Composition for use in treatment of cancer, inflammatory diseases, or obesity, and use thereof

Abstract

The present invention addresses the problem of providing a drug for use in the treatment of triple-negative breast cancer, providing a blood marker for triple-negative breast cancer, and providing a drug for use in the treatment of inflammatory diseases or obesity. A medicinal composition for use in the treatment of triple-negative breast cancer, inflammatory diseases, or obesity can be produced by employing, as an active ingredient, an inhibitor of the ZCCHC24 protein or a drug for inhibiting the expression of the ZCCHC24 gene. The inhibitor of the ZCCHC24 protein or a drug for inhibiting the expression of the ZCCHC24 gene can be used in combination with a BET inhibitor.

Description

Compositions and uses thereof for treating cancer, inflammatory disease, or obesity

[0001] The present invention relates to a composition for use in the treatment of cancer, inflammatory diseases, or obesity. More specifically, the present invention relates to a composition for use in the treatment of cancer, inflammatory diseases, or obesity, particularly triple-negative breast cancer, comprising an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene as an active ingredient.

[0002] Breast cancer is one of the most common cancers in women, and triple-negative breast cancer (TNBC) accounts for approximately 20% of all breast cancers. Because of its biologically aggressive nature, extremely high recurrence rate within three years, and significantly shorter survival time after recurrence compared to other types, treatments have been sought for this type of cancer. Hormone therapy and molecular targeted drugs targeting HER2, which are used to treat other types of breast cancer, are ineffective, and there is a strong need to develop new treatments based on an understanding of the pathogenesis of TNBC.

[0003] What makes triple-negative breast cancer particularly difficult to treat is the existence of a cancer stem-like population. This cancer stem-like population is particularly drug-resistant and has high tumorigenic potential, resulting in treatment resistance. Previously, cell surface markers for this cancer stem-like population, such as CD44, ALDH1, NRP1, and CD133, have been reported. Furthermore, transcriptional regulators regulating this population, such as ZEB1, ID2, and NFκB, have been reported. Furthermore, in other cancer types, RNA-binding proteins such as Musashi-2 have been reported to regulate the expression of cancer stem cells in pancreatic cancer and brain tumors to maintain their stemness. However, the genetic network, including the RNA hierarchy, in the breast cancer stem cell-like population remained unclear.

[0004] PDL1 is one of the most important effector molecules in this breast cancer stem-like population. It has been reported recently that the expression of CD44, a particularly important breast cancer stem-like marker, is crucial for maintaining PDL1 expression, and that PDL1 expression is highly elevated in the cancer stem-like fraction. Clinical trials of immune checkpoint inhibitors targeting PDL1 and its T cell receptor, PD-1, have been conducted in triple-negative breast cancer. The preoperative treatment of TNBC with atezolibuumab, which targets PDL1, has been reported. However, functional analysis in breast cancer is still insufficient, and further evidence is urgently needed. In particular, deletions of the 3' untranslated region (3'UTR) have been observed in many tumors, and it has been revealed that this region is downregulated by the RNA-binding protein TTP. However, the RNA-level gene network that maintains PDL1 expression remains unclear.

[0005] WO2017 / 151554A1WO2020 / 104777A1

[0006] Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016;13(11):674-90.Marra A, Trapani D, Viale G, Criscitiello C, Curigliano G. Practical classification of triple-negative breast cancer: intratumoral heterogeneity, mechanisms of drug resistance, and novel therapies. NPJ Breast Cancer. 2020;6:54Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK, Lovgren K, et al. The CD44+ / CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res. 2008;10(3):R53.Liu TJ, Sun BC, Zhao XL, Zhao XM, Sun T, Gu Q, et al. CD133+ cells with cancer stem cell characteristics associates with vasculogenic mimicry in triple-negative breast cancer. Oncogene. 2013;32(5):544-53Ma F, Li H, Wang H, Shi X, Fan Y, Ding X, et al. Enriched CD44(+) / CD24(-) population drives the aggressive phenotypes presented in triple-negative breast cancer (TNBC). Cancer Lett.2014;353(2):153-9Tominaga K, Minato H, Murayama T, Sasahara A, Nishimura T, Kiyokawa E, et al. Semaphorin signaling via MICAL3 induces symmetric cell division to expand breast cancer stem-like cells. Proc Natl Acad Sci US A. 2019;116(2):625-30.

[0007] One of the objects of the present invention is to provide a composition for use in the treatment of cancer, inflammatory diseases, or obesity, particularly triple-negative breast cancer (TNBC).

