HK40066971B - Treatment and diagnosis of colon cancer - Google Patents

Description

Treatment and diagnosis of colon cancer

This application is a divisional application of patent application No. 201910112579.3, filed on October 31, 2013, with the earliest priority date of October 31, 2012, entitled "Treatment and Diagnosis of Colon Cancer" (the parent application No. 201380068468.8).

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 61/720,912, filed October 31, 2012, and U.S. Provisional Application No. 61/786,500, filed March 15, 2013. The contents of these applications are incorporated herein by reference in their entirety.

Government interests

The invention disclosed herein was made with government support, at least in part, under grant number 1DP2 OD006506-01 from the National Institutes of Health. Therefore, the United States government holds certain rights to this invention.

Invention Field

This invention relates to the diagnosis and treatment of colon cancer.

Background of the Invention

Colorectal cancer, often called colon cancer or bowel cancer, is a cancer that arises from the uncontrolled growth of cells in the colon, rectum, or appendix. See, for example, Cancer Genome Atlas Network, "Comprehensive molecular characterization of human colon and rectal cancer," Nature 487:330–337 (July 19, 2012). It is the second most commonly diagnosed malignancy and the second leading cause of cancer death in the United States. For example, the five-year survival rate for patients diagnosed with colorectal cancer in the early localized stage is 92%, while if the cancer is allowed to spread to nearby organs or lymph nodes, the survival rate drops to 64%, and to 7% in patients with distant metastases.

The prognosis of colorectal cancer is directly related to the extent of tumor penetration through the intestinal wall and the presence or absence of joint involvement. Therefore, early detection and treatment are crucial. Currently, diagnosis is aided by screening tests for fecal occult blood, sigmoidoscopy, colonoscopy, and the use of double-contrast barium enemas. Treatment options, determined by the type and stage of cancer, include surgery, radiation therapy, and/or chemotherapy. However, recurrence after surgery (the most common form of treatment) is a major problem and often the final cause of death. Despite research into treatments for this disease, colorectal cancer remains difficult to diagnose and treat. Thus, new agents and methods are needed for the detection and treatment of colorectal cancer.

Invention Overview

This invention addresses the aforementioned needs by providing agents and methods for diagnosing and treating colon cancer.

In one aspect, the present invention features a method for treating colon cancer (e.g., metastatic colon cancer) in a subject with this need. The method includes increasing the expression level of a microRNA selected from miR-483-5p and miR-551a in the subject. The increasing step can be carried out by administering a nucleic acid encoding miR-483-5p or miR-551a to the subject. The nucleic acid can be an oligonucleotide, such as a synthetic nucleic acid. The nucleic acid can be in a vector, such as a vector selected from the group consisting of plasmids, viruses, granules, artificial chromosomes, and other media derived from bacterial and/or viral sources. Examples of viruses include adeno-associated virus (AAV) or POX virus. The adeno-associated virus or POX virus can be modified to improve activity, stability, or specificity. In some examples, the nucleic acid contains a sequence of any one of SEQ ID NO: 1-10 or its complement. The method may also include administering additional therapeutic agents as disclosed herein to the subject.

In a second aspect, the present invention provides an isolated RNA interference (RNAi) agent that inhibits the expression of creatine kinase brain type (CKB) or creatine transporter channel SLC6a8 (SLC6a8). The agent contains a first nucleotide sequence homologous to or complementary to a region of the gene encoding the CKB or SLC6a8 protein. In some examples, the first nucleotide sequence comprises the sequence of any one of SEQ ID NO: 11-18.

The present invention also provides an isolated nucleic acid comprising an isolated nucleic acid encoding a sequence of the aforementioned RNAi drug and a vector containing the nucleic acid. The vector may be a vector selected from the group consisting of plasmids, viruses, granules, artificial chromosomes, and other media derived from bacteria and/or viruses. Preferably, the vector is a viral vector, such as an AAV viral vector. A host cell having the aforementioned RNAi drug, nucleic acid, or vector is also provided. Additionally, a pharmaceutical composition is provided having (a) a pharmaceutically acceptable vector, and (b) the aforementioned RNAi drug, nucleic acid, or vector. The RNAi drug, nucleic acid, or vector can be combined with other agents such as liposomal compounds or polyethyleneamine for efficient delivery. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent.

In a third aspect, the invention is characterized by a method for treating cancers such as colon cancer (e.g., metastatic colon cancer) or pancreatic cancer in a subject with this need. The method includes reducing the expression level or activity of CKB (or SLC6a8) in the subject. This reduction step can be carried out by administering a nucleic acid, a small molecule compound, or both to the subject. In one example, the reduction step is carried out by administering cyclocreatine or beta-guanidinopropionic acid to the subject. In other examples, the reduction step is carried out by administering an agent selected from the group consisting of the RNAi agents described above, nucleic acids, and vectors. In some embodiments, the method further includes administering an additional therapeutic agent, such as beta-guanidinopropionic acid, to the subject.

The invention also features a method for treating cancers such as colon cancer or pancreatic cancer in a subject with this need. The method includes reducing the level of creatine in the subject by inhibiting the creatine transporter channel SLC6a8. The method includes administering beta-guanidinopropionic acid and optionally additional therapeutic agents to the subject. Examples of additional therapeutic agents include one or more selected from the group consisting of cyclic creatine as described above, RNAi agents, nucleic acids, and vectors. Other examples of therapeutic agents include 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, gemcitabine, cetuximab, taxol, avastin, leucovorin, regorafenib, zaltrap, topoisomerase I inhibitors, NKTR-102, titantinib, PX-866, sorafenib, linifanib, kinase inhibitors, telatinib, XL281 (BMS-908662), robatamuumab, and IGF1-R inhibitors.

In a fourth aspect, the present invention provides a method for determining whether a subject has or is at risk of having metastatic colorectal cancer. The method includes: (i) obtaining a sample from the subject; (ii) measuring the expression level of a microRNA selected from miR-483-5p and miR-551a in the sample; and (ii) comparing the expression level to a predetermined reference value. If the expression level is lower than the predetermined reference value, the subject is determined to have or be at risk of having metastatic colorectal cancer. The predetermined reference value may be obtained from a control subject who does not have metastatic colorectal cancer. The sample may be a body fluid sample or a biopsy tumor sample. The method can also be used to determine whether a subject has or is at risk of having recurrent metastatic colorectal cancer, or to determine whether a subject has or is at risk of having colorectal cancer or metastatic colorectal cancer resistant to chemotherapy agents or targeted therapy. More specifically, if the expression level is lower than the predetermined reference value, the subject is determined to have or be at risk of having recurrent metastatic colorectal cancer, or colorectal cancer or metastatic colorectal cancer resistant to chemotherapy agents or targeted therapy.

In a fifth aspect, the present invention provides an array having (i) a support having multiple unique positions, and (ii) any combination of at least one nucleic acid having a sequence complementary to the expression product (e.g., mRNA or associated cDNA) of a gene encoding miR-483-5p, miR-551a, or CKB or SLC6a8. For example, the nucleic acid may be complementary to one of SEQ ID NO: 1-18 or hybridized to one of SEQ ID NO: 1-18 under strict hybridization conditions. Each nucleic acid is immobilized at a unique position on the support.

This invention also provides a kit for diagnosing the metastatic potential of colorectal cancer in a subject, the potential for recurrence of metastatic colorectal cancer, the potential for rapid progression of metastatic colorectal cancer, or the potential for metastatic colorectal cancer to exhibit resistance to chemotherapy. The kit comprises reagents that specifically bind to the expression products (e.g., mRNA, cDNA, and peptides) of genes encoding miR-483-5p, miR-551a, or CKB or SLC6a8. The reagents may be probes having sequences complementary to the miR-483-5p and miR-551a sequences. For example, each probe may have a sequence complementary to one of SEQ ID NO:1-18 or hybridizing to one of SEQ ID NO:1-18 under strict hybridization conditions. The kit may also contain reagents or the arrays described above for performing hybridization assays or PCR assays.

In a sixth aspect, the present invention provides a method for identifying compounds that can be used to treat colon cancer or to inhibit cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization. The method includes (i) obtaining test cells expressing microRNAs selected from miR-483-5p and miR-551a; (ii) exposing the test cells to the test compound; (iii) measuring the expression level of the microRNAs in the test cells; (iv) comparing the expression level to a control level; and (v) if the comparison indicates that the expression level is higher than the control level, selecting the test compound as a candidate for treating colon cancer or for inhibiting cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization.

The present invention also provides a method for identifying compounds that can be used to treat colon cancer or to inhibit cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization. The method includes (i) obtaining test cells capable of expressing a polypeptide or mRNA of a gene selected from CKB or SLC6a8; (ii) exposing the test cells to the test compound; (iii) measuring the expression level of the gene in the test cells; (iv) comparing the expression level to a control level; and (v) if the comparison indicates that the expression level is lower than the control level, selecting the test compound as a candidate for treating colon cancer or for inhibiting cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization.

In the above method, the control level can be obtained from the same control cells as the test cells, except that the control cells have not been exposed to the test compound. The test cells can be cells from a colon cancer cell line (e.g., the LS-174T human colon cancer line). In some embodiments, gene expression levels can be measured using a reporter construct, wherein a reporter gene (e.g., a gene encoding luciferase, GFP, or LacZ) is operatively linked to the promoter of a gene encoding miR-483-5p, miR-551a, CKB, or SLC6a8.

The present invention also provides a method for treating breast cancer, gastric cancer, pancreatic cancer, esophageal cancer, liver cancer, gallbladder cancer, prostate cancer, sarcoma cancer, melanoma, or lung cancer in a subject with this need. The method includes administering beta-guanidinopropionic acid, etc., to the subject.

Details of one or more embodiments of the invention are set forth below. Other features, objects, and advantages of the invention will be apparent from the description and claims.

This invention relates to the following:

1. A method for treating colon cancer in a subject with this need, comprising reducing the expression level or activity of creatine kinase brain type (CKB) or creatine transporter channel SLC6a8 (SLC6a8) in said subject.

2. The method of item 1, wherein the reduction step is performed by administering cyclocreatine to the subject.

3. The method of item 2 further includes administering an additional therapeutic agent to the subject.

4. The method of item 3, wherein the additional therapeutic agent is selected from the group consisting of: beta-guanidinopropionic acid, 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, gemcitabine, cetuximab, taxol, and avastin.

5. The method of any one of items 1-4, wherein the cancer is metastatic.

6. A method for treating cancer in a subject with this need, comprising administering beta-guanidinopropionic acid to the subject, wherein the cancer is selected from the group consisting of: colon cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, prostate cancer, lung cancer, and melanoma.

7. A method for treating colon cancer in a subject with this need, comprising increasing the expression level of microRNAs selected from miR-483-5p and miR-551a in the subject.

8. The method of item 1, wherein the colon cancer is metastatic.

9. The method of item 7 or 8, wherein the enhancement step is performed by administering a nucleic acid encoding miR-483-5p or miR-551a to the subject.

10. The method of item 9, wherein the nucleic acid is an oligonucleotide.

11. The method of item 10, wherein the oligonucleotide is a synthetic nucleic acid.

12. The method of item 9, wherein the nucleic acid is in a vector.

13. The method of item 12, wherein the vector is selected from the group consisting of plasmids, viruses, entrapments and artificial chromosomes.

14. The method of item 13, wherein the virus is an adeno-associated virus (AAV) or a POX virus.

15. The method of any one of claims 9-14, further comprising administering an additional therapeutic agent to the subject.

16. The method of any one of items 9-14, wherein the nucleic acid comprises the sequence of any one of SEQ ID NO:1-10 or its complement.

17. A method for determining whether a subject has or is at risk of having metastatic colorectal cancer, comprising:

Samples were obtained from the subjects;

The expression levels of microRNAs selected from miR-483-5p and miR-551a in the samples were measured; and

Compare this expression level with a predetermined reference value;

Therefore, if the expression level is lower than the predetermined reference value, the subject is determined to have or be at risk of having metastatic colon cancer.

18. A method for determining whether a subject has or is at risk of metastatic colorectal cancer recurrence, comprising:

Samples were obtained from the subjects;

The expression levels of microRNAs selected from miR-483-5p and miR-551a in the samples were measured; and

The expression level is compared with a predetermined reference value, and if the expression level is lower than the predetermined reference value, the subject is determined to have or be at risk of metastatic colorectal cancer recurrence.

19. A method for determining whether a subject has or is at risk of having recurrence of metastatic colon cancer resistant to a targeted chemotherapy agent, comprising:

Samples were obtained from the subjects;

The expression levels of microRNAs selected from miR-483-5p and miR-551a in the samples were measured; and

The expression level was compared with a predetermined reference value.

Therefore, if the expression level is lower than the predetermined reference value, the subject is determined to have or be at risk of having colon cancer or metastatic colon cancer that is resistant to the targeted chemotherapy agent.

20. The method of any one of items 17-19, wherein the predetermined reference value is obtained from a control subject without metastatic colorectal cancer.

21. The method of any one of items 17-19, wherein the sample is a body fluid sample.

22. The method of any one of items 17-19, wherein the sample is a tumor sample.

23. An array comprising (i) a support having a plurality of unique positions, and (ii) any combination of at least one nucleic acid having a sequence complementary to the expression product of a gene encoding miR-483-5p, miR-551a, or CKB or SLC6a8, wherein each nucleic acid is immobilized at a unique position on the support.

