Prophylactic or therapeutic against exacerbation or recurrence of cancer, and screening method therefor

Abstract

Provided is a prophylactic or therapeutic which is against the exacerbation or recurrence of cancer due to cells including cancer stem cell that are in a dormant state during chemotherapy, and which has a YAP inhibitor as an active ingredient. Also provided is a screening method comprising: a step (a) for causing a chemotherapeutic agent to act on a cancer organoid, and killing the majority of the cancer organoid as a result; a step (b) for further incubating the cancer organoid, and as a result thereof, commencing propagation of cells including cancer stem cells that exist in the cancer organoid and were in a dormant state; and a step (c) for evaluating the propagation of the cells including the cancer stem cells that were in the dormant state, wherein steps (a) and (b) are performed in the presence of a test substance, and the suppression of proliferation of the cells including the cancer stem cells that were in the dormant state, as compared to a case in which the test substance was not present, indicates that the test substance is a prophylactic or therapeutic against the exacerbation or recurrence of the cancer due to the cells including the cancer stem cells that were in the dormant state during chemotherapy.

Description

Preventive or therapeutic agent for cancer recurrence or recurrence and screening method therefor

[0001] The present invention relates to a preventive or therapeutic agent for cancer recurrence or recurrence, and a screening method therefor. This application claims priority to U.S. Provisional Application No. US 63 / 350,414 filed June 9, 2022, the contents of which are incorporated herein by reference.

[0002] In Japan, colorectal cancer ranks first among all cancers, and in 2020 it was the leading cause of death for women and the third highest for men, casting a large shadow over society. Recent advances in chemotherapy have improved treatment outcomes for advanced, unresectable cancers, but the number of deaths from colorectal cancer remains high.

[0003] Incidentally, Patent Document 1 describes a method for screening anticancer agents that target LGR5-positive cancer stem cells and are effective against cancer stem cells. It also describes that even if LGR5-positive cancer stem cells are killed, tumor size eventually increases again, that LGR5-negative cancer cells have the ability to dedifferentiate into LGR5-positive cancer stem cells, and that the combined use of a cancer stem cell-specific anticancer agent and an existing anticancer agent is effective in cancer treatment.

[0004] Furthermore, Non-Patent Document 1 describes that in pancreatic adenocarcinoma (PDAC), the mRNA and protein levels of COL17A1 (collagen type XVIIα1 chain) are elevated, that PDAC patients with high COL17A1 expression have a lower overall survival rate than patients with low COL17A1 expression, that knockdown of COL17A1 significantly suppresses the proliferation and invasion of PDAC cells, and that cells overexpressing COL17A1 have significantly increased proliferation and invasion capabilities.

[0005] International Publication No. 2018 / 181276

[0006] Mao F., et al., High Expression of COL17A1 Predicts Poor Prognosis and Promotes the Tumor Progression via NF-kappaB Pathway in Pancreatic Adenocarcinoma, Journal of Oncology, Article ID 8868245, 2020.

[0007] Cancer chemotherapy has the problem of relapse or recurrence due to the presence of cancer cells that exhibit resistance to chemotherapeutic agents. The present invention aims to elucidate the mechanism of cancer relapse or recurrence after chemotherapy, and to provide a preventive or therapeutic agent for cancer relapse or recurrence after chemotherapy, as well as a screening method for the same.

[0008] The present invention includes the following aspects. [1] A preventive or therapeutic agent for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, comprising a YAP inhibitor as an active ingredient. [2] The preventive or therapeutic agent according to [1], wherein the cancer stem cells that are dormant during chemotherapy are LGR5-positive, p27-positive cancer stem cells. [3] The preventive or therapeutic agent according to [1] or [2], wherein the cancer relapse or recurrence is cancer relapse or recurrence after chemotherapy. [4] The preventive or therapeutic agent according to any of [1] to [3], wherein the YAP inhibitor is an inhibitor of YAP signal activation. [5] A pharmaceutical composition for preventing or treating cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, comprising the preventive or therapeutic agent according to any of [1] to [4] and a pharmaceutically acceptable carrier. [6] A method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells containing cancer stem cells in cancer tissue that are dormant during chemotherapy, comprising the steps of: (a) applying a chemotherapeutic agent to cancer organoids, resulting in the death of most of the cells in the cancer organoids; (b) further incubating the cancer organoids, resulting in cells containing cancer stem cells that were dormant in the cancer organoids beginning to proliferate; and (c) evaluating the proliferation of the cells containing cancer stem cells that were dormant; wherein steps (a) or (b) are performed in the presence of a test substance, and the suppression of proliferation of the cells containing cancer stem cells that were dormant compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells containing cancer stem cells in cancer tissue that are dormant during chemotherapy.[7] A method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in a dormant state during chemotherapy, including cancer stem cells in cancer tissue, comprising: (a) applying a chemotherapeutic agent to cancer organoids, resulting in the death of most of the cells in the cancer organoids; (b) further incubating the cancer organoids, resulting in the cells in the cancer organoids, including cancer stem cells, that were dormant, beginning to proliferate; and (c) evaluating the activation of YAP signaling in the cells containing cancer stem cells that were dormant during chemotherapy. Steps (a) or (b) are performed in the presence of a test substance, and the suppression of YAP signaling compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in a dormant state during chemotherapy, including cancer stem cells in cancer tissue. [8] A method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance, and evaluating the proliferation of the LGR5-positive, p27-positive cancer stem cells, wherein the suppression of proliferation of the LGR5-positive, p27-positive cancer stem cells compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy. [9] A method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance, and evaluating the activation of YAP signaling in the LGR5-positive, p27-positive cancer stem cells, wherein the suppression of the activation of YAP signaling compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[10] A method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance, and evaluating the expression of COL17A1 in the LGR5-positive, p27-positive cancer stem cells, wherein an increase in the expression of COL17A1 compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[11] A method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, comprising the steps of: applying a chemotherapeutic agent to cancer organoids in the presence of a test substance; and evaluating the expression of COL17A1 in the cancer organoids, wherein an increase in the expression of COL17A1 compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0009] According to the present invention, it is possible to provide a preventive or therapeutic agent for recurrence or recurrence of cancer after chemotherapy, and a screening method for the same.