[0008] The present inventors focused on PDL1 expression and performed an RNA-binding protein screening using reporter cells in which PDL1 protein expression was tagged with a HiBiT tag to identify RNA-binding proteins that regulate cancer stemness. As a result, the present inventors successfully identified the RNA-binding protein ZCCHC24, whose function is unknown. Furthermore, they found that ZCCHC24 strongly localizes in breast cancer stem-like fractions, broadly characterizing cancer stemness and contributing to tumorigenesis. They then successfully identified a TNBC therapeutic targeting ZCCHC24. In other words, the present inventors found that ZCCHC24 protein inhibitors or drugs that suppress ZCCHC24 gene expression could be used as therapeutic agents for TNBC, and identified a Wee1 / Chk1 inhibitor as a specific drug that suppresses ZCCHC24 gene expression. The present inventors also discovered a novel breast cancer stem cell fraction (hereinafter also referred to as "Stem II") that exhibits particularly high expression of ZCCHC24, and identified NRP1(+)NCAM1(-) as the cell surface marker combination that defines this Stem II cell fraction. Furthermore, they found that this Stem II population has high tumorigenicity and exhibits the characteristics of breast cancer stem cells, and that ZCCHC24 is important for maintaining the Stem II population and tumorigenicity. In other words, the present inventors have identified a novel cancer stem cell population (Stem II population) as a therapeutic target for the TNBC therapeutic drug of the present invention that targets ZCCHC24. Furthermore, the present inventors have found that inhibitors of the ZCCHC24 protein or drugs that suppress ZCCHC24 gene expression are effective in improving inflammatory diseases and obesity.

[0009] The present invention is based on these findings and includes the following aspects: [1] A composition for use in treating triple-negative breast cancer, for use in treating patients with an NRP1(+)NCAM1(-) cell fraction, comprising as an active ingredient a ZCCHC24 protein inhibitor or an agent that suppresses expression of the ZCCHC24 gene. [2] The composition for use in treating triple-negative breast cancer according to [1], wherein the agent that suppresses expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor. [3] The composition for use in treating triple-negative breast cancer according to [1] or [2], for use in combination with a BET inhibitor. [4] The composition for use in treating triple-negative breast cancer according to any one of [1] to [3], wherein the ZCCHC24 protein inhibitor is selected from the group consisting of a neutralizing antibody, a nucleic acid drug, and a small molecule compound. [5] Wee1 / Chk1 inhibitors include PD407824, AZD-1775, ZN-c3, Debio-0123, IMP-7068, GDC-0575, ESP-01, PNT-737, BEBT-260, AZD7762, LY2603618, MK-8776, CHIR-124, PF-477736, rosovitine, SNS-032, dinaciclib, flavopiridol, and AT7519. [6] The composition for use in treating triple-negative breast cancer according to any one of [1] to [5], further comprising a BET inhibitor. [7] The composition for use in treating triple-negative breast cancer according to [6], wherein the BET inhibitor is selected from the group consisting of JQ1, perabresive, BMS-986158, INCB-057643, ODM-207, PLX-2853, ABBV-744, BI-894999, BPI-23314, CC-90010, FT-1101, JAB-8263, mibebrane, SF-1126, and SYHA-1801.[8] Wee1 / Chk1 inhibitors include PD407824, AZD-1775, ZN-c3, Debio-0123, IMP-7068, GDC-0575, ESP-01, PNT-737, BEBT-260, AZD7762, LY2603618, MK-8776, CHIR-124, PF-477736, rosovitine, SNS-032, dinaciclib, and flavonoids. The composition for use in treating triple-negative breast cancer according to [5], wherein the agent that suppresses the expression of the ZCCHC24 gene is selected from the group consisting of piridol, AT7519, purvalanol A, RO-3306, SU9516, XL413, NU6027, P276-00, AZD5438, PHA-793887, JNJ-7706621, BMS-265246, MK-8776, and R547. [9] The composition for use in treating triple-negative breast cancer according to [1], wherein the agent that suppresses the expression of the ZCCHC24 gene is a nucleic acid medicine.

[10] The composition for use in treating triple-negative breast cancer according to [1], wherein the ZCCHC24 protein inhibitor comprises a 19-25 base miRNA having a sequence consisting of: (i) a seed sequence complementary to at least a portion of WGUWHWWA, (ii) an adenine or uracil base on the 5' side of the seed sequence, and (iii) a scrambled sequence on the 3' side of the seed sequence, and wherein the full-length GC content is 40-60%.

[11] A composition for use in treating triple-negative breast cancer, comprising as an active ingredient a ZCCHC24 protein inhibitor or a drug that suppresses expression of the ZCCHC24 gene (excluding mirciclib and adavosertib).

[12] A composition for use in treating triple-negative breast cancer, comprising as an active ingredient a ZCCHC24 protein inhibitor or a nucleic acid drug that suppresses expression of the ZCCHC24 gene.

[13] A composition for treating triple-negative breast cancer, which contains as an active ingredient an inhibitor of ZCCHC24 protein or a drug that suppresses expression of the ZCCHC24 gene, and is used in combination with a BET inhibitor.