24. A kit for diagnosing the metastatic potential of colon cancer in a subject, comprising a reagent that specifically binds to the expression product of a gene encoding miR-483-5p, miR-551a, or CKB or SLC6a8.

Brief description of the attached diagram

Figures 1A-E are a set of images and photographs showing that miR-483-5p, miR-551a, and CKB are clinically relevant and can be therapeutically inhibited. a, Quantification of miR-483-5p and miR-551a levels in 37 primary tumor samples and 30 liver metastasis samples by quantitative real-time PCR. b, Quantification of CKB expression levels in 37 primary tumor samples and 30 liver metastasis samples by quantitative real-time PCR. c, Liver metastases in mice injected with LvM3b cells and treated one day after cell injection with a single dose of AAV dual-expressing miR-483-5p and miR-551a. d, Bioluminescent measurements of liver metastases in mice injected with 5 x ^10⁵ LvM3b cells and treated daily with cyclocreatine for two weeks. Mice were euthanized at the end of treatment, and livers were harvested for ex vivo imaging. e. Bioluminescence assay of liver metastases in mice injected with 5 x ^10⁵ LvM3b cells and treated daily with the creatine transporter inhibitor beta-guanidinopropionic acid (B-GPA) for up to two weeks. Error bars, sem; all P values are based on one-sided Student's t-test. *P<0.05;**P<0.001;***P<0.0001.

Figure 2 shows a set of images and photographs illustrating the inhibition of colorectal cancer metastasis by B-GPA treatment. Bioluminescence measurements of liver metastases were performed in mice injected with 5 x ^10⁵ LvM3b cells and treated daily with B-GPA for three weeks. Mice were euthanized at three weeks, and livers were harvested for bioluminescence imaging and gross histology. Error bars represent SEM values; all P values are based on one-sided Student's t-test. *P < 0.05.

Figures 3A-C are a set of images and photographs showing the creatine transporter SLC6a8 required for metastasis in colorectal and pancreatic cancer. a) Liver metastasis in highly invasive LvM3b cells expressing the creatine transporter channel SLC6a8 using short hairpins. Liver metastasis was monitored by bioluminescence imaging, and mice were euthanized three weeks after inoculation with cancer cells. Liver samples were harvested for gross histology. b) Liver metastasis in mice injected with 5 x ^10⁵ SW480 cells transduced with shRNA targeting SLC6a8. Metastasis progression was monitored by bioluminescence imaging. Mice were euthanized 28 days after injection, and livers were harvested for bioluminescence imaging and gross histology. c) Liver metastasis in mice injected with 5 x ^10⁵ PANC1 pancreatic cancer cells transduced with shRNA targeting SLC6a8. Metastasis progression was monitored by bioluminescence imaging, and mice were euthanized as described above. Error bars represent SEM; all P-values are based on a one-sided Student's t-test. *P<0.05;**P<0.001;***P<0.0001.

Figure 4 shows the upregulation of SLC6a8 in liver metastases compared to primary tumors. SLC6a8 expression in 36 primary tumors and 30 liver metastases was quantified by quantitative real-time PCR. Error bars represent s.e.m.; all P values are based on one-sided Student’s t-test. *P<0.05.

Figure 5 shows a set of images and photographs illustrating the inhibition of the survival of disseminated PANC1 pancreatic cancer cells in the liver by B-GPA treatment in vivo. Bioluminescence imaging of immunodeficient mice injected with 5 x ^10⁵ PANC1 cells after 48 hours of pretreatment with 10 mM B-GPA is presented. Mice were imaged on day 1 post-injection and the signal was normalized relative to day 0. P values were based on a one-sided Student's t-test. *P < 0.05.

Figure 6 shows the enhancement of gemcitabine's cytotoxicity against PANC1 pancreatic cancer cells by B-GPA. Cell viability of PANC1 pancreatic cancer cells was determined after treatment with stepwise increasing doses of gemcitabine alone or stepwise increasing doses of gemcitabine in combination with 10 mM B-GPA. Cell viability was measured using the WST-1 reagent. Error bars represent the standard error of the mean.

Figure 7 shows the enhancement of 5'-fluorouracil's cytotoxicity against LS-LvM3b colorectal cancer cells by B-GPA. Cell viability of Ls-LvM3b cells was determined after treatment with either stepwise increasing doses of 5'-fluorouracil alone or stepwise increasing doses of 5'-fluorouracil in combination with 10 mM B-GPA. Cell viability was measured using the WST-1 reagent. Error bars represent the standard error of the mean.

Invention Details

This invention is based, in at least part, on the unexpected discovery that a synergistic miRNA-protein network is dysregulated in the liver colonization of metastatic colorectal cancer, and that miRNAs (e.g., miR-483-5p and miR-551a) inhibit metastatic colorectal cancer survival by synergistically targeting creatine kinase-dependent energies. Therefore, this invention provides novel agents and methods for the diagnosis and treatment of colorectal cancer, particularly metastatic colorectal cancer.

Colonization of organs by diffuse cancer cells represents the final, clinically significant, and least understood stage of cancer progression. The liver is a highly common organ for this type of metastatic colonization in many cancer types. To understand the molecular basis of liver colonization, an in vivo selection model of liver colonization in colorectal cancer was established. It is a powerful system because it couples competitive in vivo organ selection with small RNA profiling to functionally assess the in vivo roles of 611 microRNAs during liver colonization in parallel. As disclosed in this paper, endogenous miR-483-5p and miR-551a were identified as potent inhibitors of liver metastatic colonization in multiple colorectal cancer populations with diverse mutation backgrounds. These miRNAs are epigenetically silenced in metastatic cells and in human liver metastases and inhibit metastasis by targeting brain-type creatine kinase (CKB).

As disclosed in this paper, CKB promotes metastasis by enhancing the survival of diffuse cancer cells in the liver (where they encounter hepatic hypoxia). Colon cancer survival depends on the production of CKB from creatine phosphate, which serves as an energy reservoir for generating ATP required to withstand hepatic hypoxia. Consistent with this, inhibiting creatine uptake by cancer cells via creatine transporter knockdown also reduces metastasis. Therapeutic administration of miR-483-5p and miR-551 via adeno-associated virus delivery dramatically inhibited colon cancer metastasis. Furthermore, therapeutic targeting of CKB with small molecule inhibitors significantly inhibited colon cancer metastasis. These results highlight the importance of metabodynamics in maintaining metastatic survival during cancer progression. The findings disclosed in this paper have important implications for the treatment of gastrointestinal malignancies that preferentially colonize the liver and claim more than 500,000 lives annually. Moreover, the in vivo screening/selection method disclosed in this paper has the potential for comprehensive and rapid identification of coding and non-coding genes regulating colonization of any cancer type in any organ.

As disclosed herein, a group of miRNAs were identified as deregulated in human metastatic colorectal cancer lineages using the methods described above. As disclosed herein, miR-483-5p and miR-551a act as potent endogenous inhibitors of colorectal cancer metastasis via a converging targeting of the metabolic gene creatine kinase brain type (CKB). These miRNAs have demonstrated significant prognostic potential in identifying patients with metastatic recurrence of colorectal cancer, and therapeutic delivery of these miRNAs significantly inhibited colorectal cancer metastasis.

Members of the miRNA-protein network disclosed herein can be used as targets for the treatment of metastatic colorectal cancer. Additionally, these members can be used as biomarkers to determine whether a subject has or is at risk of developing metastatic colorectal cancer, or to determine the prognosis or monitor patients with the condition. Therefore, this invention covers methods for treating metastatic colorectal cancer by targeting one or more of the aforementioned members, methods for determining the efficacy of therapeutic regimens for inhibiting cancer, and methods for identifying anticancer agents. Methods for diagnosing whether a subject has or is at risk of developing metastatic colorectal cancer are also provided, as well as methods for screening subjects considered at risk of developing the condition. This invention also covers various kits suitable for implementing the above methods.

Treatment

As disclosed in this paper, miR-483-5p and miR-551a have been identified as endogenous metastasis inhibitors of cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization in colorectal cancer and other cancer types, while CKB and the creatine transport channel SLC6a8 act as metastasis promoters in the same process. These miRNAs or proteins not only strongly predict human metastatic outcomes but also provide targets for the treatment of cancers such as colorectal cancer and other cancer types.

Therefore, this invention provides a method for treating cancers such as colon cancer and other cancer types using relevant agents (including microRNAs, RNAi agents targeting CKB, RNAi agents targeting SLC6a8, vectors encoding such RNAi agents (e.g., AAV), cyclocreatine, and guanidinopropionic acid) via increasing the expression or activity levels of one or more metastasis inhibitors in the subject. This increase can be achieved by forcing one or more metastasis inhibitors to express, etc. Alternatively, treatment can be achieved by decreasing the expression or activity levels of one or more metastasis promoters. Examples of other cancer types include solid tumors, particularly carcinomas. Exemplary solid tumors include, but are not limited to, lung cancer, breast cancer, bone cancer, ovarian cancer, gastric cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testicular cancer, liver cancer, parotid gland cancer, bile duct cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, kidney cancer, bladder cancer, prostate cancer, thyroid cancer, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, mesothelioma, melanoma, myeloma, lymphoma, glioma, glioblastoma, neuroblastoma, Kaposi's sarcoma, and sarcoma. Specifically, the method of the present invention can be used to treat gastric cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, and breast cancer.

Forced expression of transfer inhibitors

Both miR-483-5p and miR-551a, and the nucleic acids encoding them, can be used as transfer inhibitors, thereby enabling the implementation of the invention by overexpressing them in target cells or subjects in need.

"Overexpression" refers to the expression of RNA or polypeptide encoded by nucleic acid introduced into a host cell, wherein the RNA or polypeptide or protein is either not normally present in the host cell, or the RNA or polypeptide is present in the host cell at a higher level than is normally expressed from the endogenous gene encoding the RNA or polypeptide.

All naturally occurring, genetically engineered, and chemically synthesized forms of the above-described inhibitors can be used to practice the invention disclosed herein. To express the above-described inhibitors, the invention provides nucleic acids encoding any of the above-described inhibitors. Preferably, the nucleic acid sequence is isolated and/or purified. Nucleic acids refer to DNA molecules (e.g., but not limited to cDNA or genomic DNA), RNA molecules (e.g., but not limited to mRNA), or DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded. "Isolated nucleic acid" is a nucleic acid whose structure is not identical to any naturally occurring nucleic acid or any fragment of a naturally occurring genomic nucleic acid. The term therefore encompasses, for example, (a) DNA, which has a partial sequence of a naturally occurring genomic DNA molecule but whose flanks are not two coding sequences flanking a portion of the molecule located in the genome of the organism in which it is naturally occurring; (b) nucleic acids, which are integrated into a vector or prokaryotic or eukaryotic genomic DNA in such a way that the resulting molecule is distinct from any naturally occurring vector or genomic DNA; (c) isolated molecules, such as cDNA, genomic fragments, fragments produced by polymerase chain reaction (PCR), or restriction fragments; and (d) recombinant nucleotide sequences, which are part of a heterozygous gene (i.e., a gene encoding a fusion protein).

The terms “RNA,” “RNA molecule,” and “ribonucleic acid molecule” are used interchangeably herein and refer to polymers of ribonucleotides. The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., through DNA replication or DNA transcription, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively).

This invention also provides recombinant constructs having one or more of the nucleotide sequences described herein. Examples of such constructs include vectors, such as plasmids or viral vectors, wherein the nucleic acid sequences of this invention have been inserted in either the forward or reverse orientation. In a preferred embodiment, the construct further comprises a regulatory sequence, including a promoter operatively linked to the sequence. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are also described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press).

Examples of expression vectors include chromosomal, non-chromosomal, and synthetic DNA sequences, such as derivatives of simian virus 40 (SV40), bacterial plasmids, bacteriophage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmid and bacteriophage DNA, and viral DNA such as vaccinia virus, adenovirus, avian sinus virus, and pseudorabies virus. However, any other vector may be used, provided it is replicable and viable in the host. Suitable nucleic acid sequences can be inserted into the vector using various procedures. Generally, a nucleic acid sequence encoding one of the inhibitors described above can be inserted into a suitable restriction endonuclease site using procedures known in the art. Such procedures and related subcloning procedures are within the scope of those skilled in the art.

The nucleic acid sequence in the aforementioned expression vector is preferably operatively linked to a suitable transcriptional regulatory sequence (promoter) to direct RNA synthesis. Examples of such promoters include: retroviral long terminal repeat (LTR) or SV40 promoters, E. coli lac or trp promoters, bacteriophage lambda PL promoters, and other promoters for the expression of known regulatory genes in prokaryotic or eukaryotic cells or viruses. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The vector may contain suitable sequences for amplifying expression. Additionally, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or tetracycline or ampicillin resistance, as in E. coli.

A suitable host can be transformed using a vector containing the appropriate nucleic acid sequence as described above, as well as an appropriate promoter or regulatory sequence, to allow the host to express the inhibitors described above. Such vectors can be used in gene therapy. Examples of suitable expression hosts include bacterial cells (e.g., *Escherichia coli*, *Streptomyces*, *Salmonella typhimurium*), fungal cells (yeast), insect cells (e.g., *Drosophila* and *Spodoptera frugiperda* (Sf9)), animal cells (e.g., CHO, COS, and HEK 293), adenoviruses, and plant cells. The selection of a suitable host is within the scope of those skilled in the art. In some embodiments, the present invention provides a method for producing the above-described inhibitors by transfecting host cells with an expression vector having a nucleotide sequence encoding one of said inhibitors.

