WO2020071392A1 - Proliferation inhibitor of poor-prognosis cancer cells - Google Patents

Abstract

The present inventor found that, in a cancer disease, cancer cells showing a large expression amount (production amount) of fibrillarin are associated with poor prognosis (a low survival rate). Also, the present inventor found that the proliferation of cancer cells can be inhibited by reducing the expression amount (production amount) of fibrillarin. As a result, it becomes possible to inhibit the proliferation of poor-prognosis cancer cells to thereby treat a cancer disease by using the inhibitor of the expression or activity of fibrillarin according to the present invention. Typical examples of the inhibitor of the expression or activity of fibrillarin include an siRNA, an shRNA, an antisense, etc.

Description

Cancer cell growth inhibitor with poor prognosis

The present invention relates to an agent for suppressing the growth of cancer cells having a poor prognosis. In particular, effective anti-proliferative agents for various types of cancers with high fibrillarin expression (colorectal cancer, ovarian cancer, breast cancer, lung adenocarcinoma, glioma, kidney cancer, etc.) The present invention relates to a therapeutic agent for a cancer disease to be used. Further, the present invention relates to a method for examining the prognosis of cancer.

Fibrillarin is a component of snoRNP (small nucleolar small ribonucleic acid protein) located in the nucleolus in the cell nucleus, and plays a role in rRNA methylation and is transcribed from DNA. It is known to carry out subsequent methylation of the rRNA precursor. And the amino acid sequences of many vertebrate fibrillarins are known and they are widely conserved among vertebrates.
In particular, human-derived fibrillarin is a protein consisting of 321 amino acids (National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov/), monkey, cow, dog, rat, and rat. It has about 90% or more homology in amino acid sequence with fibrillarin of a mammal such as a mouse. Its functions are known to be involved in ribosome biosynthesis, biosynthesis of small nucleolar ribonucleic acid proteins, and mRNA processing (Patent Document 1).

A typical compound that suppresses or inhibits the above function of fibrillarin is an anti-fibrillarin antibody. Further, it has been reported that a peptide fragment having no fibrillin activity, which is derived from a fibrillin protein, often acts as a fibrillin activity inhibitor (Patent Document 1). Recently, yamamarin and its derivatives, which are peptides derived from the silkworm, form a complex with fibrillarin (Non-patent Document 1 and Patent Document 2), inhibit the action of fibrillarin, exhibit a cell growth inhibitory effect, and exhibit mitochondrial NADH. It has been reported to inhibit respiration (Non-Patent Documents 2 and 3).
The cell growth inhibitory activity caused by the inhibition of fibrillarin action suppresses cell growth by shortening the S phase corresponding to the DNA replication phase and prolonging the quiescent phase, and causes cell proliferation by inducing apoptosis. It does not suppress. That is, it is reported that cell growth is suppressed by controlling the cell cycle (Patent Document 2).
Substances that suppress the expression and activity of fibrillarin are agents for suppressing or killing the proliferation of undifferentiated stem cells of ES cells and iPS cells (Patent Document 1), and polycythemia vera caused by the proliferation of erythroid progenitor cells Alternatively, it has been reported that it can be used as a therapeutic agent (Patent Document 2) for essential thrombocythemia caused by proliferation of megakaryocyte precursor cells. However, even if fibrillarin is controlled, it is considered that it is difficult to control only the cell cycle and to induce the death of cancer cells, etc. Was not considered.

JP 2009-07186 A JP 2011-051917 A

Masakatsu Kitadamiya, May 26, 2014 Kaken Research Report, "Elucidation and application of molecular basis of cell growth inhibitory activity of bioactive peptides derived from natural silkworm" Research Support Project for Interdisciplinary Research for Creation of Biological Industry (Fiscal Year Ended in 2011) Research Results, p. Natural Silkworm / Insect Biotech 82 (2), 73-78 (2013)

An object of the present invention is to provide a cancer cell proliferation inhibitor having a poor prognosis and a therapeutic agent for the cancer cell disease. Another object of the present invention is to provide a test method that can easily predict the prognosis of cancer.

