JPWO2016159377A1 - Cancer screening drug screening method - Google Patents

Abstract

The present invention provides a method for screening a cancer therapeutic drug as a molecular target drug targeting any protein without identifying the true target protein from among a huge number of target protein candidates. For the purpose. The present invention includes (i) a step of measuring the expression level of a transposable element in contact with or non-contact with a test substance in a target cancer cell, and (ii) in contact with the test substance, By providing a method of screening for a therapeutic agent for cancer, comprising a step of selecting the test substance as a therapeutic agent for cancer when the expression level of the transposable element is increased as compared to non-contact Solve the above problems.

Description

  The present invention relates to a method for screening for a therapeutic drug for cancer.

  In recent years, it has been shown that molecular targeting drugs, that is, therapeutic drugs that directly target proteins related to important intracellular signals related to cancer cell growth and progression, are effective in cancer treatment. Searches for proteins that can become new drug discovery targets are being actively pursued. Examples of target proteins for cancer treatment include proteins encoded by genes that are unique or overexpressed in cancer cells. Although it is possible to find many genes encoding such target candidate proteins by exhaustive analysis such as microarray method, the true target gene encoding a protein that can actually be a target for cancer treatment is selected from among them. It takes undue experimentation to identify.

  In addition, a technique for reprogramming somatic cells has been developed and attempts have been made to reinitialize cancer cells (Non-patent Document 1). Such attempts have been focused on changing the properties of cancer cells themselves. There are no reports on screening for cancer drugs using cancer cell reprogramming.

Kosaka T, et al, Cancer Sci. 104: 1017-1026, 2013

  An object of the present invention is to provide a method for screening for a therapeutic drug for cancer.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that when initializing cancer cells, the target cells (that is, important intracellular signals related to cancer cell growth / progression). Repression of the expression of genes that are known to be related) enhances reprogramming, in other words, the proteins encoded by these target genes maintain their characteristics as cancer cells, And found that it has the effect of suppressing the fate change of cancer cells. Furthermore, the present inventors have found that the enhanced initialization depends on the expression of transposable elements. Therefore, when cancer cells expressing a target protein of an existing molecular target drug were brought into contact with the molecular target drug to suppress the activity of the target protein, it was confirmed that the expression of the transposable element was increased. Therefore, when the expression of a transposable element in a cancer cell for which an effective target protein is known is increased under contact with the test substance, it can be confirmed that the test substance has a function of suppressing the activity of the target protein. Became clear. In addition, even when targeting any cancer cell, if the test substance increases the expression of the transposable element in the cancer cell, the test substance is a cell involved in the growth / progress of the cancer cell. Since it can be presumed to have a function of suppressing the activity of some protein related to the internal signal, it becomes possible to directly screen candidate substances for cancer therapeutic agents without identifying the target protein (target gene).
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention provides the following method.
[1] A method for screening for a therapeutic drug for cancer, comprising the following steps;
(I) a step of measuring the expression level of a transposable element in contact with or non-contact with a test substance in a target cancer cell; and (ii) under non-contact in contact with a test substance. A step of selecting the test substance as a cancer therapeutic agent when the expression level of the transposable element increases in comparison.
[2] The method according to [1], wherein the transposable element is a retrotransposon or a fragment thereof.
[3] The method according to [2], wherein the transposable element is at least one sequence selected from endogenous retrovirus, LINE, SINE, and fragments thereof.
[4] The method according to [2], wherein the transposable element is at least one sequence selected from HERV-H, HERV-W, L1-ORF2, ERVK and fragments thereof.
[5] A method for identifying a protein that can be a drug discovery target of a cancer therapeutic drug, comprising the following steps;
(I) Expression of a transposable element in a cancer cell containing a gene encoding a test protein in a form in which expression can be controlled under conditions for expressing the gene or under conditions where expression of the gene is suppressed A step of measuring the amount, and (ii) when the expression level of the transposable element is increased under the condition in which the expression of the gene is suppressed as compared with the condition under which the gene is expressed. A process of selecting proteins that can be targets for drug discovery for cancer treatment.

  According to the present invention, it is not necessary to identify a target gene, and it becomes possible to directly screen for a therapeutic drug for cancer that becomes a molecular target drug.

