cells express the gene. Alternatively cells can be transfected to expression vectors of FAM161A polypeptide, so as to express the gene.

In the preferred embodiments, control cells that do not express FAM161A polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing FAM161A associating cancer, using the FAM161A polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express a FAN161A polypeptide or a functional fragment thereof, and control cells that do not express a FAM161A polypeptide or a functional fragment thereof in the presence of the test substance;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test compound that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance.

When the biological activity to be detected in the screening method of the present invention is binding activity to CSNK2A2 polypeptide, it can be detected, for example, by detecting the binding between FAM161A polypeptide and CSNK2A2 polypeptide in the presence of a test substance. Details will be described under the section entitled " Screening for a Substance that Alters the Binding Between FAM161A and CSNK2A2".

The phrase "suppress the biological activity" as defined herein are preferably at least 10% suppression of the biological activity of FAM161A in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression.
In the preferred embodiments, control cells that do not express FAM161A polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing FAM161A associating disease, using the FAM161A polypeptide or fragments thereof including the steps as follows:
a) culturing cells that express a FAM161A polypeptide or a functional fragment thereof, and control cells that do not express a FAM161A polypeptide or a functional fragment thereof in the presence of the test substance;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test compound that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance.

As revealed herein, suppressing the biological activity of FAM161A polypeptide reduces cell growth. Thus, by screening for a candidate substance that inhibits the biological activity of FAM161A polypeptide, candidate substance that have the potential to treat and/or prevent cancers can be identified. The potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance, compounds or agent for cancers. For example, when a substance that inhibits the biological activity of a FAM161A polypeptide also inhibits the activity of a cancer, it may be concluded that such a substance has a FAM161A specific therapeutic effect.

III. Screening For a Substance that Alters the Expression of FAM161A:
In the context of the present invention, a decrease in the expression of FAM161A gene by siRNA results in the inhibition of cancer cell proliferation. Accordingly, the present invention provides a method of screening for a substance that inhibits the expression of FAM161A gene. A substance that inhibits the expression of FAM161A gene is expected to suppress the proliferation of cancer cells, and thus is useful for treating or preventing cancer relating to FAM161A, particularly wherein the cancer is lung cancer. Therefore, the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells, and a method for screening a substance for treating or preventing cancer relating to FAM161A, wherein the cancer is lung cancer. In the context of the present invention, such screening may include, for example, the following steps:
(a) contacting a candidate substance with a cell expressing FAM161A gene;
(b) detecting an expression level of the FAM161A gene in the cell of the step (a); and
(c) selecting the candidate substance that reduces the expression level of FAM161A gene the expression level of the FAM161A gene detected in the absence of the test substance.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance in the context of the treatment, prevention or inhibition of a cancer associated with over-expression of FAM161A gene, the method including steps of:
(a) contacting a candidate substance with a cell expressing FAM161A gene; and;
(b) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance reduces the expression level of FAM161A gene as compared to a control.

The method of the present invention will be described in more detail below.
Cells expressing the FAM161A gene include, for example, cell lines established from lung cancer or cell lines transfected with FAM161A gene expression vectors; any of such cells can be used for the above screening of the present invention. The expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining and flow cytometry analysis. The phrase "reduce the expression level" as defined herein are preferably at least 10% reduction of expression level of FAM161A gene in comparison to the expression level in absence of the substance, more preferably at least 25%, 50% or 75% reduced level and most preferably at 95% reduced level. The substance herein includes chemical substance, double-strand nucleotide, and so on. The preparation of the double-strand nucleotide is in aforementioned description. In the method of screening, a substance that reduces the expression level of FAM161A gene can be selected as candidate substances to be used for the treatment or prevention of lung cancer.

Alternatively, the screening method of the present invention may include the following steps:
(a) contacting a candidate substance with a cell into which a vector, including the transcriptional regulatory region of FAM161A gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) selecting the candidate substance that reduces the expression or activity of said reporter gene.

According to the present invention, the therapeutic effect of the test substance in terms of inhibiting the cell growth or a candidate substance for treating or preventing FAM161A associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing a FAM161A associated disease.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance in terms of treating, preventing, or inhibiting a cancer associated with over-expression of FAM161A gene, the method including steps of:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of FAM161A gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression or activity of said reporter gene.

In the context of the present invention, such screening may include, for example, the following steps:
a) contacting a test substance with a cell into which a vector, composed of the transcriptional regulatory region of the FAM161A gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
b) detecting the expression or activity of said reporter gene; and
c) correlating the expression level of b) with the therapeutic effect of the test substance.

In the context of the present invention, the therapeutic effect may be correlated with the expression or activity of said reporter gene. For example, when the test substance reduces the expression or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

Suitable reporter genes and host cells are well known in the art. Illustrative reporter genes include, but are not limited to, luciferase, green fluorescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on.The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of FAM161A. The transcriptional regulatory region of FAM161A or CSNK2A2 herein includes the region from transcriptional start site to at least 500 bp upstream, preferably 1000 bp, more preferably 5000 or 10000 bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of any one of these genes. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).

The vector containing the said reporter construct is infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on). "reduces the expression or activity" as defined herein are preferably at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.

IV. Screening For A Substance That Alters The Binding Between FAM161A And CSNK2A2:
In the present invention, the direct interaction of FAM161A polypeptide with CSNK2A2 polypeptide was shown by immunoprecipitation assay (Fig.5B).
Accordingly, the present invention provides a method of screening for a substance that inhibits the binding between FAM161A polypeptide and CSNK2A2 polypeptide.
Substances that inhibit the binding between a FAM161A polypeptide and a CSNK2A2 polypeptide can be screened by detecting a binding level between a FAM161A polypeptide and a CSNK2A2 polypeptide as an index. Therefore, the present invention provides a method for screening a substance for inhibiting the binding between a FAM161A polypeptide and a CSNK2A2 polypeptide using a binding level between a FAM161A polypeptide and a CSNK2A2 polypeptide as an index.

In the present invention, it is further demonstrated that a FAM161A polypeptide activates of MAPK cascade through the stabilization of a CSNK2A2 polypeptide, and such abnormally activation of MAPK cascade cause a cell carcinogenesis and cancer cell proliferation.
Therefore, substances that inhibit binding between a FAM161A polypeptide and a CSNK2A2 polypeptide are expected to be suppressing cancer cell proliferation through destabilization of a CSNK2A2 polypeptide. Accordingly, the present invention also provides a method for screening a candidate substance for inhibiting or reducing a growth of cancer cells expressing the FAM161A and CSNK2A2 genes, e.g., lung cancer cell, and therefore, a candidate substance for treating or preventing lung cancers.

Of particular interest to the present invention are the following methods of [1] to [4]:
[1] A method of screening for a substance that inhibits a binding between a FAM161A polypeptide and a CSNK2A2 polypeptide, said method including the steps of:
(a) contacting a FAM161A polypeptide or functional equivalent thereof with a CSNK2A2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptide(s) or the functional equivalent(s);
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level;
[2] A method of screening for a substance useful in connection with the treatment or prevention of cancer ,or capable of inhibiting cancer cell growth, said method including the steps of:
(a) contacting a FAM161A polypeptide or functional equivalent thereof with a CSNK2A2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptide(s) or the functional equivalent(s);
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level;
[3] The method of [1] or [2], wherein the functional equivalent of FAM161A polypeptide includes a CSNK2A2 binding domain of the FAM161A polypeptide;
[4] The method of [1] or [2], wherein the functional equivalent of CSNK2A2 polypeptide includes a FAM161A-binding domain of the CSNK2A2 polypeptide.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance connection with the treatment or prevention of cancer , or capable of inhibiting lung cancer cell growth, the method including steps of:
(a) contacting a FAM161A polypeptide or functional equivalent thereof with a CSNK2A2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptide(s) or the functional equivalent(s);
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduce the binding level.

Further, in another embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance in connection with the treatment or prevention of cancer, or capable of inhibiting lung cancer cell growth, the method including steps of:
(a) contacting a FAM161A polypeptide or functional equivalent thereof with a CSNK2A2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting binding between the polypeptide(s) or the functional equivalent(s); and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance inhibits binding between the polypeptides.

In the context of the present invention, functional equivalents of a FAM161A and CSNK2A2 polypeptide are polypeptides that have a biological activity equivalent to a full length of human FAM161A polypeptide (e.g., SEQ ID NO: 18) or a full length of human CSNK2A2 polypeptide (e.g., SEQ ID NO: 20), respectively. Particularly, the functional equivalent of FAM161A polypeptide is a polypeptide fragment containing a CSNK2A2-binding domain of the FAM161A polypeptide. Similarly, the functional equivalent of CSNK2A2 polypeptide is a polypeptide fragment containing a FAM161A-binding domain of the CSNK2A2 polypeptide.

Those of skill in the art will recognize that any of a number of standard methods may be used to screen for substances that inhibit the binding of FAM161A polypeptide to CSNK2A2 polypeptide.
A polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof. Any test substance aforementioned can be used for screening.
Likewise, those of skill in the art will readily recognize that a number of conventional methods may be used to detect the binding between a FAM161A polypeptide and CSNK2A2 polypeptide. Examples of such methods include, but are not limited to, immunoprecipitation, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)), a two-hybrid system utilizing cells ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"), affinity chromatography and a biosensor using the surface plasmon resonance phenomenon.

In some embodiments, the present screening method may be carried out in a cell-based assay using cells expressing both of a FAM161A polypeptide and a CSNK2A2 polypeptide. Cells expressing FAM161A polypeptide and CSNK2A2 polypeptide include, for example, cell lines established from cancer, e.g. lung cancer. Alternatively the cells may be prepared by transforming cells with nucleotides encoding FAM161A gene and CSNK2A2 gene. Such transformation may be carried out using an expression vector encoding both FAM161A gene and CSNK2A2 gene, or expression vectors encoding either FAM161A gene or CSNK2A2 gene. The present screening can be conducted by incubating such cells in the presence of a test substance. The binding of FAM161A polypeptide to CSNK2A2 polypeptide can be detected by immunoprecipitation assay using an anti-FAM161A antibody or anti- CSNK2A2 antibody.

According to the present invention, the therapeutic effect of a candidate substance on inhibiting the cell growth or a candidate substance suitable for use in connection with the treatment or prevention of cancer associated with FAM161A and CSNK2A2 (e.g., lung cancer) may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance for treating or preventing lung cancer, using a FAM161A polypeptide or functional equivalent thereof including the steps of:
(a) contacting a FAM161A polypeptide or functional equivalent thereof with a CSNK2A2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptide(s) or the functional equivalent(s);
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the binding level of (c) with the therapeutic effect of the test substance.
In the present invention, the therapeutic effect may be correlated with the binding level between a FAM161A polypeptide and a CSNK2A2 polypeptide. For example, when the test substance suppresses the binding level between the polypeptides as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the binding level between the polypeptides as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

V. Screening for a Substance Using the CSNK2A2 Polypeptide Level as an Index
As demonstrated in Examples, in HEK293T and COS7 cells co-transfected with FAM161A and CSNK2A2 gene, exogenous CSNK2A2 protein level was up-regulated as compared to that in cells transfected with only CSNK2A2 gene or mock instead of FAM161A gene (Fig.7A). Similarly, knockdown of FAM161A gene expression by siRNA against FAM161A gene resulted in upregulation of endogenous CSNK2A2 protein level (Fig.7B). Those results indicates that FAM161A polypeptide stabilizes CSNK2A2 polypeptide. Thus, the CSNK2A2 polypeptide level can be used as an index of the expression level and/or activity of FAM161A polypeptide in cells.

Therefore, the present invention also provides a method for screening a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth using a CSNK2A2 polypeptide level as an index. In the context of the present invention, such methods may includes following steps:
(a) contacting a test substance with a cell expressing a FAM161A gene and a CSNK2A2 gene;
(b) detecting the CSNK2A2 polypeptide level in the cell of step (a); and
(c) selecting the test substance that decreases the CSNK2A2 polypeptide level
in comparison with the CSNK2A2 polypeptide level detected in the absence of the test substance.

According to the present invention, the therapeutic effect of the test substance of suppressing the activity to promote cell proliferation, or a candidate substance for either or both of treating and preventing cancer relating to FAM161A may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance for treating or preventing cancer relating to FAM161A, using the FAM161A polypeptide or functional equivalents thereof including the steps as follows:
(a) contacting a test substance with a cell expressing a FAM161A gene and a CSNK2A2 gene;
(b) detecting the CSNK2A2 polypeptide level in the cell of step (a), and
(c) correlating the CSNK2A2 polypeptide level of (b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing or inhibiting cancer associated with over-expression of FAM161A (e.g., lung cancer), the method including steps of:
(a) contacting a test substance with a cell expressing a FAM161A gene and a CSNK2A2 gene;
(b) detecting the CSNK2A2 polypeptide level in the cell of step (a), and
(c) correlating the potential therapeutic effect of the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the CSNK2A2 polypeptide level as compared to the CSNK2A2 polypeptide level detected in the absence of the test substance.

In the context of present invention, the therapeutic effect may be correlated with the CSNK2A2 polypeptide level in a cell. For example, when the test substance reduces the CSNK2A2 polypeptide level in a cell as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the CSNK2A2 polypeptide level in a cell as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

In the present invention, it is revealed that CSNK2A2 polypeptide activates MAPK cascade to induce cancer cell growth. Thus, by screening for a candidate substance that reduces the CSNK2A2 polypeptide level, a candidate substance that has the potential to treat or prevent lung cancer can be identified. Potential of these candidate substances to treat or prevent lung cancer may be evaluated by second and/or further screening to identify therapeutic substance for lung cancer.
Additional details of the present screening method are described bellow.

Any cells can be used for the present screening methods, as long as the cells express FAM161A gene and CSNK2A2 gene. In preferred embodiments, isolated and/or purified cells expressing both of the peptides may be used for the screening method of the present invention. Such cells may be established cell lines, known to express FAM161A gene and CSNK2A2 gene, for example, cell lines established from lung cancer. Alternatively, the cells may be cells transformed with any of FAM161A gene and CSNK2A2 gene. One skilled in the art can prepare expression vectors for these genes and conduct cellular transformation by those vectors using conventional methods. Details of those genes are described above, in the section entitled " Genes and Polypeptides ".