[0010] FIG. 1 is a schematic diagram illustrating the experimental system of Experimental Example 1. FIG. 2 is a graph showing the results of Experimental Example 2. FIG. 3 is a graph showing the results of measuring the proportion of cells that underwent cell division over time in Experimental Example 4. FIG. 4 is a photograph showing the results of Experimental Example 4. FIG. 5 is a graph showing the results of Experimental Example 5. FIG. 6 is a volcano plot showing the results of comparing the transcriptomes of LGR5-positive p27-positive cells and LGR5-positive p27-negative cells in Experimental Example 6. FIG. 7 is a fluorescent micrograph showing the results of Experimental Example 7. FIG. 8 is a schematic diagram showing the experimental schedule and a graph showing the change in tumor size in mice over time in Experimental Example 8. FIG. 9 is a micrograph and a graph showing the results of Experimental Example 9. FIG. 10 is a confocal micrograph and a graph showing the results of Experimental Example 9. FIG. 11 is a confocal micrograph and a graph showing the results of Experimental Example 10. FIG. 12 is an image showing the results of a capillary-based immunoassay in Experimental Example 11. FIG. 13 is a confocal microscope photograph showing the results of Experimental Example 11. FIG. 14 is a graph showing the results of Experimental Example 11. FIG. 15 is a schematic diagram showing the experimental schedule and micrographs showing the results of immunostaining in Experimental Example 12. FIG. 16 is a graph showing the change in tumor size in mice over time in Experimental Example 12. FIG. 17 is a schematic diagram showing the experimental schedule and graph showing the change in tumor size in mice over time in Experimental Example 13. FIG. 18 is a schematic diagram showing the experimental schedule and graph showing the change in tumor size in mice over time in Experimental Example 14. A schematic diagram showing the mechanism by which dormant LGR5-positive, p27-positive cancer stem cells are released from their dormant state, resulting in tumor regrowth, and a schematic diagram illustrating how the dormant state of cancer stem cells is maintained by COL17A1, and how FAK-YAP signaling is activated after chemotherapy, resulting in the release of dormancy.

[0011] [Preventive Agent for Cancer Relapse or Recurrence] In one embodiment, the present invention provides an agent for preventing or treating cancer relapse or recurrence caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy, which agent contains a YAP inhibitor as an active ingredient.

[0012] As described below in the Examples, the inventors have demonstrated that cancer stem cells that remain dormant during chemotherapy are LGR5-positive, p27-positive cancer stem cells. The inventors have also demonstrated that chemotherapy-resistant LGR5-positive, p27-positive cancer stem cells remain dormant without undergoing cell division under chemotherapy, and proliferate after chemotherapy. The term "dormant state" refers to the absence of cell division, and may refer to the G0 phase of the cell cycle. The inventors have demonstrated that the proliferation of dormant LGR5-positive, p27-positive cancer stem cells after chemotherapy is the cause of cancer relapse or recurrence after chemotherapy.

[0013] The inventors also elucidated the mechanism of regrowth of cells, including LGR5-positive, p27-positive cancer stem cells, involved in cancer relapse or recurrence, and demonstrated that a YAP inhibitor exhibits preventive or therapeutic effects against cancer relapse or recurrence in vitro and in vivo. Therefore, the present invention can provide a preventive or therapeutic agent for cancer relapse or recurrence after chemotherapy. By administering the preventive or therapeutic agent of this embodiment to a cancer patient after chemotherapy, cancer relapse or recurrence after chemotherapy can be prevented or treated.

[0014] As used herein, cancer recurrence refers to the re-growth of cancer cells that were not completely killed after cancer treatment, and cancer recurrence refers to the re-occurrence of cancer at the primary site after cancer was once completely cured by cancer treatment, or the metastasis of cancer cells to the re-growth of cancer cells at a site other than the primary site.

[0015] The preventive or therapeutic agent of this embodiment can prevent or treat recurrence or recurrence of cancer after chemotherapy. The preventive or therapeutic agent of this embodiment can also be described as an agent for suppressing recurrence or recurrence of cancer after chemotherapy and / or an agent for suppressing regrowth of cancer, or an agent for preventing the release of dormancy of cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy.

[0016] In the preventive or therapeutic agent of this embodiment, examples of cancer include cancers that show recurrence or recurrence after chemotherapy, more specifically, colorectal cancer, gastric cancer, esophageal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, and liver cancer.

[0017] Chemotherapy refers to a method of treating cancer using a chemotherapeutic agent. Chemotherapeutic agents refer to cytotoxic anticancer agents among anticancer agents. Examples of cytotoxic anticancer agents include topoisomerase inhibitors, platinum compounds, antitumor antibiotics, alkylating agents, antimetabolites, and microtubule-active agents.

[0018] Examples of topoisomerase inhibitors include irinotecan, camptothecin, nogitecan, doxorubicin, amrubicin, etoposide, and the like.

[0019] Examples of platinum agents include cisplatin, carboplatin, nedaplatin, and oxaliplatin.

[0020] Examples of antitumor antibiotics include actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin stimalamer, daunorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, and mitoxantrone.

[0021] Alkylating agents include ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, ranimustine, and the like.

[0022] Examples of the antimetabolite include pemetrexed, tegafur, gimeracil, oteracil, tegafur-uracil, 5-fluorouracil (5-FU), gemcitabine, and the like.

[0023] Microtubule-active agents include paclitaxel, docetaxel, vinorelbine, vindesine, and the like.

[0024] In the preventive agent of this embodiment, the YAP inhibitor is not particularly limited as long as it is a substance that inhibits the activation of YAP signaling. It is known that when focal adhesion kinase (FAK) is phosphorylated and activated, it mediates the nuclear translocation of the transcription coactivator Yes-associated protein (YAP). It is known that the nuclear-translocated YAP couples with the transcription factor TEAD together with TAZ to activate the transcription of genes involved in cell proliferation and survival.

[0025] As used herein, activation of YAP signaling means phosphorylating FAK, translocating YAP into the nucleus, binding YAP, TAZ, and TEAD, and inducing target gene expression.

[0026] Therefore, a YAP inhibitor may be an inhibitor of FAK phosphorylation, an inhibitor of the binding of YAP, TAZ, and TEAD, or a substance that suppresses the expression of YAP or TAZ.

[0027] Examples of inhibitors of FAK phosphorylation (FAK inhibitors) include PF573228 (CAS number: 869288-64-2), PF-00562271 Besylate (CAS number: 939791-38-5), PF-562271 (CAS number: 717907-75-0), PF-562271 HCl (CAS number: 939791-41-0), PRT062607 (P505-15) HCl (CAS number: 1370261-97-4), TAE226 (NVP-TAE226) (CAS number: 761437-28-9), GSK215 (CAS number: 2743427-26-9), and ALK. Inhibitor 1 (CAS number: 761436-81-1), BI-4464 (CAS number: 1227948-02-8), GSK2256098 (CAS number: 1224887-10-8), PF-431396 (CAS number: 717906-29-1), PND-1186 (VS-4718) (CAS number: 1061353-68-1), Masitinib mesylate (CAS number: 1048007-93-7), AMP-945 (CAS number: 1393653-34-3), Y15 (CAS number: 4506-66-5), Defactinib (VS-6063) (CAS number: 1073154-85-4), Solanesol (Nonaisoprenol) (CAS number: 13190-97-1), and the like.