The present inventor has found that, in the case of a cancer cell having a high fibrillarin expression level (output), the prognosis of a cancer patient after starting treatment is poor (low survival rate). Therefore, suppression of the fibrillarin expression level (production rate) using fibrillarin siRNA was examined.
The present inventor has found that, when the expression level (production amount) of fibrillarin is suppressed using siRNA, the proliferation of cancer cells can be suppressed. Furthermore, by administering fibrillarin siRNA to the transplanted cancer cells, growth suppression of the cancer cells could be achieved. That is, by suppressing the expression or activity of fibrillarin, the growth of cancer cells, particularly cancer cells with a poor prognosis (cancer cells having high fibrillarin expression (production)) can be suppressed, Cancer treatment is possible.
The present inventors have completed the present invention based on the above findings.

That is, the gist of the present invention is as follows.
[1] A cancer cell growth inhibitor comprising a fibrillarin expression or activity inhibitor as an active ingredient.
[2] The cancer cell growth inhibitor according to [1], wherein the cancer cell is a high fibrillarin-expressing cancer cell.
[3] The cancer cell according to [2], wherein the high fibrillarin-expressing cancer cell is a cancer cell whose fibrillarin RNA expression level is equal to or greater than the median fibrillarin RNA expression level determined from a patient database for each cancer type. A cancer cell growth inhibitor.
[4] the cancer cell is any one of a colon cancer cell, an ovarian cancer cell, a breast cancer cell, a lung adenocarcinoma cell, a glioma cancer cell, and a kidney cancer cell, [1] or The cancer cell growth inhibitor according to [2].
[5] The cancer cell growth inhibitor according to [4], wherein the cancer cell is a kidney cancer cell.
[6] The proliferation of a cancer cell according to any one of [1] to [5], wherein the inhibitor of fibrillarin expression or activity is fibrillarin siRNA, shRNA, or antisense, or a modified form thereof. Inhibitors.
[7] the fibrillarin siRNA comprises an RNA nucleotide sequence (SEQ ID NO: 1: ggucgaggcgggagcucuua) corresponding to the nucleotide sequence at positions 103 to 121 of the human fibrillarin gene, or 1 or 2 in SEQ ID NO: 1 The cancer cell growth inhibitor according to [6], wherein the cancer cell growth inhibitor comprises an RNA base sequence having a substituted, inserted, or deleted nucleotide and suppresses human fibrillarin expression.
[8] The cancer cell growth inhibitor according to [7], wherein the fibrillarin siRNA has 19 to 25 bases.
[9] A cancer therapeutic agent, comprising the cancer cell growth inhibitor according to any of [1] to [8] as an active ingredient.
[10] SEQ ID NO: 1: A fibrillarin siRNA or a modified form thereof comprising a nucleotide sequence of up to 25 nucleotides including the nucleotide sequence of ggucggggcggggcucuua.
[11] A method for suppressing cancer cell growth, which comprises administering to a cancer patient an inhibitor of the expression or activity of fibrillarin in an amount effective for suppressing cancer cell growth.
[12] A method for treating cancer, comprising administering to a cancer patient an inhibitor of the expression or activity of fibrillarin in an amount effective for treating cancer.
[13] Use of a fibrillarin expression or activity inhibitor for the production of a cancer cell growth inhibitor.
[14] Use of a fibrillarin expression or activity inhibitor for the manufacture of a therapeutic agent for cancer.
[15] An inhibitor of fibrillarin expression or activity for use in suppressing the growth of cancer cells.
[16] An inhibitor of fibrillarin expression or activity for use in treating cancer.
[17] a step of measuring the fibrillarin expression level in the cancer tissue of the cancer patient, a step of comparing the measured value with a median fibrillarin expression level determined in advance for each type of cancer, A method of determining that the prognosis is poor when the value is equal to or more than the median value, and determining that the prognosis is good if the measured value is lower than the median value.