CSNK2A2 polypeptide level can be detected by methods well-known in the art. For example, antibodies against a CSNK2A2 polypeptide may be used as suitable reagents for the detection. Alternatively, cells to be used in the screening may be transformed with a fusion gene containing CSNK2A2 gene and a gene of commercially available epitope, and after contacting with a test substance, CSNK2A2 polypeptide level may be detected using an antibodies against the epitope. Examples of commercially available epitope include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such. Antibodies against those epitopes are also commercially available. Any immunological techniques, for example, ELISA, immunoblotting and such can be used for the detection of CSNK2A2 polypeptide level. To clarify CSNK2A2 polypeptide level stabilized by FAM161A polypeptide, protein synthesis in the cell to be used for the screening may be inhibited by addition of protein synthesis inhibitor such as cycloheximide before contacting a test substance.

VI. Screening for a Substance Using the ERK1/2 Phosphorylation Level as Index
In the present invention, it has been revealed that CSNK2A2 polypeptide, which is stabilized thorough the interaction with FAM161A polypeptide, has the serine/threonine phosphorylation activity of ERK1/2 (Fig. 7C). ERK1/2 are known to belong to MAP kinase family and act in a signaling cascade that regulates various cellular processes such as proliferation, differentiation, and cell cycle progression in response to a variety of extracellular signals. Therefore, increased phosphorylation of ERK1/2 by stabilized CSNK2A2 polypeptide may cause abnormally activation of MAPK signaling cascade to induce carcinogenesis and cancer cell proliferation.

Thus, a substance that reduces the phosphorylation level of an ERK1 and/or ERK2 polypeptide by CSNK2A2 polypeptide may be a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth. Accordingly, the present invention provides a method for screening a substance for reducing the phosphorylation level of ERK1 and/or ERK2 polypeptide by CSNK2A2 polypeptide, which includes the following steps:
(a) contacting a CSNK2A2 polypeptide or functional equivalent thereof with ERK1 and/or ERK2 polypeptide or functional equivalent thereof in the presence of a test substrate under a condition that allows phosphorylation of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
(b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
(c) comparing the phosphorylation level detected in the step (b) with the phosphorylation level detected in the absence of the test substance; and
(d) selecting the test substrate that reduces the phosphorylation level of ERK1 and/or ERK2 polypeptide as compared to that detected in the absence of the test substrate .

Furthermore, the present invention also provides a method for screening a candidate substance for either or both of treating and preventing cancer, or inhibiting or reducing cancer cell growth, including the following steps:
(a) contacting a CSNK2A2 polypeptide or functional equivalent thereof with ERK1 and/or ERK2 polypeptide or functional equivalent thereof in the presence of a test substrate under a condition that allows phosphorylation of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
(b) detecting the phosphorylation level of theERK1 and/or ERK2 polypeptide or the functional equivalent;
(c) comparing the phosphorylation level detected in the step (b) with the phosphorylation level detected in the absence of the test substance; and
(d) selecting the test substrate that reduces the phosphorylation level of ERK1 and/or ERK2 polypeptide as compared to that detected in the absence of the test substrate .

In the context of the present invention, the conditions that allow phosphorylation of the ERK1 and/or ERK2 polypeptide or the functional equivalent may be provided with an incubation of CSNK2A2 polypeptide or functional equivalent thereof and ERK1 and/or ERK2 polypeptide in the presence of a phosphate donor, e.g., ATP.

Alternatively, such condition may prepare by culturing cells expressing CSNK2A2 polypeptide or functional equivalent thereof, and ERK1 and/or ERK2 polypeptide or functional equivalent thereof. Thus, the present invention also provides a method including the steps as follows:
(a) contacting a cell expressing CSNK2A2 polypeptide and ERK1 and/or ERK2 polypeptide with a test substance;
(b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide; and
(c) selecting the test substance that reduces the phosphorylation level of the ERK1 and/or ERK2 polypeptide as compared to the phosphorylation level detected in the absence of the test substance.

Cells expressing CSNK2A2 polypeptide and ERK1 and/or ERK2 polypeptide include, for example, cell lines established from cancer, e.g. lung cancer and cells transfected with a vector capable of expressing CSNK2A2 polypeptide and ERK1 and/or ERK2 polypeptide, or expression vectors capable of expressing CSNK2A2 polypeptide and ERK1 and/or ERK2 polypeptide respectively. In order to stabilize CSNK3A2 polypeptide in a cell, the cell that further express FAM161A polypeptide may be preferably used in the method of the present invention.

Polypeptides to be used for the screening can be recombinant polypeptides or proteins derived from natural sources, or a partial peptide thereof. Preferably, the CSNK2A2 polypeptide to be used for the screening is a polypeptide including a catalytic domain of serine/threonine phosphorylation. In more preferred embodiments, the CSNK2A2 polypeptide to be used in the screening is a polypeptide including an amino acid sequence of SEQ ID NO: 20, and more preferably a polypeptide having the amino acid sequence of SEQ ID NO: 20. In the context of the present screening method, functional equivalents of the CSNK2A2 polypeptide may be polypeptides that retain a kinase activity of CSNK2A2 polypeptide. Such polypeptides may include a kinase domain of CSK2A2 polypeptide. An example of a kinase domain of CSK2A2 polypeptide includes, but is not limited to, polypeptide having an amino acid sequence of 40-325 of SEQ ID NO: 20.

On the other hands, the ERK1 and ERK2 polypeptide to be used for the screening is preferably a polypeptide including at least one serine/threonine phosphorylation site. The ERK1 polypeptides to be used in the screening are a polypeptide including an amino acid sequence of SEQ ID NOs: 22, 24 or 26, and more preferably polypeptides having the amino acid sequence of SEQ ID NOs: 22, 24 or 26. Similarly, the ERK2 polypeptides to be used in the screening are a polypeptide including an amino acid sequence of SEQ ID NOs: 29, and more preferably polypeptides having the amino acid sequence of SEQ ID NO: 29. In the context of the present screening method, functional equivalents of the ERK1 or ERK2 polypeptide may be polypeptides that retain a capability of being phosphorylated by a CSK2A2 polypeptide. Such polypeptides may include at least one phosphorylation of ERK1 or ERK2 polypeptide. For example, the phosphorylation sites of ERK1 polypeptide include Thr202 and Tyr204 of SEQ ID NOs: 22, 24 or 26. Also, the phosphorylation sites of ERK2 polypeptide include Thr185, Tyr187 and Tyr205 of SEQ ID NO: 29. Accordingly, preferred examples of functional equivalents of ERK1 or ERK2 polypeptide include polypeptides containing a fragment of ERK1 polypeptide (SEQ ID NOs: 22, 24 or 26) or ERK2 polypeptide (SEQ ID NO: 29) having the above-mentioned phosphorylation sites. Preferably, the fragment of ERK1 or ERK2 polypeptide having phosphorylation sites may have more than 10 amino acids, more than 20 amino acids, more than 30 amino acids, more than 50 amino acids or more than 100 amino acids.

As a method for detection of the phosphorylation level of ERK1 and/or ERK2 polypeptide, many methods well known in the art can be used. For example, the phosphorylation of ERK1 and/or ERK2 polypeptide may be detected by ELISA or immunoblot analysis using an antibody against phosphorylated ERK1 and/or ERK2 polypeptide. Such antibodies are commercially available.

Alternatively, an ERK1 and/or ERK2 polypeptide may incubated with a CSNK2A2 polypeptide under a labeled phosphate donor. When the labeled phosphate donor was used, the phosphorylation level of the ERK1 and/or ERK2 polypeptide can be detected via tracing the label. For example, radio-labeled ATP (e.g. ³²P-ATP) may be used as phosphate donor, and radio activity incorporated in ERK1 and/or ERK2 polypeptide may correlate with the phosphorylation level of the ERK1 and/or ERK2 polypeptide.

Prior to the detection of phosphorylated ERK1 and/or ERK2 polypeptide, ERK1 and/or ERK2 polypeptide may be separated from other elements, or cell lysate of ERK1 and/or ERK2 expressing cells. For instance, gel electrophoresis may be used for separation of ERK1 and/or ERK2 polypeptide. Alternatively, ERK1 and/or ERK2 polypeptide may be captured by contacting ERK1 and/or ERK2 polypeptide with a carrier having an anti-ERK1 and/or ERK2 antibody.

According to the present invention, the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing CSNK2A2 associating cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing CSNK2A2 associating cancer, including the steps as follows:
(a) contacting a CSNK2A2 polypeptide or a functional equivalent thereof with an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof in the presence of a test substance under a condition that allows phosphorylation of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
(b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent; and
(c) correlating the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent with therapeutic effect of the test substance.

In the present invention, the therapeutic effect may be correlated with the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent. For example, when the test substance reduces the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent as compared to a level detected in the absence of the test substance, the test substance may be identified as the substance having no significant therapeutic effect.
Also, the substances isolated by the above-mentioned screening method are candidates for antagonists of the CSNK2A2 polypeptide.

By screening for candidate substances that (i) bind to the FAM161A polypeptide; (ii) suppress/reduce the biological activity (e.g., the cell-proliferating activity, binding activity to a CSNK2A2 polypeptide) of the FAM161A polypeptide; (iii) reduce the expression level of FAM161A gene, (iv) inhibit or reduce the binding between FAM161A polypeptide and CSNK2A2 polypeptide, (v) reduce the phosphorylation level of ERK1 and/or ERK2 polypeptide by CSNK2A2 polypeptide, candidate substances that have the potential to treat or prevent cancers (e.g., lung cancer, ) can be identified. The therapeutic potential of these candidate substances may be evaluated by second and/or further screening to identify therapeutic substance for cancers. For example, when a substance that binds to the FAM161A polypeptide inhibits the growth of cancer cell that overexpresses FAM161A gene, it may be concluded that such a substance has the FAM161A -specific therapeutic effect.

Kit for Measuring a Phosphorylation Activity of CSNK2A2
The present invention further provides a kit for measuring a phosphorylation activity of a CSNK2A2 polypeptide. In the present invention, ERK1 polypeptide and ERK2 polypeptide was identified as a novel substrate of CSNK2A2 polypeptide. Thus, the present invention provides a kit for measuring a phosphorylation activity of a CSNK2A2 polypeptide, containing an ERK1 polypeptide and/or ERK2 polypeptide or a functional equivalent thereof as a substrate of CSNK2A2 polypeptide. Such kit can be used for measuring CSNK2A2-mediated phosphorylation activity in a sample containing a CSNK2A2 polypeptide or a CSNK2A2 polypeptide purified or isolated from a sample.

Furthermore, the present invention provides a kit for detecting for the ability of a test substance to inhibit phosphorylation of ERK1 and/or ERK2 polypeptide by a CSNK2A2 polypeptide, containing a CSNK2A2 polypeptide and an ERK1 and/or ERK2 polypeptide as a substrate for CSNK2A2 polypeptide.
The above-mentioned kits of the present invention find a use for identifying a substance that reduces a phosphorylation level of an ERK1 and/or ERK2 polypeptide by a CSNK2A2 polypeptide. Furthermore, the kits of the present invention are useful for screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting lung cancer cell growth.

Specifically, the present invention provides the following kits of [1] to [4]:
[1] A kit for measuring a phosphorylation activity of a CSNK2A2 polypeptide, wherein the kit includes the following components (a) and (b):
(a) an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof;
(b) a reagent for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
[2] A kit for detecting for the ability of a test substance to inhibit phosphorylation of ERK1 and/or ERK2 polypeptide by a CSNK2A2 polypeptide, wherein the kit includes the following components of (a) to (c):
(a) a CSNK2A2 polypeptide or a functionally equivalent thereof;
(b) an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof;
(c) a reagent for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
[3] The kit of [1] or [2], wherein the functional equivalent of the ERK1 or ERK2 polypeptide includes a fragment of the ERK1 or ERK2 polypeptide having at least one phosphorylation site; and
[4] The kit of any one of [1] to [3], wherein the kit further includes a phosphate donor.

Details of the kits of the present invention will be described bellow.
ERK1 and ERK2 polypeptide contained in the kits of the present invention may either the full length of ERK1 and ERK2 polypeptide. For example, ERK1 polypeptide may be a polypeptide including or having the amino acid sequence of SEQ ID NOs: 22, 24 or 26, and ERK2 polypeptide may be a polypeptide including or having the amino acid sequence of SEQ ID NO: 29. In the context of the present kits, the functional equivalent of ERK1 or ERK2 polypeptide refers to a modified polypeptide, a fragment or a modified fragment of the full length of ERK1 or ERK2 polypeptide, capable of being phosphorylated by a CSNK2A2 polypeptide. Preferably, the functional equivalents of ERK1 or ERK2 polypeptide retains at least one phosphorylation site capable of being phosphorylated by CSNK2A2 polypeptide. For example, the phosphorylation sites of ERK1 polypeptide include Thr202 and Tyr204 of SEQ ID NOs: 22, 24 or 26. Also, the phosphorylation sites of ERK2 polypeptide include Thr185, Tyr187 and Tyr205 of SEQ ID NO: 29. Accordingly, functional equivalents of the ERK1 or ERK2 polypeptide may contain a contiguous sequence of the amino acid sequence of SEQ ID NO: 22, 24, 26 or 29 including the above-mentioned phosphorylated sites, having more than 10 amino acid residues. Alternatively, such functional equivalents may have more than 20 amino acids, more than 30 amino acids, more than 50 amino acids or more than 100 amino acids.

CSNK2A2 polypeptide contained in the kits of the present invention may either the full length of CSNK2A2 polypeptide such as a polypeptide containing or having the amino acid sequence of SEQ ID NOs: 20. In the context of the present kits, the functional equivalent of CSNK2A2 polypeptide refers to a modified polypeptide, a fragment or a modified fragment of the full length of CSNK2A2 polypeptide, having a kinase activity for ERK1 or ERK2 polypeptide. Such functional equivalents may includes a polypeptide containing a kinase of CSNK2A2 polypeptide. An example of a kinase domain of CSK2A2 polypeptide includes, but are not limited to, polypeptide having an amino acid sequence of 40-325 of SEQ ID NO: 20.

Reagents for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide may be any reagents that is able to detect of phosphorylation level of the ERK1 and/or ERK2 polypeptide. For example, antibodies against a phosphorylated ERK1 and/or ERK2 polypeptide may be preferably used as a such reagent. The anti-phosphorylated ERK1 and/or ERK2 antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used as the reagent, so long as the fragment retains the binding ability to the phosphorylated ERK1 and/or ERK2 polypeptide. Methods to prepare these kinds of antibodies are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof. Furthermore, the antibody may be labeled with signal generating molecules via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods may be employed for the present invention. For example, radiolabels, chromogenic labels, fluorescent labels and such may be preferably used for labeling the antibody. When the kit contains an anti-phosphorylated ERK1 and/or ERK2 antibody with label, the kit may further contain reagent(s) for detecting a signal generated by the label. Alternatively, the antibodies may be conjugated with such enzyme that catalyses a chromogenic reaction, for example, peroxidase, alkaline phosphatase and such. When the kit contains an anti-phosphorylated ERK1 and/or ERK2 antibody conjugated with the enzyme, the kit may further contain a chromogenic substrate for the enzyme. Alternatively, a secondary antibody labeled or conjugated with an enzyme that catalyses a chromogenic reaction may be contained in the kit of the present invention.