FIG. 1A shows a schematic diagram describing a method for inducing EWS / ATF1 expression and introducing reprogramming factors. FIG. 1B shows a phase contrast microscopic image of sarcoma cell line G1297 after introduction of reprogramming factor when EWS / ATF1 expression was induced (DOX 0.2 μg / ml) or not (DOX 0 μg / ml). . FIG. 1C shows the results of measuring the number of colonies formed after introduction of reprogramming factor when EWS / ATF1 expression was induced (DOX 0.2 or 0.1 μg / ml) or not (DOX 0 μg / ml). . FIG. 1D shows RT-PCR of EWS / ATF1 expression induction by adding 0.2 μg / ml DOX to iPS cell lines (C-1, C-2, C-3 and C-4) established from sarcoma cell line G1297. The result confirmed by is shown. FIG. 1E shows the results of chromosome microarray (CGH array) analysis in the sarcoma cell line G1297 and an iPS cell line established from the same. FIG. 1F shows the results of measuring the expression of Nanog in the iPS cell lines (C-1, C-2, C-3 and C-4) by RT-PCR (left figure) and staining the iPS cell line with Nanog and DAPI. The stained image (right figure) is shown. FIG. 1G shows a stained image of teratoma formed by transplanting iPS cells established from sarcoma cell line G1297 subcutaneously into nude mice. FIG. 1H shows a photograph of a chimeric mouse generated by injecting iPS cells established from the sarcoma cell line G1297 into a blastocyst. FIG. 1I shows the case where EWS / ATF1 expression was induced (DOX 0.2 or 0.1 μg / ml) or not induced (DOX 0 μg / ml) on the fourth day after introduction of reprogramming factor (4F) into sarcoma cell line G1297. The result of FACS which measured the positive rate of SSEA1 in the 10th day after 4F introduction | transduction in is shown. FIG. 1J shows that EWS / ATF1 expression was induced on mouse embryonic fibroblasts (MEF) 4 days after introduction of reprogramming factor (4F) (DOX 0.2 or 0.1 μg / ml) or not (DOX (0 μg / ml) shows the results of FACS in which the positive rate of SSEA1 was measured 10 days after 4F introduction. FIG. 1K shows 4F introduction or GFP when EWS / ATF1 expression was induced (ON) or not induced (OFF) on day 4 after introduction of reprogramming factor (4F) or GFP into sarcoma cell line G1297. The results of microarray analysis using RNA recovered from cells on day 6 after introduction are shown. FIG. 1L shows a schematic diagram describing EWS / ATF1 expression induction and a method for introducing MYOD1. Figure 1M shows the results of RT-PCR measurement of Myogenin expression in cells in which MYOD1 was introduced into the sarcoma cell line G1297, with or without EWS / ATF1 expression induction (ON). Show. FIG. 1N shows staining with Myosin heavy chain (MHC) and DAPI, when EWS / ATF1 expression was induced (ON) or not induced (OFF) in cells in which MYOD1 was introduced into the sarcoma cell line G1297. The image (left figure) and the result of measuring the expression of Myosin heavy chain by RT-PCR (right figure) are shown. Fig. 1 O shows 6 days after MYOD1 introduction or GFP introduction when EWS / ATF1 expression was induced (ON) or not induced (OFF) 4 days after MYOD1 introduction or GFP introduction into sarcoma cell line G1297 The results of microarray analysis using RNA recovered from eye cells are shown. FIG. 2A shows the transcription initiation site (TSS) of a gene induced by EWS / ATF1 when EWS / ATF1 expression is induced (ON) or not induced (OFF) in the sarcoma cell line G1297 (left figure). 2 shows the results of ChIP-seq analysis and FAIRE-seq analysis using antibodies against H3K4me3 and H3K27Ac at EWS / ATF1-binding sites (right figure). In the figure, the red color indicates the amount of histone that has been H3K4me3 (trimethylated at the 4th lysine residue of histone H3) and H3K27Ac (acetylated at the 27th lysine residue of histone H3). Means the amount or amount of unbound to histones. FIG. 2B shows ChIP-seq analysis using antibodies against H3K4me3 and H3K27Ac in chromosome 12 with and without EWS / ATF1 expression induced in sarcoma cell line G1297 (ON) or without (OFF) and FAIRE -seq Shows the result of analysis. In the figure, EWS / ATF1 (HA) indicates the binding region of EWS / ATF1, and H3K4me3, H3K27Ac, and FAIRE indicate the amount of H3K4me3-histone, the amount of H3K27Ac-histone, and the amount of unbound histone, respectively. Shows the peak. FIG. 2C shows the result of ChIP-seq analysis using an antibody against H3K9me3 in the vicinity of the transposable element with or without induction of EWS / ATF1 in the sarcoma cell line G1297. Indicates. In the figure, the vertical axis shows the logarithm of RPM when EWS / ATF1 expression is induced (ON) with respect to RPM (read per million) when EWS / ATF1 is not induced (OFF). Therefore, a high value means that H3K9me3 is enhanced in this region when EWS / ATF1 expression is induced (ON). Fig. 2D shows the expression of L1 (left figure) and MMERVK10c (right figure) with RT-PCR when EWS / ATF1 expression was induced (ON) or not induced (OFF) in sarcoma cell line G1297. The measurement results are shown. FIG. 2E shows H3K9me3 in the vicinity of the transcription start site (TSS) of the gene induced by EWS / ATF1 when EWS / ATF1 expression was induced (ON) or not induced (OFF) in the sarcoma cell line G1297. The result of the ChIP-seq analysis using the antibody with respect to is shown. In the figure, the vertical axis shows the logarithm of RPM when EWS / ATF1 expression is induced (ON) with respect to RPM (read per million) when EWS / ATF1 is not induced (OFF). FIG. 2F shows Reduced Representation Bisulfite Sequencing (RRBS) at the CpG site in the ERVK region when EWS / ATF1 expression was induced (ON) or not (OFF) in ES cells, somatic cells, and sarcoma cell line G1297. ) Shows the result of the analysis. FIG. 3A shows the results of measuring the amount of H3K9me3 at the Oct3 / 4, Nanog and Sox2 binding sites of ES cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). In the figure, shading indicates the amount of H3K9me3. FIG. 3B shows the results of measuring the amount of H3K9me3 at the MYOD1 binding site of myotube cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). In the figure, shading indicates the amount of H3K9me3. FIG. 3C shows the results of measuring the amount of Oct3 / 4 at the Oct3 / 4 binding site of ES cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). FIG. 3D shows the introduction of 4F in the sarcoma cell line G1297 with or without induction of EWS / ATF1 expression (ON) or 4 days after introduction of reprogramming factor (4F) or GFP. The result of having analyzed the expression of the gene adjusted by Oct3 / 4 among the RNA collect | recovered from the cell of the 6th day after GFP introduction by a microarray is shown. FIG. 3E shows that when EWS / ATF1 expression was induced (ON) or not induced (OFF) in cells introduced with MYOD1 into the sarcoma cell line G1297, expression of EWS / ATF1 was induced (ON), and Suv39h1 was induced by RNAi. The result of having measured the expression of Myogenin in the case of suppressing the expression of Suv39h2 or Ezh2 by RT-PCR is shown. Fig. 3F shows the case where EWS / ATF1 expression was induced (ON) on day 4 after introduction of MYOD1 or GFP into the sarcoma cell line G1297, and Suv39h1 expression was suppressed (siSuv39h1) or not (siControl). The results of microarray analysis using RNA recovered from cells 6 days after MYOD1 introduction or GFP introduction are shown. FIG. 3G shows the gene ontology (GO) term of the gene whose expression increased when Suv39h1 expression was suppressed in the result of FIG. 3F. FIG. 4A shows a schematic diagram describing the method of introduction of reprogramming factor, MYOD1 or GFP and siRNA into the MP-CCS-SY cell line. FIG. 4B shows that when the reprogramming factor (+ 4F) or GFP was introduced into the MP-CCS-SY cell line, expression of EWS / ATF1 was suppressed by RNAi (siEWS / ATF1) or expression was not suppressed (siControl). ) Results of PODXL expression measured by RT-PCR (left figure), and when MYOD1 or GFP was introduced into the MP-CCS-SY cell line, RNAi suppressed EWS / ATF1 expression (siEWS / The result (right figure) which measured the expression of Myogenin in the case of ATF1) case or not suppressing expression (siControl) is shown. FIG. 4C shows a schematic diagram describing the introduction of reprogramming factors into HCC827 or SK-BR3 cell lines and the method of adding each drug. Fig. 4D shows the case where DMSO, 5FU or Lapatinib (Lap) was added to the SK-BR3 cell line when reprogramming factor was introduced (Dox (4F) +) or not (Dox (4F)-) Of PODXL expression by RT-PCR (left figure) and when reprogramming factor was introduced into HCC827 cell line (Dox (4F) +) or not (Dox (4F)-) The result (right figure) which measured the expression of PODXL by RT-PCR when DMSO, 5FU, or Gefitinib (Gef) was added in is shown. Fig. 4E shows the results when Lapatinib (Lap) or DMSO was added to the SK-BR3 cell line when reprogramming factor was introduced (Dox (4F) +) or not (Dox (4F)-). Results of microarray analysis (left figure) and Gefitinib (Gef) or DMSO when reprogramming factor was introduced into HCC827 cell line (Dox (4F) +) or not (Dox (4F)-) The result (right figure) of the microarray analysis in the case of adding is shown. FIG. 4F shows the results of measuring the expression of L1 OFR2 (left figure) and HERV-W (right figure) by RT-PCR when Lapatinib (Lap) or DMSO was added in the SK-BR3 cell line. FIG. 4G shows the results of measuring the expression of L1 OFR2 (left figure) and HERV-W (right figure) by RT-PCR when Gefitinib (Gef) or DMSO was added in the HCC827 cell line. Fig. 4H shows the results when Lapatinib (Lap) or DMSO was added to the SK-BR3 cell line when reprogramming factor was introduced (Dox (4F) +) or not (Dox (4F)-). The result of having performed the microarray analysis about the HERVH related gene is shown. FIG. 4I shows the results of microarray analysis of the expression of all genes or HERVH-related genes when Lapatinib (Lap) or DMSO was added to the SK-BR3 cell line (left figure) and Gefitinib (Gef) to the HCC827 cell line Or the result (right figure) which performed microarray analysis about the expression of all the genes at the time of DMSO addition or a HERVH related gene is shown. FIG. 5A shows a growth curve when doxycycline (DOX) 0, 0.1, 0.2, 1 or 2 μg / ml was added to sarcoma cell line G1297 and cultured. FIG. 5B shows that when the reprogramming factor was introduced into the sarcoma cell line G1297 (+ 4F) or not (-4F), expression of EWS / ATF1 was induced (ON) or not (OFF) ) Shows the results of measuring the amount of Oct3 / 4 in each case by Western blotting. FIG. 5C shows a phase contrast microscopic image of iPS cells established after introduction of reprogramming factors when EWS / ATF1 expression was not induced (DOX 0 μg / ml). FIG. 5D shows the results of measuring the expression levels of Oct3 / 4 and Nanog by RT-PCR when EWS / ATF1 expression was induced (ON) or not induced (OFF) in the sarcoma cell line G1297. . In the figure, ESC (ES cell) was used as a positive control. FIG. 5E shows a schematic diagram describing the method of induction of EWS / ATF1 expression and introduction of reprogramming factors. FIG. 5F shows that EWS / ATF1 expression was induced (ON) or not induced (OFF) in cells (+ 4F) or cells not introduced (GFP) into the sarcoma cell line G1297. The results of measuring the expression of Cadh1 (left figure) and Epcam (right figure) by RT-PCR are shown. FIG. 6A shows ChIP using antibodies against H3K4me3 and H3K27Ac at the transcription start site (TSS) of all genes when EWS / ATF1 expression was induced (ON) or not induced (OFF) in sarcoma cell line G1297. -Seq analysis and FAIRE-seq analysis results are shown. FIG. 6B shows ChIP-seq using an antibody against H3K9me3 in the vicinity of the transcription start site (TSS) of ERVK when EWS / ATF1 expression was induced (ON) or not induced (OFF) in sarcoma cell line G1297. The result of the analysis is shown. In the figure, the vertical axis shows the logarithm of RPM when EWS / ATF1 expression is induced (ON) with respect to RPM (read per million) when EWS / ATF1 is not induced (OFF). Therefore, a high value means that H3K9me3 in the region is enhanced when EWS / ATF1 expression is induced (ON). FIG. 6C shows the results of ChIP-seq analysis using an antibody against H3K9me3 in chromosome 15 when EWS / ATF1 expression was induced (ON) or not induced (OFF) in the sarcoma cell line G1297. The transcription region of LTR / ERVK is shown in the figure below. FIG. 6D shows the transcription initiation site of the gene induced by EWS / ATF1 in the vicinity of the transposable element when EWS / ATF1 expression was induced (ON) or not (OFF) in the sarcoma cell line G1297. The result of the ChIP-seq analysis (random mapping method) using the antibody with respect to H3K9me3 in (TSS) is shown. In the figure, the vertical axis shows the logarithm of RPM when EWS / ATF1 expression is induced (ON) with respect to RPM (read per million) when EWS / ATF1 is not induced (OFF). Therefore, a high value means that H3K9me3 is enhanced in this region when EWS / ATF1 expression is induced (ON). FIG. 7A shows the transcription initiation site (TSS), EWS / ATF1 binding site when EWS / ATF1 expression was induced (ON) or not induced (OFF) in ES cells, somatic cells, and sarcoma cell line G1297. The result of Reduced Representation Bisulfite Sequencing (RRBS) analysis in CpG site in Oct3 / 4 binding site, Sox2 binding site and Nanog binding site is shown. FIG. 7B shows the results of measuring the amount of H3K9me3 at the Ascl1-binding site of neural stem cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). In the figure, shading indicates the amount of H3K9me3. FIG. 7C shows ChIP-seq analysis using antibodies against H3K4me3 and H3K27Ac at the Oct3 / 4 binding site of ES cells when EWS / ATF1 expression was induced (ON) or not (OFF) and FAIRE- Results of seq analysis (left figure) and results of ChIP-seq analysis and FAIRE-seq analysis using antibodies against H3K4me3 and H3K27Ac at the MyoD1 binding site of myotube cells (right figure) are shown. FIG. 8A shows a schematic diagram describing a method for inducing EWS / ATF1 expression and introducing a reprogramming factor for ChIP analysis. FIG. 8B shows 6 days after introduction of MyoD1 or GFP when EWS / ATF1 expression was induced (ON) or not induced (OFF) 4 days after introduction of MyoD1 or GFP into sarcoma cell line G1297. The result of having analyzed the expression of the gene adjusted by MyoD1 by the microarray among RNA collect | recovered from the said cell is shown. FIG. 8C shows the expression of histone modifying enzymes (Suv39h1, Suv39h2, Eed, Suz12, and Ezh2) in the sarcoma cell line G1297 when EWS / ATF1 expression was induced (ON) or not (OFF). The result confirmed by PCR is shown. In each histone modifying enzyme, the left bar shows EWS / ATF1 expression induced (EWS / ATF1 ON), and the right bar shows EWS / ATF1 expression not induced (EWS / ATF1 OFF). FIG. 9A is a schematic diagram illustrating the method of induction of expression of EWS / ATF1 for RT-PCR analysis, introduction of MyoD1, and introduction of each siRNA, and the histone modifying enzymes (Suv39h1, Suv39h2, Ezh2, Suz12 and Eed). The effect of expression suppression is shown. FIG. 9B shows the results of measuring the expression of L1 promoter by RT-PCR when Suv39h1 or Ezh2 expression was suppressed by RNAi in sarcoma cell line G1297. FIG. 9C shows a PiggyBac vector construct for introducing the reprogramming factor and a phase contrast image and a fluorescence image of the cell after introduction into SK-BR3. FIG. 10 shows DMSO, 5FU (1 mM or 10 mM) or Gefitinib (Gef) in the case where the reprogramming factor was introduced into the A549 cell line (Dox (4F) +) or not (Dox (4F) −). The result of having measured the expression of PODXL in the case where 10 mM or 50 mM) was added by RT-PCR is shown. FIG. 11A shows a schematic diagram describing the method of introduction of the reprogramming factor (4F) by retrovirus and the introduction of anticancer agents (Alectinib, Imatinib) into H2228 and KBM7. FIG. 11B shows that when reprogramming factors are introduced into H2228 and KBM7, Alectinib (0 μM (control), 1 μM or 10 μM) is added to H2228, and Imatinib (0 μM (control) is added), 1 μM or The expression of L1 when 10 μM) is added is measured by RT-PCR, and the transcription level is normalized by the GAPDH value. FIG. 12A shows that when the expression of EWS / ATF1 was induced in the mouse sarcoma cell line G1297 (DoxON) or not induced (DoxOFF), the reprogramming factor was introduced (+ 4F) or not, respectively. The result of the RNA-seq analysis in the ERVK locus of the chromosome 8 in the case is shown. FIG. 12B shows the results of measuring the expression of ERVK in the case of DoxON and DoxOFF by RT-PCR and normalizing the transcription level with β-actin (Actb) value.