[0028] Examples of inhibitors of the binding of YAP, TAZ, and TEAD include TEAD inhibitors (TEADi) such as MYF-01-37 (CAS number: 2416417-65-5), K-975 (CAS number: 2563855-03-6), MGH-CP1 (CAS number: 896657-58-2), VT103 (CAS number: 2290608-13-6), and TED-347 (CAS number: 2378626-29-8).

[0029] Substances that suppress the expression of YAP or TAZ include siRNA, shRNA, and the like against YAP or TAZ mRNA.

[0030] The prophylactic or therapeutic agent of this embodiment may be formulated as a pharmaceutical composition mixed with a pharmaceutically acceptable carrier. That is, in one embodiment, the present invention provides a pharmaceutical composition for preventing or treating cancer relapse or recurrence caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy, comprising the prophylactic or therapeutic agent described above and a pharmaceutically acceptable carrier.

[0031] The pharmaceutical composition can be administered orally in the form of, for example, tablets, capsules, elixirs, microcapsules, etc., or parenterally in the form of injections, suppositories, etc.

[0032] The pharmaceutically acceptable carrier may be any carrier typically used in the preparation of pharmaceutical compositions, including, for example, binders such as hypromellose, dextrin, macrogol 400, gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as lactose hydrate, D-mannitol, starch, crystalline cellulose, and alginic acid; and solvents for injections such as water, ethanol, and glycerin.

[0033] The pharmaceutical composition of this embodiment may contain additives, such as lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and rhizome oil; stabilizers such as carmellose sodium, hydrogenated oil, light anhydrous silicic acid, povidone, glycerin fatty acid esters, benzyl alcohol, and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; and colorants such as yellow ferric oxide, red ferric oxide, black ferric oxide, and titanium oxide.

[0034] The pharmaceutical composition can be formulated by appropriately combining the above-mentioned YAP inhibitor with the above-mentioned pharmaceutically acceptable carriers and additives, and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. In the pharmaceutical composition, one YAP inhibitor may be used alone, or two or more YAP inhibitors may be used in combination.

[0035] An appropriate daily dose of a prophylactic or therapeutic agent or pharmaceutical composition is an amount containing the minimum effective dose of the active ingredient (YAP inhibitor) to produce a therapeutic effect. The effective minimum dose depends on various factors, including the activity of the active ingredient contained in the pharmaceutical composition, functional group modifications that determine its lipid solubility and water solubility, the administration route, the administration time, the excretion rate of the specific active ingredient used, the treatment period, other drugs used concomitantly, the age, sex, weight, disease, health condition, and medical history of the patient, and other factors well known in the medical arts.

[0036] Typically, the dosage of the prophylactic or therapeutic agent or pharmaceutical composition is an amount containing about 0.0001 to about 100 mg / kg body weight of the active ingredient (YAP inhibitor) per day. The prophylactic or therapeutic agent or pharmaceutical composition may be administered once a day or in divided doses about 2 to 4 times a day.

[0037] [Method for screening preventive or therapeutic agents for cancer relapse or recurrence] (First embodiment) In one embodiment, the present invention provides a method for screening preventive or therapeutic agents for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy. The method comprises the steps of: (a) applying a chemotherapeutic agent to cancer organoids, resulting in the death of most of the cells in the cancer organoids; (b) further incubating the cancer organoids, resulting in the cells in the cancer organoids that were dormant, including cancer stem cells, beginning to proliferate; and (c) evaluating the proliferation of the cells that were dormant, including cancer stem cells. Steps (a) or (b) are performed in the presence of a test substance, and the suppression of proliferation of the cells that were dormant, including cancer stem cells, compared to the absence of the test substance, indicates that the test substance is a preventive or therapeutic agent for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0038] The screening method of this embodiment allows screening for a preventive or therapeutic agent for cancer relapse or recurrence by cells containing cancer stem cells that are in a dormant state during chemotherapy in cancer tissue. In the screening method of this embodiment, the cancer relapse or recurrence, chemotherapy, dormancy, etc. are the same as those described above. The cancer stem cells that are in a dormant state under chemotherapy are preferably LGR5-positive, p27-positive cells.

[0039] In the screening method of this embodiment, step (a) or (b) is carried out in the presence of a test substance. That is, the step of making a chemotherapeutic agent act on cancer organoids can be carried out in the presence of a test substance. Alternatively, after making a chemotherapeutic agent act on cancer organoids, the step of further incubating cancer organoids in the absence of a chemotherapeutic agent can be carried out in the presence of a test substance. Alternatively, both the step of making a chemotherapeutic agent act on cancer organoids and the step of further incubating cancer organoids after making a chemotherapeutic agent act can be carried out in the presence of a test substance.

[0040] Organoids are small organs formed by the accumulation of cells, and have structures and functions similar to those of organs in vivo. In recent years, research into the creation of various organoids from pluripotent stem cells and biological tissues has been actively conducted, and for example, intestinal organoids, liver organoids, kidney organoids, brain organoids, etc. have been created. Cancer organoids include organoids derived from cancer tissues of patients. Cancers include those mentioned above.

[0041] The test substance is not particularly limited, and for example, a natural compound library, a synthetic compound library, an existing drug library, etc. can be used.

[0042] In step (c), the evaluation of proliferation of cells including cancer stem cells may refer to the proliferation of cells derived from cancer stem cells. That is, the evaluation of proliferation of cells may include cancer stem cells that maintain stemness and cells differentiated from cancer stem cells.

[0043] The proliferation of cells, including cancer stem cells, can be evaluated by any suitable method, including, for example, measuring the number of cells under a microscope, measuring the number of cells by measuring ATP, or measuring the number of cells using a tetrazolium compound such as MTT. If the proliferation of organoids is suppressed compared to that in the absence of the test substance, it can be determined that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, that are dormant during chemotherapy.

[0044] (Second embodiment) In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in a cancer tissue, including cancer stem cells, that are dormant during chemotherapy, comprising the steps of: (a) applying a chemotherapeutic agent to cancer organoids, resulting in the death of most of the cells in the cancer organoids; (b) further incubating the cancer organoids, resulting in the cells, including cancer stem cells, that were dormant in the cancer organoids beginning to proliferate; and (c) evaluating the activation of YAP signaling in the cells, including cancer stem cells, that were dormant during chemotherapy; wherein steps (a) or (b) are performed in the presence of a test substance, and suppression of YAP signaling compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells in a cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0045] The screening method of the second embodiment differs from the screening method of the first embodiment mainly in that, in step (c), activation of YAP signaling in cells containing cancer stem cells that were in a dormant state is evaluated.