[14] A composition for treating triple-negative breast cancer, comprising as an active ingredient an inhibitor of ZCCHC24 protein or a drug that suppresses expression of the ZCCHC24 gene, wherein the drug that suppresses expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor, The composition, wherein the Wee1 / Chk1 inhibitor is selected from the group consisting of PD407824, AZD-1775, ZN-c3, Debio-0123, IMP-7068, GDC-0575, ESP-01, PNT-737, BEBT-260, AZD7762, LY2603618, MK-8776, CHIR-124, PF-477736, rosovitine, SNS-032, dinaciclib, flavopiridol, AT7519, purvalanol A, RO-3306, SU9516, XL413, NU6027, P276-00, AZD5438, PHA-793887, JNJ-7706621, BMS-265246, MK-8776, and R547.

[15] A method for testing for breast cancer in a subject, comprising: a) measuring the amount of ZCCHC24 protein in a body fluid sample derived from the subject; and b) comparing the amount of ZCCHC24 protein with a predetermined standard value, wherein a finding that the ZCCHC24 protein amount is higher than the predetermined standard value indicates the possibility of breast cancer.

[16] A method for testing for breast cancer in a subject, comprising: determining whether a cell sample derived from the subject has an NRP1(+)NCAM1(-) cell fraction, wherein the presence of the subcellular fraction indicates the possibility of breast cancer.

[17] A method for testing the state of breast cancer in a subject, comprising: determining whether a cell sample derived from the subject has an NRP1(+)NCAM1(-) cell fraction, wherein the presence of the subcellular fraction indicates a high level of malignancy of breast cancer.

[18] A method for examining the state of breast cancer in a subject, comprising determining whether a cell sample derived from the subject has an NRP1(+)NCAM1(-) cell fraction, and identifying the subject as a candidate for administration of a composition for use in treating triple-negative breast cancer if the cell sample has the NRP1(+)NCAM1(-) cell fraction.

[19] A composition for treating an inflammatory disease, comprising as an active ingredient an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene.

[20] The composition for treating an inflammatory disease according to

[19] , wherein the agent that suppresses expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor.

[21] The composition for treating an inflammatory disease according to

[19] , wherein the agent that suppresses expression of the ZCCHC24 gene is a nucleic acid medicine.

[22] The composition for treating an inflammatory disease according to

[19] , wherein the inhibitor of ZCCHC24 protein comprises a 19-25 base miRNA having a sequence with a GC content of 40-60% over its entire length, the miRNA consisting of: (i) a seed sequence complementary to at least a portion of WGUWHWWA; (ii) an adenine or uracil base on the 5' side of the seed sequence; and (iii) a scrambled sequence on the 3' side of the seed sequence.

[23] A composition for treating obesity, comprising as an active ingredient an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene.

[24] The composition for treating obesity according to

[23] , wherein the agent that suppresses expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor.

[25] The composition for treating obesity according to

[23] , wherein the agent that suppresses expression of the ZCCHC24 gene is a nucleic acid medicine.

[26] The composition for treating obesity according to

[23] , wherein the inhibitor of ZCCHC24 protein comprises a 19-25 base miRNA having a sequence with a GC content of 40-60% over its entire length, the miRNA consisting of: (i) a seed sequence complementary to at least a portion of WGUWHWWA, (ii) an adenine or uracil base on the 5' side of the seed sequence, and (iii) a scrambled sequence on the 3' side of the seed sequence.

[27] A method for screening drugs for use in treating triple-negative breast cancer, inflammatory diseases, or obesity, comprising: (i) preparing cells that express ZCCHC24; (ii) contacting the cells with a test substance; (iii) measuring the expression level of ZCCHC24; and (iv) selecting a substance that reduces the expression level of ZCCHC24.

[28] A method for treating and / or preventing triple-negative breast cancer, an inflammatory disease, or obesity in a subject in need of treatment, comprising administering to the subject an effective amount of an inhibitor of the ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene.

[29] Use of an inhibitor of the ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene in the manufacture of a pharmaceutical for treating and / or preventing triple-negative breast cancer, an inflammatory disease, or obesity.