The present invention provides a method for screening for a therapeutic agent for cancer, comprising the following steps;
(I) a step of measuring the expression of a transposable element in contact with a test substance in a target cancer cell; and (ii) the transposer from a condition in which the cancer cell is not in contact with the test substance. A step of selecting the test substance as a cancer therapeutic agent when the expression of a bull element increases.

Cancer Cell In the present invention, cancer means a malignant tumor, including carcinoma (malignant tumor derived from epithelial cells), sarcoma, and other leukemias, and is not limited to a specific cancer. The cancer cell used in the present invention is a cell constituting a malignant tumor, and may be a cell line or a cell isolated from a living body. In addition, cancer cells used in the present invention are cancer cells (for example, EWS / ATF1 fusion gene forced expression) whose effective target protein is known (ie, known to be sensitive to an existing molecular target drug). Cancer cells, gefitinib-sensitive mutant EGFR-expressing cancer cells, lapatinib-sensitive HER2-amplified cancer cells, alectinib-sensitive EML4-ALK fusion gene-expressing cancer cells, imatinib-sensitive chronic myeloid leukemia cells, etc.) Alternatively, cancer cells whose effective target protein is unknown may be used.

Test substance In the screening method of the present invention, any test substance can be used, and any known compound and novel compound may be used. For example, cell extract, cell culture supernatant, microbial fermentation product, marine organism origin Extract, plant extract, nucleic acid (especially siRNA), purified protein or crude protein, peptide, non-peptide compound, synthetic low molecular weight compound, natural compound and the like. In the present invention, the test substance may also be (1) a biological library method, (2) a synthetic library method using deconvolution, (3) “one-bead one-compound” live Can be obtained using any of a number of approaches in combinatorial library methods known in the art, including rally methods, and (4) synthetic library methods using affinity chromatography sorting. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem 37: 1233-51). Compound libraries are available in solution (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555- 6) Bacteria (US Pat. No. 5,223,409), Spores (US Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; US Patent Application No. 2002103360).

  The contact between the cancer cell and the test substance may be performed by appropriately adding the test substance to the culture solution for cancer cells. In the present invention, the culture solution for cancer cells is used for culturing animal cells. The medium can be prepared as a basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. Is included. The medium may contain serum or may be serum-free. If necessary, the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules One or more substances such as compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like may also be included.

Other culture conditions such as culture temperature and CO ₂ concentration can be set as appropriate. The culture temperature is not particularly limited and is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 5%. The O ₂ concentration is 1-20%. The O ₂ concentration may be 1 to 10%.

  Although the contact time of a cancer cell and a test substance is not specifically limited, 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 1.5 days, 2 days or more are illustrated.

  Epigenetic modification is a method of analyzing methylation of DNA by a method of concentrating methylated DNA, a method using base substitution by bisulfite treatment, or a method using a restriction enzyme sensitive to methylation, Perform chromatin immunoprecipitation (ChIP) with modified histone antibodies, analyze the concentrate with RT-PCR or next-generation sequencer (ChIP-seq analysis), extract the open chromatin region on the genome with FAIRE, and generate the next generation Analysis can be performed by a method using a sequencer (FAIRE-seq analysis) or the like.