[0046] As described later in the Examples, the inventors have revealed that in cells, including cancer stem cells, in cancer tissue that were dormant during chemotherapy, activation of YAP signaling after chemotherapy causes the cells to regrow, resulting in recurrence or recurrence of cancer.

[0047] Therefore, a test substance that suppresses the activation of YAP signaling in cells, including cancer stem cells, in cancer tissue after chemotherapy can be determined to be a preventive or therapeutic agent for the recurrence or recurrence of cancer.

[0048] In the screening method of this embodiment, the recurrence or recurrence of cancer, chemotherapy, dormant state, YAP signaling, test substance, etc. are the same as those described above. The dormant cancer stem cells are preferably LGR5-positive, p27-positive cells.

[0049] Activation of YAP signaling can be evaluated, for example, by measuring pFAK, measuring nuclear translocation of YAP, or measuring the expression levels of YAP target genes (YAP / TAZ / TEAD target genes).

[0050] Suppression of YAP signal activation in the presence of a test substance may mean that, in the presence of the test substance, the amount of pFAK present is reduced, the amount of YAP translocated into the nucleus is reduced, the expression level of a YAP target gene is reduced, etc. Examples of YAP target genes include LATS2, CTGF, and CYR61, as compared to the absence of the test substance.

[0051] (Third embodiment) In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for the relapse or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance, and evaluating the proliferation of the LGR5-positive, p27-positive cancer stem cells, wherein inhibition of proliferation of the LGR5-positive, p27-positive cancer stem cells compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the relapse or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0052] As described later in the Examples, the inventors have demonstrated that cancer stem cells that remain dormant during chemotherapy are LGR5-positive, p27-positive cancer stem cells. The inventors have also demonstrated that COL17A1-mediated cell-extracellular matrix adhesion is required for the maintenance of the dormant state of LGR5-positive, p27-positive cancer stem cells.

[0053] Dissociating LGR5-positive, p27-positive cancer stem cells into single cells disrupts cell-extracellular matrix adhesion, resulting in activation of FAK-YAP signaling (YAP signaling) in LGR5-positive, p27-positive cancer stem cells, which are released from their dormant state.

[0054] Therefore, a test substance that inhibits the proliferation of LGR5-positive, p27-positive cancer stem cells dissociated into single cells can be determined to be an agent for preventing or treating the recurrence or recurrence of cancer.

[0055] The method for dissociating LGR5-positive, p27-positive cancer stem cells into single cells is not particularly limited, and may be an enzymatic dissociation method, a physical dissociation method, or a combination of these. When enzymatic dissociation is performed, enzymes that can be used include collagenase, dispase I, liberase, trypsin, TrypLE Express (Thermo Fisher Scientific), etc. Physical dissociation can be performed by mechanical shearing, for example, by pipetting.

[0056] In the screening method of this embodiment, the recurrence or recurrence of cancer, chemotherapy, dormant state, YAP signaling, test substance, etc. are the same as those described above.

[0057] (Fourth Embodiment) In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for the relapse or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance, and evaluating activation of YAP signaling in the LGR5-positive, p27-positive cancer stem cells, wherein suppression of the activation of YAP signaling compared to that in the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the relapse or recurrence of cancer caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0058] The screening method of the fourth embodiment differs from the screening method of the third embodiment mainly in that activation of YAP signaling in LGR5-positive, p27-positive cancer stem cells is evaluated.

[0059] As described later in the Examples, the inventors have revealed that in LGR5-positive, p27-positive cancer stem cells that were in a dormant state during chemotherapy, activation of YAP signaling after chemotherapy causes the cells to regrow, resulting in recurrence or recurrence of cancer.

[0060] The inventors also demonstrated that COL17A1-mediated cell-extracellular matrix adhesion is required for maintaining the dormant state of LGR5-positive, p27-positive cancer stem cells. Dissociating LGR5-positive, p27-positive cancer stem cells into single cells disrupts cell-extracellular matrix adhesion. As a result, FAK-YAP signaling (YAP signaling) is activated in LGR5-positive, p27-positive cancer stem cells, releasing them from dormancy.

[0061] Therefore, a test substance that suppresses activation of YAP signaling in LGR5-positive, p27-positive cancer stem cells dissociated into single cells can be determined to be a preventive or therapeutic agent for cancer recurrence or recurrence.

[0062] In the screening method of this embodiment, the recurrence or recurrence of cancer, chemotherapy, dormant state, YAP signaling, test substance, dissociation into single cells, etc. are the same as those described above.

[0063] The activation of YAP signaling can be evaluated in the same manner as in the second embodiment, and can be performed, for example, by measuring pFAK, measuring nuclear translocation of YAP, or measuring the expression levels of YAP target genes (YAP / TAZ / TEAD target genes).

[0064] Suppression of YAP signal activation in the presence of a test substance may mean that, in the presence of the test substance, the amount of pFAK present is reduced, the amount of YAP translocated into the nucleus is reduced, the expression level of a YAP target gene is reduced, etc. Examples of YAP target genes include LATS2, CTGF, and CYR61, as compared to the absence of the test substance.

[0065] (Fifth Embodiment) In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance, and evaluating the expression of COL17A1 in the LGR5-positive, p27-positive cancer stem cells, wherein an increase in the expression of COL17A1 compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0066] As will be described later in the Examples, the inventors have revealed that COL17A1-mediated cell-extracellular matrix adhesion is required for maintaining the dormant state of LGR5-positive, p27-positive cancer stem cells.

[0067] The inventors also demonstrated that COL17A1 expression is lost and FAK-YAP signaling is activated in cancer cells treated with a chemotherapeutic agent.

[0068] Dissociating LGR5-positive, p27-positive cancer stem cells into single cells disrupts cell-extracellular matrix adhesion, resulting in activation of FAK-YAP signaling in LGR5-positive, p27-positive cancer stem cells, which are released from their dormant state.

[0069] Therefore, a test substance that increases the expression of COL17A1 in LGR5-positive, p27-positive cancer stem cells dissociated into single cells can be determined to be an agent for preventing or treating cancer recurrence or recurrence.

[0070] In the screening method of this embodiment, the recurrence or recurrence of cancer, chemotherapy, dormant state, YAP signaling, test substance, dissociation into single cells, etc. are the same as those described above.

[0071] (Sixth embodiment) In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy, the screening method comprising the steps of: applying a chemotherapeutic agent to cancer organoids in the presence of a test substance; and evaluating the expression of COL17A1 in the cancer organoids, wherein an increase in the expression of COL17A1 compared to that in the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for cancer relapse or recurrence caused by cells in cancer tissue, including cancer stem cells, that are dormant during chemotherapy.