Reduce the expression or activity level of transfer promoters

As mentioned above, inhibitory agents that reduce the expression or activity levels of CKB or SLC6a8 can be used in the treatment of colorectal cancer. These inhibitory agents (i.e., inhibitors) can be nucleic acids, peptides, antibodies, or small molecule compounds. In one example, the inhibitor functions at the levels of transcription, mRNA stability, translation, protein stability/degradation, protein modification, and protein binding.

Nucleic acid inhibitors can encode small interfering RNAs (e.g., RNAi agents) that target one or more of the aforementioned genes, such as CKB or SLC6a8, and inhibit their expression or activity. The term "RNAi agent" refers to RNA or its analogues that have sufficient sequence complementarity to the target RNA to direct RNA interference. Examples also include DNA that can be used to prepare the RNA. RNA interference (RNAi) refers to a sequence-specific or selective process in which a target molecule (e.g., a target gene, protein, or RNA) is downregulated. Generally, interfering RNAs ("iRNAs") are double-stranded short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), or single-stranded microRNAs (miRNAs) that cause the catalytic degradation of specific mRNAs and can also be used to reduce or suppress gene expression.

The term "short interfering RNA" or "siRNA" (also known as "small interfering RNA") refers to an RNA agent of about 10-50 nucleotides in length that can direct or mediate RNA interference. It is preferably a double-stranded agent, more preferably about 15-25 nucleotides in length, and more preferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The strand optionally has an overhanging end containing, for example, one, two, or three nucleotides (or nucleotide analogs). Naturally occurring siRNAs are generated from longer dsRNA molecules (e.g., >25 nucleotides in length) via cellular RNAi systems (e.g., Dicer or its homologs).

The term “miRNA” or “microRNA” refers to an RNA agent of about 10-50 nucleotides in length that can direct or mediate RNA interference, preferably a single-stranded agent, more preferably about 15-25 nucleotides in length, and more preferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Naturally occurring miRNAs are generated from stem-loop precursor RNA (i.e., pre-miRNA) via Dicer. As used herein, the term “Dicer” includes Dicer and any Dicer or homolog capable of processing dsRNA structures into siRNA, miRNA, siRNA-like, or miRNA-like molecules. The term microRNA (or “miRNA”) may be used interchangeably with the term “temporally sequential RNA” (or “stRNA”) based on the fact that naturally occurring microRNAs (or “miRNAs”) are found to be expressed temporally (e.g., during development).

As used herein, the term "shRNA" refers to an RNA agent with a stem-loop structure containing first and second regions with complementary sequences, the degree of complementarity and orientation of the regions being sufficient to allow base pairing to occur between the regions, and the second and second regions being connected by a loop region from which base pairing is absent between the nucleotides (or nucleotide analogs) of the loop region.

RNAi, characterized by the degradation of RNA molecules (e.g., intracellularly), is within the scope of this invention. Degradation is catalyzed by an enzymatic RNA-induced silencing complex (RISC). An RNA agent having a sequence sufficiently complementary to the target RNA sequence (e.g., the CKB or SLC6a8 genes described above) to guide RNAi means that the RNA agent has at least 50% homology (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% homology) with the target RNA sequence, thereby making them sufficiently complementary to each other to hybridize and trigger the destruction of the target RNA via an RNAi system (e.g., the RISC complex) or process. An RNA agent having a "sequence sufficiently complementary to the target RNA sequence to guide RNAi" also means that the RNA agent has a sequence sufficient to trigger translational repression of the target RNA via an RNAi system or process. The RNA agent may also have a sequence sufficiently complementary to the target RNA encoded by the target DNA sequence, thereby silencing the target DNA sequence on the chromosome. In other words, RNA agents have sequences sufficient to induce transcriptional gene silencing, such as downregulating gene expression at or near the target DNA sequence, for example by inducing changes in chromatin structure at or near the target DNA sequence.

The aforementioned polynucleotides can be delivered using polymerizable, biodegradable microparticles or microcapsules known in the art. Another method for achieving polynucleotide uptake is using liposomes prepared by standard methods. Polynucleotides can be incorporated alone or co-incorporated with tissue-specific antibodies into these delivery media. Alternatively, molecular conjugates consisting of plasmids or other carriers attached to poly-L-lysine via electrostatic or covalent forces can be prepared. Poly-L-lysine binds to ligands that bind to receptors on target cells (Cristiano, et al., 1995, J. Mol. Med. 73:479). Alternatively, tissue-specific targeting can be achieved using tissue-specific transcriptional regulatory elements known in the art. Delivering naked DNA (i.e., without a delivery mediator) to intramuscular, intradermal, or subcutaneous sites is another means of achieving in vivo expression.

siRNA, miRNA, and asRNA (antisense RNA) molecules can be designed using methods known in the art. Procedures known in the art can be used to design siRNA, miRNA, and asRNA molecules with sufficient sequence specificity to provide unique degradation of any RNA, including but not limited to those available on the websites of AMBION, Inc. and DHARMACON, Inc. Those skilled in the art can routinely perform systematic testing on several design species to optimize siRNA, miRNA, and asRNA sequences. Considerations when designing short interfering nucleic acid molecules include, but are not limited to, biophysical, thermodynamic, and structural considerations, base preference at specific positions in the sense strand, and homology. These considerations are well known in the art and provide guidance for designing the aforementioned RNA molecules.

The antisense polynucleotide (preferably DNA) of the present invention can be any antisense polynucleotide, as long as it has a base sequence that is complementary or substantially complementary to the sequence of the gene encoding a component of the aforementioned network. The base sequence can be at least about 70%, 80%, 90%, or 95% homologous to the complement of the gene encoding the polypeptide. These antisense DNAs can be synthesized using a DNA synthesizer.

The antisense DNA of this invention may contain altered or modified sugars, bases, or linkages. Antisense DNA, as well as the RNAi described above, may also be provided in specific forms such as liposomes, microspheres, or applicable to gene therapy, or may be provided in combination with attached modules. Such attached modules include polycations such as polylysine that act as charge neutralizers for the phosphate backbone, or hydrophobic modules such as lipids (e.g., phospholipids, cholesterol, etc.) that enhance interactions with the cell membrane or increase nucleic acid uptake. Preferred examples of lipids to be attached are cholesterol or its derivatives (e.g., cholesterol chloroformate, bile acids, etc.). These modules may be attached to the 3' or 5' end of nucleic acids and may also be attached to nucleic acids via bases, sugars, or molecular nucleosides. Other modules may be capping groups specifically positioned at the 3' or 5' end of nucleic acids to prevent degradation by nucleases (e.g., exonucleases, RNases, etc.). Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol, tetraethylene glycol, etc. The inhibitory effect of antisense DNA can be examined in vivo and in vitro using the cell line or animal-based gene expression system of the present invention.

Nucleic acids encoding one or more of the aforementioned RNAi agents or peptide inhibitors (discussed below) can be cloned into vectors for in vitro or in vivo delivery to cells. For in vivo use, delivery can target specific tissues or organs (e.g., the liver or colon). Targeted delivery involves the use of vectors (e.g., organ-guided peptides) targeting specific organs or tissues after systemic administration. For example, a vector could have a covalent conjugate of avidin and a monoclonal antibody targeting a liver-specific protein.

In some embodiments, the present invention provides a method for expressing the above-mentioned metastasis inhibitors in vivo. Such methods achieve their therapeutic effects by introducing a nucleic acid sequence encoding any of the said factors into human or non-human animal cells or tissues in which inhibition of cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization is desired. Delivery of the nucleic acid sequence can be achieved using recombinant expression vectors such as chimeric viruses or gel dispersion systems. Preferably, targeted liposomes are used for therapeutic delivery of the nucleic acid sequence.

The various viral vectors disclosed herein that can be used for gene therapy include adenoviruses, adeno-associated viruses (AAVs), herpesviruses, vaccinia viruses, or preferably RNA viruses such as retroviruses and lentiviruses. Preferably, the retroviral vector is a lentivirus or a derivative of a mouse or avian retrovirus. Examples of retroviral vectors that can insert a single foreign gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). Many other retroviral vectors can incorporate multiple genes.

All these vectors can transfer or incorporate selectable marker genes, thereby enabling the identification and generation of transduced cells. Retroviral vectors can be made target-specific by attaching, for example, sugars, glycolipids, or proteins. Preferred targeting is achieved using target-specific antibodies or hormones that have receptors within the target. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to the viral envelope, thereby allowing target-specific delivery of the retroviral vector.

Another targeted system for nucleic acid delivery is the colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, microcells, mixed microcells, and liposomes. The preferred colloidal system of this invention is the liposome. Liposomes are artificial membrane mediators that can be used as delivery mediators both in vitro and in vivo. RNA, DNA, and intact viral particles can be encapsulated within an aqueous interior and delivered to cells in a biologically active form. Methods using liposome mediators for efficient gene transfer are known in the art. The composition of liposomes is typically a combination of phospholipids, often combined with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids that can be used for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include lecithinylcholine, dipalmitoylphosphatidylcholine, and distearatelphosphatidylcholine. Targeting of liposomes is also possible and is known in the art based on, for example, organ specificity, cell specificity, and organelle specificity.

When used in vivo, a reversible delivery-expression system is desirable. For this purpose, Cre-loxP or FLP/FRT systems and other similar systems can be used for the reversible delivery-expression of one or more of the aforementioned nucleic acids. See WO2005/112620, WO2005/039643, US Applications 20050130919, 20030022375, 20020022018, 20030027335, and 20040216178. Specifically, the reversible delivery-expression system described in US Application No. 20100284990 can be used to provide selective or emergency shutdown.

In another example, the aforementioned inhibitory agent or inhibitor may be a polypeptide or protein complex, such as an antibody or its antigen-binding portion, which inhibits or interferes with the activity of CKB or SLC6a8.

The term “antibody” refers to an immunoglobulin molecule or its immunologically active portion, i.e., the antigen-binding portion. Examples include, but are not limited to, proteins having at least one or two heavy (H) chain variable regions ( _VH ) and at least one or two light (L) chain variable regions ( _VL ). The _VH and _VL regions can be further subdivided into hypervariable regions called “complementarity-determining regions” (“CDRs”), which are scattered within more conserved regions called “framework regions” (FRs). As used herein, the term “immunoglobulin” refers to a protein composed of one or more polypeptides essentially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes.

The term "antigen-binding portion" (or "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CKB or SLC6a8). Antigen-binding function of antibodies has been shown to be performed by fragments of full-length antibodies. Examples of binding fragments covered in the term "antigen-binding portion" of an antibody include (i) Fab fragments, a monovalent fragment composed of _VL , _VH , _CL , and _CH1 domains; (ii) F(ab') ₂ fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region; (iii) Fd fragments composed of _VH and _CH1 domains; (iv) Fv fragments composed of _VL and _VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), composed of VH _domains ; and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains ( _VL and _VH ) of the Fv fragment are encoded by separate genes, these two domains can be joined together using a recombinant approach via a synthetic adapter, which allows for the preparation of a single protein chain in which the _VL and _VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of the antibody. These antibody fragments are obtained using common techniques known to those skilled in the art and are screened for use in the same manner as intact antibodies.

Antibodies that specifically bind to one of the aforementioned target proteins (e.g., CKB or SLC6a8) can be prepared using methods known in the art. The antibody can be a polyclonal or monoclonal antibody. In one embodiment, the antibody can be recombinant, for example, produced by phage display or combinatorial methods. In another embodiment, the antibody is a fully human antibody (e.g., an antibody prepared in mice that has been genetically engineered to produce an antibody derived from a human immunoglobulin sequence), a humanized antibody, or a non-human antibody, such as, but not limited to, rodent (mouse or rat), goat, primate (e.g., but not limited to monkey), rabbit, or camel antibodies. Examples of methods for generating humanized antibody forms include, but are not limited to, CDR grafting (Queen et al., U.S. Pat. No. 5, 585, 089; Riechmann et al., Nature 332: 323 (1988)), chain tampering (U.S. Pat. No. 5, 565, 332); and veneering or resurfacing (EP 592, 106; EP 519, 596); Padlan, Molecular Immunology 28 (415): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)). Examples of methods for generating fully human antibodies include, but are not limited to, generating antibodies from mice that express human immunoglobulin genes and using phage display technology to generate and screen human immunoglobulin gene libraries.

"Isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CKB or SLC6a8 is substantially free of antibodies that specifically bind to antigens other than these types). Moreover, isolated antibodies may be substantially free of other cellular material and/or chemicals.

As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to preparations of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the frame and CDR regions are derived from human germline immunoglobulin genes. Furthermore, if the antibody contains a constant region, that constant region is also derived from human germline immunoglobulin genes. The human antibodies of this invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) is grafted onto a human frame sequence.