Transposable element In the present invention, a transposable element means a gene sequence called a mobile genetic element or transposon that is moved or copied on a genome and inserted into the genome. A preferred transposable element used as an index for screening for a therapeutic drug for cancer of the present invention is a retrotransposon. In the present invention, a retrotransposon is a sequence copied to RNA, and thus the copied RNA can be used as an index. In the present invention, retrotransposons include LTR (long terminal repeat) type retrotransposons (endogenous retroviruses) and non-LTR type retrotransposons. As LTR type retrotransposon, HERV-E, HERV-F, HERV-H, HERV-K, HERV-L, HERV-T, HERV-W, HERV-FRD, ERV9, HML-1, HML-2, HML- 3, a gene selected from the group consisting of HML-4, HML-5, HML-6, HML-9HML-10 and ERVK is exemplified, and as a non-LTR type retrotransposon, LINE (long interspersed repetitive sequence) or SINE (Short interspersed repetitive sequence) is exemplified. Examples of LINE include LINE-1 (L1) retrotransposon encoding endonuclease / reverse transcriptase of open reading frame (ORF) 1 and ORF2.

  The sequence of the transposable element can be obtained from the NCBI database as appropriate. Using this sequence information, the expression level of the transposable element or its fragment can be analyzed by reverse transcriptase PCR or quantitative reverse transcriptase PCR. , Northern blot analysis, immunohistochemistry, array analysis, RNA-seq analysis and combinations thereof. The expression of the transposable element or a fragment thereof can be evaluated by analysis of epigenetic modifications such as DNA methylation and histone modification.

Kit for Screening for Cancer Therapeutic Agent In the present invention, the kit for screening for a cancer therapeutic agent includes a primer or a probe used for measuring the expression level of the above-described transposable element.

  In the present invention, the kit for screening for a therapeutic drug for cancer may further include a document or instructions describing the measurement procedure. Alternatively, it may further include a panel of various cancer cells having different effective target proteins (wherein the effective target protein has the same meaning as described above).

In another aspect of the present invention, a method for identifying a protein that can be a drug discovery target of a cancer therapeutic agent is provided. The identification method includes the following steps:
(I) a transposer under a condition for expressing a candidate gene or a condition in which the expression of the gene is suppressed in a cancer cell containing a gene (candidate gene) encoding a test protein in a form capable of controlling expression. A step of measuring the expression level of the bull element, and (ii) when the expression level of the transposable element is increased under the condition where the expression of the candidate gene is suppressed as compared with the condition of expressing the gene, The method includes a step of selecting the test protein as a protein that can be a drug discovery target of a therapeutic drug for cancer.
Here, candidate genes are identified as a result of exhaustive analysis of gene expression in cancer cells using a microarray or the like, and are highly expressed in genes or cancer cells that are specifically expressed in cancer cells. Genes that are present.
The expression controllable form means a form capable of ON / OFF of the expression of the candidate gene. For example, the candidate gene is an inducible promoter (eg, metallothionein promoter (induced by heavy metal ions), heat shock protein promoter. (Induced by heat shock), Tet-ON / Tet-OFF promoter (induced by addition or removal of tetracycline or its derivative), steroid-responsive promoter (induced by steroid hormone or its derivative), etc.) Expression vectors and the like.
Confirmation / evaluation of expression of the transposable element is the same as the screening method for cancer therapeutic agents described above.

  The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by these examples.

  In order to investigate the effects of oncogenes on cancer cell reprogramming, the EWS / ATF1 fusion gene described in Yamada K, et al, J Clin Invest. 123: 600-610, 2013 is induced in a doxycycline (Dox) -dependent manner. A possible mouse sarcoma cell line (also referred to as G1297 strain) was used. The sarcoma cells have been confirmed to stop growth in vitro and to regress tumors in vivo due to EWS / ATF1 expression cessation.

  Induction of EWS / ATF1 expression by addition of 0.2 μg / ml doxycycline does not affect the growth of mouse ES cells, so EWS / ATF1 does not affect the expression level of the introduced reprogramming factor. This was confirmed (FIGS. 5A, 5B and 5D).

Subsequently, it was confirmed whether or not G1297 was initialized by introducing an initialization factor into G1297. In sarcoma cells expressing EWS / ATF1, no iPS cell-like colonies were observed even when reprogramming factors (4F: Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced by retrovirus. On the other hand, when the expression of EWS / ATF1 was stopped, iPS cell-like colonies were confirmed by the expression of 4F (FIGS. 1A to 1C). An iPS cell-like cell line was established by picking up this iPS cell-like colony (FIG. 5C). The induction of iPS cells was performed by the following method. Oct3 / 4, Sox2, Klf4 and c-Myc were each introduced into Plat-E cells using a pMXs-based retroviral vector, and the virus-containing supernatant was collected and filtered through a 0.45 μm cellulose acetate filter. G1297 was seeded at 8 × 10 ⁵ cells per 60-mm dish, infected with the virus-containing supernatant, and replaced with LIF-containing ES medium on the third day after infection.

  The established iPS cell-like cell line was confirmed to express EWS / ATF1 in a doxycycline-dependent manner, similar to the parent sarcoma cell (FIG. 1D). Furthermore, some chromosomal abnormalities were also observed, confirming that the iPS cell-like cell line was derived from sarcoma cells (FIG. 1E). When the expression of pluripotency-related genes such as Nanog and endogenous Oct3 / 4 (Pou5f1) in iPS cell-like cells derived from the sarcoma cells was compared with ES cells, there was no significant difference (Fig. 1F and FIG. 5D). RT-PCR was performed using the following method. Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany). Quantitative real-time PCR analysis was performed using Go-taq qPCR Master Mix (Promega, Madison, USA). Transcription levels were normalized with β-actin or GAPDH values.

  The efficiency of sarcoma cell reprogramming was 0.06%, which was shown to be lower than that of MEF. When the obtained iPS cell-like cells were administered subcutaneously to immunodeficient mice, teratomas were formed, and chimeric mice could be created by injection into blastocysts (FIGS. 1G and 1H).