[0072] The screening method of the sixth embodiment differs mainly from the screening method of the fifth embodiment in that, rather than disrupting cell-extracellular matrix adhesion by dissociating cancer organoids into single cells, cell-extracellular matrix adhesion is disrupted by applying a chemotherapeutic agent to the cancer organoids to eliminate the expression of COL17A1.

[0073] As described above, the inventors have demonstrated that in cancer cells treated with a chemotherapeutic agent, COL17A1 expression is lost, FAK-YAP signaling is activated, and the dormant state of LGR5-positive, p27-positive cancer stem cells is released.

[0074] Therefore, a test substance that increases the expression of COL17A1, which is decreased by the action of a chemotherapeutic agent, can be determined to be an agent for preventing or treating the recurrence or recurrence of cancer.

[0075] In the screening method of this embodiment, the test substance, cancer recurrence or recurrence, chemotherapy, dormant state, YAP signal, test substance, etc. are the same as those described above.

[0076] [Other Embodiments] In one embodiment, the present invention provides a method for preventing or treating relapse or recurrence of cancer caused by cancer stem cells that remain dormant during chemotherapy, comprising the step of administering an effective amount of a YAP inhibitor to a cancer patient after chemotherapy.

[0077] In one embodiment, the present invention provides a YAP inhibitor for use in preventing or treating cancer relapse or recurrence in cancer patients after chemotherapy, caused by cancer stem cells that remain dormant during chemotherapy.

[0078] In one embodiment, the present invention provides use of a YAP inhibitor for the manufacture of an agent for preventing or treating cancer relapse or recurrence caused by cancer stem cells that remain dormant during chemotherapy.

[0079] In each of these embodiments, the recurrence or recurrence of cancer, chemotherapy, dormancy, YAP inhibitors, etc. are the same as those described above.

[0080] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0081] [Experimental Example 1] (Establishment of in vivo tracing technology for cancer stem cells) A technology for observing human cancer stem cells over time at the single-cell level using in vivo imaging was established. Figure 1 is a schematic diagram illustrating this experimental system. First, human colon cancer-derived organoids containing LGR5-positive cancer stem cells labeled with a cell lineage marker were prepared. For details on cell lineage markers, see, for example, Shimokawa M., et al., Visualization and targeting of LGR5+ human colon cancer stem cells, Nature, 545 (7653), 187-192, 2017.

[0082] These organoids express ubiquitously intranuclear GFP. Furthermore, upon tamoxifen treatment, random recombination occurs at multiple loxP sites within the rainbow cassette. As a result, LGR5-positive cells are stochastically labeled with yellow fluorescent protein (YFP), red fluorescent protein (RFP), or blue fluorescent protein (CFP).

[0083] Once a cell has undergone recombination to express one of the fluorescent proteins, the cell and its progeny continue to express the same fluorescent protein. This allows cells derived from a single cell to be tracked and analyzed. Hereinafter, organoids labeled with the above-mentioned cell lineage markers may be referred to as "LGR5-CreER / Rainbow organoids."

[0084] LGR5-CreER / Rainbow organoids were xenografted subcutaneously into immunodeficient NOG mice. In vivo imaging of the subcutaneously implanted organoids was then performed using an imaging window device. This system enabled the observation of live tumor cells at single-cell resolution using multiphoton microscopy for over 30 days.

[0085] [Experimental Example 2] (In vivo tracing of cancer stem cells) The presence of three patterns of clones was revealed by tracking the labeled LGR5-positive clones in the xenografted mice of Experimental Example 1. Figure 2 is a graph showing the results of measuring cell proliferation of each of the three patterns of cells.

[0086] The upper panel of Figure 2 shows the pattern of clones that continued to expand (n = 23). A clone consisting of four or more cells, without a 50% or greater reduction in size, and that could be tracked for six days or more was defined as a continuing to expand clone. The continuing to expand clones showed stable proliferation, consistent with the typical cancer stem cell model. Approximately 24% of the clones were continuing to expand.

[0087] The middle panel of Figure 2 shows the pattern of gradually decreasing clones (n = 51). A gradually decreasing clone was defined as a clone consisting of three or fewer cells that decreased within six days or a clone consisting of four or more cells that decreased in size by more than 50%. A gradually decreasing clone initially expanded but then decreased in size. Approximately 54% of the clones were gradually decreasing clones.

[0088] The lower panel of Figure 2 shows the pattern of dormant clones (n = 20). Clones consisting of three or fewer cells that could be tracked for six days or more were defined as dormant clones. Dormant clones rarely divided and maintained a small clone size throughout the observation period, indicating that they were dormant cancer cells. 21% of the clones were dormant clones.

[0089] As described above, the various dynamics of LGR5-positive colon cancer cells in vivo could be faithfully visualized with single-cell resolution.

[0090] [Experimental Example 3] (Identification of a marker for dormant clones) Ki67 protein is a marker for cell proliferation. It is known that Ki67 is present in all cell cycle phases (G1, S, G2, and M phases) of proliferating cells, but is not present in the resting phase (G0 phase) in which cell proliferation is quiescent.

[0091] To identify markers for dormant clones, i.e., LGR5-positive, Ki67-negative human colorectal cancer stem cells, we screened the expression patterns of cell cycle inhibitor proteins, p21, p57, and p27, which are expressed in LGR5-positive colorectal cancer stem cells. The results revealed that LGR5-positive, Ki67-negative colorectal cancer stem cells primarily express p27, and p27 expression indicates a slowly dividing or quiescent population.

[0092] Analysis of four different human colorectal cancer organoid lines (CCO7, CCO20, CCO25, and CCO32) confirmed the presence of LGR5-positive, p27-positive cells in all of these organoids. Furthermore, DNA / RNA analysis confirmed that the LGR5-positive, p27-positive cells were primarily in the G0 phase.

[0093] These results demonstrate that LGR5-positive colon cancer dormant clones express p27.

[0094] [Experimental Example 4] (LGR5-positive, p27-positive cells were resistant to chemotherapeutic agents) The growth kinetics of LGR5-positive, p27-positive cells and LGR5-positive, p27-negative cells in human colon cancer organoids was tracked by in vitro live imaging.

[0095] Figure 3 is a graph showing the results of measuring the proportion of cells that underwent cell division over time using in vitro live imaging. As a result, more than 80% of LGR5-positive, p27-negative cells underwent cell division and proliferated, whereas more than two-thirds of LGR5-positive, p27-negative cells remained as single cells.