[0010] Reanalysis of human breast cancer scRNA-seq data identified ZCCHC24 as a novel RNA-binding protein with unknown function that is highly expressed in cell fractions exhibiting breast cancer stem cell-like expression. Genetic screening for RNA-binding proteins using PC9-KI cell lines carrying a HiBiT tag knocked into the C-terminus of PDL1 identified ZCCHC24 as a gene that upregulates PDL1 expression (Figures 2A and 2B). Western blotting confirmed this elevated expression (Figure 2C). Furthermore, comparison of multiple cell lines revealed high ZCCHC24 expression in the triple-negative breast cancer cell line MDAMB231 (Figure 2D). Furthermore, knockdown of ZCCHC24 reduced both PDL1 mRNA and protein expression (Figures 2E and 2F). Wu et al., EMBO J. 2020. Reanalysis of single-cell RNA-seq data from five human breast cancer specimens revealed that the cell fractions were divided into 23 clusters (Figure 3A). Violin plots revealed that ZCCHC24 was specifically expressed in cluster 9 (Figure 3B). Cluster 9 strongly expressed vimentin, which contributes to epithelial-mesenchymal transition (EMT) and cancer stem cell maintenance (Figure 3C). Cancer stem cell markers included CD44 high, CD24 negative, NRP1 positive, and ZEB1 positive, suggesting that ZCCHC24 is strongly expressed in the cancer stem cell-like fraction (Figure 3D). RNA-seq / qPCR analysis of the human breast cancer cell line MDAMB231 revealed that knockdown of ZCCHC24 significantly reduced the expression of genes characteristic of cancer stemness in breast cancer, particularly CD44, NRP1, and ZEB1. Furthermore, we confirmed by qPCR that knockdown of ZCCHC24 reduced the expression of CD44, NRP1, and ZEB1 in human breast cancer patient-derived cell lines (PDX). RNA-Seq analysis of ZCCHC24-knockdown cells revealed significantly decreased expression of genes that characterize cancer stemness in breast cancer, PDL1, and cytokines and chemokines important for cancer survival (Figure 5A).FACS analysis of MDAMB231 revealed that ZCCHC24 knockdown reduced the percentage of breast cancer stem cells, defined as CD44-positive and NRP1-positive (Figures 5B and 5C). Furthermore, in another triple-negative breast cancer cell line, HCC38, ZCCHC24 knockdown reduced the expression of cancer stem cell-like cell surface markers, such as CD44 and NRP1, at the mRNA level (Figure 5D). FACS analysis also revealed a significant decrease in the CD44-positive and NRP1-positive cell fractions (Figures 5E and 5F). Merging the eCLIP-Seq results from ZCCHC24-overexpressing cells with the RNA-Seq DEGs identified 364 genes as target genes whose expression is regulated by direct binding of ZCCHC24 (Figure 6A). Classification of the binding regions of these target genes by RSeqC revealed that ZCCHC24 binds to the 3'UTR and CDS regions (Figure 6B). Peak pattern analysis of these target genes revealed that ZCCHC24 directly binds to the 3'UTR of genes such as PDL1, IL6, IL8, CXCL1, CD44, and NRP1 (Figure 6C). GO term and pathway analysis of these target genes revealed that ZCCHC24 regulates pathways such as negative regulation of apoptosis and cell proliferation, as well as focal adhesion and actin cytoskeleton regulation (Figures 6D and 6E). PAR-CLIP analysis of MDAMB231 revealed that ZCCHC24 recognizes a novel, unique motif sequence, (A / U)GU(A / U)U(A / U)U, and binds to the 3'UTR of the mRNAs of genes characteristic of cancer stemness, such as ZEB1, CD44, and NRP1. BRIC-Seq analysis of MDAMB231 showed that knockdown of ZCCHC24 tended to significantly reduce the RNA stability of genes that characterize breast cancer stemness, such as ZEB1, CD44, and NRP1.Actinomycin D assays revealed that ZCCHC24 knockdown reduced the mRNA stability of target genes, including CD274 (PDL1), CXCL8 (IL8), IL6, CD44, NRP1, and ZEB1, in MDAMB231 (Figure 9A). It also reduced the mRNA stability of CD44 and NRP1 in HCC38 (Figure 9B). Sphere formation assays using MDAMB231 demonstrated significant reductions in sphere formation ability with ZCCHC24 knockdown (Figures 10A and 10B). Extreme dilution assays (ELDAs) for MDAMB231 and HCC38 demonstrated a tendency for ZCCHC24 knockdown to decrease the number of breast cancer stem cells per cell count. In vivo tumor formation assays in subcutaneously transplanted MDAMB231 and PDX models revealed a tendency for ZCCHC24 knockdown to significantly reduce tumorigenicity. Single-cell RNA-seq analysis of subcutaneously implanted PDX models revealed that ZCCHC24 knockdown significantly reduced the stem cell-like population, which exhibited cancer stem cell-like expression of NRP1(+)ZEB1(+)CD24(-)EPCAM(-). Furthermore, among the two major cell populations, the