The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity, wherein both the framework and CDR region are derived from variable regions of human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody can be produced via a hybridoma comprising B cells obtained from a transgenic nonhuman animal (e.g., a transgenic mouse) having a genome containing human heavy chain transgenes and light chain transgenes, said B cells being fused to immortalized cells.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated through recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) or hybridomas prepared therefrom (further elaborated below); (b) antibodies isolated from host cells transformed to express human antibodies (e.g., from transfected tumors); (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which the frame and CDR regions are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be subject to in vitro mutagenesis (or in vivo somatic cell mutagenesis when using animals transgenic for human Ig sequences), and the amino acid sequences of the _VH and _VL regions of the recombinant antibody, although derived from and associated with human germline _VH and _VL sequences, are sequences that are not naturally present in the in vivo germline reservoir of human antibodies.

As used herein, “isotype” refers to an antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The phrases “antibody that recognizes an antigen” and “antigen-specific antibody” are used interchangeably with the term “antibody that specifically binds to an antigen.” As used herein, the term “high affinity” for IgG antibodies means that the antibody has a _KD of ^10⁻⁷ M or less, preferably ^10⁻⁸ M or less, more preferably ^10⁻⁹ M or less, and even more preferably ^10⁻¹⁰ M or less for the target antigen. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for IgM isotypes means that the antibody has a _KD of ^10⁻⁷ M or less, more preferably ^10⁻⁸ M or less.

In one example, the composition contains a monoclonal antibody that neutralizes CKB or SLC6a8. In one embodiment, the antibody may be a fully human antibody, a humanized antibody, or a non-human antibody, such as, but not limited to, rodent (mouse or rat), goat, primate (e.g., but not limited to monkey), rabbit, or camel antibodies. In one embodiment, one or more amino acids of the monoclonal antibody may be substituted to modify its physical properties. These properties include, but are not limited to, binding specificity, binding affinity, immunogenicity, and antibody isotype. Pharmaceutical compositions containing the above-described fully human or humanized forms of the antibody may be used to treat colon cancer or to inhibit cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization.

As used herein, “subject” refers to a human or a non-human animal. Examples of non-human animals include all vertebrates, such as mammals, including non-human mammals, non-human primates (especially higher primates), dogs, rodents (e.g., mice or rats), guinea pigs, cats, and rabbits, as well as non-mammals such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is a laboratory animal or an animal suitable as a disease model. Subjects to be treated for a condition can be identified by standard diagnostic techniques for that condition. Optionally, the subject may be tested for mutations, expression levels, or activity levels of one or more of the CKB, SLC6a8, miR-483-5p, and miR-551a described above using methods known in the art or described above prior to treatment. A subject is a candidate for treatment of the present invention if the subject has a specific mutation in a gene, or if the gene expression or activity level in a sample from the subject is, for example, greater than (in the case of CKB or SLC6a8) the gene expression or activity level in a sample from a normal human.

To confirm inhibition or treatment, techniques known in the art can be used to confirm inhibition of cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization before and/or after the application step. Exemplary techniques include CT scans or PET scans of body organs.

As used herein, “treatment” or “treatment” refers to the administration of a compound or agent to a subject suffering from a condition with the aim of curing, alleviating, relieving, remedying, delaying the onset of the condition, preventing or improving the condition, its symptoms, secondary disease states, or susceptibility to the condition. “Effective amount” or “therapeutic effective amount” refers to the amount of a compound or agent that produces a medically desired outcome in the subject being treated. Treatment methods can be performed in vivo or ex vivo, alone or in combination with other drugs or treatments. Therapeutic effective amounts can be administered, applied, or dosed once or multiple times, and are not intended to be limited to a particular formulation or route of administration.

Therapeutic agents can be administered in vivo or ex vivo, alone or in combination with other drugs or treatments, i.e., cocktail therapy. As used herein, the term "co-administration" means administering at least two agents or treatments to a subject. For example, in the treatment of tumors, particularly malignant tumors, agents can be used alone or in combination with, for example, chemotherapeutic agents, radiotherapy, apoptosis-inducing agents, and/or immunotoxins or coaguligands. In some embodiments, the co-administration of two or more agents/therapies is simultaneous. In other embodiments, the first agent/therapy is administered before the second agent/therapy. Those skilled in the art will understand that the dosage forms and/or routes of administration of the various agents/therapies used can vary.

In vivo methods, a compound or drug is administered to a subject. Typically, the compound is suspended in a pharmaceutically acceptable carrier (such as, but not limited to, physiological saline) and administered orally or intravenously, or via subcutaneous, intramuscular, intrathecal, intraperitoneal, rectal, intravaginal, intranasal, intragastric, intratracheal, or intrapulmonary injection or implantation.

The required dose depends on the chosen route of administration; the nature of the formulation; the nature of the patient's disease; the subject's size, weight, surface area, age, and sex; other medications administered; and the attending physician's judgment. A suitable dose is in the range of 0.01–100 mg/kg. Variations in required doses are to be expected, taking into account the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration may be expected to require a higher dose than intravenous injection. These variations in dose levels can be adjusted using standard empirical optimization methods known in the art. Encapsulation of the compound in a suitable delivery carrier (e.g., polymeric microparticles or implantable devices) can improve delivery efficiency, especially for oral delivery.

Composition

Within the scope of this invention are compositions containing a suitable carrier and one or more of the above-described therapeutic agents. The composition may be a pharmaceutical composition containing a pharmaceutically acceptable carrier, a dietary composition containing a dietaryly acceptable suitable carrier, or a cosmetic composition containing a cosmetically acceptable carrier.

The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects when administered to a subject. The meaning of "acceptable" for a carrier in a pharmaceutical composition also includes that it is compatible with the active ingredient and capable of stabilizing it. One or more solubilizers can be used as drug carriers for delivering the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible mediators, adjuvants, additives, and diluents to obtain compositions that can be used as a dosage form. Other examples of carriers include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

The above-described compositions in any of the above forms can be used to treat colon cancer. The effective dose refers to the amount of active compound/pharmaceutical required to impart a therapeutic effect to the treated subject. As will be appreciated by those skilled in the art, the effective dose can vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of co-administration with other therapeutic treatments.

The pharmaceutical compositions of the present invention can be administered parenterally, orally, nasally, rectally, topically, or buccally. As used herein, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

Sterile injectable compositions may be solutions or suspensions in nontoxic, parenteral-acceptable diluents or solvents. These solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, non-volatile oils are commonly used as solvents or suspension media (e.g., synthetic mono- or diglycerides). Fatty acids (e.g., but not limited to oleic acid and its glyceride derivatives) may be used in the preparation of injections, as may natural, pharmaceutically acceptable oils (e.g., but not limited to, olive oil or castor oil, and their polyoxyethylene versions). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as, but not limited to, carboxymethyl cellulose or similar dispersants. For formulation purposes, other commonly used surfactants may also be used, such as, but not limited to, Tween or Spans or other similar emulsifiers or bioavailability enhancers, which are typically used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms.

Compositions for oral administration can be in any orally acceptable dosage form, including capsules, tablets, emulsions, and aqueous suspensions, dispersants, and solutions. In the case of tablets, common carriers include, but are not limited to, lactose and corn starch. Lubricants, such as, but not limited to, magnesium stearate, are also commonly added. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oil phase in combination with an emulsifier or suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may be added.

The pharmaceutical compositions for surface application according to the present invention can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Optionally, the surface preparation can be in the form of a patch or dressing impregnated with the active ingredient, which may optionally include one or more excipients or diluents. In some preferred embodiments, the surface preparation includes materials capable of enhancing the adsorption or penetration of the active agent through the skin or other infected areas.

The surface composition contains a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A "cosmetically acceptable" or "dermatologically acceptable" composition or ingredient refers to one suitable for contact with human skin without undue toxicity, incompatibility, instability, allergic reactions, etc. The carrier allows the active agent and optional ingredients to be delivered to the skin at a suitable concentration. Therefore, the carrier can function as a diluent, dispersant, solvent, etc., to ensure that the active material is applied and evenly distributed at a suitable concentration on the selected target. The carrier can be solid, semi-solid, or liquid. The carrier can be in the form of an emulsion, cream, or gel, especially one with sufficient thickness or yield point to prevent precipitation of the active material. The carrier can be inert or have dermatological benefits. It should also be physiologically and chemically compatible with the active ingredients of the present invention and should not unduly impair the stability, efficacy, or other benefits associated with the composition.

Diagnostic and prognostic methods

These genes can be used to determine whether a subject has or is at risk of developing metastatic colorectal cancer. Alternatively, they can be used to determine the prognosis of this type of disease in subjects.

Diagnostic methods

In one aspect, the present invention provides qualitative and quantitative information to determine whether a subject has or is susceptible to metastatic colorectal cancer, is susceptible to recurrence of metastatic colorectal cancer, is susceptible to chemotherapy-resistant colorectal cancer, or other diseases characterized by cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization. Subjects with such conditions or predisposition to such conditions can be identified based on the expression levels, patterns, or profiles of the aforementioned genes or products (mRNA, microRNA, or peptides) in test samples from the subject. In other words, the product can be used as a biomarker to indicate the presence or absence of the condition. The diagnostic and prognostic assays of the present invention include methods for assessing the expression levels of the product. This method allows for the detection of the condition. For example, a relatively elevated expression level of one or more promoters (i.e., CKB or SLC6a8) indicates the presence of the condition. Conversely, a lower expression level or lack of expression indicates the absence of the condition. Similarly, a lower expression level or lack of expression of one or more inhibitors (i.e., miR-483-5p or miR-551a) indicates the presence of the condition, while a relatively elevated expression level indicates the absence of the condition.

The presence, level, or absence of mRNA, microRNA, or peptide products in a test sample can be assessed by obtaining the test sample from the test subject and contacting the test sample with a compound or agent that can detect nucleic acids (e.g., RNA or DNA probes) or peptides. A “test sample” includes tissues, cells, and biological fluids isolated from the subject, as well as tissues, cells, and fluids present within the subject. The expression level of a target gene can be measured in many ways, including measuring the RNA encoded by the gene.

Expressed RNA samples can be isolated from biological samples using any of many well-known procedures. For example, biological samples can be lysed in a guanidine-based lysis buffer (optionally containing additional components to stabilize the RNA). In some embodiments, the lysis buffer may contain purified RNA as a control to monitor the recovery and stability of RNA from cell cultures. Examples of such purified RNA templates include kanamycin-positive control RNA from PROMEGA (Madison, WI) and 7.5 kb poly(A)-tailed RNA from LIFE TECHNOLOGIES (Rockville, MD). The lysate can be used immediately or stored, for example, at -80°C.

Optionally, total RNA can be purified from cell lysates (or other sample types) using silica-based separation in an automated, compatible, 96-well format, such as the RNEASY purification platform (QIAGEN, Inc., Valencia, CA). Other RNA isolation methods are also covered, such as extraction with silica-coated beads or guanidine salts. Those skilled in the art can design other methods for RNA isolation and preparation.

The method of the present invention can be carried out using crude samples (e.g., blood, serum, plasma, or cell lysates), thereby eliminating the need for RNA isolation. Optional RNAse inhibitors are added to the crude sample. When using crude cell lysates, it should be noted that, depending on the sample, genomic DNA may supply one or more copies of a target sequence, such as a gene. In cases where the target sequence originates from one or more highly expressed genes, the signal from the genomic DNA may be insignificant. However, for genes expressed at low levels, background can be eliminated by treating the sample with DNase or by using primers that target the splice junction for subsequent initiation of cDNA or amplification products.

RNA levels corresponding to genes in cells can be measured in situ and in vitro. RNA isolated from test samples can be used for hybridization or amplification assays, including Southern or Northern blotting, PCR, and probe arrays. Preferred diagnostic methods for detecting RNA levels involve contacting isolated RNA with a nucleic acid probe that hybridizes to gene-encoded RNA. The probe can be a full-length nucleic acid or a portion thereof, such as an oligonucleotide at least 10 nucleotides in length that is specific enough to hybridize to the RNA under stringent conditions.

In one form, RNA (or cDNA prepared from it) is immobilized on a surface and contacted with a probe, for example, by running isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane such as nitrocellulose. In another form, a probe is immobilized on a surface and RNA (or cDNA) is contacted with the probe, for example, in a gene chip array. Skilled technicians can adapt known RNA detection methods to detect RNA levels.

Nucleic acid amplification can be used to estimate the level of RNA (or cDNA prepared from) the gene to be examined in a sample, for example by standard PCR (US Patent No. 4,683,202), RT-PCR (Bustin S.J Mol Endocrinol. 25:169-93, 2000), quantitative PCR (Ong Y. et al., Hematology. 7:59-67, 2002), real-time PCR (Ginzinger D. Exp Hematol. 30:503-12, 2002), and in situ PCR (Thaker V. Methods MolBiol. 115:379-402, 1999), or any other nucleic acid amplification method, followed by detection of the amplified molecule using techniques known in the art. In another embodiment, the method of the present invention further includes contacting a control sample with a compound or agent capable of detecting the RNA of the gene and comparing the presence of RNA in the control sample with the presence of RNA in the test sample.

The methods and biomarkers described above can be used to assess a subject's risk of developing colorectal cancer. Specifically, the invention is applicable to subjects in a high-risk group who already possess some level of risk, thereby providing crucial insights for early detection. Changes in the levels of the aforementioned gene products associated with colorectal cancer can be detected before or at an early stage in the formation of a transformed or neoplastic phenotype in the subject's cells. The invention therefore also provides a method for screening subjects at risk of developing colorectal cancer or metastatic recurrence of colorectal cancer, comprising assessing the level of at least one disease-related gene product or combination of gene products in a biological sample obtained from the subject. Thus, a change in the level of a gene product or combination of gene products in a biological sample compared to the corresponding gene level in a control sample indicates a subject's risk of developing the disease. Biological samples used for such screening may include tissue samples, which may be normal or suspected of being cancerous. Subjects with changes in the levels of one or more gene products associated with colorectal cancer are candidates for further monitoring and testing. Such further testing may include histological examination of the tissue sample or other techniques in the art.