[0096] Human colon cancer organoids were treated with the topoisomerase inhibitor camptothecin (CPT) (50 nM). Subsequently, LGR5-positive p27-positive cells, LGR5-positive p27-negative cells, LGR5-negative p27-positive cells, and LGR5-negative p27-negative cells were sorted using a cell sorter and collected, and 1,000 cells of each were cultured.

[0097] Figure 4 shows photographs of each cell line on day 13 after sorting. The numbers in Figure 4 indicate the number of colonies (mean ± standard error). The scale bar is 1 mm. As a result, LGR5-positive, p27-positive cells after camptothecin treatment showed colony formation, but LGR5-positive, p27-negative cells after camptothecin treatment did not show colony formation. This result indicates that LGR5-positive, p27-positive cells are resistant to chemotherapeutic agents.

[0098] Experimental Example 5 (In Vivo Imaging of LGR5-Positive, p27-Positive Cells) Whether chemotherapeutic-resistant LGR5-positive, p27-positive cells contribute to tumor recurrence or recurrence after chemotherapy was investigated in vivo. Specifically, irinotecan was administered to mice xenografted with human colon cancer organoids, and then in vivo imaging and gene lineage tracing of LGR5-positive, p27-positive cells and LGR5-positive, p27-negative cells were performed over time.

[0099] 5 is a graph showing the results of measuring the survival rate of LGR5-positive, p27-positive cells and LGR5-positive, p27-negative cells after administration of irinotecan. As a result, it was confirmed that LGR5-positive, p27-positive cells survived chemotherapy. On the other hand, LGR5-positive, p27-negative cells disappeared during the observation period.

[0100] These results suggest that LGR5-positive, p27-positive cells function as dormant cancer stem cells resistant to chemotherapy agents, and contribute to the recurrence or recurrence of cancer after chemotherapy.

[0101] [Experimental Example 6] (The cell-extracellular matrix interface controlled the dormancy of cancer stem cells) The inventors found that when LGR5-positive, p27-positive cells were dissociated into single cells and passaged, they lost p27 expression and underwent cell division.

[0102] When dissociated into single cells, they are separated from the extracellular matrix and neighboring cells. Therefore, we investigated whether cell-extracellular matrix adhesion or cell-cell adhesion is necessary for the maintenance of LGR5-positive, p27-positive cells. Specifically, we sorted and recovered two types of cells, including LGR5-positive, p27-positive single cells and LGR5-positive, p27-positive cells that maintained cell-cell adhesion, and observed them. As a result, both types of cells underwent cell division in the same manner.

[0103] This result suggests that cell-extracellular matrix adhesion, rather than cell-cell adhesion, contributes to the maintenance of the dormant state.

[0104] FIG. 6 is a volcano plot showing the results of comparing the transcriptomes of LGR5-positive p27-positive cells and LGR5-positive p27-negative cells.

[0105] Among the genes whose expression levels were specifically elevated in LGR5-positive, p27-positive cells, the inventors focused on COL17A1, a structural component of hemidesmosomes that strengthens the adhesion between epithelial cells and basement membranes or Matrigel.

[0106] [Experimental Example 7] (COL17A1 maintained cancer stem cell dormancy) To investigate the functional role of COL17A1 in cancer stem cell dormancy, human colon cancer organoids (CCO7 strain and CCO32 strain) in which COL17A1 was knocked out using CRISPR-Cas9 were generated.

[0107] 7 shows fluorescence micrographs showing the expression of LGR5 and p27 in human colon cancer organoids expressing COL17A1 and in human colon cancer organoids in which COL17A1 has been knocked out. In FIG. 7, "COL17A1 WT" indicates a human colon cancer organoid expressing COL17A1, and "COL17A1 KO" indicates a human colon cancer organoid in which COL17A1 has been knocked out. The scale bar is 100 μm.

[0108] Interestingly, the results revealed that COL17A1 KO organoids maintained LGR5 expression, but lost dormant LGR5-positive, p27-positive cells.

[0109] It was also confirmed that p27-positive cells were restored when COL17A1 expression was rescued by doxycycline (Dox)-induced COL17A1 overexpression.

[0110] These results suggest that COL17A1 regulates the dormancy of cancer stem cells.

[0111] [Experimental Example 8] (Investigation of chemotherapy sensitivity of COL17A1 KO organoids) The chemotherapy sensitivity of COL17A1 KO human colon cancer organoids was investigated. The results revealed that COL17A1 KO organoids were more sensitive to camptothecin than COL17A1 WT organoids and COL17A1 single allele KO (heterozygous KO) organoids. It was suggested that the loss of LGR5-positive and p27-positive cells in COL17A1 KO organoids makes them vulnerable to chemotherapy.

[0112] Next, irinotecan was administered to mice xenografted with COL17A1 KO human colon cancer organoids, and tumor size was measured over time.

[0113] The upper panel of Figure 8 is a schematic diagram showing the schedule of this experiment. The lower panel of Figure 8 is a graph showing the change in tumor size over time. In Figure 8, "Vehicle" indicates a control administered with the solvent, and "IRI" indicates administration of irinotecan. Furthermore, "*" indicates a significant difference at p<0.05.

[0114] The results showed that tumor regeneration after irinotecan administration was suppressed in COL17A1 KO xenografts more than in COL17A1 WT xenografts, possibly reflecting the depletion of dormant cancer cells capable of regrowing after chemotherapy in COL17A1 KO xenografts.

[0115] In addition, a similar experiment was performed using colon cancer organoids derived from a different patient, and results similar to those of this experimental example were obtained.

[0116] These results suggest that COL17A1 prevents the proliferation of dormant cancer cells and that its loss increases the efficacy of chemotherapy.

[0117] [Experimental Example 9] (COL17A-FAK-YAP pathway controlled cancer stem cell dormancy) We analyzed how COL17A1 controls dormancy in human colon cancer organoids. We considered the possibility that COL17A1 loss disrupts hemidesmosome adhesion, and that focal adhesion signaling may be activated instead. Therefore, in COL17A1 KO organoids, we investigated the activation of vinculin and phosphorylated FAK, which are components of focal adhesion signaling. Ｙ３９７ The expression of (pFAK) and activation of YAP signaling downstream of FAK (nuclear translocation of YAP) were analyzed.

[0118] The left side of Figure 9 is a representative confocal microscope photograph showing the results of analyzing the expression of vinculin and pFAK, and the activation of YAP signaling. The scale bar is 100 μm. The right side of Figure 9 is a graph showing the results of measuring the expression of vinculin and pFAK, and the activation of YAP signaling, based on the photograph on the left side of Figure 9. In the right side of Figure 9, "N.S." indicates no significant difference.

[0119] As a result, in COL17A1 KO organoids, increased expression of both vinculin and pFAK was confirmed, suggesting activation of the focal adhesion signal. Activation of the downstream YAP signal was also observed.