The inhibitor of fibrillarin expression or activity of the present invention can suppress the growth of cancer cells even in malignant cancer cells with high expression of fibrillarin and poor prognosis, and kill cancer cells be able to. As a result, by using the inhibitor of the expression or activity of fibrillarin of the present invention, it has become possible to treat cancer diseases even with poor prognosis and low survival rates.

It is a figure which shows the expression level of the fibrillarin gene in the cancer tissues of various cancer-bearing patients. It is a figure which shows the relationship between the fibrillarin expression level in colorectal cancer and the transition of the patient survival rate. It is a figure which shows the relationship between the fibrillarin expression level in ovarian cancer and the transition of the patient survival rate. It is a figure which shows the relationship between the fibrillarin expression level in breast cancer and the transition of the patient survival rate. It is a figure which shows the relationship between the change of the fibrillarin expression level and the patient survival rate in lung gland cancer. It is a figure which shows the relationship between the fibrillarin expression level in glioma and the transition of the patient survival rate. It is a figure showing the fibrillarin expression level (production level) suppression effect of fibrillarin siRNA in a renal cell carcinoma cell line. FIG. 2 is a diagram showing a test method for suppressing the growth of xenograft renal cell carcinoma by suppressing the fibrillarin expression level (production level). FIG. 3 is a view showing the results of a xenograft renal cell carcinoma growth suppression test by suppressing the fibrillarin expression level (production level).

The “inhibitor of fibrillarin expression or activity” of the present invention refers to a drug capable of suppressing the expression of fibrillarin in cancer cells or a drug capable of suppressing or inhibiting the function or activity of fibrillarin.
Examples of the agent capable of suppressing the expression of fibrillarin, that is, the agent capable of suppressing the expression of the fibrillarin gene include antisense, siRNA, and shRNA targeting fibrillarin (for example, human fibrillarin) (above, nucleic acids that target or bind to mRNA) Compounds), miRNAs, decoys, aptamers, nucleic acid compounds such as CpG oligonucleotides, and low molecular weight compounds that regulate the transcription of rRNA such as actinomycin D.
The number of bases of the nucleic acid compound such as antisense or siRNA to be used may be 14 to 45, and a suitable number of bases can be used depending on the kind of the nucleic acid compound. For example, the number of bases in antisense is 14 to 30, the number of bases in siRNA is 19 to 25, the number of bases in miRNA is 19 to 25, the number of bases in decoy is 16 to 24, and the number of bases in aptamers is 26 to 45. And the number of bases of the CpG oligonucleotide is preferably 16 to 24, respectively.

The nucleic acid sequence of the nucleic acid compound for suppressing the expression of fibrillarin is not particularly limited, and an appropriate nucleic acid sequence can be used in accordance with the type of the nucleic acid compound to be used. For example, in the case of siRNA, a human fibrillarin gene sequence consisting of an RNA base sequence corresponding to the base sequence at positions 103 to 121 (SEQ ID NO: 1: ggucggggcgggagcucuua) may be mentioned. In addition, those comprising an RNA base sequence of 19 to 25 bases including the base sequence of SEQ ID NO: 1 can also be mentioned. Furthermore, shRNAs containing these nucleotide sequences can also be used, and can be used as siRNAs for suppressing the expression of fibrillarin in cells.
In addition, examples of the antisense RNA include those having a base sequence of SEQ ID NO: 1 or a base sequence of 19 to 30 bases including the base sequence. Antisense DNAs include those consisting of the nucleotide sequence at positions 103 to 121 (SEQ ID NO: 2: ggtcgaggcgggaggctttta) in the gene sequence of human fibrillarin, or those consisting of a nucleotide sequence of 19 to 30 nucleotides including the nucleotide sequence Is mentioned.
Also, the siRNA may be an RNA consisting of an RNA base sequence in which one or two, particularly one base has been substituted, inserted or deleted in SEQ ID NO: 1, or a 19 to 25 RNA base sequence containing the same. Can also be used as long as the expression of fibrillarin is suppressed. In addition, the antisense RNA includes an RNA base sequence of SEQ ID NO: 1 in which one to two, particularly one base has been substituted, inserted, or deleted, or a 19 to 30 RNA base sequence including the same. Can be used as long as the expression of fibrillarin is suppressed. The antisense DNA includes a DNA base sequence in which one to two, particularly one base in SEQ ID NO: 2 has been substituted, inserted, or deleted, or a 19 to 30 DNA base sequence including the same. Can also be used as long as the expression of fibrillarin is suppressed. Suppressing the expression of fibularin includes not only stopping expression, but also reducing the expression level.