When the kit further contains a labeled phosphate donor (e.g. ³²P-ATP), the reagents for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide may be reagents for detecting signal generated by the label. For example, when the ERK1 and/or ERK2 polypeptide is labeled with a radiolabel, the reagents for the detection of phosphorylation level may be liquid scintillators, reagents for autoradiography and the like.

The kit may contain more than one of the aforementioned reagents. Furthermore, the kit may include a solid matrix for binding an anti-phosphorylated ERK1 and/or ERK2 antibody, a medium or buffer and container for incubating the polypeptides under a suitable condition for phosphorylation, and positive and negative control samples. The kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, etc.) with instructions for use. These substances and such may be included in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.

Double-Stranded Molecules
As used herein, the term "isolated double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene and includes, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).

As used herein, the term "target sequence" refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppress of translation of the whole mRNA of the target gene if a double-stranded nucleic acid molecule containing the sequence is introduced into a cell expressing the gene. A nucleotide sequence within mRNA or cDNA sequence of a target gene can be determined to be a target sequence when a double-stranded molecule including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. When a target sequence is shown by cDNA sequence, a sense strand sequence of a double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence.

A double-stranded molecule is composed of a sense strand having a sequence corresponding to a target sequence and an antisense strand having a complementary sequence to the target sequence, such that the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule. Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleotides that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in a DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region is replaced with base "u"s. On the other hand, when a target sequence is shown in an RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region is replaced with "t"s. For example, when a target sequence is the RNA sequence shown in SEQ ID NO: 11, 12, 13 or 14 and the 3' side half region of the sense strand of the double-stranded molecule is composed of DNA, "a sequence corresponding to a target sequence" is "5'-GGUACAUAAAGCGCTCAAA-3'" (for SEQ ID NO: 11), "5'-GUACUUGAGUACTTCAACA-3'" (for SEQ ID NO: 12), "5'-GAUUAUAGCTTGGACATGT-3'" (for SEQ ID NO: 13), or
"5'- GAGUUUGGGCTGTATGTTA-3'" (for SEQ ID NO: 14).

Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleotides that constitutes the antisense strand. For example, when a target sequence is the RNA sequence shown in SEQ ID NOs: 11, 12, 13 or 14 and the 5' side region of the antisense strand of the double-stranded molecule is composed of DNA, " a complementary sequence to a target sequence " is "5'-TTTGAGCGCTUUAUGUACC-3'" (for SEQ ID NO: 11) , "5'-TGTTGAAGTACUCAAGUAC-3' " (for SEQ ID NO: 12), "5'-ACATGTCCAAGCUAUAAUC-3' " (for SEQ ID NO: 13) or "5'-TAACATACAGCCCAAACUC-3'" (for SEQ ID NO: 14).

On the other hand, when a double-stranded molecule is composed of RNA, the sequence corresponding to a target sequence shown in SEQ ID NOs: 11,12,13 or 14 is the ribonucleotide sequence shown in SEQ ID NOs: 11,12,13 or 14, and the complementary sequence to the target sequence is "5'-UUUGAGCGCUUUAUGUACC-3'" (for SEQ ID NO: 11) , "5'-UGUUGAAGUACUCAAGUAC-3' " (for SEQ ID NO: 12), "5'-ACAUGUCCAAGCUAUAAUC-3' " (for SEQ ID NO: 13) or "5'-UAACAUACAGCCCAAACUC-3'" (for SEQ ID NO: 14).

A double-stranded molecule may has one or two 3'overhang(s) having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.

As used herein, the term "siRNA" refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a FAM161A or a CSNK2A2 sense nucleic acid sequence (also referred to as "sense strand"), a FAM161A or a CSNK2A2 antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.

As used herein, the term "dsRNA" refers to a construct of two RNA molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.

The term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, composed of first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".

As used herein, the term "siD/R-NA" refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a FAM161A or a CSNK2A2 sense nucleic acid sequence (also referred to as "sense strand"), a FAM161A or a CSNK2A2 antisense nucleic acid sequence (also referred to as "antisense strand") or both (nucleotide "t" is replaced with "u" in RNA). The siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.

As used herein, the term "dsD/R-NA" refers to a construct of two molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).

The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop structure, composed of a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".

As used herein, an "isolated nucleic acid" is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In the context of the present invention, examples of isolated nucleic acid include DNA, RNA, and derivatives thereof. Examples of isolated nucleic acid include DNA, RNA, and derivatives thereof, for example, a cDNA molecule, substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Double-stranded molecules (e.g., siRNA and the like) against target gene(s) can be used to reduce the expression level of said gene(s). Herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)) as described in "Definitions". In the context of the present invention, a double-stranded molecule against FAM161A or CSNK2A2 in which an antisense strand hybridizes to the FAM161A or CSNK2A2 mRNA, induces degradation of the FAM161A or CSNK2A2 mRNA by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the protein. As demonstrated herein, the expression of FAM161A or CSNK2A2 in lung cancer cell lines is inhibited by dsRNA (Fig. 3AB). Accordingly, the present invention provides isolated double-stranded molecules that are capable of inhibiting the expression of a FAM161A or a CSNK2A2 gene when introduced into a cell expressing the gene. The target sequence of double-stranded molecules may be designed by an siRNA design algorithm such as that mentioned below.

Examples of FAM161A target sequences include, for example, nucleotide sequences such as SEQ ID NO: 11 and SEQ ID NO: 12, and examples of CSNK2A2 target sequences include, for example, nucleotide sequences such as SEQ ID NO:13 and SEQ ID NO: 14.
Double stranded molecules of particular interest in the context of the present invention are set forth in[1] to [18] below:
[1] An isolated double-stranded molecule that, when introduced into a cell, inhibits the expression of FAM161A or CSNK2A2 gene and cell proliferation, such molecules composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
[2] The double-stranded molecule of [1], wherein said double-stranded molecule acts on mRNA, matching a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14;
[3] The double-stranded molecule of [2], wherein the sense strand contains a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14;
[4] The double-stranded molecule of any one of [1] to[3], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 100 nucleotides pairs in length;
[5] The double-stranded molecule of any one of [1] to [4], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 75 nucleotide pairs in length;
[6] The double-stranded molecule of [5], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 50 nucleotide pairs in length;
[7] The double-stranded molecule of [6], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 25 nucleotide pairs in length;
[8] The double-stranded molecule of [7] , wherein the sense strand hybridize with antisense strand at the target sequence to form the double-stranded molecule having a length of between about 19 and about 25 nucleotide pairs in length;
[9] The double-stranded molecule of any one of [1] to [8], composed of a single polynucleotide having both the sense and antisense strands linked by an intervening single-strand;
[10] The double-stranded molecule of [9], having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3, wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to the target sequence;
[11] The double-stranded molecule of any one of [1] to [10], composed of RNA;
[12] The double-stranded molecule of any one of [1] to [10], composed of both DNA and RNA;
[13] The double-stranded molecule of [12], wherein the molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[14] The double-stranded molecule of [13] wherein the sense and the antisense strands are composed of DNA and RNA, respectively;
[15] The double-stranded molecule of [12], wherein the molecule is a chimera of DNA and RNA;
[16] The double-stranded molecule of [15], wherein a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of the sense strand and a region flanking to the 3'-end of the antisense strand are RNA;
[17] The double-stranded molecule of [16], wherein the flanking region is composed of 9 to 13 nucleotides; and
[18] The double-stranded molecule of any one of [1] to [17], wherein the molecule contains one or two 3' overhang(s).

The double-stranded molecule of the present invention is described in more detail below.
Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Patent No. 6,506,559, herein incorporated by reference in its entirety). For example, a computer program for designing siRNAs is available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html).
Such a computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.

Selection of Target Sites:
1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. Basically, BLAST, which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/, is used (Altschul SF et al., Nucleic Acids Res 1997 Sep 1, 25(17): 3389-402).
3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.

Using the above protocol, the target sequences of the isolated double-stranded molecules of the present invention were designed as:
SEQ ID NO: 11 and 12 for FAM161A gene, and
SEQ ID NO: 13 and 14 for CSNK2A2 gene.

Double-stranded molecules targeting the above-mentioned target sequences were respectively examined for their ability to suppress the growth of cells expressing the target genes. Accordingly, the present invention provides double-stranded molecules targeting any of the sequences selected from among:
SEQ ID NOs: 11 (at the position 894-912nt of SEQ ID NO: 17) and 12 (at the position 1885-1903nt of SEQ ID NO: 17) for FAM161A, and SEQ ID NOs: 13 (at the position 773-791nt of SEQ ID NO: 19) and 14 (at the position 793-811nt of SEQ ID NO: 19) for CSNK2A2 gene.
The double-stranded molecule of the present invention may be directed to a single target FAM161A or CSNK2A2 gene sequence or may be directed to a plurality of target FAM161A or CSNK2A2 gene sequences.

A double-stranded molecule of the present invention targeting the above-mentioned targeting sequence of FAM161A or CSNK2A2 gene include isolated polynucleotides that contain any of the nucleic acid sequences corresponding to target sequences and/or complementary sequences to the target sequences. Examples of polynucleotides targeting FAM161A or CSNK2A2 gene include those containing the sequence corresponding to SEQ ID NO: 11, 12, 13 or 14 and/or complementary sequences to these nucleotide sequences.

In an embodiment, a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and another polypeptide has a complementary sequence to the target sequence, i.e., antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule. Examples of such double-stranded molecules include dsRNA and dsD/R-NA . In an another embodiment, a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand. Generally, the sense strand and the antisense strand are linked by a intervening strand, and hybridize to each other to form a hairpin loop structure. Examples of such double-stranded molecule include shRNA and shD/R-NA. In preferred embodiments, double-stranded molecules targeting the FAM161A or CSNK2A2 gene may have a sequence selected from among SEQ ID NOs: 11, 12, 13 and 14 as a target sequence. Accordingly, preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 11, 12, 13 or 14 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 11, 12, 13 or 14 and a complementary sequence thereto.

In other words, a double-stranded molecule of the present invention includes a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule. In the double-stranded molecule including the polynucleotides, a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence and complementary sequence thereto is replaced with "u". Alternatively, a part of the polynucleotide of either or both of the strands may be DNA, and when the target sequence is defined with a RNA sequence, the nucleotide "u" within the target sequence and complementary sequence thereto is replaced with "t".

In one embodiment of the present invention, such a double-stranded molecule of the present invention includes a stem-loop structure, composed of the sense and antisense strands. The sense and antisense strands may be joined by a loop. Accordingly, the present invention also provides the double-stranded molecule including a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.

In the present invention, double-stranded molecules targeting the FAM161A and/or CSNK2A2 gene may have a sequence selected from among SEQ ID NOs: 11, 12, 13 and 14 as a target sequence. Accordingly, preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 11, 12, 13 and 14 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 11, 12, 13 and 14 and a complementary sequence thereto.

However, the present invention is not limited to these examples, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of FAM161A or CSNK2A2 gene. Herein, the phrase "minor modification" as used in connection with a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleotide(s) to the sequence.

In the context of the present invention, the term "several" as applies to nucleotide substitutions, deletions, additions and/or insertions may mean 3 to 7, preferably 3 to 5, more preferably 3 or 4, even more preferably 3 nucleic acid residues.

According to the present invention, a double-stranded molecule of the present invention can be tested for its suppression ability using the methods utilized in the Examples. In the Examples herein below, double-stranded molecules composed of sense strands of various portions of FAM161A or CSNK2A2 mRNA and antisense strands complementary thereto were tested in vitro for their ability to decrease production of a FAM161A or a CSNK2A2 gene product in lung cancer cell lines according to standard methods. For example, reduction in a FAM161A or a CSNK2A2 gene product in cells transfected with the candidate double-stranded molecule compared to that in cells transfected no oligonucleotide or control siRNA (e.g., siRNA against EGFP or LUC) can be detected by, e.g. RT-PCR using primers for FAM161A or CSNK2A2 mRNA mentioned under Examples in the section entitled "Semi-quantitative RT-PCR". Candidate target sequences that decrease the production of a FAM161A or a CSNK2A2 gene product in vitro cell-based assays can then be tested for their inhibitory effects on cell growth. Target sequences that inhibit cell growth in vitro cell-based assay may then be tested for their in vivo suppression ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of a FAM161A or a CSNK2A2 gene product and decreased cancer cell growth.

When the isolated polynucleotide is RNA or derivatives thereof, base "t" should be replaced with "u" in the nucleotide sequences. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide, and the term "binding" means the physical or chemical interaction between two polynucleotides. When the polynucleotide includes modified nucleotides and/or non-phosphodiester linkages, these polynucleotides may also bind each other as same manner. Generally, complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. However, the present invention extends to complementary sequences that include mismatches of one or more nucleotides. In addition, the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In an especially preferred embodiment, where the strands of the duplex are fully complementary, such duplexes contain no mismatches.

The complementary or antisense polynucleotide is preferably less than 3692 nucleotides in length for FAM161A or less than 1674 nucleotides in length for CSNK2A2. Preferably, the polynucleotide is less than 500, 200, 100, 75, 50, or 25 nucleotides in length for all of the genes. The isolated polynucleotides of the present invention are useful for forming double-stranded molecules against a FAM161A or a CSNK2A2 gene or preparing template DNAs encoding the double-stranded molecules. When the polynucleotides are used for forming double-stranded molecules, the polynucleotide may be longer than 19 nucleotides, preferably longer than 21 nucleotides, and more preferably has a length of between about 19 and 25 nucleotides.

Accordingly, the present invention provides the double-stranded molecules including a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence corresponding to a target sequence. In preferable embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.

The double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells. The identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, a double-stranded molecule of the present invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the FAM161A or CSNK2A2 gene under stringent conditions. Herein, the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence" or "target nucleic acid" or "target nucleotide". In the present invention, the nucleotide sequence of the "target sequence" can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.

The double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. It is well known in the art to introduce chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. A person skilled in the art will the wide array of chemical modifications that may be incorporated into the present molecules (See WO03/070744; WO2005/045037). For example, in one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include, but are not limited to, phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (See US20060122137).

In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Examples of such modifications include, but are not limited to, chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (See WO2004/029212). In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (See WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides may be replaced with deoxyribonucleotides (See Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. However, the present invention should not construed as limited to these examples; any of a number of conventional chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.