As used herein, the term "diagnosis" means the detection of a disease or condition, or the determination of its stage or extent. Typically, the diagnosis of a disease or condition is based on the assessment of one or more factors and/or symptoms that indicate the disease. That is, a diagnosis may be based on the presence, absence, or quantity of factors that indicate the presence or absence of a disease or condition. Each factor or symptom considered as an indicator for a diagnosis of a particular disease need not be uniquely associated with that particular disease; different diagnoses can be inferred from a particular diagnostic factor or symptom. Similarly, it is possible that factors or symptoms indicating a particular disease may be present in individuals who do not have that particular disease. The diagnostic methods described may be used independently or in combination with other diagnostic and/or staging methods known in the medical field for a particular disease or condition (e.g., colon cancer).

Prognostic methods

The diagnostic methods described above can identify subjects who have, or are at risk of developing, recurrent colorectal cancer or metastatic colorectal cancer. Additionally, changes in the expression levels and/or trends of the aforementioned genes in biological samples, such as peripheral blood samples, can provide early indicators of recovery or its absence. For example, further increases (or decreases) or persistent alterations in the expression levels of promoter genes (or inhibitor genes) indicate a poorer prognosis, i.e., no improvement or decline in health (or still a poor prognosis). Therefore, these genes allow for the assessment of recovery after colorectal cancer treatment. Analysis of this selected group of genes, or a subset thereof, indicates the outcome of the disease.

The prognostic assays described herein can be used to determine whether a subject is suitable for administration of a drug (e.g., an agonist, antagonist, peptide mimic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat colon cancer or other conditions associated with cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization. For example, such assays can be used to determine whether a subject is suitable for administration of a chemotherapy agent.

Thus, the present invention also provides a method for monitoring treatment of proliferative disorders in subjects. For this purpose, gene expression levels of genes disclosed herein can be measured in test samples from the subject before, during, or after treatment. The magnitude of change in these levels compared to baseline is then assessed. A decrease in the expression of the aforementioned promoter genes (e.g., CKB or SLC6a8) after treatment indicates that the subject can be further treated with the same treatment. Similarly, an increase in inhibitors (e.g., miR-483-5p or miR-551a) also indicates that the subject can be further treated with the same treatment. Conversely, a further increase or persistently high expression level of one or more promoter genes (or a further decrease or persistently low or no expression level of one or more inhibitor genes) indicates a lack of improvement or health decline.

Information obtained from the above-described assays can be used to predict, identify, and clinically manage diseases and other harmful conditions affecting the health of individual subjects. In a preferred embodiment, the aforementioned diagnostic assays provide information that can be used to predict, identify, and manage colon cancer, metastatic colon cancer, and other diseases characterized by cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization. More specifically, this information assists clinicians in designing chemotherapy or other treatment regimens to eradicate such diseases from the body of the affected subject.

As used herein, the term “prognosis” refers to the prediction of the likely course and outcome of a clinical symptom or disease. Prognosis is typically determined by estimating factors or symptoms of the disease that indicate a favorable or unfavorable course or outcome. The phrase “determine prognosis” as used herein refers to the process by which a skilled technician can predict the course or outcome of a disease in a patient. The term “prognosis” does not refer to the ability to predict the course or outcome of a symptom with 100% accuracy; rather, a skilled technician will understand the term “prognosis” to mean an increased likelihood that a particular course or outcome will occur; that is, a certain course or outcome is more likely to occur in patients presenting with the condition (compared to those who do not present with the condition).

As used herein, the terms “favorable prognosis” and “positive prognosis,” or “unfavorable prognosis” and “negative prognosis,” are relevant terms used to predict the likely course and/or possible outcome of a disease or condition. A favorable or positive prognosis is a better predictive outcome for a disease than an unfavorable or negative prognosis. In a general sense, a “favorable prognosis” is a relatively better outcome than many other possible prognoses associated with a particular disease, while an unfavorable prognosis predicts a relatively worse outcome than many other possible prognoses associated with a particular disease. Typical examples of favorable or positive prognoses include better-than-average cure rates, lower tendency to metastasize, longer expected lifespan, and differentiation from a cancerous process to a benign process. For example, a positive prognosis is a 50% chance that a patient has a particular cancer that will be cured after treatment, compared to an average of only a 25% chance of being cured for patients with the same cancer.

The terms “determination/identification,” “measurement,” “evaluation,” and “testing” are used interchangeably and include both quantitative and qualitative measurements, and include determining the presence of a characteristic trait or feature. Evaluation can be relative or absolute. “Evaluating the presence of…(target)” includes determining the amount of the target present, and determining whether it is present or absent.

Array

This invention also provides biochips or arrays. The biochip/array may contain a solid or semi-solid substrate having attached probes or multiple probes as described herein. The probes may be capable of hybridizing with target sequences under stringent hybridization conditions. The probes may be attached to spatially defined addresses on the substrate. More than one probe per target sequence, overlapping probes, or probes targeting different segments of a specific target sequence may be used. The probes may be capable of hybridizing with target sequences associated with a single disease as understood by those skilled in the art. The probes may be synthesized first and then attached to the biochip, or they may be synthesized directly on the biochip.

As used herein, the terms "attachment" or "immobilization" involving nucleic acids (e.g., probes) and solid supports can mean that the binding between the probe and the solid support is sufficiently stable under conditions of binding, washing, analysis, and removal. Binding can be covalent or non-covalent. Covalent bonds can be formed directly between the probe and the solid support or can be formed via cross-linking agents or by incorporating specific reactive groups onto the molecules of the solid support, the probe, or both. Non-covalent binding can be one or more of electrostatic, hydrophilic, and hydrophobic interactions. Examples of non-covalent binding include the covalent attachment of molecules such as streptavidin to a support and the non-covalent binding of biotinylated probes to streptavidin. Immobilization may also involve combinations of covalent and non-covalent interactions.

The solid matrix can be a material that can be modified to contain discrete individual sites suitable for probe attachment or association and can be processed by at least one detection method. Examples of such matrices include glass and modified or functionalized glass, plastics (including acrylic resins, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon J, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, and plastics. The matrix may allow optical detection without perceptible fluorescence.

The matrix can be planar, although other construction mechanisms can also be used. For example, a probe can be placed on the inner surface of a tube for flow-through sample analysis to minimize sample volume. Similarly, the matrix can be flexible, such as elastic foam, including closed-cell foam made of specific plastics.

Arrays/biochips and probes can be derivatized with chemical functional groups for subsequent attachment of both. For example, biochips can be derivatized with chemical functional groups including, but not limited to, amino, carboxyl, oxygen, or thiol groups. Using these functional groups, probes can be attached directly or indirectly using the functional groups on the probes via adapters. Probes can be attached to solid supports via their 5' ends, 3' ends, or via internal nucleotides. Probes can also be non-covalently attached to solid supports. For example, biotinylated oligonucleotides can be prepared that can bind to surfaces covalently coated with streptavidin, thereby inducing attachment. Alternatively, probes can be synthesized on surfaces using techniques such as photopolymerization and photolithography. Detailed descriptions of methods for linking nucleic acids to solid matrices can be found, for example, in U.S. Patent Nos. 5837832, 6087112, 5215882, 5707807, 5807522, 5958342, 5994076, 6004755, 6048695, 6060240, 6090556, and 6040138.

In some embodiments, the expressed transcript (e.g., transcripts of microRNA or polypeptide genes described herein) is represented in a nucleic acid array. In such embodiments, a set of binding sites includes probes with different nucleic acids complementary to different sequence segments of the expressed transcript. Examples of such nucleic acids may be 15 to 200 bases, 20 to 100 bases, 25 to 50 bases, or 40 to 60 bases in length. In addition to the sequence complementary to its target sequence, each probe sequence may also include one or more adapter sequences. The adapter sequence is the sequence between the sequence complementary to its target sequence and the support surface. For example, the nucleic acid array of the present invention may have one probe specific to each target microRNA gene. However, if desired, the nucleic acid array may contain at least 2, 5, 10, 100, 200, 300, 400, 500, or more probes specific to some expressed transcript (e.g., transcripts of microRNA genes described herein).

Reagent test kit

In another aspect, the present invention provides kits embodying methods, compositions, and systems for analyzing peptide and microRNA expression as described herein. Such kits may contain the nucleic acids described herein along with any or all of the following: assay reagents, buffers, probes and/or primers, and sterile saline or another pharmaceutically acceptable emulsion and suspension base. Additionally, the kits may contain instructional materials containing instructions (e.g., protocols) for practicing the methods described herein. For example, the kit may be a kit for amplifying, detecting, identifying, or quantifying target mRNA or microRNA sequences. For this purpose, the kit may contain suitable primers (e.g., hairpin primers), forward primers, reverse primers, and probes.

In one example, the kit of the present invention comprises one or more microarray slides (or alternative microarray formats) pre-loaded with multiple different nucleic acids (each corresponding to one of the genes described above). Alternatively, the kit may comprise multiple polynucleotides suitable for the probe and a selection of markers, or other polynucleotide sequences, for customized use with the included polynucleotide sequences, as determined by a qualified professional. Typically, at least one included polynucleotide sequence corresponds to a control sequence, such as a normalized gene. Exemplary markers include, but are not limited to, fluorophores, dyes, radiolabels, and enzyme tags linked to nucleic acid primers.

In one embodiment, a kit is provided for amplifying nucleic acids corresponding to an expressed RNA sample. Such kits contain kit primers suitable for use in any of the amplification methods described above. Alternatively/additionally, the kit is suitable for amplifying signals corresponding to hybridization between a probe and a target nucleic acid sample (e.g., stored on a microarray).

Additionally, optionally, the kit includes one or more materials and/or reagents required for preparing biological samples for gene expression analysis. Furthermore, optionally included in the kit are one or more enzymes suitable for amplifying nucleic acids, including various polymerases (RT, Taq, etc.), one or more deoxynucleotides, and buffers providing the reaction mixture required for amplification.

Typically, kits are used to analyze gene expression patterns, using mRNA or microRNA as a starting template. The RNA template can be presented as total cellular RNA or isolated RNA; the two sample types yield comparable results. In other embodiments, the methods and kits described in this invention allow for the quantification of other products of gene expression, including tRNA, rRNA, or other transcripts.

Optionally, the kit of the present invention also includes software that accelerates the generation, analysis, and/or storage of data and facilitates database retrieval. The software includes logical instructions, instruction sets, or suitable computer programs that can be used to collect, store, and/or analyze data. The provided software allows for data comparison and relationship analysis.

Optionally, the kit contains different containers for each kit and/or enzyme. Each component is generally intended for aliquoting the sample in its respective container. The containers for the kit may optionally include at least one vial, ampoule, or test tube. Flasks, bottles, or other container devices in which reagents can be placed and/or aliquoted may also be used. Each container of the kit is preferably kept under strict conditions for commercial sale. Suitable larger containers may include injection or blow-molded plastic containers containing the desired tubular vials. Optionally, instructions for use, such as written guidance or video demonstrations detailing the use of the kit of the present invention, are provided with the kit.

In another aspect, the present invention provides the use of any composition or kit described herein for practicing any method or assay described herein, and/or any apparatus or kit for practicing any assay or method described herein.

As used herein, “test sample” or “biological sample” can mean a biological tissue or fluid sample containing nucleic acids. Such samples include, but are not limited to, tissues or body fluids isolated from animals. Biological samples can also include tissue sections, such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, feces, tears, mucus, urine, exudate, amniotic fluid, ascites, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. Biological samples can be provided by taking cell samples from animals, but can also be provided by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose) or by performing the methods described herein in vivo. Archived tissues, such as those with a history of treatment or outcome, can also be used.

The term "body fluid" or "body fluid" refers to any fluid derived from the body of an animal. Examples of body fluids include, but are not limited to, plasma, serum, blood, lymph, cerebrospinal fluid, synovial fluid, urine, saliva, mucus, phlegm, and sputum. Body fluid samples can be collected by any suitable method. Body fluid samples may be used immediately or preserved for later use. Any suitable preservation method known in the art may be used to preserve body fluid samples: for example, samples may be frozen at about -20°C to about -70°C. Suitable body fluids are cell-free fluids. "Cell-free" fluids include body fluid samples in which cells are absent or present in such low amounts that the measured miRNA level reflects its level in the liquid portion of the sample rather than the cellular portion. Such cell-free body fluids are generally produced by treating cellular body fluids (by, for example, centrifugation or filtration) to remove cells. Cell-free body fluids typically do not contain intact cells; however, some may contain cell debris or cell remnants. Examples of cell-free fluids include plasma or serum, or bodily fluids from which cells have been removed.