When the nucleic acid compound is RNA, it may be designed so that it can be produced in vivo. For example, a DNA encoding the RNA can be inserted into an expression vector for mammalian cells. Examples of such an expression vector include a virus vector and an animal cell expression plasmid.

Further, when the nucleic acid compound is RNA, it may be chemically modified to improve stability. Examples of the chemically modified RNA include RNA containing nucleic acid analogs such as phosphorothioate, morpholino phosphorodiamidate, boranophosphate, and LNA (Locked Nucleic Acid), and 2′-O-methylated RNA and 2′-O-. Methoxyethylated RNA and the like can be mentioned.

The drug capable of suppressing or inhibiting the function or activity of fibrillin refers to a drug that suppresses or inhibits the function of fibrillin and a drug that suppresses or inhibits the activity of fibrillin. It should be noted that fibrillarin has a function of modifying the methylation of rRNA, and the rRNA is matured by the methylation modification and participates in the formation of liposomes, which are protein factories. Therefore, if the methylation modification of rRNA is inhibited, ribosome formation was not possible, indicating that the function of fibrillarin was inhibited.
Note that human rRNA has about 200 methylation modification sites. The introduced methyl group generally has the effect of providing a local hydrophobic environment and weakening hydrogen bonds. 2′-O-methylation of ribose has a role of fixing the twisted structure of ribose to C3′-end type, and contributes to local structure formation of rRNA. Thus, the chemical nature of the methylation modification of rRNA plays various roles in the biosynthesis and function of ribosomes (Biochemistry Vol. 85, No. 10, pp. 896-908, 2013). The mechanism of this rRNA methylation modification reaction is as follows. That is, snoRNA having a common sequence called Box C / D forms a base pair with rRNA, and four proteins of fibrillarin, Nop58p, Nop56p, and Snu13p (15.5k in human) (mainly fibrillarin) are formed. A bound Box C / D snoRNP complex is formed, which serves as a guide for determining the methylation site of the target rRNA, and the rRNA is methylated. The donor of the methyl group to be introduced is S-adenosylmethionine (SAM), and methylation modification is performed by the action of methyltransferase.
Therefore, an agent that suppresses or inhibits the function of fibrillarin refers to an agent that suppresses or inhibits the above-described mechanism of methylation modification. For example, nucleic acid compounds such as aptamers or peptides can be mentioned.

An agent that suppresses or inhibits the activity of fibrillin refers to a molecule that binds to the target of fibrillin but has no activity, and thus suppresses or inhibits the activity of fibrillin, or binds or interacts with fibrillin to inhibit the activity of fibrillin. Or a drug that inhibits. For example, the fibrillin-derived peptide fragment described in Patent Document 1 binds to the fibrillin target but has no fibrillin activity, and thus competes with the fibrillin to inhibit its activity. In addition, yamamarin (C-terminal amidated peptide (Asp-Ile-Leu-Arg-Gly) -NH ₂ ) described in Non-patent Document 1 and Patent Document 2 and N-terminal acylation thereof (particularly, C6 to The 28-acylated derivative binds to fibrillarin and inhibits its activity. Antibodies to fibrillarin also bind to and inhibit its activity.

「The“ cancer cell ”of the present invention refers to a cancer cell highly expressing fibrillarin. Depending on the type of cancer cell, as shown in FIG. 1, the expression level of fibrillarin varies depending on the type of cancer cell. Cells. The median fibrillarin expression level of various cancer cells refers to the median fibrillarin expression level in cancer tissues of at least 50, especially 50 to 150, cancer patients. It may be calculated from data collected in the Cancer Genome Atlas Project by the Human Genome Research Institute.