The double-stranded molecules of the present invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. For example, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability and are thus contemplated herein. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule composed of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule containing both DNA and RNA on either or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule.

The hybrid of a DNA strand and an RNA strand may either have a DNA sense strand is DNA coupled to an RNA antisense strand, or vice versa, so long as the resulting double stranded molecule can inhibit expression of the target gene when introduced into a cell expressing the gene. In a preferred embodiment, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may have either or both sense and antisense strands composed of DNA and RNA, so long as the resulting double-stranded molecule has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. In order to enhance stability of the double-stranded molecule, the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.

A preferred chimera type double-stranded molecule contains an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of RNA. Preferably, the upstream partial region indicates the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. Alternatively, regions flanking to 5'-end of sense strand and/or 3'-end of antisense strand may be referred to as the upstream partial region. That is, in preferred embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand are composed of RNA. For instance, a chimera or hybrid type double-stranded molecule of the present invention may include following combinations.

sense strand:
5'-[---DNA---]-3'
3'-(RNA)-[DNA]-5'
:antisense strand,
sense strand:
5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5'
:antisense strand, and
sense strand:
5'-(RNA)-[DNA]-3'
3'-(---RNA---)-5'
:antisense strand.

The upstream partial region preferably is a domain composed of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the double-stranded molecule is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).

In the context of the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin composed of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA includes the sense strand containing a sequence corresponding to target sequence and an antisense containing a complementary sequence corresponding to the target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA molecules, which are then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the target sequence of the dsRNA or dsD/R-NA.

A loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Such loop sequence may be joined to 5' or 3' end of a sense strands to form the hairpin loop structure. Thus, the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A]. The target sequence may be selected from among, for example, nucleotide sequences of SEQ ID NOs: 11 and 12 for FAM161A and SEQ ID NOs: 13 and 14 for CSNK2A2.

The present invention is not limited to these examples, and the target sequence in [A] may be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted FAM161A or CSNK2A2 gene. The region [A] hybridizes to [A'] to form a loop composed of the region [B]. The intervening single-stranded portion [B], i.e., loop sequence may be preferably 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from among the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). Furthermore, loop sequence composed of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26;
UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and
UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.

Examples of preferred double-stranded molecules of the present invention having hairpin loop structure are shown below. In the following structure, the loop sequence can be selected from among AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto:
GGUACAUAAAGCGCUCAAA -[B]- UUUGAGCGCUUUAUGUACC (for target sequence SEQ ID NO: 11);
GUACUUGAGUACUUCAACA -[B]- UGUUGAAGUACUCAAGUAC (for target sequence SEQ ID NO: 12);
GAUUAUAGCUUGGACAUGU -[B]- ACAUGUCCAAGCUAUAAUC (for target sequence SEQ ID NO: 13);
GAGUUUGGGCUGUAUGUUA -[B]- UAACAUACAGCCCAAACUC (for target sequence SEQ ID NO: 14).

Additionally, several nucleotides can be added to 3'end of the sense strand and/or the antisense strand of the target sequence, as 3' overhangs so as to enhance the inhibition activity of the double-stranded molecules,. The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotides form (a) single strand(s) at the 3'end of the sense strand and/or antisense strand of the double-stranded molecule. The preferred examples of nucleotides to be added include "t" and "u", but are not limited to. In cases where double-stranded molecules consists of a single polynucleotide to form a hairpin loop structure, a 3' overhang sequence may be added to the 3' end of the single polynucleotide.

The method for preparing the double-stranded molecule is not particularly limited though it is preferable to use one of the standard chemical synthetic methods known in the art. According to one chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. In one specific annealing embodiment, the synthesized single-stranded polynucleotides are mixed in a molar ratio of preferably at least about 3:7, more preferably about 4:6, and most preferably substantially equimolar amount (i.e., a molar ratio of about 5:5). Next, the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down. The annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Examples of purification methods include methods utilizing agarose gel electrophoresis. Remaining single-stranded polynucleotides may be optionally removed by, e.g., degradation with appropriate enzyme.

Alternatively, the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence. The regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules are described below.

The regulatory sequences flanking FAM161A or CSNK2A2 sequences may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. The double-stranded molecules can be transcribed intracellularly by cloning FAM161A or CSNK2A2 gene templates into a vector containing, e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.

Vectors Encoding a Double-Stranded Molecule
Also included in the present invention are vectors containing one or more of the double-stranded molecules described herein, and a cell containing such a vector.
Of particular interest to the present invention are the vectors of [1] to [11] set forth below:
[1] A vector, encoding a double-stranded molecule that, when introduced into a cell, inhibits in the expression of FAM161A or CSNK2A2 and cell proliferation, such molecules composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule.
[2] The vector of [1], encoding the double-stranded molecule acts on mRNA, matching a target sequence selected from among SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14;
[3] The vector of [1], wherein the sense strand contains a sequence corresponding to a target sequence selected from among SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14;
[4] The vector of any one of [1] to [3], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 100 nucleotide pairs in length;
[5] The vector of [4], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 75 nucleotide pairs in length;
[6] The vector of [5], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 50 nucleotide pairs in length;
[7] The vector of [6], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 25 nucleotide pairs in length;
[8] The vector of [7], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[9] The vector of any one of [1] to [8], wherein the double-stranded molecule is composed of a single polynucleotide having both the sense and antisense strands linked by an intervening single-strand;
[10] The vector of [9], encoding the double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to the target sequence;
[11] The vector of any one of [1] to [10], wherein the double-stranded molecule contains one or two 3' overhang(s).

A vector of the present invention preferably encodes a double-stranded molecule of the present invention in an expressible form. Herein, the phrase "in an expressible form" indicates that the vector, when introduced into a cell, will express the molecule carried, contained or encoded therein. In a preferred embodiment, the vector includes one or more regulatory elements necessary for expression of the double-stranded molecule. Accordingly, in one embodiment, the expression vector encodes the nucleic acid sequences of the present invention and is adapted for expression of said nucleic acid sequences. Such vectors of the present invention may be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.

Vectors of the present invention can be produced, for example, by cloning a FAM161A or CSNK2A2 sequence into an expression vector so that regulatory sequences are operatively-linked to the FAM161A or CSNK2A2 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5). For example, RNA molecule that is the antisense strand to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA). After transcribed the sense and antisense strands hybridize to each other in vivo to generate a double-stranded molecule constructs for silencing of the gene. Alternatively, two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and antisense strands and then forming a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); accordingly, a single transcript of a vector may contain both the sense and complementary antisense sequences of the target gene.

The present invention contemplates a vector that includes each or both of a combination of polynucleotides, including a sense strand nucleic acid and an antisense strand nucleic acid, wherein the antisense strand includes a nucleotide sequence which is complementary to said sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said vector, when introduced into a cell expressing the FAM161A or CSNK2A2 gene, inhibits expression of said gene.

The vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See also, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).

The vectors of the present invention include, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell. Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.

Methods of Inhibiting Cancer Cell Growth and Treating and/or Preventing Cancer
The ability of certain siRNA to inhibit NSCLC has been previously described in WO 2005/89735, the entire contents of which are incorporated by reference herein. In the present invention, two different dsRNA for FAM161A or CSNK2A2 were tested for their ability to inhibit cell growth. The two dsRNA for FAM161A (Fig. 3A) and the two dsRNA for CSNK2A2 (Fig. 3B) that effectively knocked down the expression of the gene in lung cancer cell lines coincided with suppression of cell proliferation.

Accordingly, the present invention provides methods for inhibiting lung cancer cell growth, by inducing dysfunction of the FAM161A or CSNK2A2 gene via inhibiting the expression of FAM161A or CSNK2A2. FAM161A or CSNK2A2 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention that specifically target the FAM161A or CSNK2A2 gene or the vectors of the present invention that can express any of the double-stranded molecules.

Such ability of the present double-stranded molecules and vectors to inhibit cell growth of lung cancerous cell indicates that they can be used for methods for treating lung cancer, as well as treating or preventing a post-operative, secondary, or metastatic recurrence thereof. Thus, the present invention provides methods to treat patients with cancer by administering a double-stranded molecule against a FAM161A or a CSNK2A2 gene or a vector expressing the molecules without adverse effect because those genes were hardly detected in normal organs without testis (Fig. 1B, Fig. 2C).

Of particular interest to the present invention are the methods of [1] to [34] set forth below:
[1] A method for inhibiting cancer cell growth and treating and/or preventing cancer, wherein the cancer cell or the cancer expresses a FAM161A and/or CSNK2A2 gene, such method including the step of administering at least one isolated double-stranded molecule against FAM161A or CSNK2A2 gene, or vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing FAM161A or a CSNK2A2 gene, inhibits the expression of the FAM161A or CSNK2A2 genes as well as the cell proliferation, wherein the double-stranded molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
[2] The method of [1], wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 11,12, 13 and 14;
[3] The method of [1], wherein the sense strand contains the sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14;
[4] The method of any one of [1] to [3], wherein the cancer is lung cancer;
[5] The method of any one of [1] to [4], wherein plural kinds of the double-stranded molecules are administered;
[6] The method of any one of [1] to [5], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[7] The method of [6], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[8] The method of [7], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[9] The method of [8], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotides pairs in length;
[10] The method of [9], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[11] The method of any one of [1] to [10], wherein the double-stranded molecule is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[12] The method of [11], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, [B] is the intervening single strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to the target sequence;
[13] The method of [1] to [12], wherein the double-stranded molecule is an RNA;
[14] The method of [1] to [12], wherein the double-stranded molecule contains both DNA and RNA;
[15] The method of [14], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[16] The method of [15] wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[17] The method of [14], wherein the double-stranded molecule is a chimera of DNA and RNA;
[18] The method of [17], wherein a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand are composed of RNA;
[19] The method of [18], wherein the flanking region is composed of 9 to 13 nucleotides;
[20] The method of any one of [1] to [19], wherein the double-stranded molecule contains one or two 3' overhangs;
[21] The method of any one of [1] to [20], wherein the double-stranded molecule is contained in a composition which includes, in addition to the molecule, a transfection-enhancing substance and pharmaceutically acceptable carrier.
[22] The method of [1], wherein the double-stranded molecule is encoded by a vector;
[23] The method of [22], wherein the double-stranded molecule encoded by the vector acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14.
[24] The method of [22], wherein the sense strand of the double-stranded molecule encoded by the vector contains the sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14.
[25] The method of any one of [22] to [24], wherein the lung cancer is NSCLC or SCLC;
[26] The method of any one of [22] to [25], wherein plural kinds of the double-stranded molecules are administered;
[27] The method of any one of [22] to [26], wherein the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotides;
[28] The method of [27], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[29] The method of [28], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[30] The method of [29], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having has a length of less than about 25 nucleotide pairs in length;
[31] The method of [30], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[32] The method of any one of [22] to [31], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[33] The method of [32], wherein the double-stranded molecule encoded by the vector has the general formula 5'-[A]-[B]-[A']-3 or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, [B] is a intervening single-strand is composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[34] The method of any one of [22] to [33], wherein the double-stranded molecule encoded by the vector is contained in a composition which includes, in addition to the molecule, a transfection-enhancing agent and pharmaceutically acceptable carrier.

Therapeutic methods of the present invention are described in more detail below.
The growth of cells expressing a FAM161A or a CSNK2A2 gene may be inhibited by contacting the cells with a double-stranded molecule against a FAM161A or a CSNK2A2 gene, a vector expressing the molecule or a composition containing the same. The cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule. Cell growth may be measured by any of a number of methods known in the art, e.g., using the MTT cell proliferation assay.

The growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention. Exemplary cells include lung cancer cells.

Thus, patients suffering from or at risk of developing a disease related to the over-expression of a FAM161A or CSNK2A2 gene may be treated with the administration of at least one of the present double-stranded molecules, at least one vector expressing at least one of the molecules or at least one composition containing at least one of the molecules. For example, patients suffering from lung cancer may be treated according to the present methods. The type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed. Lung cancer may be diagnosed, for example, with Carcinoembryonic antigen (CEA), CYFRA, pro-GRP and so on, as lung cancer marker, or with Chest X-Ray and/or Sputum Cytology. More preferably, patients treated by the methods of the present invention are selected by detecting the expression of FAM161A or CSNK2A2 in a biopsy sample from the patient by RT-PCR or immunoassay. Preferably, before the treatment of the present invention, the biopsy specimen from the subject is confirmed for FAM161A or CSNK2A2 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.

According to the present method, to inhibit cell growth and thereby treat cancer through the administration of plural kinds of the double-stranded molecules (or vectors expressing or compositions containing the same), each of the molecules may have different structures but act on an mRNA that matches the same target sequence of FAM161A or CSNK2A2 gene. Alternatively, plural kinds of double-stranded molecules may act on an mRNA that matches a different target sequence of same gene. For example, the method may utilize double-stranded molecules directed to FAM161A or CSNK2A2 gene.

For inhibiting cell growth, a double-stranded molecule of present invention may be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts. Alternatively, as described above, a DNA encoding the double-stranded molecule may be introduced into cells as a vector. For introducing the double-stranded molecules and vectors into the cells, transfection-enhancing agent, such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), may be employed.

As noted in the "Definitions" section above, a treatment is deemed "efficacious" if it leads to clinical benefit such as, reduction in expression of FAM161A or CSNK2A2 gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.

To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels". While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.

The treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. For example, reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.

Those of skill in the art understand that a double-stranded molecule of the invention degrades FAM161A or CSNK2A2 mRNA in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, as compared to standard cancer therapies, the present invention requires the delivery of significantly less double-stranded molecule at or near the site of cancer in order to exert therapeutic effect.

One skilled in the art can readily determine the optimal effective amount of the double-stranded molecule of the invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.

The present methods can be used to inhibit the growth or metastasis of lung cancer expressing at least one FAM161A or CSNK2A2; especially the lung cancer is NSCLC and/or SCLC. In particular, a double-stranded molecule containing a target sequence of FAM161A or CSNK2A2 (i.e., SEQ ID NOs: 11, 12, 13 and 14) is particularly preferred for the treatment of lung cancer.

For treating lung cancer, the double-stranded molecule of the invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule. Alternatively, the double-stranded molecule of the invention can be administered to a subject in combination with another therapeutic method designed to treat lung cancer. For example, the double-stranded molecule of the invention can be administered in combination with therapeutic methods currently employed for treating lung cancer or preventing lung cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).

In the context of the present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector that expresses the double-stranded molecule.

Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as lung tumor tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.

Preferably, the liposomes encapsulating the present double-stranded molecule include a ligand molecule that can deliver the liposome to the cancer site. Ligands that bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.