[0120] Furthermore, in COL17A1 OE organoids, in which COL17A1 was forcibly expressed in COL17A1 KO organoids, the effects of increased expression of vinculin and pFAK were negated.

[0121] Gene set enrichment analysis revealed that YAP target genes were enriched in COL17A1 KO organoids compared with COL17A1 OE organoids, suggesting that COL17A1 deficiency induces FAK-YAP activation.

[0122] Next, to investigate whether FAK-YAP activation affects the dormant state of LGR5-positive p27-positive cells, we analyzed the YAP status of human colon cancer organoids.

[0123] The left side of Figure 10 shows representative confocal micrographs showing the results of analyzing the nuclear translocation of LGR5, p27, and YAP in camptothecin-treated human colon cancer organoids (CCO7 and CCO32 strains) and non-camptothecin-treated human colon cancer organoids. The scale bar is 100 μm. The right side of Figure 10 is a graph showing the results of measuring the percentage of cells containing nuclear-translocated YAP based on the photograph on the left side of Figure 10.

[0124] The results showed that the nuclear localization of YAP was lower in LGR5-positive p27-positive cells than in LGR5-positive p27-negative cells, suggesting that YAP signaling negatively regulates dormancy in LGR5-positive p27-positive cells. Furthermore, camptothecin treatment was suggested to activate YAP signaling in LGR5-positive p27-positive cells.

[0125] Experimental Example 10 (Study of YAP signaling inhibitor) A YAP signaling inhibitor was administered to COL17A1 KO organoids to examine its effect on cancer stem cell dormancy. The YAP signaling inhibitor used was MYF-01-37 (CAS number: 2416417-65-5), a small molecule TEAD inhibitor (TEADi) that disrupts the YAP / TAZ-TEAD protein-protein interaction (20 μM, 2 days).

[0126] The left side of Figure 11 shows representative confocal micrographs depicting the results of analyzing LGR5 and p27 expression in human colon cancer organoids (COL17A1 KO or COL17A1 WT) treated with a YAP signal inhibitor. The scale bar is 100 μm. The right side of Figure 11 shows a graph depicting the results of measuring the percentage of LGR5-positive, p27-positive cells based on the photograph on the left side of Figure 11.

[0127] The results showed that blocking YAP signaling in COL17A1 KO organoids resulted in the reappearance of p27-positive cells.

[0128] This result suggested that expression of COL17A1 inhibits activation of the FAK-YAP signal, thereby suppressing re-entry of p27-positive cells into the cell cycle.

[0129] [Experimental Example 11] (Chemotherapy suppressed COL17A1 expression) Live imaging confirmed that LGR5-positive, p27-positive cells exhibited resistance to chemotherapy, remained, and were reactivated after chemotherapy. However, it was unclear how chemotherapy released cancer stem cells from their dormant state.

[0130] Therefore, we investigated whether the COL17A1-FAK-YAP pathway is involved in releasing cancer stem cells from their dormant state after chemotherapy.

[0131] FIG. 12 is an image showing the results of a capillary-based immunoassay that detected COL17A1, pFAK, and total FAK in human colon cancer organoids (CCO7 and CCO32 lines) treated with camptothecin (50 nM).

[0132] The results showed that COL17A1 expression was lost and FAK phosphorylation occurred in human colon cancer organoids treated with camptothecin, indicating that the COL17A1-FAK-YAP pathway is involved in the release of cancer stem cells from their dormant state after chemotherapy.

[0133] To further clarify the role of FAK-YAP signaling in releasing cancer stem cells from dormancy after chemotherapy, FAK-YAP signaling was altered during and after camptothecin treatment.

[0134] Figure 13 shows representative confocal micrographs of human colon cancer organoids (CCO32 strain) treated with camptothecin (10 nM, 24 hours) in the presence or absence of a FAK inhibitor, followed by analysis of LGR5 and p27 expression. The scale bar is 100 μm. PF573228 (CAS number: 869288-64-2) was used as the FAK inhibitor (10 μM). In Figure 13, "FAKi" refers to the FAK inhibitor.

[0135] 14 is a graph showing the results of measuring the percentage of LGR5-positive, p27-positive cells in human colon cancer organoids treated with or without camptothecin for 5 days in the presence or absence of a FAK inhibitor. In FIG. 14, "N.S." indicates no significant difference.

[0136] The results showed that co-treatment of camptothecin with a FAK inhibitor inhibited cell cycle reentry of LGR5-positive, p27-positive cells after exposure to camptothecin. These results suggest that FAK inhibition suppresses the release of cancer stem cells from their dormant state after chemotherapy and prevents cancer recurrence or recurrence after chemotherapy.

[0137] [Experimental Example 12] (Study of YAP inhibitor 1) It was investigated whether YAP inhibition can counteract tumor regrowth after chemotherapy by preventing the release of dormancy from LGR5-positive, p27-positive cells.

[0138] The upper panel of Figure 15 is a schematic diagram showing the experimental schedule. Human colon cancer organoids with doxycycline-induced YAP / TAZ knockdown were prepared and subcutaneously xenografted into immunodeficient NOG mice. Irinotecan was then administered.

[0139] The lower panel of Figure 15 is a photomicrograph showing the results of immunostaining of the grafts from each group 3 days after administration of irinotecan. The scale bar is 100 µm. In the lower panel of Figure 15, "Control" indicates the results of the group that was not administered with irinotecan or doxycycline, "Dox" indicates the results of the group that was administered with doxycycline only, "Irinotecan" indicates the results of the group that was administered with irinotecan only, and "Irinotecan + Dox" indicates the results of the group that was administered with irinotecan and doxycycline.

[0140] As a result, administration of irinotecan was confirmed to induce nuclear translocation of YAP in vivo, but it was shown that nuclear translocation of YAP was suppressed in the group in which doxycycline was administered together with irinotecan to knockdown YAP / TAZ.

[0141] Figure 16 is a graph showing the change in tumor size over time in mice in each group. As a result, in Figure 16, "Control," "Dox," "Irinotecan," and "Irinotecan + Dox" have the same meanings as in the lower part of Figure 15. In addition, "*" indicates a significant difference at p<0.05.

[0142] The results showed that the therapeutic effect of irinotecan was transient, but that in the group in which irinotecan was administered together with doxycycline to knock down YAP / TAZ, the antitumor effect of irinotecan and the maintenance of the dormant state of cancer stem cells were enhanced.

[0143] [Experimental Example 13] (Study of YAP inhibitors 2) We investigated how inhibition of YAP signaling affects tumor regrowth after chemotherapy. The upper panel of Figure 17 is a schematic diagram showing the schedule of this experiment. Human colon cancer organoids with doxycycline-induced YAP / TAZ knockdown were prepared and xenografted subcutaneously into immunodeficient NOG mice. Subsequently, irinotecan was administered once a week for three doses, followed by long-term administration of doxycycline to inhibit YAP signaling. Furthermore, doxycycline administration was discontinued.