As used herein, the term "gene" refers to a natural (e.g., genome) or synthetic gene that contains transcriptional and/or translational regulatory sequences and/or coding regions and/or untranslated sequences (e.g., introns, 5'- and 3' untranslated sequences). The coding region of a gene can be a nucleotide sequence encoding an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA, or antisense RNA. A gene can also be mRNA or cDNA corresponding to a coding region (e.g., exons and miRNAs), optionally containing a 5'- or 3' untranslated sequence linked thereto. A gene can also be an in vitro generated amplified nucleic acid molecule containing all or part of the coding region and/or a 5'- or 3' untranslated sequence linked thereto. The term also includes pseudogenes, which are known functional abnormalities of genes that have lost their protein-coding ability or are no longer expressed in cells.

As used herein, “expression profile” refers to a genome expression profile, such as a microRNA expression profile. Profiles can be generated using any conventional means for determining nucleic acid sequence levels, such as quantitative hybridization of microRNA, cRNA, etc., quantitative PCR, ELISA for quantification, etc., allowing for the analysis of differential gene expression between two samples. The sample is a subject or patient sample, such as cells or an aggregate thereof, such as tissue. Samples are collected using any convenient method known in the art. The target nucleic acid sequence is a predictive nucleic acid sequence, including those described herein, wherein the expression profile may contain expression data for 5, 10, 20, 25, 50, 100 or more (inclusive) of the listed nucleic acid sequences. The term “expression profile” can also mean measuring the abundance of a nucleic acid sequence in the sample being measured.

"Differential expression" refers to qualitative or quantitative differences in temporal and/or cellular gene expression patterns in cells and tissues. Thus, differentially expressed genes can qualitatively alter their expression, including, for example, activation or inactivation in normal versus diseased tissues. Genes may be turned on or off in a particular state relative to another state, thus allowing comparison of two or more states. Qualitatively regulated genes will exhibit expression patterns within a state or cell type, which can be detected using standard techniques. Some genes will be expressed in one state or cell type rather than both. Alternatively, differences in expression can be quantitative, such as expression being regulated, with upregulation resulting in increased transcript levels or downregulation resulting in decreased transcript levels. The degree of expression difference only needs to be large enough to be quantified via standard characterization techniques such as expression arrays, quantitative reverse transcription PCR, Northern blotting, and RNase protection.

As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” refers to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Thus, nucleic acid also encompasses the complementary strand of the single strand shown. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Thus, nucleic acid also encompasses substantially the same nucleic acids and their complements. A single strand provides a probe that can hybridize with a target sequence under strict hybridization conditions. Thus, nucleic acid also encompasses probes that hybridize under strict hybridization conditions.

Nucleic acids can be single-stranded or double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids can be DNA (both genomic and cDNA), RNA, or hybrids (where the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine). Nucleic acids can be obtained through chemical synthesis or recombinant methods.

The term "primer" refers to any nucleic acid that can hybridize at its 3' end to a complementary nucleic acid molecule and provides a free 3' hydroxyl terminus that can be extended by a nucleic acid polymerase. As used herein, amplification primers are a pair of nucleic acid molecules that can anneal to the 5' or 3' region of a gene (positive and negative strands, respectively, or vice versa) and contain a short region between them. Under suitable conditions and using suitable reagents, such primers allow the amplification of nucleic acid molecules having nucleotide sequences flanked by the primers. For in situ methods, cell or tissue samples can be prepared and immobilized on a support such as a glass slide, and then contacted with a probe that can hybridize to RNA. Other methods for amplifying nucleic acids corresponding to expressed RNA samples include those described, for example, in U.S. Patent No. 7,897,750.

As used herein, the term "probe" refers to an oligonucleotide capable of binding to a target nucleic acid with a complementary sequence via one or more types of chemical bonds, typically via complementary base pairing, usually formed via hydrogen bonds. Depending on the stringency of the hybridization conditions, a probe can bind to a target sequence that lacks complete complementarity to its own sequence. Any number of base pair mismatches can exist, interfering with hybridization between the target sequence described herein and a single-stranded nucleic acid. However, if the number of mutations is so great that hybridization cannot occur even under minimally stringent hybridization conditions, then the sequence is not a complementary target sequence. Probes can be single-stranded or partially single-stranded and partially double-stranded. The strandiness of the probe is determined by the structure, composition, and properties of the target sequence. Probes can be directly or indirectly labeled, such as with biotin, which can then be bound by a streptavidin protein complex.

As used herein, the terms “complement” or “complement” in reference to nucleic acids may refer to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairings between nucleotides or nucleotide analogs of a nucleic acid molecule. A complete complement or complete complement may refer to 100% complementary base pairings between nucleotides or nucleotide analogs of a nucleic acid molecule.

As used herein, “strict hybridization conditions” refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes with a second nucleic acid sequence (e.g., a target), as in a complex mixture of nucleic acids. Strict conditions are sequence-dependent and vary under different conditions, and can be suitably selected by those skilled in the art. Strict conditions may be selected to be about 5-10 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength pH. Tm can be the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence to reach equilibrium (at defined ionic strength, pH, and nucleic acid concentration) (because of the excess of the target sequence, at Tm, 50% of the probe is in an equilibrium occupied state). Strict conditions may be conditions where the salt concentration is below about 1.0 M sodium ions, such as about 0.01-1.0 M sodium ion concentration (or other salts), at pH 7.0 to 8.3, and the temperature is at least about 30 °C for short probes (e.g., about 10-50 nucleotides) and at least about 60 °C for long probes (e.g., more than about 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times the background hybridization. Exemplary strict hybridization conditions include: 50% formamide, 5xSSC, and 1% SDS, incubated at 42°C; or 5xSSC, 1% SDS, incubated at 65°C, followed by washing at 65°C with 0.2xSSC and 0.1% SDS. However, several factors besides temperature, such as salt concentration, can affect hybridization strictness, and those skilled in the art can appropriately select these factors to achieve similar strictness.

As used herein, the term "reference value" refers to a value that is statistically relevant to a particular outcome when compared to a measured outcome. In a preferred embodiment, the reference value is determined from a statistical analysis of studies comparing microRNA or protein expression to known clinical outcomes. The reference value can be a threshold score or a cutoff score value. Typically, a reference value will be a threshold above which (or below) one outcome is more likely, and below which another outcome is more likely.

In one implementation, the reference level can be one or more miRNA or peptide levels, expressed as the average level of miRNAs or peptides in samples taken from a control group of healthy (disease-free) subjects. In another implementation, the reference level can be the level in the same subject at different times, such as before the current assay, e.g., before the subject develops disease or before treatment is initiated. Generally, samples are normalized using universal factors. For example, cell-free body fluid samples are normalized by volumetric fluid volume, and cellular samples are normalized by protein content or cell count. Nucleic acid samples can also be normalized relative to an internal control nucleic acid.

As disclosed herein, differences in the levels of one or more peptides or RNAs (mRNAs or microRNAs) indicate a disease or its stage. The phrase "difference in level" refers to a difference in the amount of a specific biomarker, such as a nucleic acid, in a sample compared to a control or reference level. For example, a specific biomarker may be present in a sample from a patient with a neoplastic disease by an amount that is either increased or decreased compared to a reference level. In one embodiment, "difference in level" can be a difference in the amount of a specific biomarker present in the sample compared to a control (e.g., a reference value) of at least about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 75%, 80%, 100%, 150%, 200%, or greater. In one embodiment, "difference in level" can be a statistically significant difference in the amount of a biomarker present in the sample compared to a control. For example, a difference can be statistically significant if the measured level of a biomarker falls outside approximately 1.0, 1.5, 2.0, or 2.5 standard deviations of the mean of any control or reference group. For miRNA measurements, the level can be measured as a Ct value from real-time PCR, which can be normalized to a ΔCt value as described in the examples below.

Drug screening

This invention provides a method for identifying compounds that can be used to treat colon cancer or inhibit cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization.

Candidate compounds to be screened (e.g., proteins, peptides, peptide mimics, peptide-like molecules, antibodies, small molecules, or other drugs) can be obtained using a variety of combinatorial library methods known in the art. These libraries include: peptide libraries; peptide-like libraries (libraries of molecules with peptide functionality but novel non-peptide backbones resistant to enzymatic degradation); spatially addressable parallel solid-phase or solution-phase libraries; synthetic libraries obtained by selection via deconvolution or affinity chromatography; and one-bead-one-compound libraries. See, for example, Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145. Examples of methods used for synthesizing molecular libraries can be found in, for example, DeWitt et al., 1993, PNAS USA 90:6909; Erb et al., 1994, PNAS USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994 J. Med. Chem. 37:1233. The compound library can be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or in beads (Lam, 1991, Nature 354:82-84), on microarrays (Fodor, 1993, Nature 364:555-556), in bacteria (US Patent No. 5,223,409), in spores (US Patent No. 5,223,409), or in plasmids (Cull et al., 1992, PNAS). (USA 89:1865-1869) or on bacteriophages (Scott and Smith 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, PNASUSA 87:6378-6382; Felici 1991, J.Mol.Biol. 222:301-310; and US Patent No. 5,223,409).

To identify usable compounds, the test compound can be contacted with a system containing test cells expressing a reporter gene, which is encoded by a nucleic acid operatively linked to a promoter of a marker gene selected from the aforementioned transfer promoters or inhibitors. This system can be an in vitro cell line model or an in vivo animal model. The cells may naturally express the gene or may be modified to express a recombinant nucleic acid. The recombinant nucleic acid may contain a nucleic acid encoding a reporter polypeptide and a heterologous promoter. The expression levels of the miRNA, polypeptide, or reporter polypeptide can then be measured.

For peptides, expression levels can be determined at the mRNA level or the protein level. Methods for measuring mRNA levels in cell, tissue, or body fluid samples are well known in the art. To measure mRNA levels, cells can be lysed, and the levels of mRNA in the lysates, or RNA purified or semi-purified from the lysates, can be determined by, for example, hybridization assays (using detectable labeled gene-specific DNA or RNA probes) and quantitative or semi-quantitative RT-PCR (using suitable gene-specific primers). Alternatively, quantitative or semi-quantitative in situ hybridization assays can be performed using tissue sections or undysed cell suspensions with detectable (e.g., fluorescent or enzyme-labeled) DNA or RNA probes. Other mRNA quantification methods include RNA protection assays (RPA) and SAGE. Methods for measuring protein levels in cell or tissue samples are also known in the art.

To determine the efficacy of candidate compounds in treating colon cancer or inhibiting cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization, levels obtained as described above can be compared with control levels (e.g., in the absence of candidate compounds). A compound is identified as effective if (i) the level of a metastasis inhibitor is higher than the control or reference value, or (ii) the level of a metastasis promoter is lower than the control or reference value. The efficacy of such identified compounds can be further validated using the in vitro cell culture models or in vivo animal models disclosed in the examples below.

Example

Example 1

This embodiment describes the materials and methods used in Examples 2-15 below.

In vivo selection

1 x ^10⁶ LS174T cells expressing luciferase reporter were suspended in 20 μl of a 1:1 PBS/Matrigel mixture and injected intrahepatically into the livers of NOD-SCID mice. Metastatic nodules were allowed to grow over a period of 3–4 weeks, and the growth was monitored by bioluminescence imaging. The formed nodules were excised and dissociated into single-cell suspensions by digestion with collagenase and hyaluronidase. Cells were allowed to expand in vitro and then re-injected into mice. After three cycles of in vivo selection, highly metastatic LvM3a and LvM3b-derived cell lines were established.

Lenti-miR library filtering

Cells were transduced with a lentiviral library of 611 miRNAs (System Biosciences) at low multiple of infection (MOI) to overexpress a single miRNA per cell. The transduced population was then intrahepatically injected into NOD-SCID mice for in vivo selection of miRNAs that, upon overexpression, promoted or inhibited metastatic liver colonization. Prior to injection, genomic DNA PCR amplification and lentiviral miRNA inserts were performed from cells in liver nodules according to the manufacturer's protocol. miRNA array profiling allowed for quantification of miRNA inserts before and after in vivo selection.

Organ-type section culture system

Cells to be injected were labeled with red or green cell tracking agents (Invitrogen) and inoculated into the livers of NOD-SCID mice via intrasplenic injection. The livers were then harvested and sectioned into 150 μm sections using a McIlwain tissue mincer (Ted Pella). These sections were plated on organoid tissue culture inserts (Millipore) and cultured in William’s E medium supplemented with Hepatocyte Maintenance Supplement Pack (Invitrogen). After specified time intervals, the liver sections were fixed in paraformaldehyde and imaged using multiphoton microscopy.

In vivo cysteine activation assay

To measure caspase activity in vivo, VivoGlo caspase 3/7 substrate (Z-DEVD-aminoluciferin sodium salt, Promega) was used. Luciferin is inactive until caspase-3, activated by apoptosis, cleaves the DEVD peptide. DEVD-luciferin was injected into mice carrying colorectal cancer cells expressing luciferase. Bioluminescence imaging was performed upon activation by apoptosis to measure caspase activity in vivo. Five hours after the measurement of caspase activity in vivo, mice were injected with routine luciferin for standardization purposes.

Adeno-associated virus therapy

miR-483-5p and miR-551a were cloned as polycistronic sequences (consisting of two tandem miRNA precursor and flanking genomic sequences) into the BglII and NotI sites of scAAV.GFP (plasmid 21893, Addgene). The genomic sequences encoding miR-483-5p and miR-551a (SEQ ID NOs: 5 and 6), the corresponding precursor sequences (underlined, SEQ ID NOs: 3 and 4), and the corresponding mature microRNA sequences (underlined and bolded, SEQ ID NOs: 1 and 2) are listed below. Adeno-associated viruses were packaged, purified, and titrated using the AAV-DJ Helper-Free expression system from Cell Biolabs.

miR-551a:

miR-483-5p:

The corresponding RNA sequences (SEQ ID NOs:7-10) for SEQ ID NOs:1-4 are listed below.