Particularly preferably, the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure. In one embodiment, a liposome of the invention can include both opsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, target tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM.sub.1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
The opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH. sub. 3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.

Vectors expressing a double-stranded molecule of the present invention are discussed above. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express a double-stranded molecule of the invention, to an area of cancer in a patient are within the skill of the art.

The double-stranded molecule of the invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites. For example, the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravesical or intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of the cancer.

The double-stranded molecule of the invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the substance directly into the tissue is at or near the site of cancer preferred. Multiple injections of the substance into the tissue at or near the site of cancer are particularly preferred.
One skilled in the art can also readily determine an appropriate dosage regimen for administering the double-stranded molecule of the invention to a given subject.

For example, the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days. In a preferred dosage regimen, the double-stranded molecule is injected at or near the site of cancer once a day for seven days. Where a dosage regimen includes multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.

Compositions Containing a Double-Stranded Molecule
In addition to the above, the present invention also provides pharmaceutical compositions that include at least one of the present double-stranded molecules or the vectors coding for the molecules.

In the context of the present invention, the term "composition" is used to refer to a product including that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such terms, when used in relation to the modifier "pharmaceutical" (as in "pharmaceutical composition"), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, in the context of the present invention, the term "pharmaceutical composition" refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" or "physiologically acceptable carrier", as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material.

The term "active ingredient" herein refers to a substance in composition that is biologically or physiologically active. Particularly, in the context of pharmaceutical composition, the term "active ingredient" refers to a substance that shows an objective pharmacological effect. For example, in case of pharmaceutical compositions for use in the treatment or prevention of cancer, active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly. Preferably, such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on. Before being formulated, the "active ingredient" may also be referred to as "bulk", "drug substance" or "technical product".

Of particular interest to the present invention are the following compositions [1] to [34]:
[1] A composition for inhibiting cancer cell growth, or treating and/or preventing cancer, wherein the cancer and the cancer cell express FAM161A and/or CSNK2A2 gene, including at least one isolated double-stranded molecule against FAM161A or CSNK2A2 gene, or vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing FAM161A or CSNK2A2 gene, inhibits the expression of FAM161A or CSNK2A2 gene as well as the cell proliferation wherein the double-stranded molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
[2] The composition of [1], wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14;
[3] The composition of [1], wherein the double-stranded molecule, wherein the sense strand contains a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14;
[4] The composition of any one of [1] to [3], wherein the cancer is lung cancer;
[5] The composition of any one of [1] to [4], wherein the composition contains plural kinds of the double-stranded molecules;
[6] The composition of any one of [1] to [5], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[7] The composition of [6], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[8] The composition of [7], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[9] The composition of [8], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[10] The composition of [9], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[11] The composition of any one of [1] to [10], wherein the double-stranded molecule is composed of a single polynucleotide containing the sense strand and the antisense strand linked by an intervening single-strand;
[12] The composition of [11], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand sequence contains a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand contains a sequence complementary to the target sequence;
[13] The composition any one of [1] to [12], wherein the double-stranded molecule is an RNA;
[14] The composition of any one of [1] to [12], wherein the double-stranded molecule is DNA and/or RNA;
[15] The composition of [14], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[16] The composition of [15], wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[17] The composition of [14], wherein the double-stranded molecule is a chimera of DNA and RNA;
[18] The composition of [17], wherein a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand are composed of RNA;
[19] The composition of [18], wherein the flanking region is composed of 9 to 13 nucleotides;
[20] The composition of any one of [1] to [19], wherein the double-stranded molecule contains 3' overhangs;
[21] The composition of any one of [1] to [20], wherein the composition includes a transfection-enhancing agent and pharmaceutically acceptable carrier.
[22] The composition of [1], wherein the double-stranded molecule is encoded by a vector and contained in the composition;
[23] The composition of [22], wherein the double-stranded molecule encoded by the vector acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14.
[24] The composition of [23], wherein the sense strand of the double-stranded molecule encoded by the vector contains the sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14.
[25] The composition of any one of [22] to [24], wherein the lung cancer is NSCLC or SCLC;
[26] The composition of any one of [22] to [25], wherein the composition contains the vector encodes plural kinds of double-stranded molecules or a plural kinds of vectors, each of the vectors encoding a different double-stranded molecule;
[27] The composition of any one of [22] to [26], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[28] The composition of [27], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[29] The composition of [28], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[30] The composition of [29], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[31] The composition of [30], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[32] The composition of any one of [22] to [31], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[33] The composition of [32], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, [B] is a intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[34] The composition of any one of [22] to [33], wherein the composition includes a transfection-enhancing agent and pharmaceutically acceptable carrier.

Suitable compositions of the present invention are described in additional detail below.
The double-stranded molecules of the invention are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Thus, the compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees

In the context of the present invention, suitable pharmaceutical formulations of the present invention include those suitable for oral, rectal, nasal, topical (including buccal, sub-lingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Other formulations include implantable devices and adhesive patches that release a therapeutic agent. When desired, the above-described formulations may be adapted to give sustained release of the active ingredient. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.

The present pharmaceutical formulations contain at least one of the double-stranded molecules or vectors encoding them of the present invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt of the molecule, mixed with a physiologically acceptable carrier medium. Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

According to the present invention, the composition may contain plural kinds of the double-stranded molecules, each of the molecules may be directed to the same target sequence, or different target sequences of FAM161A or CSNK2A2. For example, the composition may contain double-stranded molecules directed to the FAM161A or CSNK2A2 genes or gene products. Alternatively, for example, the composition may contain double-stranded molecules directed to one, two or more target sequences FAM161A or CSNK2A2.

Furthermore, the present composition may contain a vector coding for one or plural double-stranded molecules. For example, the vector may encode one, two or several kinds of the present double-stranded molecules. Alternatively, the present composition may contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.

Moreover, the present double-stranded molecules may be contained as liposomes in the present composition. See under the section entitled "Methods of treating cancer using the double-stranded molecule" for details of liposomes.

Pharmaceutical compositions of the invention can also include conventional pharmaceutical excipients and/or additives. Examples of suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.

For solid compositions, conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
For example, a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, preferably 25-75%, of one or more double-stranded molecule of the invention. A pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, preferably 1-10% by weight, of one or more double-stranded molecule of the invention encapsulated in a liposome as described above, and propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.

In addition to the above, the present composition may contain other pharmaceutically active ingredients, so long as they do not inhibit the in vivo function of the double-stranded molecules of the present invention. For example, the composition may contain chemotherapeutic agents conventionally used for treating cancers. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives. Furthermore, it should be understood that, in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.

In another embodiment, the present invention provides for the use of the double-stranded nucleic acid molecules of the present invention in manufacturing a pharmaceutical composition for use in treating cancer characterized by the expression of FAM161A or CSNK2A2. For example, the present invention relates to a use of double-stranded nucleic acid molecule inhibiting the expression of a FAM161A or a CSNK2A2 gene in a cell, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence selected from among SEQ ID NOs: 11, 12, 13 and 14, for manufacturing a pharmaceutical composition for use in treating cancer expressing FAM161A or CSNK2A2.

The present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a lung cancer characterized by the expression of FAM161A or CSNK2A2, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of FAM161A or CSNK2A2 in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence selected from among SEQ ID NOs: 11, 12, 13 and 14 as active ingredients.

In another embodiment, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating cancer characterized by the expression of FAM161A or CSNK2A2, wherein the method or process includes a step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of FAM161A or CSNK2A2 in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence selected from among SEQ ID NOs: 11, 12, 13 and 14.

Inhibitory Polypeptides of FAM161A
The present invention also relates to inhibitory polypeptides that inhibit the interaction of FAM161A with CSNK2A2 polypeptide. As demonstrated in Examples, a partial N-terminal FAM161A polypeptide could suppress cancer cell growth, possibly through dominant negative effect. Thus, the protein-protein interacting inhibition between CSNK2A2 polypeptide and FAM161A polypeptide may be used as a novel strategy for the development of anti-cancer drugs. Accordingly, the present invention also provides inhibitory polypeptides that inhibits the binding between CSNK2A2 polypeptide and FAM161A polypeptide. Furthermore, the present invention also provides polynucleotides and vectors that encode such polypeptides.

Of particular interest to the present invention are the following items [1] to [5].
[1] A polypeptide including a CSNK2A2-binding domain of a FAM161A polypeptide.;
[2] The polypeptide of [1], wherein the polypeptide is selected from among:
a) a polypeptide including an amino acid sequence of SEQ ID NO: 32; and
b) a polypeptide that includes an amino acid sequence of SEQ ID NO: 32 in which one or more amino acids are substituted, deleted, inserted, and/or added.
[3] The polypeptide of [1] or [2], which is modified with a cell-membrane permeable substance.
[4] A polynucleotide encoding the polypeptide of [1] or [2].
[5] A vector encoding the polypeptide of [1] or [2].

The polypeptides of the present invention are described in more detail below.
The inhibitory polypeptide of the present invention contains a CSNK2A2-binding domain of a FAM161A polypeptide. In a preferred embodiment, the polypeptide of the present invention includes an amino acid sequence of SEQ ID NO: 32.

Furthermore, in some embodiments, the polypeptides of the present invention may include polypeptides homologous (i.e., share sequence identity) to a polypeptide having the amino acid sequence of SEQ ID NO: 32. In the present invention, polypeptides homologous to the polypeptide having the amino acid sequence of SEQ ID NO: 32 are those which contain any mutations selected from addition, deletion, substitution and insertion of one or several amino acid residues and are functionally equivalent. Herein, the phrase "functionally equivalent" refers to having the function to bind to CSNK2A2 polypeptide, consequently inhibit cancer cell proliferation. Therefore, the inhibitory polypeptide of the present invention preferably have amino acid mutations in the amino acid sequence of SEQ ID NO: 32. Alternatively, the inhibitory polypeptide of the present invention may be composed an amino acid sequence having at least 80% or higher, preferably 90% or higher, or more preferably 95% or higher, and further more preferably 98% or 99% or higher homology to the amino acid sequence of SEQ ID NO: 32. Amino acid sequence homology can be determined using algorithms well known in the art, for example, BLAST or ALIGN set to their default settings.

The polypeptides of the present invention can be chemically synthesized as described above (See section above entitled "Screening For An Anti-Cancer Substance I. Protein Based Screening Methods").

The polypeptides of the present invention can be also synthesized by known genetic engineering techniques. For example, a polynucleotide encoding the polypeptide of the present invention is introduced into an appropriate host cell to prepare a transformed cell. The polypeptides of the present invention can be obtained by recovering polypeptides produced by this transformed cell. In a preferred embodiment, the polypeptide encoding the polypeptide may be a vector encoding the polypeptide. Such polynucleotides and vectors can be prepared by conventional methods (See, "Screening for an Anti-cancer Substance I. Protein based screening methods"). Alternatively, the polypeptide of the present invention can be synthesized with an in vitro translation system, in which necessary elements for protein synthesis are reconstituted in vitro.

When genetic engineering techniques are used, the polypeptide of the present invention can be expressed as a fused protein with a peptide having a different amino acid sequence. A vector expressing a desired fusion protein can be obtained by linking a polynucleotide encoding the polypeptide of the present invention to a polynucleotide encoding a different peptide so that they are in the same reading frame, and then introducing the resulting nucleotide into an expression vector. The fusion protein is expressed by transforming an appropriate host with the resulting vector. Different peptides to be used in forming fusion proteins include the following peptides: FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10), 6xHis having six His (histidine) residues, 10xHis, Influenza hemagglutinin (HA) , Human c-myc fragment, VSV-GP fragment, p18 HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, GST (glutathione-S-transferase), HA (Influenza hemagglutinin), Immunoglobulin constant region, beta-galactosidase, and MBP (maltose-binding protein).

The polypeptides of the present invention can be obtained by treating the fusion protein thus produced with an appropriate protease, and then recovering the desired polypeptide. To purify the polypeptide, the fusion protein is captured in advance with affinity chromatography that binds with the fusion protein, and then the captured fusion protein can be treated with a protease. With the protease treatment, the desired polypeptide is separated from affinity chromatography, and the desired polypeptide with high purity is recovered.

In some embodiment, the polypeptides of the present invention may include modified polypeptides. In the present invention, the term "modified" refers, for example, to binding with other substances. Accordingly, in the present invention, the polypeptides of the present invention may further include other substances such as cell-membrane permeable substance. The other substances include organic compounds such as peptides, lipids, saccharides, and various naturally-occurring or synthetic polymers. The polypeptides of the present invention may have any modifications so long as the polypeptides retain the inhibitory function. In some embodiments, the inhibitory polypeptides of the present invention can directly compete with FAM161A polypeptide binding to CSNK2A2 polypeptide. Modifications can also confer additive functions on the polypeptides of the invention. Examples of the additive functions include targetability, deliverability, and stabilization.

Preferred examples of modifications in the present invention include, for example, the introduction of a cell-membrane permeable substance. Usually, the intracellular structure is cut off from the outside by the cell membrane. Therefore, it is difficult to efficiently introduce an extracellular substance into cells. Cell membrane permeability can be conferred on the polypeptides of the present invention by modifying the polypeptides with a cell-membrane permeable substance. As a result, by contacting the polypeptide of the present invention with a cell, the polypeptide can be delivered into the cell to act thereon.

The "cell-membrane permeable substance" refers to a substance capable of penetrating the mammalian cell membrane to enter the cytoplasm. For example, a certain liposome fuses with the cell membrane to release the content into the cell. Meanwhile, a certain type of polypeptide penetrates the cytoplasmic membrane of mammalian cell to enter the inside of the cell. For polypeptides having such a cell-entering activity, cytoplasmic membranes and such in the present invention are preferable as the substance. Specifically, the present invention includes polypeptides having the following general formula.
[R]-[D];
wherein,
[R] represents a cell-membrane permeable substance; [D] represents an amino acid sequence of the inhibitory polypeptide of the present invention. In the above-described general formula, [R] and [D] can be linked directly or indirectly through a linker. Peptides, compounds having multiple functional groups, or such can be used as a linker. Specifically, amino acid sequences containing -GGG- can be used as a linker. Alternatively, a cell-membrane permeable substance and a polypeptide containing a selected sequence can be bound to the surface of a minute particle. [R] can be linked to any positions of [D]. Specifically, [R] can be linked to the N terminal or C terminal of [D], or to a side chain of amino acids constituting [D]. Furthermore, more than one [R] molecule can be linked to one molecule of [D]. The [R] molecules can be introduced to different positions on the [D] molecule. Alternatively, [D] can be modified with a number of [R]s linked together.