[0144] The lower panel of Figure 17 is a graph showing the change in tumor size over time in mice in each group. In the lower panel of Figure 17, "Irinotecan" indicates the results for the group administered with irinotecan alone, and "Irinotecan+Dox" indicates the results for the group administered with irinotecan and doxycycline.

[0145] As a result, in the group that did not receive doxycycline after administration of irinotecan, a transient therapeutic effect was observed, with cancer regrowth occurring from around day 40. Furthermore, administration of doxycycline after administration of irinotecan minimized tumor size. Furthermore, tumor regrowth was observed when doxycycline administration was discontinued.

[0146] [Experimental Example 14] (Study of YAP inhibitor 3) With a view to clinical application of YAP targeting therapy, the in vivo application of small molecule TEAD inhibitors (TEADi) as drugs to be used adjunctively in chemotherapy was studied.

[0147] The upper panel of Figure 18 is a schematic diagram showing the schedule of this experiment. Human colon cancer organoids (CCO32 strain) were xenografted subcutaneously into immunodeficient NOG mice. Subsequently, irinotecan was administered once a week for three doses, followed by long-term administration of a TEAD inhibitor to inhibit YAP signaling. Thereafter, administration of the TEAD inhibitor was discontinued. MYF-01-37 (CAS number: 2416417-65-5) was used as the TEAD inhibitor.

[0148] The lower panel of Figure 18 is a graph showing the change in tumor size over time in mice in each group. In the lower panel of Figure 18, "Vehicle" indicates the result of the group administered with only the solvent, "TEADi" indicates the result of the group administered with only the TEAD inhibitor, "IRI" indicates the result of the group administered with only irinotecan, and "IRI+TEADi" indicates the result of the group administered with both irinotecan and the TEAD inhibitor.

[0149] The results showed that the TEAD inhibitor alone did not have a tumor-suppressing effect, but administration of the TEAD inhibitor after irinotecan minimized tumor size. Furthermore, in the group that did not receive the TEAD inhibitor after irinotecan, a transient therapeutic effect was observed, with cancer regrowth occurring from around day 40.

[0150] [Proof of Concept] The upper panel of Figure 19 is a schematic diagram illustrating the mechanism by which dormant LGR5-positive, p27-positive cancer stem cells are released from their dormant state and tumors regrow, as revealed by the above experimental results. Chemotherapy kills cancer cells, but LGR5-positive, p27-positive cancer stem cells survive. In LGR5-positive, p27-positive cancer stem cells that survive chemotherapy, FAK-YAP signaling is activated, p27 expression becomes negative, and they regrow.

[0151] The lower panel of Figure 19 is a schematic diagram illustrating how COL17A1 maintains the dormant state of cancer stem cells, and how FAK-YAP signaling is activated after chemotherapy, resulting in the release of the dormant state.

[0152] According to the present invention, it is possible to provide a preventive or therapeutic agent for recurrence or recurrence of cancer after chemotherapy, and a screening method for the same.

Claims

1. A preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy, comprising a YAP inhibitor as an active ingredient.

2. The method according to claim 1 , wherein the cancer stem cells in a dormant state during chemotherapy are LGR5-positive, p27-positive cancer stem cells.

3. The preventive or therapeutic agent according to claim 1 or 2, wherein the relapse or recurrence of cancer is relapse or recurrence of cancer after chemotherapy.

4. The preventive or therapeutic agent according to claim 1 or 2, wherein the YAP inhibitor is an inhibitor of YAP signal activation.

5. A pharmaceutical composition for preventing or treating cancer recurrence or recurrence caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy, comprising the prophylactic or therapeutic agent according to claim 1 or 2 and a pharma- ceutical acceptable carrier.

6. A method for screening a preventive or therapeutic agent for relapse or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy, comprising: A step (a) of applying a chemotherapeutic agent to a cancer organoid, resulting in the death of most of the cells of the cancer organoid; (b) further incubating the cancer organoids, so that cells, including cancer stem cells, present in the cancer organoids and in a dormant state begin to proliferate; (c) evaluating the proliferation of cells containing cancer stem cells that were in a dormant state; Including, A screening method in which the steps (a) or (b) are carried out in the presence of a test substance, and the fact that the proliferation of cells containing cancer stem cells that were in a dormant state is inhibited compared to the absence of the test substance indicates that the test substance is an agent for preventing or treating the recurrence or recurrence of cancer caused by cells in cancer tissue that contain cancer stem cells that are in a dormant state during chemotherapy.

7. A method for screening a preventive or therapeutic agent for relapse or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy, comprising: A step (a) of applying a chemotherapeutic agent to a cancer organoid, resulting in the death of most of the cells of the cancer organoid; (b) further incubating the cancer organoids, so that cells, including cancer stem cells, present in the cancer organoids and in a dormant state begin to proliferate; (c) evaluating activation of YAP signaling in the cells containing the dormant cancer stem cells; Including, A screening method in which step (a) or (b) is performed in the presence of a test substance, and the activation of the YAP signal is suppressed compared to the absence of the test substance, indicating that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy.

8. A method for screening a preventive or therapeutic agent for relapse or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy, comprising: Incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance; Evaluating the proliferation of the LGR5-positive, p27-positive cancer stem cells; Including, A screening method in which the inhibition of proliferation of the LGR5-positive, p27-positive cancer stem cells compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy.

9. A method for screening a preventive or therapeutic agent for relapse or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy, comprising: Incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance; Evaluating activation of YAP signaling in the LGR5-positive, p27-positive cancer stem cells; Including, A screening method in which the activation of the YAP signal is suppressed compared to the absence of the test substance, indicating that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy.

10. A method for screening a preventive or therapeutic agent for relapse or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy, comprising: Incubating LGR5-positive, p27-positive cancer stem cells dissociated into single cells in the presence of a test substance; Evaluating the expression of COL17A1 in the LGR5-positive, p27-positive cancer stem cells; Including, A screening method in which an increase in the expression of COL17A1 compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy.

11. A method for screening a preventive or therapeutic agent for relapse or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are in a dormant state during chemotherapy, comprising: A step of exposing the cancer organoid to a chemotherapeutic agent in the presence of a test substance; Evaluating the expression of COL17A1 in the cancer organoid; Including, A screening method in which an increase in the expression of COL17A1 compared to the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for the recurrence or recurrence of cancer caused by cells, including cancer stem cells, in cancer tissue that are dormant during chemotherapy.