  From the above, it was confirmed that in EWS / ATF1 expression-dependent sarcoma cells, pluripotent cells can be obtained by reprogramming by suppressing the expression of EWS / ATF1. This result shows that EWS / ATF1 acts suppressively in the reprogramming of EWS / ATF1 expression-dependent sarcoma cells.

  Subsequently, the mechanism of cancer cell reprogramming failure via EWS / ATF1 was analyzed. First, a phenomenon in the early stage of initialization was confirmed by FACS analysis of the generation of SSEA1-positive cells (FIG. 1I). As a result, it was confirmed that the number of SSEA1-positive cells decreased depending on the Dox concentration. This suggests that EWS / ATF1 inhibits from the early stage of reprogramming. In addition, when the expression of markers for mesenchymal-epithelial transition, which is the first event in fibroblast reprogramming, was examined by microarray, it was confirmed that these genes were significantly increased after suppression of EWS / ATF1 expression. It was. On the other hand, the expression of EWS / ATF1 does not suppress the generation of SSEA1-positive cells due to reprogramming in MEF (Fig. 1J). Was suggested.

  In order to investigate the influence of oncogene expression on the transcriptional response when 4F is introduced, sarcoma cells when 4F is introduced (4F-sarcoma cells) or sarcoma cells when GFP is introduced as a negative control The microarray analysis was performed for each of the cases where EWS / ATF1 was expressed and not expressed (FIG. 5E). Microarray analysis was performed using Mouse Gene 1.0 ST Array (Affymetrix Inc., Santa Clara, USA). All data analysis was performed using the GeneSpring GX software program (version 12; Agilent Technology, Santa Clara, USA).

  As a result, it was confirmed that many genes were increased or decreased by suppressing the expression of EWS / ATF1 in 4F-sarcoma cells (FIG. 1K). Genes with altered expression were also elevated or decreased in 4F-sarcoma cells that expressed EWS / ATF1, but the difference in changes was small compared to 4F-sarcoma cells that did not express EWS / ATF1. (FIG. 5F). These results suggest that EWS / ATF1 expression suppresses fate changes in sarcoma cells.

  Furthermore, the influence of the expression of EWS / ATF1 on skeletal muscle differentiation induction by introduction of MYOD1 was examined (FIG. 1L). As a result, it was confirmed that the expression of Myogenin (MYOG), an early skeletal muscle differentiation marker, was significantly upregulated after suppression of EWS / ATF1 expression in MYOD1-introduced sarcoma cells (MYOD1-Sarcoma cells) (Fig. 1M). In addition, MHC (myosin heavy chain) positive cells were significantly increased in MYOD1-sarcoma cells in which EWS / ATF1 was not expressed (FIG. 1N). Analysis of the initial transcription response by MYOD1 introduction by microarray analysis showed that the number of gene changes by MYOD1 introduction increased in cells that did not express EWS / ATF1 compared to sarcoma cells that expressed EWS / ATF1 (Fig. 1 O). . It has been shown that skeletal muscle differentiation-inducing genes (eg, Myogenin) are greatly upregulated by introduction of MYOD1 in sarcoma cells in which EWS / ATF1 expression is suppressed. On the other hand, it was confirmed that the differentiation transformation of myocytes was suppressed by EWS / ATF1 expression.

  These results suggest that EWS / ATF1 expression limits the transcriptional response to exogenous transcription factors that alter cell fate.

  Since such transcriptional regulation is thought to be deeply related to epigenetic regulation, we investigated epigenetic modifications regulated by EWS / ATF1 expression in EWS / ATF1 expression-dependent sarcoma cells.

  First, the effect of EWS / ATF1 expression was examined using ChIP-seq analysis and FAIRE-seq analysis (FIG. 2A and FIG. 6A). Although the expression of the target gene of EWS / ATF1 is up-regulated as described above, at the transcription start site (TSS) of the gene induced by EWS / ATF1, H3K4me3 is an indicator of the activation promoter. No change was observed. However, the expression of EWS / ATF1 was confirmed to promote H3K27Ac, which is an indicator of the activation enhancer, at the EWS / ATF1 binding site away from TSS but not TSS. These results suggest that enhancer activity is associated with increased transcription at the distal regulatory site.

  FAIRE-seq analysis confirmed that the open chromatin peak due to EWS / ATF1 expression is at the EWS / ATF1 binding site, and that this open chromatin peak disappears due to EWS / ATF1 expression suppression (FIG. 2B). . This result suggests that activation by epigenetic modification at the EWS / ATF1 binding site is closely related to increased transcription.

  Subsequently, the analysis was conducted focusing on inhibitory histone markers. EWS / ATF1 expression was found to increase H3K9me3 in the transposable elements (ERVs, LINEs and SINEs) of EWS / ATF1 expression-dependent sarcoma cells (FIGS. 2C, 6B, 6C and 6D). ). Based on this, EWS / ATF1 expression-dependent sarcoma cells suppressed EWS / ATF1 expression and confirmed the expression of the transposable element, confirming that the expression of the transposable elements L1 and MMERVK10c increased. (FIG. 2D). Furthermore, when H3K9me3 was confirmed in the TSS region, it was found that appropriate amplification was caused by the expression of EWS / ATF1 (FIG. 2E). On the other hand, when RRBS (Reduced Representation Bisulfite Sequencing) analysis was performed, the difference in the level of DNA methylation due to the presence or absence of EWS / ATF1 expression at ERVs, TSS region, Oct3 / 4 binding site, Nanog binding site and Sox2 binding site Not found (Figure 2F and Figure 7A).

  These results suggest that sarcoma cells expressing EWS / ATF1 have complex histone methylation modifications because EWS / ATF1 induces both active and inhibitory histone modifications.

  Since transposable elements have binding sites for major transcription factors that play an important role in the transcriptional network that characterizes cells, the effect of EWS / ATF1 expression was examined. It has been reported that the core regulatory network for maintaining the pluripotency of ES cells consists of coordinated activation through Oct3 / 4, Nanog, and Sox2. Thus, when the influence of EWS / ATF1 on H3K9me3 modification at the binding sites of Oct3 / 4, Nanog and Sox2 in ES cells was examined, it was found that H3K9me3 increased (FIG. 3A). Similarly, it was also found that H3K9me3 is enhanced by expression of EWS / ATF1 at Ascl1 binding sites in neural stem cells and MyoD1 binding sites in myotubes (FIGS. 3B and 7B).