Stem II cell fraction, which expresses ZCCHC24 as well as genes such as ZEB1 and NRP1, was significantly reduced (Figure 13A). Furthermore, scRNA analysis of subcutaneously implanted NOG mice with patient-derived breast cancer specimens also confirmed that the NRP1(+)ZEB1(+)CD24(-)EPCAM(-) fraction was largely separated into two, and that ZCCHC24 was specifically expressed in the NRP1(+)ZEB1(+)NCAM1(-) fraction (Stem II fraction) (Figure 13B). Single-cell RNA-seq analysis of the PDX subcutaneous transplant model confirmed that ZCCHC24 was specifically expressed in the NRP1(+)NCAM1(-) Stem II cell fraction. The same samples were sorted by FACS (Stem I cell fraction: NRP1(+)NCAM1(+), Stem II cell fraction: NRP1(+)NCAM1(-)).The recovered Stem I cell fraction (NRP1(+)NCAM1(+)), Stem II cell fraction (NRP1(+)NCAM1(-)), and their mixture (Stem I + Stem II) were reinjected into mice. The Stem II cell fraction exhibited higher tumorigenicity than the Stem I cell fraction and the Stem I + II cell fraction. Reanalysis of tumor samples formed by reinjection of Stem I, Stem II, and Stem I + Stem II cell fractions using scRNAseq revealed that the tumors were fractionated into ECAM-high (Stem I), ECAM-low (Stem II), and epithelial, similar to the tumors before reinjection. Compound screening using reporter cells labeled with a HiBiT tag inserted at the C-terminus of ZCCHC24 identified 11 upregulators (compounds with a Z-score > 3.0) and 9 downregulators (compounds with a Z-score < -3.0) (Figures 17A and 17B). BET inhibitors and topoisomerase inhibitors were specifically identified as up-regulators (Fig. 17C), while microtubule polymerization inhibitors and Wee1 / Chk1 inhibitors, which are important in the DNA damage response pathway, were identified as down-regulators (Fig. 17D). EC20 values ​​of the BET inhibitor JQ1 relative to the maximum increase in ZCCHC24 expression. 50 The IC50 of MDAMB231 for the maximum inhibition of cell viability by JQ1 was approximately 50 nM, and it was found to increase ZCCHC24 expression (Fig. 18A). 50 The concentration of ZEB1 was approximately 0.1 μM (Fig. 18B). Regarding the transcription factor ZEB1, a database of histone marker ChIP and previous ChIP-Seq data using ZEB1 revealed a region between the first and second exons where histone markers H3K4Me1 and H3K27Ac showed peaks, and ZEB1 peaks were also observed (Fig. 18C). Furthermore, administration of JQ1 dramatically increased ZEB1 expression (Fig. 18D). Furthermore, the increase in ZCCHC24 expression induced by JQ1 was significantly reduced by ZEB1 knockdown (Fig. 18E). The EC value of the Wee1 / Chk1 inhibitor PD407824 for maximum suppression of ZCCHC24 expression was 0.1 μM. 50The IC50 concentration was approximately 50 nM (Fig. 19A). On the other hand, although PD407824 inhibited cell activity to a certain extent, it did not exceed the IC50 concentration. 50 The suppression of cell activity was not significant enough to allow calculation of the α-tocopherol level (Figure 19B). Furthermore, when JQ1 and PD407824 were administered separately, JQ1 administration did not result in a decrease in the cancer stem cell-like population, whereas PD407824 administration and the combined administration of JQ1 and PD407824 tended to decrease the cell population (Figures 19C and 19D). Furthermore, when the inhibitory effect of 100 nM JQ1 and 1 μM PD407824 on cell proliferation was examined, an additive effect was confirmed (Figure 19E). Intraperitoneal injection of LPS into ZCCHC24 knockout mice demonstrated a prolonged survival time after LPS injection compared with wild-type mice. These findings suggest that ZCCHC24 plays an important role as an RNA-binding protein regulating inflammation. When ZCCHC24 knockout mice were fed a high-fat diet (HFD), weight gain was significantly suppressed compared with wild-type mice. This suggests that ZCCHC24 may also play an important role in obesity. The structures of miRNAs (miR-PBE1 and miR-PBE2) with sequences complementary to at least a portion of the ZCCHC24 protein binding target sequence are shown. Transfection of miR-PBE1 and / or miR-PBE2 into the MDAMB231 cell line suppressed the expression of genes involved in breast cancer stemness and cancer cell invasion, such as ZEB1, IL-6, ITGB1, and THBS1. Transfection of TNBC patient-derived cells (PDX) with miR-PBE1 and / or miR-PBE2 suppressed the expression of genes involved in breast cancer stemness and cancer cell invasion, such as IL-6, CD44, ITGB1, and NRP1. Transfection of miR-PBE1 into the human chondrocyte cell line SW1353 suppressed the expression of MMP13 and IL1-β, which are important effector molecules in knee osteoarthritis.