GACCCACUCUUGGUUUCCA(SEQ ID NO:7)

GGGGACUGCCGGGUGACCCUGGAAAUCCAGAGUGGGUGGGGCCAGU CUGACCGUUUCUAGGCGACCCACUCUUGGUUUCCAGGGUUGCCCUG GAAA(SEQ ID NO:8)

GAAGACGGGAGGAAAGAAGGGAG(SEQ ID NO:9)

GAGGGGGAAGACGGGAGGAAAGAAGGGAGUGGUUCCAUCACGCCUCCUCACUCCUCCUCCCGUCUUCUCCUC(SEQ ID NO:10)

CKB, SLC6a8 knocked down

pLKO vectors expressing shRNA hairpins targeting CKB and SLC6a8 were purchased from Sigma-Aldrich. Two independent hairpins that produced optimal transcript knockdown levels were used in all experiments. The DNA and RNA sequences of these hairpins are listed below:

The following primers were used for quantitative qRT-PCR of SLC6a8: forward primer: 5’-GGC AGC TAC AAC CGC TTCAAC A-3’ and reverse primer: 5’-CAG GAT GGA GAA GAC CAC GAA G-3’ (SEQ ID No. 21 and 22, respectively).

Cyclocreatine and Beta-guanidinopropionic acid treatment

Mice were treated with 10 mg of cyclocreatine or saline-mediated treatment via intraperitoneal injection. Treatment began one day after tumor cell inoculation and continued until the mice were euthanized. Beta-guanidinopropionic acid was then administered intraperitoneally at a dose of 200 μL of 0.5 M solution. The treatment regimen was the same as for cyclocreatine treatment.

Example 2

As a first step in identifying molecular regulators of liver colonization in colorectal cancer, in vivo selection was performed on the LS-174T human colorectal cancer line by repeatedly injecting cancer cells into immunodeficient mice to enhance liver colonization, followed by surgical resection of liver colonies and cell dissociation. More specifically, liver colonization of ⁵ x 10⁵ LS-parental, LvM3a, and LvM3b cells was examined by bioluminescence after direct intrahepatic injection. Mice were imaged on day 21 post-injection, and livers were extracted for in vitro imaging and gross morphological examination. Photon flux ratios were obtained and compared among the groups. The third-generation liver colonizers LS-LvM3a and LS-LvM3b were found to exhibit a dramatically enhanced liver colonization capacity (>50-fold) relative to their parental lines after intrahepatic injection. Importantly, these derivatives also exhibited significantly enhanced (>150-fold) liver metastatic capacity after portal vein injection in the metastatic colonization assay—revealing that liver colonization is a key step in the progression of colorectal cancer metastasis. For these bioluminescence assays, all p-values for the corresponding photon flux ratios for each group were based on a one-sided Student's t-test and were found to be less than 0.05, 0.001, or 0.0001.

To systematically identify microRNA regulators of metastatic progression, a library of lentiviral particles (each encoding one of 611 human microRNAs) was transduced into the LS-LvM3b colonization population LS-174T parental line and the SW620 colon cancer population. These cancer populations, containing cancer cells expressing each of the 661 miRNAs, were then intrahepatically injected into mice to allow selection of cells capable of colonizing the liver. Genomic PCR amplification of miRNA sequences, reverse transcription, and miRNA sequencing analysis of miRNA inserts allowed for the quantification of miRNA insert representation. Several miRNAs were identified as exhibiting drop-out in the liver colonization background of both cell lines, consistent with the inhibition of liver colonization of colon cancer cells by overexpression of these miRNAs.

Example 3

In this embodiment, assays were performed to examine the silencing of endogenous levels of any of these miRNAs relative to syngeneic poorly metastatic cells in highly metastatic derivatives. Indeed, miR-483-5p and miR-551a were found to be silenced relative to their parent lines in highly metastatic LS-LVM3a and LS-LVM3b liver colonies and relative to their syngeneic parent lines in the metastatic SW620 derivative. Consistent with the inhibitory effects of these miRNAs on liver colonization, overexpression of miR-483-5p or miR-551a strongly inhibited metastatic colonization of LS-LVM3b cells, while inhibition of endogenous miR-483-5p or miR-551a significantly enhanced liver metastatic colonization in the poorly metastatic parent lines LS-174T and SW480.

Example 4

In this embodiment, assays were performed to investigate the mechanisms by which these miRNAs exert their anti-metastatic effects. The effects of these miRNAs on metastatic progression are not secondary to the regulation of proliferation, as miR-551a inhibition does not affect in vitro proliferation, while miR-483-5p inhibition increases proliferation. Furthermore, overexpression of these miRNAs does not alter the invasiveness or apoptosis rate of cancer cells. To determine the mechanisms by which these miRNAs affect metastasis, assays were performed to identify the time points at which cells overexpressing these miRNAs exhibited progression defects during the metastatic process. Surprisingly, it was noted that as early as 24 hours after cell injection into the portal circulation for the liver metastatic colonization assay, cells overexpressing these miRNAs outnumbered those expressing control hairpins in terms of representativeness.

Example 5

To elucidate the mechanism by which these miRNAs inhibit liver metastatic colonization, an in vitro liver organoid section culture system was developed. This system allows for the study of early events during liver metastasis following single-cell diffusion of colon cancer cells in the liver microenvironment. Consistent with previous studies of significant selection of cell survival during metastatic colonization, a substantial reduction in cell number in the liver microenvironment was observed as a function of time. Highly metastatic LvM3b colonized cells significantly outperformed their poorly metastatic parent lines in terms of persistence in the liver microenvironment—consistent with their positive role in intrahepatic persistence during metastatic progression.

Next, assays were performed to investigate whether the effect of this miRNA regulatory network on cancer cell persistence was due to reduced cancer cell survival during metastatic progression. To quantify cell death in vivo, bioluminescent luciferin reporters with caspase-3/7 activity were used.

More specifically, SW480 cells with suppressed endogenous miR-483-5p or miR-551a were subsequently introduced into the livers of immunodeficient mice via intrasplenic injection. The relative in vivo caspase activity in these cells was then monitored using caspase-3-activated DEVD-luciferin. It was found that miRNA inhibition significantly reduced in vivo caspase activity in colon cancer cells early in liver colonization, revealing a phenotype where cancer survival is inhibited by these miRNAs.

These in vivo findings are supported by an organoid section culture system. In short, the survival of SW480 cells with endogenous miR-483-5p or miR-551a was inhibited in organoid cultures (n=8) by pretreatment with LNA. Five x ^10⁵ cells were labeled with cell tracking agents green (LS-Parental) or red (LvM3b) and introduced into the liver via intrasplenic injection. Immediately after injection, liver tissue was excised and 150-µm sections were prepared using a tissue mincer. Cell viability in the organoid cultures was monitored for up to 4 days using multiphoton microscopy. Dye exchange assays were performed to eliminate the influence of dye bias. Representative images from day 0 and day 3 are shown. Overexpression of microRNAs in LS-LvM3b cells was found to inhibit colon cancer persistence, while inhibition of endogenous levels of both microRNAs enhanced persistence in poorly metastatic SW480 cells. These findings explain why miR-483-5p and miR-551a inhibit hepatic metastatic colonization and metastatic cell survival (a phenotype exhibited by highly metastatic colon cancer cells).

Example 6

In this embodiment, assays were performed to identify downstream effectors of these miRNAs. Transcriptotyping analysis identified transcripts downregulated by overexpression of each microRNA and containing a 3'-UTR or coding sequence (CDS) element complementary to that miRNA. Interestingly, creatine kinase brain type (CKB) was identified as a putative target of two miRNAs, suggesting that these miRNAs exhibiting common in vivo and organoid phenotypes may mediate their action through common target genes. Indeed, quantitative PCR validation revealed the inhibition of CKB transcript levels upon overexpression of the aforementioned microRNAs. CKB expression levels in highly metastatic LvM3b cells were found to be inhibited by overexpression of miR-483-5p and miR-551a. Furthermore, endogenous miR-483 and miR-551a were found to inhibit endogenous CKB protein levels. For example, CKB expression was found to be upregulated in poorly metastatic SW480 cells, where endogenous miR-483-5p and miR-551aa were inhibited by LNA. Mutagenesis and luciferase-based reporter assays revealed that miR-483-5p and miR-551a directly target the 3'UTR or CDS of CKB. For this purpose, luciferase reporter assays of the CKB coding sequence and 3'-UTR were performed. miR-483-5p and miR-551a were found to target complementary regions in the 3'-UTR and coding sequence of CKB, respectively. Assays were also performed with mutations in the complementary regions and at least three times.

Example 7

In this embodiment, a assay was performed to examine whether CKB was sufficient and necessary for the liver metastatic colonization of colon cancer.

In short, liver metastasis was examined in mice injected intrasplenically with ⁵ x 10⁵ poorly metastatic SW480 cells and CKB-overexpressing cells. Mice were euthanized 28 days post-injection, and the livers were harvested for bioluminescence imaging. Similarly, liver metastasis was also examined in mice injected intrasplenically with 5 x ^10⁵ highly aggressive LvM3b (expressing either control hairpins or CKB-targeting hairpins). These mice were euthanized 21 days post-injection as described above.

Overexpression of CKB in poorly metastatic SW480 cells was found to promote liver metastasis by more than 3-fold, while CKB knockdown in metastatic LS-LvM3b cells and SW480 cells (via independent hairpin knockdown in each lineage) potently inhibited liver metastasis by more than 5-fold. Consistent with the effects of miRNAs, CKB overexpression was sufficient to significantly enhance the ability of colon cancer cells to persist in the liver microenvironment and enhance their representation in the liver, while CKB knockdown significantly reduced intrahepatic persistence. Therefore, studies were conducted to examine the survival of control SW480 cells and CKB-overexpressing SW480 cells in organoid liver sections (n=8), and organoid culture sections of LvM3b cells expressing control hairpins or CKB-targeting hairpins (n=8). Images taken on day 0 and day 2 showed that CKB overexpression was sufficient to significantly enhance the ability of cancer cells. In these assays, p-values were less than 0.001 or 0.0001 based on a one-sided Student’s test.

To investigate whether CKB acts directly downstream of miR-483-5p and miR-551a, the coding sequence of CKB was overexpressed in cells overexpressing miR-483-5p or miR-551a. Briefly, assays were performed to examine metastatic progression in mice injected with ⁵ x 10⁵ LvM3b cells (with and without CKB overexpression) overexpressing miR-483-5p and miR-551a. Liver metastasis was monitored by bioluminescence imaging, and mice were euthanized 35 days post-injection. It was found that CKB overexpression was sufficient to rescue the suppressed liver metastatic phenotype in cells overexpressing miR-483-5p and miR-551a. Conversely, knockdown of CKB in cells exhibiting suppression of endogenous miR-483-5p or miR-551a prevented the enhanced metastatic effect seen with suppression of miR-483-5p or miR-551a. To this end, assays were performed to examine liver metastases in mice injected with 5 x ^10⁵ SW480 cells (with and without CKB knockdown) containing LNA-inhibited endogenous miR-483-5p and miR-551a. Mice were euthanized after 28 days, and livers were harvested for in vitro bioluminescence imaging. Results from the above-described gain-and-loss-of-function assays and epistatic analysis revealed that CKB is a direct target of miR-483-5p and miR-551a and acts as a downstream effector of these miRNAs in the regulation of metastatic progression in colorectal cancer. In these assays, p-values were less than 0.05 or 0.001 based on one-sided Student's t-tests.

To further confirm the role of CKB, relative in vivo caspase activity was examined in control SW480 cells and CKB-overexpressing cells in mouse liver. Activity was measured using bioluminescence, employing caspase-3-activated DEVD-luciferin and normalized to the bioluminescent signal from conventional luciferin (n=3). Similar relative in vivo caspase-3 activity was also examined in SW480 cells expressing either control or CKB-targeting hairpins, introduced into mouse liver via intrasplenic injection. Caspase activity was measured on days 1, 4, and 7 post-injection. Consistent with the above findings, CKB overexpression significantly decreased during the initial phase of liver colonization, while CKB knockdown significantly enhanced in vivo caspase-3/7 activity. In these assays, p-values were less than 0.05 or 0.001 based on one-sided Student’s t-tests. These findings reveal that CKB is a promoter of colon cancer survival during liver metastatic colonization.

Example 8

CKB is known to regulate rapidly moving high-energy phosphate reservoirs in tissues such as the brain and kidneys, which catalyze the transfer of high-energy phosphate groups from phosphocreatine to ADP, thereby generating ATP and creatine. It has been hypothesized that ATP generation from phosphocreatine-CKB may provide an energy advantage to colon cancer cells during liver colonization. To determine whether the end product ATP catalyzed by CKB could rescue the metastasis inhibition seen in CKB knockdown, CKB-knocked cells were loaded with ATP prior to injection in an experimental metastasis assay. Briefly, liver metastasis was examined in 5 x ^10⁵ LvM3b mice injected with and without CKB knockdown and pretreated with 100 μM ATP or a mediator. Metastasis burden was monitored by bioluminescence imaging, and mice were euthanized 21 days post-injection. ATP loading of cells was found to significantly enhance the suppressed metastasis phenotype in CKB-depleted cells by more than 10-fold. The rescue by ATP was specific, as ATP loading did not enhance metastatic activity in cells expressing short hairpin controls.