Among cancer cells, as shown in FIGS. 2 to 7, for example, colon cancer, ovarian cancer, breast cancer, lung cancer (especially lung adenocarcinoma), glioma, and kidney cancer have If the expression level of fibrillin in cancer tissue is high, cancer cells are likely to metastasize, resulting in a poor post-operative prognosis and a low survival rate.

Therefore, a cancer cell with high fibrillarin expression can be regarded as a cancer cell of a cancer patient exhibiting a median survival rate or a lower survival rate of various cancer patients. The median survival rate of various cancer patients is the median survival rate of at least 50, especially 50 to 150, cancer patients. What is necessary is just to determine from the data collected by the genome atlas project.
The survival rate is defined as, for example, the survival rate after 50 to 3000 days, especially 300 to 1500 days after the start of cancer treatment (including surgical treatment including surgery such as cancer resection, radiation therapy, chemotherapy, etc.). It can be. Survival rates range from 50-150 days (especially 50 days) for colorectal cancer, 50-150 days (especially 50 days) for ovarian cancer, and 50-150 days ( After 100 days), 1000-3000 days (especially 1500 days) for lung cancer (especially lung adenocarcinoma), and 300-1000 days (especially 500 days) for glioma. it can.
The inhibitor of the expression or activity of fibrillarin of the present invention is more effective for cancer cells expressing fibrillarin at a high level with poor prognosis.

The "cancer therapeutic agent" of the present invention may contain additives and carriers as long as an effective amount of an inhibitor of the expression or activity of fibrillarin exists in such a manner as to exert its function. For example, depending on the route of administration and the mode of drug release, the drug may be coated with a time-disintegrating material, or may be incorporated into an appropriate drug release system. It may be formulated with a targeting agent for delivery to the target cancer tissue.
The cancer therapeutic agent of the present invention can be administered by various routes including both oral and parenteral, such as, but not limited to, oral, intravenous, intramuscular, subcutaneous, topical (particularly, intratumoral), lymphatic or Intralymph node, intrathecal, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, etc. It can be formulated into a dosage form suitable for the route. As such a dosage form and a preparation method, any known one can be appropriately adopted (see, for example, Standard Pharmacology, edited by Yoshiteru Watanabe et al., Nankodo, 2003).
An effective amount of an inhibitor of fibrillarin expression or activity is, for example, an amount that suppresses cancer growth, reduces symptoms, or delays or stops progression, and preferably inhibits cancer progression. Or the amount that cures the cancer. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined by an in vitro test using cultured cells or the like, or a test in a model animal such as mouse, rat, dog or pig, and such a test method is well known to those skilled in the art. . Further, the dose of the additive or the carrier is also known to those skilled in the art, or can be appropriately determined by the above-described test and the like.

The present invention provides a method for suppressing cancer cell proliferation, which comprises administering to a cancer patient an inhibitor of the expression or activity of fibrillarin in an amount effective for suppressing cancer cell growth. Suppressing the growth of cancer cells refers to reducing the degree of proliferation of cancer cells as compared to the case where no drug is administered.
The present invention also provides a method for treating cancer, which comprises administering to a cancer patient an inhibitor of the expression or activity of fibrillarin in an amount effective for treating cancer. Cancer treatment includes cancer cure, remission, amelioration, mitigation, progression delay, progression arrest, and the like.
As described above, the “cancer cell” of the present invention refers to a cancer cell with high fibrillarin expression. Therefore, the “cancer patient” of the present invention is a patient with a high fibrillarin-expressing cancer cell, that is, a prognosis. You have a bad cancer patient.
The inhibitor of fibrillarin expression or activity may be formulated and administered together with additives and carriers. The route of administration differs depending on the dosage form, and is oral, intravenous, intramuscular, subcutaneous, local (particularly, within a tumor), lymphatic vessel or lymph node, intrathecal, rectum, intraarterial, intraportal, intraventricular. Transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, etc. routes.