[0011] As described above, the present inventors have discovered that inhibitors of the ZCCHC24 protein or agents that suppress expression of the ZCCHC24 gene can be used as therapeutic agents for TNBC, inflammatory diseases, or obesity, and have identified a Wee1 / Chk1 inhibitor as a specific agent that suppresses expression of the ZCCHC24 gene. The present invention is described in detail below.

[0012] Triple-negative breast cancer (TNBC) Triple-negative breast cancer (TNBC) is a type of breast cancer characterized by estrogen receptor-negative, progesterone receptor-negative, and HER2 (human epidermal growth factor receptor 2)-negative mutations. TNBC accounts for approximately 20% of all breast cancers, has a very high recurrence rate within three years, and has a shorter survival time after recurrence than other types of breast cancer. Furthermore, hormone therapy and the molecular targeted drug Herceptin therapy, which are commonly used in breast cancer treatment, are ineffective, meaning that the only drugs that can be expected to be effective are anticancer drugs. As a result, treatment is often difficult, and many patients suffer from the side effects of anticancer drugs.

[0013] Composition for use in the treatment of TNBC One aspect of the present invention relates to a composition for use in the treatment and / or prevention of triple-negative breast cancer, which contains as an active ingredient an inhibitor of ZCCHC24 protein or a drug that suppresses expression of the ZCCHC24 gene.

[0014] ZCCHC24 (Zinc Finger CCHC-Type Containing 24) is a protein with two distinct zinc finger domains. We have discovered that (1) ZCCHC24 is an RNA-binding protein that is strongly expressed in cancer tissues and cells, particularly in breast cancer stem cells; (2) ZCCHC24 promotes tumorigenicity by upregulating the expression of a series of cancer-promoting proteins through post-transcriptional regulation; (3) reducing ZCCHC24 expression suppresses cancer growth and tumorigenicity; and (4) ZCCHC24 is a therapeutic target for intractable breast cancers, such as triple-negative breast cancer.

[0015] <Drugs that inhibit the function of ZCCHC24 protein> Therefore, for example, drugs that inhibit the function of ZCCHC24 protein (hereinafter also referred to as ZCCHC24 inhibitors or ZCCHC24 protein inhibitors) can be used to treat and / or prevent triple-negative breast cancer. ZCCHC24 inhibitors include, but are not limited to, nucleic acid drugs such as neutralizing antibodies, aptamers, miRNAs, antimiRs, and antagomiRs, as well as small molecule compounds. Note that, in this specification, the term "nucleic acid drug" with respect to drugs that inhibit the function of ZCCHC24 protein and drugs that suppress the expression of the ZCCHC24 gene described below includes nucleic acid molecules containing any modified base. Modified nucleic acids include, but are not limited to, 2'-MOE, 2'-O-MCE, LNA (2'-4'BNA), ENA, AmNA, GuNA, scpBNA, 2'-OMe, and phosphorothioate modifications.

[0016] Neutralizing antibodies Neutralizing antibodies may be polyclonal or monoclonal. They can be produced by any method known to those skilled in the art. For example, the entire ZCCHC24 protein or a fragment thereof can be used as the antigen. Antibody production can be outsourced to a contract manufacturer such as Eurofins Genomics, Inc. (Tokyo). The mammal from which the antibody is derived is not particularly limited, and human, mouse, rat, rabbit, sheep, camel, and other antibodies can be used. When used in humans, the antibody can be human, humanized, or chimeric, but human antibodies are preferred. Neutralizing antibodies may also include antibody fragments. Therefore, neutralizing antibodies may also include, for example, scFv, Fab, Fab', F(ab')2, Fv, rIgG, nanobodies, peptibodies, minibodies, and diabodies. Neutralizing antibodies may also include ZCCHC24-binding peptides obtained by phage display or other methods.

[0017] Aptamers are nucleic acid molecules (generally RNA) that form a three-dimensional structure that binds to a target. Aptamers may be used as ZCCHC24 inhibitors. Aptamers that bind to ZCCHC24 can be obtained by any method known to those skilled in the art, for example, by the SELEX method.

[0018] Novel miRNA: As the miRNA, a novel miRNA that targets the target gene of ZCCHCH24 can be used. An example of the miRNA according to this embodiment will be described below.

[0019] MicroRNAs (miRNAs) are short, non-coding RNAs (20-24 nucleotides) present in living organisms that are involved in the post-transcriptional regulation of gene expression in multicellular organisms by affecting both mRNA stability and translation. In vivo, miRNAs are incorporated into the RNA-induced silencing complex (RISC), where they recognize target mRNAs through imperfect base pairing, resulting in translational inhibition or destabilization of the target mRNA.

[0020] As shown in the Examples below, the present inventors have for the first time identified a novel target motif sequence, "WGUWHWWA" (*1 in Table 1), as the target sequence for ZCCHC24 to bind to the 3'UTR of mRNA of a group of genes that characterize cancer stemness. The target motif sequence can be, for example, "UGUAHAWA" (*2 in Table 1) or "WGUWUWUA (i.e., (A / U)GU(A / U)U(A / U)UA) (*3 in Table 1)" (in the sequence, W represents A or U, and H represents A, C, or U).

[0021] The novel miRNA of this embodiment recognizes the target sequence of ZCCHC24 as a target. As a result, the miRNA of this embodiment binds competitively with ZCCHCH24 to the target gene of ZCCHC24, thereby inhibiting the function of the ZCCHC24 protein, i.e., suppressing the effect of ZCCHC24 in promoting target gene expression. At the same time, the miRNA of this embodiment can inhibit or destabilize the translation of the mRNA of the target gene through its original miRNA function. Due to these dual effects, the miRNA of this embodiment can exert excellent effects as a ZCCHC24 inhibitor that suppresses the expression of the target gene of ZCCHC24.