For example, there have been reported a variety of naturally-occurring or artificially synthesized polypeptides having cell-membrane permeability (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). All of these known cell-membrane permeable substances can be used for modifying polypeptides in the present invention. In the present invention, for example, any substance selected from the following group can be used as the above-described cell-permeable substance:
poly-arginine; Matsushita et al., (2003) J. Neurosci.; 21, 6000-7.
Tat / RKKRRQRRR (SEQ ID NO: 34) (Frankel et al., (1988) Cell 55,1189-93.
Green & Loewenstein (1988) Cell 55, 1179-88.)
Penetratin / RQIKIWFQNRRMKWKK (SEQ ID NO: 35)
(Derossi et al., (1994) J. Biol. Chem. 269, 10444-50.)
Buforin II / TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 36)
(Park et al., (2000) Proc. Natl Acad. Sci. USA 97, 8245-50.)
Transportan / GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37)
(Pooga et al., (1998) FASEB J. 12, 67-77.)
MAP (model amphipathic peptide) / KLALKLALKALKAALKLA (SEQ ID NO: 38)
(Oehlke et al., (1998) Biochim. Biophys. Acta. 1414, 127-39.)
K-FGF / AAVALLPAVLLALLAP (SEQ ID NO: 39)
(Lin et al., (1995) J. Biol. Chem. 270, 14255-8.)
Ku70 / VPMLK (SEQ ID NO: 40)
(Sawada et al., (2003) Nature Cell Biol. 5, 352-7.)
Ku70 / PMLKE (SEQ ID NO: 41)
(Sawada et al., (2003) Nature Cell Biol. 5, 352-7.)
Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 42)
(Lundberg et al., (2002) Biochem. Biophys. Res. Commun. 299, 85-90.)
pVEC / LLIILRRRIRKQAHAHSK (SEQ ID NO: 43)
(Elmquist et al., (2001) Exp. Cell Res. 269, 237-44.)
Pep-1 / KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 44)
(Morris et al., (2001) Nature Biotechnol. 19, 1173-6.)
SynB1 / RGGRLSYSRRRFSTSTGR (SEQ ID NO: 45)
(Rousselle et al., (2000) Mol. Pharmacol. 57, 679-86.)
Pep-7 / SDLWEMMMVSLACQY (SEQ ID NO: 46)
(Gao et al., (2002) Bioorg. Med. Chem. 10, 4057-65.)
HN-1 / TSPLNIHNGQKL (SEQ ID NO: 47)
(Hong & Clayman (2000) Cancer Res. 60, 6551-6.)

In the present invention, the poly-arginine, which is listed above as an example of cell-membrane permeable substances, is constituted by any number of arginine residues. Specifically, for example, it is constituted by consecutive 5-20 arginine residues. The preferable number of arginine residues is 11.
Hereinafter, the present invention is described in more detail with reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

The methods of the present invention comprise a step of collecting samples or data from a human body or a step of using samples or data collected from a human body for performing analyses such as comparisons with standards; however, the methods may not comprise any step of assessing a physical or mental state such as a disease condition, health, or such of a human being, or assessing a prescription or a therapeutic/surgical plan based on such state.

The methods of the present invention do not comprise any step of direct assessment by a medical doctor, but relate to methods of examining information that would be used as a material when a medical doctor will assess a condition. Data obtained by the present invention, such as the expression level of the FAM161A gene, are useful for diagnoses by medical doctors; however, the methods of the present invention may also be methods in which an individual other than a medical doctor collects and presents data that are useful for diagnoses by medical doctors.

The methods of the present invention comprise a step of processing samples collected or isolated from humans (for example, blood, urine, skin, hair, cell, tissue), or a step of analyzing such sample and collecting various data. These steps may also be carried out in an in vitro system.

Materials and Methods
Cell lines and tissue samples
Fifteen human lung-cancer cell lines used in this study included five adenocarcinomas (NCI-H1781, NCI-H1373, LC319, A549, and PC-14), five squamous cell carcinomas (SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170, and LU61), one large-cell carcinoma (LX1), and four small-cell lung cancers (SBC-3, SBC-5, DMS114, and DMS273) (details are shown in Table 1). All cells were grown in monolayer in appropriate media supplemented with 10% FCS and were maintained at 37 degrees C in humidified air with 5% CO2. Human small airway epithelial cells (SAEC) used as a normal control were grown in optimized medium from Cambrex Bioscience, Inc. Primary NSCLC tissue samples as well as their corresponding normal tissues adjacent to resection margins from patients having no anticancer treatment before tumor resection had been obtained earlier with informed consent (Kikuchi T, et al. Oncogene 2003;22:2192-205., Taniwaki M, et al. Int J Oncol 2006;29:567-75.). All tumors were staged on the basis of the pathologic tumor-node-metastasis classification of the International Union Against Cancer (Table 1A; Sobin L and Wittekind CH. New York: Wiley-Liss; 2002.). Formalin-fixed primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained from 339 patients (208 adenocarcinomas, 95 squamous cell carcinomas, 21 large-cell carcinomas, and 11 adenosquamous carcinomas; 106 female and 233 male patients; median age of 66 y with a range of 29-85 y) undergoing surgery at Saitama Cancer Center. These patients that received resection of their primary cancers did not receive any preoperative treatment, and among them only patients with positive lymph node metastasis were treated with platinum-based adjuvant chemotherapies after their surgery. This study and the use of all clinical materials mentioned were approved by individual institutional ethics committees.

Semiquantitative reverse transcription-PCR
A total of 3 microgram of mRNA aliquot from each sample were reverse transcribed to single-stranded cDNAs using random primer (Roche Diagnostics) and Superscript II (Invitrogen). Semiquantitative reverse transcription-PCR (RT-PCR) experiments were carried out with the following sets of synthesized primers specific for human FAM161A or CSNK2A2 with beta-actin (ACTB)-specific primers as an internal control: FAM161A, 5'-TGCCAACACACTTTATCTCTCTG-3' (SEQ ID NO: 1) and 5'-CCAAATCCCAAGGAGTTTACAA-3' (SEQ ID NO: 2); CSNK2A2, 5'-GTGCAGACAATGCTGTGCTT-3' (SEQ ID NO: 3) and 5'-CTGTGGAGTCTGTGGTCAGC-3' (SEQ ID NO: 4); ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO: 5) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO: 6). PCR reactions were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.

Northern blot analysis
Human multiple tissue blots covering 23 tissues (BD Bioscience) were hybridized with an ³²P-labeled, 385-bp PCR product of FAM161A that was prepared as a probe using primers 5'- AGGGGCAAGTATCCCTTTTT -3' (SEQ ID NO: 7) and 5'- CCCCTGATGGAGAAAAATGA -3' (SEQ ID NO: 8), and 421-bp PCR product of CSNK2A2 that was prepared as a probe using primers 5'-GTGCAGACAATGCTGTGCTT-3' (SEQ ID NO: 9) and 5'- CTGTGGAGTCTGTGGTCAGC-3' (SEQ ID NO: 10). Prehybridization, hybridization, and washing were done following manufacturer's recommendation. The blots of FAM161A and CSNK2A2 were autoradiographed with intensifying screens at -80 degrees C for 14day and 7day.

Preparation of anti-FAM161A polyclonal antibody
Rabbit antibodies specific for FAM161A were raised by immunizing rabbits with FAM161A partial length recombinant protein (MATSHRVAKLVASSLQTPVNPITGARVAQYEREDPLKALAAAEAILEDEEEEKVAQPAGASADLNTSFSGVDEHAPISYEDFVNFPDIHHSNEEYFKKVEE) (SEQ ID NO: 30) and purified using standard protocols. The present inventors confirmed that the antibody was specific to FAM161A on immunocytochemistry using LC319 cells transfected with siRNA against FAM161A or control.

Western blotting
Tumor cells were lysed in lysis buffer; 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and Protease Inhibitor Cocktail Set III (Calbiochem). The protein content of each lysate was determined by a Bio-Rad protein assay (Bio-Rad) with bovine serum albumin as a standard. Ten micrograms of each lysate were resolved on 7.5% to 12% denaturing polyacrylamide gels (with 3% polyacrylamide stacking gel) and transferred electrophoretically onto a nitrocellulose membrane (GE Healthcare Biosciences). After blocking with 5% nonfat dry milk or 5% bovine serum albumin in TBST, the membrane was incubated for 1 hour at room temperature with a rabbit polyclonal antibody. A commercially available rabbit polyclonal anti-CSNK2A2 antibody, ERK1/2 and phosphorylated ERK1/2 were purchased SIGMA and Cell signaling technology, Inc., and were probed to be specific to human CSNK2A2 and total ERK and phosphorylated ERK1/2, by western blot analysis using lysates of lung cancer cell lines. And immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences).

Immunofluorescence analysis
Cultured cells washed twice with PBS(-), fixed in 4% paraformaldehyde solution for 60 minutes at 4 degrees C, and rendered permeable by treatment for 5 minutes with PBS(-) containing 0.1% Triton X-100. Cells were covered with CAS Block (Zymed) for 10 minutes to block nonspecific binding before the primary antibody reaction. Then the cells were incubated with antibody to for 1 hour at room temperature, followed by incubation with Alexa488-conjugated goat anti-rabbit antibodies (Molecular Probes; 1:1,000 dilution) for 1 hour. Images were captured on a confocal microscope (TCS SP2-AOBS; Leica Microsystems).

Immunohistochemistry and tissue microarray.
To investigate clinicopathologic significance of the FAM161A protein in clinical lung cancer samples that had been formalin fixed and embedded in paraffin blocks, the inventors stained the sections using Envision+ Kit/horseradish peroxidase (DakoCytomation) in the following manner. For antigen retrieval, slides were immersed in Target Retrieval Solution pH 9 (DakoCytomation) and boiled at 108 degrees C for 15 min in an autoclave. A rabbit polyclonal anti-human FAM161A antibody was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled anti-rabbit IgG [Histofine Simple Stain MAX PO (G), Nichirei] as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.

Tumor tissue microarrays were constructed with 339 formalin-fixed primary NSCLCs which had been obtained by Saitama Cancer Center with an identical protocol to collect, fix, and preserve the tissues after resection (Chin S F, et al. Mol Pathol 2003;56:275-9., Callagy G, et al. Diagn Mol Pathol 2003;12:27-34.). Considering the histological heterogeneity of individual tumors, tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide. Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block using a tissue microarrayer (Beecher Instruments). A core of normal tissue was punched from each case, and 5 micro-m sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed FAM161A positivity without prior knowledge of clinicopathologic data. Positivity for FAM161A was assessed semiquantitatively by three independent investigators without prior knowledge of the clinical follow-up data, each of whom recorded staining intensity as negative (scored as 0) or positive (1+). Cases were accepted as positive only if reviewers independently defined them as such.

Statistical analysis
Statistical analyses were done using the StatView statistical program (SaS). FAM161A immunoreactivity was assessed for association with clinicopathologic variables such as age, gender, pathologic tumor-node-metastasis stage, and histologic type using the Fisher exact test. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for FAM161A expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality. First, the inventors analyzed associations between death and possible prognostic factors including age, gender, histology, pT classification, and pN classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced FAM161A expression into the model, along with any and all variables that satisfied an entry level of P < 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.

RNA interference assay
To evaluate the biological functions of FAM161A and CSNK2A2 in lung cancer cells, small interfering RNA (siRNA) duplexes against the target genes (SIGMA) were used. The target sequences of the synthetic oligonucleotides for RNA interference were as follows:
si-FAM161A-#A, 5'-GGUACAUAAAGCGCUCAAA -3' (for target sequence SEQ ID NO: 11 );
si-FAM161A-#B, 5'-GUACUUGAGUACUUCAACA-3' (for target sequence SEQ ID NO: 12),
si-CSNK2A2-#2, 5'-GAUUAUAGCUUGGACAUGU-3' (for target sequence SEQ ID NO: 13);
si-CSNK2A2-#3, 5'-GAGUUUGGGCUGUAUGUUA-3' (for target sequence SEQ ID NO: 14),
control 1: (EGFP, enhanced green fluorescence protein [GFP] gene, a mutant of Aequorea gictoria GFP), 5'-GAAGCAGCACGACUUCUUC-3' (for target sequence SEQ ID NO: 15);
control 2 (LUC, luciferase gene from Photinus pyralis), 5'-CGUACGCGGAAUACUUCGA-3' (for target sequence SEQ ID NO: 16). A lung cancer cell lines, SBC5 and LC319, were plated onto 10-cm dishes (8.0 x 10⁵ per dish), and transfected with either of the siRNA oligonucleotides (100 nmol/L) using 30 microL of Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions. After 7 days of incubation, these cells were stained by Giemsa solution to assess colony formation, and cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Flow cytometry
SBC5 cells and LC319 cells transfected with siRNA oligonucleotides were plated at densities of 5x10⁵ per 100-mm dish. Cells were trypsinized 2 or 3 day after transfection, collected in PBS, and fixed in 70% cold ethanol for 30 min. After treatment with 100 Ag/mL RNase (Sigma-Aldrich), the cells were stained with 50 Ag/mL propidium iodide(Sigma-Aldrich) in PBS. Flow cytometry was done on a Becton Dickinson FACScan and analyzed by ModFit software (Verity Software House, Inc.). The cells selected from at least 20,000 ungated cells were analyzed for DNA content.

Confirmation of interaction FAM161A and CSNK2A2 by immunoprecipitation.
Cell extracts from COS-7 co-transfected with FAM161A or mock plasmid and CSNK2A2 or mock plasmid and SBC5 transfected with FAM161A or mock plasmid were precleared by incubation at 4 degrees C for 1 hour with 100 microliter of protein G-agarose beads in a final volume of 1 mL of immunoprecipitation buffer (0.5% NP-40, 50 mM Tris-HCl, 150 mM NaCl) in the presence of proteinase inhibitor. After centrifugation at 1000 rpm for 1 min at 4 degrees C, the supernatant was incubated at 4 degrees C with anti-Flag M2 agarose beads for 2 hours. The beads were then collected by centrifugation at 5000 rpm for 1 min and washed six times with 1 mL of each immunoprecipitation buffer. The washed beads were resuspended in 20 microliter of Laemmli sample buffer and boiled for 5 min, and the proteins were separated in 10% SDS polyacrylamide gel electrophoresis (PAGE) gels (BIO RAD). After electrophoresis, western blotting analysis system was done using a commercially available rat monoclonal antibody to HA tag of exogenous CSNK2A2 or rabbit polyclonal antibody to endogenous CSNK2A2 (Catalog No. AV53596, SIGMA).