The amount of the inhibitor of the expression or activity of fibrillarin, which is effective for suppressing the growth of cancer cells or treating cancer, depends on various conditions, for example, the type of cancer, the severity of symptoms, general health, age, and body weight. The determination can be made by those skilled in the art in consideration of, for example, gender, diet, timing and frequency of administration, concomitant medication, response to treatment, compliance with treatment, dosage form, and administration route.
The amount of the inhibitor of the expression or activity of fibrillarin, which is effective for suppressing the growth of cancer cells or for treating cancer, varies depending on the above-mentioned various conditions, but is, for example, 0.00001 mg to 100 g per day, preferably 0.0001 mg per day. It can be from 10 to 10 g, especially from 0.001 mg to 1 g, from 0.01 mg to 100 mg, especially from 0.1 mg to 10 mg. When the inhibitor of the expression or activity of brillarin is a modified nucleic acid compound, the dose is an amount calculated as a nucleic acid compound. In addition, when not administered daily or when the frequency of administration changes over time, the daily dose is calculated from the total dose.

The dosing frequency or schedule will depend on the various conditions described above, but may be, for example, many times a day (ie, 2, 3, 4, or 5 or more times a day), once a day, every few days (ie, 2, 3 Every four, five, six, seven days, etc.) several times a week (for example, two, three, four times a week, etc.) every one week, every few weeks (ie, every two, three, four weeks, etc.) It may be. Further, the administration frequency may change over time depending on the symptoms.
The period of administration can be until the cancer is cured or ameliorated.

The present invention provides a step of measuring the fibrillarin expression level of a cancer tissue of a cancer patient, a step of comparing the measured value with a median fibrillarin expression level predetermined for each type of cancer, and the measured value. Determining that the prognosis is poor when the measured value is higher than or equal to the median value, and determining that the prognosis is good when the measured value is lower than the median value.

発 現 The fibrillarin expression level, ie, the fibrillarin gene expression level, can be determined by extracting RNA from a cancer tissue collected from a cancer patient in accordance with a conventional method, for example, by RT-qPCR.

The median fibrillarin expression level of various cancer cells refers to the median fibrillarin expression level in cancer tissues of at least 50, especially 50 to 150, cancer patients. It may be determined from data collected in the Cancer Genome Atlas Project by the Human Genome Research Institute.

Poor prognosis means that the survival rate is low after the start of cancer treatment (including surgical treatment, including surgery such as cancer resection, radiation therapy, and chemotherapy). It means that the survival rate after the day of starting such cancer treatment is high.
The survival rate can be, for example, the survival rate on the 50th to 3000th day, especially the 300th to 1500th day from the day when the cancer treatment is started. In particular, 50 to 150 days (especially 50 days) from the start of cancer treatment for colorectal cancer, 50 to 150 days (especially 50 days) from the start of cancer treatment for ovarian cancer, and cancer treatment for breast cancer 50 to 150 days (especially 100 days) from the day, 1000 to 3000 days (especially 1500 days) from the start of cancer treatment for lung adenocarcinoma, and 300 to 1000 days from the start of cancer treatment for glioma. The survival rate after days (particularly 500 days) can be used.

に お い て In the method for testing the prognosis of cancer of the present invention, the fibrillarin expression level may be measured either before or after the cancer treatment start date. Above all, it is preferable to measure the fibrillarin expression level before the start of the cancer treatment. In the case of the cancer treatment by suppressing the fibrillarin expression, the fibrillarin expression level may be measured before the start of the cancer treatment.

Hereinafter, the present invention will be described more specifically with reference to Examples and Test Examples, but the present invention is not limited thereto.

(Example 1) Fibrillarin gene expression level in various cancer-bearing patients a) Materials and reagents:
Public data such as cancer genomes, epigenomes, transcriptomes, and mutation information in various tissues, collected from the National Cancer Institute's Cancer Genome Atlas Project by the US National Cancer Institute b) Methods:
RNA expression data (RPKM value: Reads per kilobase of exon per million mapped reads) of the fibrillarin gene analyzed by RNA-seq in the public data was obtained, and the relative value of the fibrillarin RNA expression level was obtained using the expression level of all genes. (AU) was calculated by the normalization method, and the relative value of the fibrillarin gene expression level for each patient was displayed as log2.
c) Result:
As shown in FIG. 1, it was revealed that the fibrillarin gene was highly expressed in many cancers.