[0022] As an example, the miRNA of this embodiment can be designed as an RNA molecule having a sequence consisting of: (i) a seed sequence that recognizes the target sequence of ZCCHCH24; (ii) an adenine or uracil base on the 5' side of the seed sequence; and (iii) a scrambled sequence on the 3' side of the seed sequence.

[0023] The seed sequence can be configured to be complementary to at least a portion of the sequence of WGUWHWWA (*1 in Table 1), the target sequence of ZCCHCH24 (also referred to herein as the "seed target sequence"). The base length of the seed sequence is generally 5 or more bases, preferably 6 or more bases, and more preferably 7 or more bases. For example, the seed sequence can be 5 to 7 bases long. The scrambled sequence may be any random sequence, but can be configured so that the GC content of the full-length miRNA is 40 to 60%, preferably 45 to 55%, and more preferably 48 to 52%. It is also preferable that the scrambled sequence be configured so as not to form complementary base pairs with transcription products other than the target mRNA. The miRNA can have a total length of 19 to 25 bases, preferably 20 to 24 bases, and more preferably 21 to 23 bases. Specific examples of miRNAs according to this embodiment are described in detail in the Examples section. Furthermore, the adenine at the 5' end of the seed sequence in the sequences described in the Examples section below may be substituted with uracil. By configuring the miRNA as described above, it is possible to improve the single-stranded nature of the miRNA, the formation of the RISC complex, the mRNA recognition ability, and the like, and thereby improve the action of the miRNA.

[0024] Small molecular weight compounds may be used as ZCCHC24 inhibitors. Small molecules that bind to ZCCHC24 can be designed by any method known to those skilled in the art, for example, by computer simulation based on the crystal structure of ZCCHC24.

[0025] <Drugs that suppress expression of the ZCCHC24 gene> Similarly to ZCCHC24 inhibitors, drugs that suppress expression of the ZCCHC24 gene (hereinafter also referred to as ZCCHC24 expression inhibitors) can be used to treat and / or prevent triple-negative breast cancer. ZCCHC24 expression inhibitors include, but are not limited to, small molecular weight compounds, peptides, proteins, antisense nucleic acids, siRNAs, miRNAs, antimiRs, antagomiRs, and other nucleic acid drugs.

[0026] Suppression of ZCCHC24 gene expression can be achieved at any level, but is not limited to, suppression of mRNA transcription, degradation of transcribed mRNA, or inhibition of mRNA-to-protein translation. Suppression of ZCCHC24 gene expression can be evaluated by measuring the amount of ZCCHC24 mRNA or protein using known methods.

[0027] Substances that can be used as ZCCHC24 expression inhibitors can be identified, for example, by the screening methods described herein. Furthermore, nucleic acid drugs such as antisense nucleic acids, siRNAs, miRNAs, antimiRs, and antagomiRs that can be used as ZCCHC24 expression inhibitors can be designed based on sequences complementary to portions of the ZCCHC24 gene sequence. The miRNA of this embodiment can be any miRNA that targets ZCCHCH24, i.e., any miRNA that inhibits and / or destabilizes the translation of ZCCHCH24 mRNA.

[0028] The present inventors have identified a Wee1 / Chk1 inhibitor as one of the compounds that reduces the expression of ZCCHC24. As used herein, the term "Wee1 / Chk1 inhibitor" refers to a substance that inhibits at least one of Wee1 and Chk1, but a substance that inhibits both Wee1 and Chk1 is also effective.

[0029] Examples of Wee1 inhibitors include PD407824, AZD-1775, ZN-c3, Debio-0123, IMP-7068, GDC-0575 (i.e., (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)cyclopropanecarboxamide (CAS No. 1196541-47-5)), MKK-1775 (i.e., 1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-1-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one (CAS No. 1196541-47-5), also commonly known as adavosertib), and No. 955365-80-7) can be used, but is not limited to these.

[0030] Examples of Chk1 inhibitors include PD407824, ESP-01, PNT-737, BEBT-260, AZD7762, LY2603618, MK-8776, CHIR-124, PF-477736, rosovitine, SNS-032, dinaciclib, flavopiridol, AT7519, purvalanol A, RO-3306, SU9516, XL413, NU6027, P276-00, AZD5438, and P Examples of Chk1 inhibitors that may be used include, but are not limited to, HA-793887, JNJ-7706621, BMS-265246, mirciclib, MK-8776, R547, or MKK-1775 (i.e., 1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-1-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one (CAS No. 955365-80-7), also commonly known as adavosertib. In one embodiment, Chk1 inhibitors exclude mirciclib and adavosertib.

[0031] WO2017 / 151554A1 exemplifies WEE1 / CHK1 inhibitors as examples of DNA damaging agents, and WO2020 / 104777A1 exemplifies WEE1 / CHK1 inhibitors as examples of DNA damage response inhibitors. These documents are incorporated herein by reference in their entireties.