Cell growth assay.
COS-7 and HEK293T cells that weakly expressed endogenous FAM161A and CSNK2A2 were plated at densities of 5.0 x 10^-5 cells/100 mm dish, transfected with plasmids designed to express FAM161A, CSNK2A2 or mock plasmids. COS-7 cells were selected in medium containing 0.4 mg/mL of geneticin (Invitrogen) and HEK293T cells were selected in medium containing 0.9 mg/mL of geneticin for 7 days, and cell numbers were assessed by MTT assay.

Identification of FAM161A-Associating Protein
Cell extracts from SBC5-FAM161A or SBC5-Mock were precleared by incubation at 4 degrees C for 1 hour with 100 microliter of protein G-agarose beads in a final volume of 1 mL of Immunoprecipitation buffer (0.5% NP-40, 50 mM Tris-HCl, 150 mM NaCl) in the presence of proteinase inhibitor. After centrifugation at 1,000 rpm for 1 min at 4 degrees C, the supernatant was incubated at 4 degrees C with anti-Flag M2 agarose beads for 2 hours. The beads were then collected by centrifugation at 5,000 rpm for 1 min and washed six times with
1 mL of each immunoprecipitation buffer. The washed beads were resuspended in 20 microliter of Laemmli sample buffer and boiled for 5 min, and the proteins were separated in 5-20% SDS polyacrylamide gel electrophoresis (PAGE) gels (BIO RAD). After electrophoresis, the gels were stained with colloidal CBB. Protein band specifically found in SBC5-FAM161A extracts immunoprecipitated with anti-Flag M2 agarose beads was excised and served for matrix-assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis (AXIMA-CFR plus, SHIMADZU BIOTECH).

Dominant-Negative Peptide Assay.
Twenty-two amino acid sequence derived from minimized CSNK2A2-binding domain in FAM161A (codons 331-373; see Fig. 6B) was covalently linked at its N-terminus to a membrane transducing 11 poly-arginine sequence (11R) as described elsewhere (Hayama S, et al. Cancer Res 2006;66:10339-48., Hayama S, et al. Cancer Res 2007; 67:4113-22.). Three dominant-negative peptides were synthesized covering the codons 331-373 region:
1P FAM161A_331-352, RRRRRRRRRRR-GGG-EEQKRAAREKQLRDFLKYKKKT (SEQ ID NO: 31);
2P FAM161A_342-363, RRRRRRRRRRR-GGG-LRDFLKYKKKTNRFKARPIPRS (SEQ ID NO: 32);
3P FAM161A_352-373, RRRRRRRRRRR-GGG-TNRFKARPIPRSTYGSTTNDKL (SEQ ID NO: 33). Peptides were purified by preparative reversephase high-performance liquid chromatography to make >95% purity. Lung cancer LC319 cells that expressed FAM161A and CSNK2A2 as well as normal human bronchial epithelial cell line BEAS-2B that did not express FAM161A and CSNK2A2 were incubated with the 11R peptides at the concentration of 10, 20, or 30 M for 5 days. The viability of cells was evaluated by MTT assay at 5 days after the treatment.

Results
Expression of FAM161A transcripts in lung cancers and normal tissues
To identify novel target molecules for the development of therapeutic agents and/or biomarkers for lung cancer, firstly a cDNA microarray composed of 27,648 genes or expressed sequence tags was screened (NPL11-12). The FAM161A transcript was identified to be overexpressed (>3-fold) in the majority of lung cancer samples examined. Furthermore, microarray expression status showed no FAM161A expression in any of 29 normal tissues except testis (data not shown). As a result, FAM161A was considered as a good candidate gene for novel molecular target. FAM161A overexpression was confirmed by semiquantitative RT-PCR experiments in 11 of 15 lung cancer tissues and in 15 of 15 lung-cancer cell lines examined (Fig. 1A). In addition, northern blot analysis with a FAM161A cDNA as a probe identified a 3.7-kb transcript only in testis among 16 normal human tissues examined (Fig. 1B). To determine the subcellular localization of endogenous FAM161A in lung cancer cells, the inventors performed immunofluoresence analysis using anti-FAM161A antibody and found its staining in the cytoplasm at Interphase and spindle fiber like localization at mitotic phase of SBC-5 cells (Fig. 1C). The specificity of FAM161A antibody was confirmed by immunocytochemistry using LC319 cells transfected with siRNA against FAM161A or control (Fig. 1E). In addition expression of FAM161A protein in five normal tissues (heart, lung, liver, kidney, and testis) as well as lung cancer tissues was examined using anti-FAM161A antibody. Positive staining of FAM161A was observed in the cytoplasm of testis cells and lung cancer cells, but not in other normal tissues (Fig. 1D)

Inhibition of growth of cancer cells by siRNA for FAM161A
To assess whether upregulation of FAM161A plays a role in growth or survival of lung-cancer cells, synthetic oligonucleotide siRNAs against FAM161A (si-FAM161A-#A and si-FAM161A-#B) along with control siRNAs (si-EGFP and si-LUC) were transfected into SBC5 and LC319 cells in which FAM161A was endogenously overexpressed (Fig. 3A). The mRNA levels of FAM161A in cells transfected with si-FAM161A-#A and si-FAM161A-#B were significantly decreased in comparison with those transfected with either control siRNAs (Fig. 3A, top). To clarify the mechanism of tumor suppression by siRNAs against FAM161A, flow cytometric analysis of the tumor cells transfected with these siRNAs was performed. A significant increase of the cells of sub-G1 fraction was found at 72 hours after the treatment (Fig. 4).

Activation of mammalian cellular proliferation by FAM161A
To examine a potential role of FAM161A in tumorigenesis, plasmids designed to express FAM161A (pCAGGSn 3FC-FAM161A) were constructed and were transfected into COS-7 and HEK293T cells. Exogenous FAM161A expression was confirmed by western-blot analysis (Fig. 6A). MTT assay was performed, and found that growth of the COS-7 and HEK293T cells transfected with FAM161A was promoted comparison to the cells transfected with the mock vector (Fig. 6B).

Identification of molecules interacting with FAM161A
To elucidate the biological mechanism of FAM161A in lung carcinogenesis, it was attempted to identify proteins that would interact with FAM161A in lung cancer cells. Cell extracts from SBC5 cells with exogenous FAM161A expressed or mock vector were immunoprecipitated with agarose with anti-FLAG antibody. Following separation by SDS-PAGE, protein complexes were colloidal CBB-stained. Protein bands, which were seen in immunoprecipitates with exogenous FAM161A, but not in those with mock vector, were excised, trypsindigested, and subjected to mass spectrometry analysis. Peptides from two independent protein bands matched to amino-acid sequences of CSNK2A2. Subsequently, the cognate interaction of exogenous FAM161A with endogenous CSNK2A2 in SBC5 cells was confirmed by immunoprecipitation experiments (Fig. 5A). To examine a role of CSNK2A2 in tumorigenesis, the present inventors constructed plasmids designed to express CSNK2A2 (pCAGGSn HC-CSNK2A2) and transfected them into COS-7 cells. The expression of exogenous CSNK2A2 in COS-7 cells, and cognate interaction of exogenous FAM161A with exogenous CSNK2A2 in the cells were confirmed by immunoprecipitation experiments (Fig. 5B).

Association of cell proliferation and CSNK2A2 expression
At first, CSNK2A2 expression profile in lung cancer cell lines was examined. Expression of CSNK2A2 proteins in 5 lung cancer cell lines was confirmed by western blot analysis using anti-CSNK2A2 antibody (Fig. 2A). In addition, northern blot analysis with a CSNK2A2 cDNA as a probe identified transcript only in testis among 16 normal human tissues examined (Fig. 2C). To determine the subcellular localization of endogenous CSNK2A2 in lung cancer cells, the inventors performed immunofluoresence analysis using anti-CSNK2A2 antibody and found its staining in the A549 and LC319 cells (Fig. 2B). To assess the association of cell proliferation and CSNK2A2 expression, synthetic oligonucleotide siRNAs against CSNK2A2 (si-CSNK2A2-#2 and si-CSNK2A2-#3) along with control siRNAs (si-EGFP and si-LUC) were transfected into LC319 cells in which CSNK2A2 was endogenously overexpressed (Fig. 3B). The mRNA levels of CSNK2A2 in cells transfected with si-CSNK2A2-#2 and si-CSNK2A2-#3 were significantly decreased in comparison with those transfected with either control siRNAs. In addition, CSNK2A2 expressed plasmid was transfected into COS-7 and HEK293T cells and performed MTT assays, and found that growth of the COS-7 and HEK293T cells transfected with CSNK2A2 was promoted comparison to the cells transfected with the mock vector (Fig. 6B).

Relation between FAM161A and CSNK2A2
To assess the relationship between the two molecules, the expression level of exogenous CSNK2A2 protein was examined in COS-7 and HEK293T cells with exogenous FAM161A transfected. To compare with FAM161A untransfected COS-7 and HEK293T cells, exogenous CSNK2A2 protein level was unregulated in COS-7 and HEK293T cells with FAM161A transfected (Fig.7A, left). To examine endogenous CSNK2A2 protein level, SBC5 cells transfected with exogenous FAM161A were used. To similar, endogenous CSNK2A2 protein level was unregulated in SBC5 and LC319 cells with FAM161A transfected compare with the cells transfected mock vector, too (Fig.7A, right). In contrast, endogenous CSNK2A2 protein level was down regulated in SBC5 and LC319 cells with si-FAM161A transfected compare with the cells transfected si-EGFP (Fig. 7B). Cell extracts from SBC-5 cells with exogenous FAM161A expressed or mock vector were immunoprecipitated with agarose with anti-FLAG antibody. Following separation by SDS-PAGE, protein complexes were colloidal CBB-stained. Protein bands, which were seen in immunoprecipitates with exogenous FAM161A, but not in those with mock vector, were excised, trypsindigested, and subjected to mass spectrometry analysis. Peptides from two independent protein bands matched to amino-acid sequences of CSNK2A2. The present inventors subsequently confirmed the cognate interaction of exogenous FAM161A with endogenous CSNK2A2 in SBC-5 and LC319 cells by Immunoprecipitation experiments (Fig. 7D, 7E).

Detection of downstream of CSNK2A2
CSNK2A2 protein had serine/threonine kinase domain, therefore, screening of CSNK2A2 substrate was performed using COS-7 cells lysate expressed exogenous CSNK2A2. For this screening, molecules of MAPK pathway were detected as substrate of CSNK2A2. Exogenous CSNK2A2 induced activation of ERK was confirmed using WB analysis. And, CSNK2A2 knocked down SBC5 and LC319 cells showed degradation of phosphorylation level of ERK (Fig. 7C).

Growth inhibition of lung cancer cells by dominant-negative peptides of FAM161A.
To investigate the effect of interaction between FAM161A and CSNK2A2 on lung cancer cell growth or survival, the inventors transfected either of five partial constructs of FAM161A with FLAG sequence at its N-terminus or C-terminus (FAM161A_1-220, FAM161A_221-440, FAM161A_441-660, FAM161A_1-440, FAM161A_221-660; Fig. 8A, top₎ into LC319 and SBC-5 cells. Immunoprecipitation with anti-FLAG antibody showed that three construct, FAM161A_221-440, FAM161A_1-440, and FAM161A_221-660 could interact with endogenous CSNK2A2 (Fig. 8B). To narrow down the interacting site on FAM161A, the inventors transfected into LC319 and SBC-5 cells either of three additional construct of FAM161A (FAM161A_221-330, FAM161A_294-373, and FAM161A_331-440; Fig. 6A, bottom) and found that FAM161A_294-373 and FAM161A_331-440 were able to interact with endogenous CSNK2A2 (Fig. 8C). These data showed that the 43-amino acid polypeptides (codons 331-373) in FAM161A could play important roles in the interaction with CSNK2A2.
To develop the bioactive cell-permeable peptides that can inhibit the functional association of FAM161A with CSNK2A2, the present inventors synthesized three different kinds of 19-amino acid polypeptides covering the CSNK2A2-binding site (codons 331-373) with a membrane-permeable 11 residues of arginine (11R) at its N-terminus (P1-FAM161A_331-352, P2-FAM161A_342-363, P3-FAM161A_352-373; Fig. 8D).

To assess the effect of these peptides on lung cancer cell growth or survival, LC319 and SBC-5 cells were treated with each of the three peptides. Addition of the P2-FAM161A_342-363 into the culture medium inhibited the complex formation between FAM161A and CSNK2A2 (Fig. 8E). To examine the effect of cell-permeable peptides on cell growth of lung cancer, the inventors performed cell growth assay using LC319 cells. As a result, LC319 cells treated with P2-FAM161A_342-363 significantly decreases in cell viability as measured by MTT assay with dose dependent manner (Fig. 8F). On the other hand, no effect on cell growth was observed when the cells were treated with the remaining two peptides (P1-FAM161A_331-352 and P3-FAM161A_352-373). The P2-FAM161A_342-363 revealed no effect on cell viability of human bronchial epithelial cell line BEAS-2B cells in which FAM161A and CSNK2A2 expression were hardly detectable (Fig. 8G, 8H). These data suggested that P2-FAM161A_342-363 peptides could inhibit a functional complex formation of FAM161A and CSNK2A2 and have no offtarget toxic effect on normal human cells that do not express FAM161A protein.

Association of FAM161A overexpression with poor prognosis for NSCLC patients.
To examine the biological and clinicopathological significance of FAM161A in prognosis of lung cancer patients, the inventors carried out immunohistochemical staining on tissue microarrays containing NSCLC tissues from 339 patients who underwent curative surgical resection. FAM161A positive staining with the anti-FAM161A polyclonal antibody was observed in the cytoplasm in lung cancer cells, but staining was negative in any of their adjacent normal lung cells or stromal cells surrounding tumor cells. The present inventors classified FAM161A expression levels on the tissue array ranging from negative (scored as 0) to positive (scored as 1+) (Fig. 9A). Of the 339 NSCLCs, FAM161A was positively stained in 153 cases (45%; score 1+) and not stained in 186 cases (55%: score 0; details are shown in Table 2A). The inventors next examined a correlation of FAM161A expression levels (positive versus negative) with various clinicopathologic variables and found that strong FAM161A expression was associated with poor prognosis for NSCLC patients after the resection of primary tumors (P = 0.0004, log-rank test; Fig.9B, Table3). In addition, the inventors examined univariate analysis to evaluate associations between patient prognosis and several clinicopathological factors including gender (male versus female), age (>=65 versus <65 years), histology (non-adenocarcinoma versus adenocarcinoma), smoking history (smoker versus non-smoker), pT stage (tumor size; T2-T3 versus T1), pN stage (lymph node metastasis; N1-N2 versus N0), and FAM161A expression (score 1+ versus 0). All those parameters except age and smoking history were significantly associated with poor prognosis (Table 2B). Multivariate analysis using the Cox proportional hazard model indicated that pT stage, pN stage, and strong FAM161A positivity were independent prognostic factors for NSCLC (Table 2B).