  On the other hand, the amounts of H3K4me3 and H3K27Ac, which are active histone markers at the Oct3 / 4 binding site of ES cells and the MyoD1 binding site of myotubes, were not changed by the expression of EWS / ATF1 (FIG. 7C). Similarly, the open chromatin peak did not change (FIG. 7C). These results suggest that EWS / ATF1 expression may enhance H3K9me3 and suppress the expression of cell fate determinants.

  To investigate the binding of EWS / ATF1 and the transduction reprogramming factor, Oct3 / 4 tagged with V5 was introduced with Sox2, Klf4 and c-Myc, and ChIP-seq analysis was performed using anti-V5 antibody ( FIG. 8A). As a result, V5-tagged Oct3 / 4 binding to the Oct3 / 4 binding site of ES cells was not suppressed by the expression of EWS / ATF1 (FIG. 3C).

  Subsequently, microarray analysis of the expression of genes adjacent to the Oct3 / 4 binding site in the presence or absence of EWS / ATF1 expression revealed that many Oct3 / 4 binding sites were found in 4F-Sarcoma cells that do not express EWS / ATF1. Adjacent genes were up- or down-regulated, but when EWS / ATF1 was expressed, the number of genes up- or down-regulated was limited (FIG. 3D). Similarly, it was confirmed that the transcriptional activation associated with the MyoD1 binding site is suppressed when EWS / ATF1 is expressed (FIG. 8B).

In order to examine the relationship between H3K9me3, the relationship between the expression level of EWS / ATF1 and the expression level of histone methyltransferase was examined. As a result, it was confirmed that Suv39h1 / Suv39h2 which is an H3K9 methyltransferase and Ezh2 which is an H3K27 methyltransferase are up-regulated by the expression of EWS / ATF1 (FIG. 8C).
Subsequently, the expression of the regulatory gene of MYOD1 when the induction of the expression of these methyltransferases by EWS / ATF1 was suppressed was examined. The experimental method and the effect of siRNA of each methyltransferase are shown in FIG. 9A. As a result, expression of L1 increased when Suv39h1 was knocked down in sarcoma cells (FIG. 9B). In addition, knocking down Suv39h1 and Suv39h2 significantly increased the induction of Myogenin expression even when EWS / ATF1 was expressed (FIG. 3E). Furthermore, when the gene changes when Suv39h1 was suppressed and MYOD1 was not introduced were analyzed by the microarray method, it was revealed that the expression level of the gene induced by MYOD1 including Myogenin increased (Fig. 3F). On the other hand, even when Ezh2 or Eed was knocked down, Myogenin expression was not induced by MYOD1. This result suggests that the expression of H3K9 methyltransferase is induced by EWS / ATF1, thereby suppressing the transcription of genes that determine cell fate in sarcoma cells.

  Next, the effect of EWS / ATF1 expression on reprogramming of human clear cell sarcoma (CCS) cell line was examined. When EWS / ATF1 was knocked down in CCS cells transfected with 4F (FIG. 4A), the expression of PODXL encoding TRA-1-60, an early marker of human reprogramming, was increased (FIG. 4B), but completely It was not possible to establish iPS cells that had been reprogrammed. Similarly, when EWS / ATF1 was knocked down in CCS cells into which MYOD1 had been introduced, an increase in MYOG expression was observed (FIG. 4B).

In addition, the effect of oncogene signal activation on cell reprogramming was investigated. Epidermal growth factor receptor (EGFR) mutant lung cancer cell line HCC827 and HER2-amplified breast cancer cell line SK-BR-3 were used in the experiments. These cancer cell lines are sensitive to EGFR-specific tyrosine kinase and HER2 tyrosine kinase inhibitors, respectively. Dox-inducible 4F was introduced into HCC827 and SK-BR-3 using a PB transposon (FIG. 9C), and the effects on the reprogramming of EGFR tyrosine kinase inhibitor gefitinib and HER2 tyrosine kinase inhibitor lapatinib were investigated (FIG. 9C). FIG. 4C). As a result, when lapatinib was treated with 50% inhibitory concentration (IC ₅₀ ) in 4F-introduced SK-BR-3, it promoted the expression of reprogramming marker PODXL (FIG. 4D). The establishment of fully reprogrammed iPS cells was not successful. On the other hand, treatment with 5-fluorouracil (5FU) at an IC ₅₀ concentration did not increase the expression of PODXL (FIG. 4D). Similarly, in HCC827 introduced with 4F, treatment with gefitinib at an IC ₅₀ concentration promoted the expression of PODXL, but treatment with 5FU at an IC ₅₀ concentration did not increase the expression of PODXL (FIG. 4D). . On the other hand, in the lung cancer cell line A549 having wild-type EGFR, the expression of PODXL was not increased by gefitinib treatment at the time of 4F introduction (FIG. 10). By microarray analysis, changes in gene expression upon introduction or non-introduction of 4F into HCC827 and SK-BR-3 were examined. Treatment with gefitinib and lapatinib, respectively, revealed that many genes including PODXL and NANOG were up- or down-regulated (FIG. 4E). These results suggested that major oncogene signals maintain cancer cell characteristics through a stable transcriptional network.