(Example 2) Correlation between expression level of fibrillarin gene in various cancer-bearing patients and survival rate of cancer-bearing patients a) Materials and reagents:
Public data such as cancer genomes, epigenomes, transcriptomes, and mutation information in various tissues, collected from the National Cancer Institute's Cancer Genome Atlas Project by the US National Cancer Institute b) Methods:
We collected the expression levels of the fibrillarin gene in cancer tissues and the number of surviving days after starting treatment in cancer-bearing patients in the public data.
For each cancer tissue of patients with colorectal cancer, ovarian cancer, breast cancer, lung adenocarcinoma, and glioma, calculate the median fibrillarin RNA expression level. Was classified as low. Each cancer patient was classified into a high group and a low group, the postoperative survival rate of each group patient was calculated over time, and graphed by the Kaplan-Meier method. Statistical analysis was performed using the Cox proportional hazard model.
c) Result:
As shown in FIGS. 2 to 6, cancer-bearing patients with high fibrillarin expression (production) in colon cancer, ovarian cancer, breast cancer, lung adenocarcinoma, and glioma cancer-bearing patients Showed poor survival. 2 to 6, the solid line indicates the median value, and the two broken lines indicate the highest value and the lowest value, respectively.

(Example 3) Fibrillarin expression level (production level) inhibitory effect of fibrillarin siRNA in renal cell carcinoma cell line a) Materials and reagents:
Renal cell carcinoma: 7860 strain fibrillarin siRNA: The nucleotide sequence of the RNA corresponding to positions 103 to 121 in the nucleotide sequence of the human fibrillarin gene (SEQ ID NO: 1: ggucgaggcgggagcucuua) is used.
b) Method:
After culturing the 7860 strain on a 24-well plate, 5 μM of fibrillarin siRNA was introduced into the cells using 5 μL of Dharmafect reagent (GE-healthcare). After culturing for 72 hours, RNA was extracted and fibrillarin expression was analyzed by RT-qPCR. The fibrillarin expression level was expressed as a relative value to the fibrillarin expression level of control cells into which siRNA was not introduced.
c) Result:
As shown in FIG. 7, the use of fibrillarin siRNA reduced the fibrillarin expression level (production level) of the renal cell carcinoma cell line to about 1/10.

(Example 4) Test for inhibiting the growth of xenograft renal cell carcinoma by suppressing the fibrillarin expression level (production level) a) Materials and reagents:
-Human kidney cancer cell line for xenotransplantation: 786 Mock
-Fibrillarin siRNA: Uses the nucleotide sequence of the human fibrillarin gene (the nucleotide sequence corresponding to the nucleotide sequence at positions 103 to 121 (SEQ ID NO: 1: ggucggggcgggagcucuua)).
-EGFP siRNA: MISSION siRNA (SIGMA) is used.
b) Method:
5 × 10 ⁶ human kidney cancer cell lines (786O mock) were injected subcutaneously into nude mice (male, 4 weeks old). According to the schedule shown in FIG. 8, fibrillarin siRNA (1 nM) mixed with atelocollagen was injected into the human kidney cancer cell line injection site. As a control, a human kidney cancer cell line was similarly injected into the opposite body side, and EGFP siRNA (1 nM) mixed with atelocollagen was injected into the human kidney cancer cell line injection site on the same schedule.
The diameter of the transplanted kidney cells was measured on the day after the start of administration of the fibrillarin siRNA, and then on days 5, 10, 17, 25, and 32.
c) Result:
As shown in FIG. 9, kidney cancer formation was suppressed at the site where fibrillarin siRNA was administered. By the 17th day from the start of administration of siRNA, the diameter of transplanted kidney cancer was clearly reduced, and it is considered that the cancer had died. On the other hand, at the site where EGFP siRNA was administered as a control, the diameter of transplanted kidney cancer was increased.