Discussion
Developments of molecular-targeting anticancer drugs are expected to be highly specific to malignant cells, with minimal risk of adverse reactions. For development of novel molecular target, the present inventors established a powerful screening system to identify proteins and their interacting proteins that were activated specifically in lung cancer cells. Firstly, the present inventors analyzed a genome-wide expression profile of 101 lung cancer samples through the genome-wide cDNA microarray system containing 27,648 genes coupled with laser microdissection. After verification of very low or absent expression of such genes in normal organs by cDNA microarray analysis and multiple-tissue northern blot analysis, the present inventors analyzed the protein expression of candidate targets among hundreds of clinical samples on tissue microarrays, investigated loss of function phenotypes using RNA interference systems, and further defined biological functions of the proteins. Through these analyses, the present inventors identified candidate genes for the development of novel diagnostic biomarkers, therapeutic drugs, and/or immunotherapy that were up-regulated in cancer cells but not expressed in normal organs, except testis, placenta, and/or fetus, and considered them to be good candidates (NPL3, 11-15). In this invention, the present inventors report that FAM161A, encoding a member of cleavage stimulation factor, was frequently overexpressed in the great majority of clinical lung cancer samples and cell lines, and that its gene products play indispensable roles in the growth and progression of lung cancer cells.

FAM161A protein encodes a 660-amino-acid protein and functional analyses don't have ever done yet. This is the first report about functional analysis of FAM161A. And CSNK2A2 that is interaction protein of FAM161A is known about one of subunit of Casein kinase 2. CK2 is well known protein complex and is considered the target of cancer therapy. The present inventors detected FAM161A associated with CSNK2A2 stability, then, the present inventors focused on the catalytic activity of CSNK2A2 in isolation, not as one of CK2 complex. By screening of CSNK2A2 downstream, the present inventors identified CSNK2A2 activated MAP kinase (MAPK) cascade. The MAPK cascade plays an important role in the intracellular signal transduction of eukaryotic cells. The abnormally activation of MAPK cascade caused a cell carcinogenesis and cancer cell proliferation.

The results disclosed herein, show that FAM161A gene is overexpressed in lung cancer with high frequency and likely to be playing an important role in the development / progression of lung cancers. Knockdown of FAM161A expression by siRNA in lung cancer cells induced suppression of cell growth. The results obtained by in vitro and in vivo assays strongly suggested that FAM161A is likely to be an important growth factor and be associated with a more malignant phenotype of lung cancer cells.

In conclusion, FAM161A and CSNK2A2 genes might play an important role in the growth/progression of lung cancers. FAM161A and CSNK2A2 interaction induced activation of MAPK cascade of cancer cells and inhibition of interaction between FAM161A and CSNK2A2 suppressed cancer cell survival and proliferation. In addition these data strongly raise the possibility of designing new anticancer drugs to specifically target the oncogenic activity of FAM161A and CSNK2A2 for the treatment of lung cancer patients.

The results herein demonstrate that the expression of the human gene FAM161A is markedly elevated in lung cancer. Accordingly, this gene can be conveniently used as diagnostic and/or prognostic marker of lung cancer and the protein encoded thereby may be used in diagnostic assays of lung cancer.

The results herein additionally identify CSNK2A2 as an interacting molecule of the FAM161A. In particular, multiple tissue northern-blot analysis identified FAM161A and CSNK2A2 expression only in testis and not in any other normal tissues. Additionally, cell growth is suppressed by a double-stranded nucleic acid molecule that specifically targets the FAM161A or CSNK2A2 gene. Thus, such double-stranded nucleic acid molecule find utility in the development of anti-cancer pharmaceuticals.

Furthermore, as the results herein demonstrate, FAM161A and/or CSNK2A2 polypeptide is a useful target for the development of anti-cancer pharmaceuticals. For example, substances that bind FAM161A and/or CSNK2A2 or block the expression of FAM161A and/or CSNK2A2, or prevent biological activity of FAM161A and/or CSNK2A2 may find therapeutic utility as anti-cancer or diagnostic agents, particularly anti-cancer agents for the treatment of lung cancer.
The results herein additionally identify the ERK1/2 polypeptide as a novel substrate for CSNK2A2 polypeptide. Accordingly, a kit that contains the ERK1/2 polypeptide as a substrate for CSNK2A2 polypeptide may find utility in connection with assays screening for candidate anti-cancer agents.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims (44)

1. A method of detecting or diagnosing cancer, or a predisposition for developing cancer in a subject, comprising determining an expression level of a FAM161A gene in a subject-derived biological sample, wherein an increase of the expression level in the subject-derived biological sample as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, wherein the expression level is determined by any one of method selected from the group consisting of:
   (a) detecting an mRNA of a FAM161A gene;
   (b) detecting a protein encoded by a FAM161A gene; and
   (c) detecting a biological activity of a protein encoded by a FAM161A gene.
2. The method of claim 1, wherein the FAM161A expression level is at least 10% greater than the normal control level.
3. The method of claim 1, wherein the subject-derived biological sample is biopsy sample.
4. A kit for detecting or diagnosing cancer, or a predisposition therefor, which comprises a reagent selected from the group consisting of:
   (a) a reagent for detecting an mRNA of a FAM161A gene;
   (b) a reagent for detecting a protein encoded by a FAM161A gene; and
   (c) a reagent for detecting a biological activity of a protein encoded by a FAM161A gene.
5. The kit of claim 4, wherein the reagent comprises a probe or a primer set to the mRNA of the FAM161A gene, or an antibody against the protein encoded by the FAM161A gene.
6. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
   (a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof;
   (b) detecting the binding activity between the polypeptide or the functional equivalent and the test substance; and
   (c) selecting the test substance that binds to the polypeptide or the functional equivalent.
7. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
   (a) contacting a test substance with a cell expressing a FAM161A gene;
   (b) detecting an expression level of the FAM161A gene in the cell of the step (a); and
   (c) selecting the test substance that reduces the expression level detected in the step (b) in comparison with the expression level of the FAM161A gene detected in the absence of the test substance.
8. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
   (a) contacting a test substance with a FAM161A polypeptide or a functional equivalent thereof;
   (b) detecting a biological activity of the polypeptide or the functional equivalent of the step (a); and
   (c) selecting the test substance that suppresses the biological activity detected in the step (b) in comparison with the biological activity detected in the absence of the test substance.
9. The method of claim 8, wherein the biological activity is a cell proliferation enhancing activity or a binding activity to CSNK2A2 polypeptide.
10. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
    (a) contacting a candidate substance with a cell into which a vector, comprising the transcriptional regulatory region of FAM161A gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
    (b) measuring the expression or activity of said reporter gene; and
    (c) selecting the test substance that reduces the expression or activity level of said reporter gene as compared to a control.
11. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
    (a) contacting a FAM161A polypeptide or a functional equivalent thereof with a CSNK2A2 polypeptide or a functional equivalent thereof in the presence of a test substance;
    (b) detecting binding between the polypeptide(s) or the functional equivalent(s); and
    (c) selecting the test substance that inhibits binding between the polypeptide(s) or the functional equivalent(s).
12. The method of claim 11, wherein the functional equivalent of FAM161A polypeptide comprise a CSNK2A2-binding domain of the FAM161A polypeptide.
13. The method of claim 12, wherein the CSNK2A2-binding domain of the FAM161A polypeptide comprise the position 342 to 363 of SEQ ID NO: 18.
14. The method of claim 11, wherein the functional equivalent of CSNK2A2 polypeptide comprise a FAM161A-binding domain of the CSNK2A2 polypeptide.
15. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
    (a) contacting a test substance with a cell expressing FAM161A gene and CSNK2A2 gene;
    (b) detecting the CSNK2A2 polypeptide level in the cell of step (a); and
    (c) selecting the test substance that decreases the CSNK2A2 polypeptide level of step (b) in comparison with the CSNK2A2 polypeptide level detected in the absence of the test substance.
16. A method of screening for a candidate substance for either or both of treating and preventing cancer , or inhibiting cancer cell growth, said method comprising the steps of:
    (a) contacting a cell expressing CSNK2A2 and ERK1 and/or ERK2 polypeptide with a test substance;
    (b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide; and
    (c) selecting the test substance that reduces the phosphorylation level of the ERK1 and/or ERK2 polypeptide in comparison with the phosphorylation level detected in the absence of the test substance.
17. A method of screening for a candidate substance for either or both of treating and preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
    (a) contacting a CSNK2A2 polypeptide or a functional equivalent thereof with an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof in the present of a test substance under a condition that allows phosphorylation of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
    (b) detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent;
    (c) comparing the phosphorylation level detected in the step (b) with the phosphorylation level detected in the absence of the test substance; and
    (d) selecting the test substance that reduces the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent as a candidate substance for treating and/or preventing cancer, or inhibiting cancer cell growth.
18. The method of claim 17, wherein the functional equivalent of ERK1 or ERK2 polypeptide comprises a fragment of the ERK1 or ERK2 polypeptide having at least one serine or threonine phosphorylation site.
19. A kit for measuring a phosphorylation activity of a CSNK2A2 polypeptide, wherein the kit comprises the following components (a) and (b):
    (a) an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof; and
    (b) a reagent for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent thereof.
20. A kit for detecting for the ability of a test substance to reduce a phosphorylation level of an ERK1 and/or ERK2 polypeptide by a CSNK2A2 polypeptide, wherein the kit comprises the following components of (a) to (c):
    (a) a CSNK2A2 polypeptide or a functional equivalent thereof;
    (b) an ERK1 and/or ERK2 polypeptide or a functional equivalent thereof; and
    (c) a reagent for detecting the phosphorylation level of the ERK1 and/or ERK2 polypeptide or the functional equivalent thereof.
21. The kit of claim 19 or 20, wherein the functional equivalent of the ERK1 or ERK2 polypeptide comprises a fragment of the ERK1 or ERK2 polypeptide having at least one serine or threonine phosphorylation site.
22. An isolated double-stranded molecule that, when introduced into a cell expressing FAM161A and/or CSNK2A2 gene, inhibits an expression of a FAM161A or CSNK2A2 gene as well as cell proliferation, said molecule comprising a sense strand and an antisense strand complementary thereto, said strands hybridized to each other to form the double-stranded molecule, the sense strand comprises the nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13 and 14.
23. The double-stranded molecule of claim 22, wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pairs in length.
24. The double-stranded molecule of claim 22 or 23, which consists of a single polynucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
25. The double-stranded molecule of claim 24, which has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from a group consisting of SEQ ID NOs: 11, 12, 13 and 14, [B] is an intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is an antisense strand including a complementary sequence to the target sequence.
26. A vector encoding the double-stranded molecule of any one of claims 22 to 25.
27. Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to a target sequence of SEQ ID NO: 11, 12, 13 or 14 and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing FAM161A and/or CSNK2A2 gene, inhibits the cell proliferation.
28. A method for either or both of treating and preventing cancer, wherein the method comprises the step of administering an pharmaceutically effective amount of at least one isolated double-stranded molecule against FAM161A or CSNK2A2 gene, or vector encoding the double-stranded molecule, wherein the double-stranded molecule that, when introduced into a cell expression FAM161A and/or CSNK2A2 gene, inhibits an expression of FAM161A or CSNK2A2 gene as well as cell proliferation.
29. The method of claim 28, wherein the double-stranded molecule is that of any one of claims 22 to 25.
30. The method of claim 28, wherein the vector is that of claim 26 or 27.
31. A composition for either or both of treating and preventing cancer, wherein the composition comprises at least one isolated double-stranded molecule against FAM161A or CSNK2A2 gene, or vector encoding the double-stranded molecule, wherein the double-stranded molecule that, when introduced into a cell expression FAM161A and/or CSNK2A2 gene, inhibits an expression of FAM161A or CSNK2A2 gene as well as cell proliferation.
32. The composition of claim 31, wherein the double-stranded molecule is that of any one of claims 22 to 25.
33. The composition of claim 31, wherein the vector is that of claim 26 or 27.
34. A method for monitoring, assessing or predicting a prognosis of a subject with cancer, wherein said method comprises a step of determining an expression level of the FAM161A gene in a subject-derived biological sample, wherein an increase of said expression level compared to a good prognosis control level of the FAM161A gene indicates a poor prognosis of said subject, wherein said expression level is determined by a method selected from the group consisting of:
    (a) detecting an mRNA of the FAM161A gene;
    (b) detecting a FAM161A polypeptide; and
    (c) detecting a biological activity of a FAM161A polypeptide.
35. A kit for use in monitoring, assessing or predicting a prognosis of a subject with cancer, wherein said kit comprises at least one reagent selected from the group consisting of:
    (a) a reagent for detecting an mRNA of the FAM161A gene;
    (b) a reagent for detecting a FAM161A protein; and
    (c) a reagent for detecting a biological activity of a FAM161A protein.
36. The kit of claim 35, wherein the reagent comprises an oligonucleotide that has a complementary sequence to a part of an mRNA of the FAM161A gene and specifically binds to said mRNA; or an antibody against the FAM161A protein.
37. A polypeptide comprising a CSNK2A2-binding domain of a FAM161A polypeptide, wherein the polypeptide lacks a biological function of the FAM161A polypeptide, and wherein the biological function is a function to control a subcellular localization of a CSNK2A2 polypeptide.
38. The polypeptide of claim 37, wherein the polypeptide is selected from the group consisting of:
    a) a polypeptide comprising an amino acid sequence of SEQ ID NO:32;
    b) a polypeptide that comprises an amino acid sequence of SEQ ID NO:32 in which one or more amino acids are substituted, deleted, inserted, and/or added; and
    c) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:32.
39. The polypeptide of claim 37 or 38, which is modified with a cell-membrane permeable substance.
40. A polynucleotide encoding the polypeptide of claim 37 or 38.
41. A vector encoding the polypeptide of claim 37 or 38.
42. A method of either or both of treating and preventing cancer in a subject, wherein the method comprises the step of administering to the subject a pharmaceutically effective amount of the polypeptide of any one of claims 37 to 39 or the vector of claim 41.
43. A composition for either or both of treating and preventing cancer, wherein the composition comprises a pharmaceutically effective amount of the polypeptide of any one of claims 37 to 39 or the vector of claim 41.
44. The method of any one of claims 1 to 3, 6 to 18, 28 to 30, 34, the kit of any one of claims 4, 5, 35 and 36 or the composition of any one of claims 31 to 33 and 43, wherein the cancer is lung cancer.

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