  Next, it was evaluated whether cancer cells other than HCC827, SK-BR-3, and G1297 can be applied to the screening method of the present invention. That is, evaluation was performed using a known cancer cell type, a non-small cell lung cancer cell line H2228 expressing the EML4-ALK fusion gene, and a chronic myeloid leukemia cell line KBM7. Here, the H2228 cell line is known to be sensitive to Alectinib, an ALK inhibitor, and the KBM7 cell line is sensitive to Imatinib, a Bcr-Abl tyrosine kinase inhibitor. A reprogramming factor (4F: Oct3 / 4, Sox2, Klf4 and c-Myc) is introduced into H2228 and KBM7 by retrovirus, and Alectinib is added to the culture medium of H2228 to 1 μM or 10 μM on the first day of infection. On the other hand, Imatinib was added to the culture medium of KBM7 so as to be 1 μM or 10 μM. 3 days after infection RPMI1640 + 10% FBS, GlutaMax, PS (penicillin, streptomycin) medium (for H2228), IMDM + 10% FBS, GlutaMax containing Alectinib (1 μM, 10 μM) and Imatinib (1 μM, 10 μM), respectively , PS medium (for KBM7), 2 days later (5 days after infection), extract total RNA using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and use Go-Taq qPCR Master Mix ( Promega, Madison, USA) was used to perform quantitative RT-PCR analysis and normalize transcription levels with GAPDH values (FIG. 11A). The result is shown in FIG. 11B. In the H2228 reprogrammed cell line, increased expression of the LINE1 (L1) transposable element was observed when 1 μM Alectinib treatment was performed. In the KBM7 reprogrammed cell line, the expression of L1 transposable element was increased depending on the concentration of Imanitib.

  From the above results, it was suggested that the cancer cells that can be used in the screening method of the present invention are not limited to specific cell types and can be applied to a wide variety of cancer cell types.

  ChIP-seq analysis revealed that H3K9me3 decreased in transposable elements when treated with lapatinib and gefitinib in SK-BR-3 and HCC827, respectively. Similarly, the expression level of transposable elements (L1 or HERV-W was increased (Fig. 4F, Fig. 4G). HERVH with or without 4F introduction into SK-BR-3 and HCC827 by microarray analysis Examination of changes in the expression of related genes revealed that HERVH-related genes were up-regulated by treatment with lapatinib and gefitinib, respectively (FIG. 4H and FIG. 4I). It was confirmed that inhibition of oncogene signals by drugs in human cancer cells due to the introduction of phenotype preferentially affects all genes (FIG. 4I).

Furthermore, from the expression analysis of the G1297 strain, an attempt was made to identify a transposable element in which expression induction was observed when EWS / ATF1 expression was suppressed. The experimental procedure is the same as the schematic diagram of FIG. 5E. Reprogramming factor (+ 4F) or GFP was introduced into G1297 strain expressing EWS / ATF1 by Dox addition by retrovirus, and Dox addition was performed 4 days after the introduction. RNA-seq analysis examined the expression of transposable elements in cells that had stopped (DoxOFF) and cells that continued to contain Dox (DoxON). As a result, as shown in FIG. 12A, an ERVK transposable element was identified, in which the expression was significantly increased by suppressing the expression of EWS / ATF1. RNA-seq analysis was performed using the following method. After extracting total RNA from each cell using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), a library was created using TruSeq Stranded Total RNA with Ribo-Zero Gold LT sample Prep kit (illumina) . After quantification of the concentration using KAPA Library Quantification kits (Nippon Genetics), sequencing was performed with Hiseq 2500 (illumina) using Hiseq PE Rapid Cluster kit v2-HS. The sequence data was analyzed using TopHat software and Cufflinks software, and visualized by IGV (Integrative Genomics Viewer). In addition, quantitative RT-PCR also showed that the expression level of the ERVK transposable element increased predominately when EWS / ATF1 expression was suppressed (FIG. 12B).
From the above results, it was shown that the identified ERVK transposable element can be applied to the screening method of the present invention.

  As described above, it was shown that the characteristics of cancer cells are maintained by the oncogene signal, that is, the initialization of cancer cells is inhibited. On the other hand, anticancer drugs targeting oncogenes have also been shown to promote early stages of cancer cell reprogramming. These results indicate that agents that inhibit oncogene signals can make cancer cells tolerant to changes in cell fate. Therefore, it is possible to detect an important oncogene signal by examining the effect on the initialization of a drug that inhibits a specific signal in cancer cells.

  On the other hand, cancer cells are adaptable to the environment and can therefore survive and continue to progress. After an initial response to a molecular target drug for cancer, the majority of cancers acquire resistance and cause disease recurrence. However, the mechanism by which cancer cells acquire resistance to drugs is not completely understood, but by inhibiting the oncogene signal, the release of slow transcriptional repression through changes in epigenetic modifications. The phenomenon that cancer cells forced to initialize from the outside can acquire adaptability supports the mechanism of resistance acquisition.

  In general, cancer cells are said to reduce the level of DNA methylation in transposable elements, but the reduction of inhibitory histone markers in transposable elements by the addition of drugs increases the adaptability of cancer cells This is not inconsistent with the above consideration. Adaptation gained through inhibition of oncogene signaling could change the fate of cancer cells to drug resistant cells, a new mechanism for survival of cancer cells when treated with molecularly targeted drugs Is expected to be offered.

  This also seems to deny the current status of identifying compounds that interfere with oncogene signaling, such as anticancer drugs. However, the findings of this study suggest that combination therapy targeting both important oncogene pathways and cancer cell adaptability can be an effective way to treat cancer patients .

  This application is based on patent application Japanese Patent Application No. 2015-77267 (filing date: April 3, 2015) filed in Japan, the contents of which are incorporated in full herein.

Claims (5)

A method of screening for a therapeutic agent for cancer, comprising the steps of:
(I) a step of measuring the expression level of the transposable element in a target cancer cell in contact with or without contact with the test substance; and (ii) under contact with the test substance without contact. A step of selecting the test substance as a cancer therapeutic agent when the expression level of the transposable element is increased as compared with.   The method of claim 1, wherein the transposable element is a retrotransposon or a fragment thereof.   The method according to claim 2, wherein the transposable element is at least one sequence selected from endogenous retrovirus, LINE, SINE and fragments thereof.   The method according to claim 2, wherein the transposable element is at least one sequence selected from HERV-H, HERV-W, L1-ORF2, ERVK and fragments thereof. A method for identifying a protein that can be a drug discovery target of a cancer therapeutic drug, comprising the following steps;
(I) Expression of a transposable element in a cancer cell containing a gene encoding a test protein in a form in which expression can be controlled under conditions for expressing the gene or under conditions where expression of the gene is suppressed A step of measuring the amount, and (ii) when the expression level of the transposable element is increased under the condition in which the expression of the gene is suppressed as compared with the condition under which the gene is expressed. A process of selecting proteins that can be targets for drug discovery for cancer treatment.