Similar studies were conducted to determine whether creatine and creatine phosphate could rescue the phenotypes observed in CKB knockdown. More specifically, assays were performed to examine liver metastases in mice injected with 5 x ^10⁵ LvM3b cells pretreated with 10 μM creatine in a CKB knockdown context. The mice were then euthanized on day 21 post-injection, as described above, and the livers were extracted for ex vivo bioluminescence imaging. Additionally, colorectal cancer metastases were examined in mice injected with 5 x ^10⁵ LvM3b cells pretreated with 10 μM creatine phosphate. Liver metastases were monitored by bioluminescence imaging, followed by euthanasia as described above. Both creatine and creatine phosphate were found to rescue metastasis inhibition.

To investigate whether colorectal cancer metastasis can be inhibited by blocking creatine transport to colorectal cancer cells, the creatine transport channel SLC6a8 was inhibited in LvM3b cells by expressing a short hairpin targeting SLC6a8. Liver metastasis in LvM3b cells was examined in the same manner described above. Knockdown of the creatine transport channel SLC6a8 was found to inhibit colorectal cancer metastasis. These findings reveal that colorectal cancer cells depend on ATP generated by CKB for survival during liver colonization.

Example 9

To determine whether this synergistic miRNA regulatory network controlling metastatic progression in colorectal cancer is pathologically relevant, the expression levels of miR-483-5p and miR-551a were analyzed in a cohort of 67 primary colorectal cancer and liver metastases obtained from patients with MSKCC. More specifically, the levels of miR-483-5p and miR-551a in 37 primary tumor samples and 30 liver metastasis samples were quantified by quantitative real-time PCR. Consistent with the metastatic inhibitory effects of these miRNAs during metastatic progression, both miR-483 and miR-551a showed significantly reduced expression levels in human liver metastases compared to primary colorectal cancer (Figure 1A; p<0.05 for miR-483-5p and p<0.05 for miR-551a; N=67).

CKB expression levels were also examined by quantitative real-time PCR in 37 primary tumor samples and 30 liver metastasis samples. Importantly, CKB expression was found to be significantly elevated in liver metastases relative to primary colorectal cancer (p<0.05), and its expression was significantly inversely correlated with the miRNAs—consistent with the direct targeting of these miRNAs in human colorectal cancer (Figure 1B). These findings are consistent with previous clinical histological analyses revealing elevated levels of CKB protein in advanced cancers.

Example 10

In this embodiment, an assay was performed to investigate the therapeutic potential of targeting this miRNA regulatory network. For this purpose, mice were injected with a high number (500k) of highly metastatic LvM3a cells, followed 24 hours later by a single intravenous dose of adenovirus-associated virus (AAV) expressing miR-483-5p and miR-551a from a single transcript. A single therapeutic dose of adenovirus-associated virus (AAV) delivering both miRNAs was found to dramatically and significantly reduce metastatic colonization by more than 5-fold (Figure 1C).

Finally, assays were performed to determine the effects of small-molecule inhibition of CKB and creatine availability restrictions on colorectal cancer metastasis. Cyclocreatine (similar to creatine phosphate) is a trans-state analog of creatine kinase. To examine the effect of cyclocreatine, bioluminescent measurements of liver metastases were performed in mice injected with 5 x ^10⁵ LvM3b cells and treated daily with cyclocreatine for two weeks. Mice were then euthanized at the end of treatment, and livers were harvested for ex vivo imaging. It was found that although cyclocreatine is a poor inhibitor of CKB (5000 μM ki), treatment with cyclocreatine significantly reduced metastatic colonization in mice and demonstrated superiority over current standard-of-care FOLFOX chemotherapy (Figure 1D).

A similar assay was performed using the creatine transporter inhibitor beta-guanidinopropionic acid (B-GPA). Bioluminescence measurements were used to examine liver metastases in mice injected with 5 x ^10⁵ LvM3b cells and treated daily with B-GPA for two weeks. Treatment of mice with this competitive inhibitor of the creatine transport pathway was found to significantly reduce metastatic colonization (Figure 1E).

Using a systematic approach, two miRNAs were identified as inhibitors of liver metastatic colonization of colorectal cancer cells. These miRNAs were found to convergently target CKB—a key gene that confers the ability of cells encountering liver hypoxia to generate ATP from a creatine phosphate reservoir. Successful targeting of this pathway using four independent therapeutic agents (more effective than current standard clinical care and exhibiting no apparent toxicity) demonstrates the promise of therapy targeting this pathway in human colorectal cancer. The combined in vivo selection/gene screening approach described above ( called Multi - Gene Screening of Human Genes through Intra-Organ Tandem Selection (MUGSHOTS)) has effectively identified potent and pathologically validated regulators of liver colonization and metastasis in colorectal cancer and has the potential to discover coding and non-coding regulators of metastatic colonization in any organ for any cancer type.

Example 11

In this embodiment, assays were performed to confirm the therapeutic potential of targeting the creatine transport channel SLC6a8 with small molecule B-GPA (an inhibitor of SLC6a8). As described above, administration of B-GPA to mice injected with LvM3b colon cancer cells resulted in inhibition of colon cancer metastasis to the liver after two weeks of treatment (Figure 1E). To confirm this therapeutic effect, mice injected with LvM3b colon cancer cells were treated daily for 3 weeks via intraperitoneal injection of B-GPA or a control medium (PBS) (Figure 2). At 3 weeks, the mice were euthanized and the livers were extracted for bioluminescence imaging and gross histology.

Daily treatment with B-GPA resulted in a significant reduction in colorectal cancer metastasis to the liver, as assessed by in vivo bioluminescence imaging in mice, bioluminescence imaging of extracted livers, and gross anatomical examination of extracted livers from treated mice (Figure 2). More specifically, the mean photon flux ratios, measured by bioluminescence imaging, were approximately 800 in the control group (without B-GPA treatment) and 100 in the treatment group. A p-value of less than 0.05 was found based on a one-sided Student’s t-test.

Example 12

In this embodiment, an assay is performed to estimate the therapeutic benefit of targeting the creatine transport channel SLC6a8 by knocking down shRNA targeting SLC6a8.

In short, mice were injected with either LvM3b colon cancer cells expressing either one of two independent short hairpin RNAs (shSLC6a8#4 or shSLC6a8#5) targeting the creatine transport channel SLC6a8, or with control RNA (empty pLKO vector, purchased from Sigma Aldrich) (Figure 3A). Liver metastasis was monitored by bioluminescence imaging, and mice were euthanized 3 weeks after cancer cell inoculation. Liver tissue was extracted for gross histology. Knockdown of SLC6a8 with the two independent shRNAs was found to inhibit colon cancer metastasis (Figure 3A).

To further confirm the therapeutic benefit of SLC6a8 knockdown, another independent colon cancer cell line (SW480 colon cancer cell line) expressing short hairpin RNA targeting SLC6a8 (shSLC6a8#2) was injected into mice (Figure 3B). SLC6a8 knockdown was found to significantly inhibit the metastasis of SW480 colon cancer cells (Figure 3B).

Finally, the therapeutic benefits of targeting SLC6a8 were investigated in pancreatic cancer cells. To accomplish this, PANC1 pancreatic cancer cells expressing either shRNA targeting SLC6a8 (shSLC6a8#5) or control RNA (empty pLKO vector) were injected into mice. Metastatic progression was monitored by bioluminescence imaging, and the mice were euthanized in the same manner described above. At 28 days, a significant reduction in pancreatic cancer metastasis was observed in cells treated with shRNA targeting SLC6a8, revealing SLC6a8 as a potential therapeutic target for pancreatic cancer.

Example 13

In this embodiment, we investigated whether the expression of the creatine transporter SLC6a8 in human colon cancer tumors is associated with metastatic progression.

To accomplish this, quantitative real-time PCR was used to quantify the expression of SLC6a8 in 36 primary colorectal cancer tumors and 30 metastatic colorectal cancer tumors (Figure 4). Indeed, SLC6a8 expression was significantly higher in metastatic tumors (approximately 1.3) than in primary tumors (approximately 0.5), further confirming the pivotal role of SLC6a8 in metastasis (Figure 4). The p-value based on the one-sided Student’s t-test was found to be less than 0.05.

Example 14

As described above, shRNA-mediated knockdown of the creatine transporter SLC6a8 showed inhibition of metastasis in two types of colon cancer and pancreatic cancer. Inhibition of SLC6a8 with the small molecule inhibitor B-GPA also demonstrated a therapeutic benefit against colon cancer metastasis in vivo. To assess whether B-GPA treatment provides a therapeutic benefit in pancreatic cancer treatment, the ability of B-GPA treatment to inhibit the survival of human pancreatic cancer cells was evaluated in vivo in mice.

In short, PANC1 pancreatic cancer cells were incubated for 48 hours with and without 10 mM B-GPA, and then injected into immunodeficient mice (5 x ^10⁵ PANC1 cells per mouse; 4 mice in each of the treatment and untreated groups). Mice were imaged using bioluminescence imaging 1 day post-injection, with the signal standardized to day 0. Treatment benefits were observed as early as 1 day post-injection, such as a significant reduction in tumor burden of pancreatic cancer cells in vivo, as assessed by bioluminescence imaging (Figure 5), demonstrating the therapeutic benefit of B-GPA treatment for pancreatic cancer. More specifically, the mean photon flux ratios in the control group (without B-GPA treatment) and the treatment group, as measured by bioluminescence imaging, were approximately 2.7 and 1.6, respectively. A p-value of less than 0.05 was found based on a one-sided Student's t-test.

Example 15

The above embodiments demonstrate the therapeutic benefits of B-GPA treatment alone for colorectal and pancreatic cancer. In this embodiment, it was investigated whether B-GPA treatment could enhance the therapeutic activity of the chemotherapeutic agents 5'-fluorouracil and gemcitabine. To achieve this, cell viability assays were performed to compare the cytotoxic activity of 5'-fluorouracil or gemcitabine alone compared to combination therapy with B-GPA.

In short, 10,000 PANC1 cells were seeded in triplicate in 96-well plates and treated for 48 hours with multiple concentrations of gemcitabine (1 nm, 10 nm, 100 nm, 1000 nm, 10000 nm, 100000 nm, and 1000000 nm) with or without 10 mM B-GPA. Cell viability was then measured using the WST-1 reagent (Roche Applied Science), with absorbance at 440 nm used as an indicator of viable cell count. As shown in Figure 6, the addition of therapeutic concentrations of B-GPA enhanced the cytotoxic activity of gemcitabine against PANC1 pancreatic cancer cells, as assessed by cell viability assays using the WST-1 reagent.

Similarly, the addition of therapeutic concentrations of B-GPA enhanced the cytotoxic activity of 5'-fluorouracil against Ls-LvM3b colon cancer cells. For this purpose, 10,000 Ls-LvM3b cells were seeded triplicate in 96-well plates and treated for 48 hours with or without 10 mM B-GPA at various concentrations of 5'-fluorouracil. Cell viability was determined in the same manner as described above, with absorbance at 440 nm used as an indicator of viable cell count. As shown in Figure 7, these results demonstrate that B-GPA enhances the therapeutic activity of commonly used chemotherapeutic agents for the treatment of colorectal and pancreatic cancer.

The foregoing embodiments and descriptions of preferred embodiments should be considered illustrative and not limiting of the scope of the invention as defined by the claims. It will be readily apparent that numerous variations and combinations of the above features can be utilized without departing from the invention as described in the claims. Such variations are not considered to depart from the scope of the invention, and all such variations are intended to be included within the scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

sequence list

<110> Rockefeller University

<120> Treatment and Diagnosis of Colon Cancer

<130> 070413.20370

<140> PCT/US13/67860

<141> 2013-10-31

<150> 61/720,912

<151> 2012-10-31

<150> 61/786,500

<151> 2013-03-15

<160> 22

<170> PatentIn version 3.5

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Claims (7)

1. Use of β-guanidinopropionic acid in the preparation of a medicament for treating cancer in subjects with this need, wherein the subjects have higher levels of creatine kinase brain type (CKB) or SLC6a8 gene expression or activity than normal individuals, wherein the β-guanidinopropionic acid is a therapeutically effective amount for reducing the gene expression or activity level of CKB or SLC6a8 for treating said cancer, wherein said cancer is pancreatic cancer. 2. The use of claim 1, wherein the cancer is metastatic. 3. The use of claim 1, wherein the subject has higher levels of CKB than normal. 4. The use of claim 3, wherein the subject has at least 10% high CKB levels. 5. The use of any one of claims 1-4, wherein the medicament further comprises an additional therapeutic agent selected from the group consisting of: 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, gemcitabine, cetuximab, paclitaxel, avastin, leucovorin, regorafenib, zaltrap, topoisomerase I inhibitors, NKTR-102, Tivantinib, PX-866, sorafenib, Linifanib, Telatinib, XL281 (BMS-908662), Robatumumab, and IGF1-R inhibitors. 6. The use of claim 5, wherein the additional therapeutic agent is gemcitabine. 7. The use of claim 5, wherein the additional therapeutic agent is 5-fluorouracil.