As described above, in the human kidney cancer cell line, the administration and administration of fibrillarin siRNA significantly suppressed the engraftment and proliferation of cancer cells in nude mice by suppressing the expression of fibrillarin. This indicates that the suppression of fibrillarin expression or the suppression of fibrillarin action in cancer cells, particularly cancer cells highly expressing fibrillarin, can suppress the growth of cancer cells. That is, it indicates that cancer treatment is possible by suppressing the expression of fibrillarin or suppressing the action of fibrillarin.

The inhibitor of the expression or action of fibrillarin of the present invention effectively suppresses the growth of cancer cells in cancer cells, particularly cancer cells in which fibrillarin is highly expressed, and provides a new mechanism of action for cancer treatment. You can do it. The inhibitor of the expression or action of fibrillarin of the present invention can be used as a single agent, and can also be used in combination with other cancer therapeutic agents.

Claims (17)

剤 A cancer cell growth inhibitor comprising a fibrillarin expression or activity inhibitor as an active ingredient. 剤 The cancer cell growth inhibitor according to claim 1, wherein the cancer cells are high fibrillarin-expressing cancer cells. 3. The cancer cell according to claim 2, wherein the fibrillarin-high-expressing cancer cell is a cancer cell whose fibrillarin RNA expression level is equal to or greater than the median fibrillarin RNA expression level determined from a patient database for each cancer type. Growth inhibitor. The cancer cell according to claim 1 or 2, wherein the cancer cell is any one of a colon cancer cell, an ovarian cancer cell, a breast cancer cell, a lung adenocarcinoma cell, a glioma cancer cell, and a kidney cancer cell. Cancer cell growth inhibitor. 剤 The cancer cell growth inhibitor according to claim 4, wherein the cancer cell is a kidney cancer cell. (6) The cancer cell growth inhibitor according to any one of (1) to (5), wherein the inhibitor of fibrillarin expression or activity is fibrillarin siRNA, shRNA or antisense, or a modified form thereof. Wherein the fibrillarin siRNA comprises an RNA base sequence (SEQ ID NO: 1: ggucgaggcggagcucuua) corresponding to the base sequence at positions 103 to 121 of the human fibrillarin gene, or one or two bases in SEQ ID NO: 1 The cancer cell growth inhibitor according to claim 6, wherein the agent comprises a substituted, inserted or deleted RNA nucleotide sequence and suppresses human fibrillarin expression. 8. The cancer cell growth inhibitor according to claim 7, wherein the fibrillarin siRNA has 19 to 25 bases. が ん A cancer therapeutic agent comprising the cancer cell growth inhibitor according to any one of claims 1 to 8 as an active ingredient. SEQ ID NO: 1: a fibrillarin siRNA comprising a base sequence of up to 25 bases including the base sequence of ggucggggcgggagcuua, or a modified form thereof. (4) A method for suppressing cancer cell growth, comprising administering to a cancer patient an inhibitor of the expression or activity of fibrillarin in an amount effective for suppressing the growth of cancer cells. (4) A method for treating cancer, comprising administering to a cancer patient an inhibitor of the expression or activity of fibrillarin in an amount effective for treating cancer. (4) Use of a fibrillarin expression or activity inhibitor for the production of a cancer cell growth inhibitor. (4) Use of a fibrillarin expression or activity inhibitor for the manufacture of a therapeutic agent for cancer. (4) An agent for suppressing the expression or activity of fibrillin for use in suppressing the growth of cancer cells. 剤 An inhibitor of fibrillarin expression or activity for use in treating cancer. Measuring the fibrillarin expression level in the cancer tissue of the cancer patient, and comparing the measured value to the median fibrillarin expression level predetermined for each type of cancer, and the measured value is equal to or greater than the median value And determining a poor prognosis when the measured value is lower than the median, and determining a good prognosis when the measured value is lower than the median.

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