[0032] BET (bromodomain and extraterminal domain) proteins are proteins that recognize acetylated histones and regulate gene transcription through the recruitment of transcription factors. Inhibition of BET proteins has been shown to have antitumor activity.

[0033] The inventors found that ZCCHC24 expression is dramatically increased by the action of BET inhibitors, which are expected to be novel therapeutic agents for triple-negative breast cancer. This indicates that ZCCHC24 is involved in the mechanism of resistance to BET inhibitor treatment. For example, Wee1 / Chk1 inhibitors are compounds that reduce ZCCHC24 expression, and their combination with existing BET inhibitors is possible for the treatment of refractory breast cancer.

[0034] Thus, in some embodiments, a composition for use in the treatment and / or prevention of triple-negative breast cancer, which contains as an active ingredient an inhibitor of the ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene, can be used in combination with a BET inhibitor. Also, in some embodiments, a composition for use in the treatment and / or prevention of triple-negative breast cancer, which contains as an active ingredient an inhibitor of the ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene, further comprises a BET inhibitor.

[0035] Examples of BET inhibitors that can be used include, but are not limited to, JQ1, pelabresib, BMS-986158, INCB-057643, ODM-207, PLX-2853, ABBV-744, BI-894999, BPI-23314, CC-90010, FT-1101, JAB-8263, mibebresib, SF-1126, or SYHA-1801.

[0036] Composition for treating patients with a Stem II cell fraction. As described above, the present inventors have also discovered a new breast cancer stem cell fraction, Stem II, which specifically expresses high levels of ZCCHC24, and identified NRP1(+)NCAM1(-) as a combination of cell surface markers that can identify this Stem II. Therefore, in some embodiments, the therapeutic target of the composition for use in treating and / or preventing triple-negative breast cancer of the present invention is a patient with a Stem II stem cell fraction identified as NRP1(+)NCAM1(-). That is, in some embodiments, the present invention relates to a composition for treating triple-negative breast cancer, for use in treating patients with an NRP1(+)NCAM1(-) cell fraction. Note that, in this specification, with respect to surface antigens, "(+)" and "positive" are synonymous and mean positive for the presence of the surface antigen. Similarly, "(-)" and "negative" mean negative for the presence of the surface antigen.

[0037] Composition for use in the treatment of inflammatory diseases The discoveries and experimental results by the inventors indicate that ZCCHC24 is a therapeutic target molecule for the treatment of a wide range of inflammatory diseases, not limited to triple-negative breast cancer, as it is a molecule that integrates and controls inflammatory cytokines such as PDL1, a costimulatory inhibitor, and IL-6, IL-8, and CXCL1 at the RNA level.

[0038] Therefore, one aspect of the present invention relates to a composition for use in the treatment and / or prevention of inflammatory diseases, comprising as an active ingredient an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene. The agent that suppresses expression of the ZCCHC24 gene may be, for example, a Wee1 / Chk1 inhibitor. Furthermore, the descriptions herein regarding compositions for treating triple-negative breast cancer also apply equally to compositions for use in the treatment and / or prevention of inflammatory diseases.

[0039] Inflammatory diseases include systemic inflammatory response syndrome (SIRS) including sepsis, rheumatism (involving TNF and IL6), psoriatic arthritis (involving TNF, IL17, and IL12 / 23), psoriasis (involving TNF, IL17, and IL12 / 23), ankylosing spondylitis (involving TNF and IL17), inflammatory bowel disease (involving TNF), SLE, atopic dermatitis (involving IL4 / IL13), asthma (involving IL4 / IL13 and IL5), COPD (involving IL5), gout (involving IL1), and knee osteoarthritis (involving MMP13 and IL1-β). Therefore, the composition of the present invention can be effectively used in the treatment and / or prevention of these diseases.

[0040] Composition for use in the treatment of obesity It has been revealed that inflammatory cytokines and chemokines such as IL-6, IL-8, CXCL1, and TNFα play an important role in the pathogenesis of obesity in inflammatory cells such as macrophages that infiltrate adipose tissue. The findings and experimental results of the present inventors indicate that ZCCHC24, as a molecule that broadly regulates these inflammatory cytokines and chemokines at the RNA level, is a therapeutic target molecule for the treatment of obesity.

[0041] Therefore, one aspect of the present invention relates to a composition for use in the treatment and / or prevention of obesity, which contains as an active ingredient an inhibitor of the ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene. The agent that suppresses expression of the ZCCHC24 gene may be, for example, a Wee1 / Chk1 inhibitor. Furthermore, the descriptions herein regarding compositions for treating triple-negative breast cancer also apply equally to compositions for use in the treatment and / or prevention of obesity.

[0042] Obesity is generally defined as "a state in which excess fat accumulates in adipose tissue, and the body mass index (BMI) is calculated by dividing body weight (kg) by height (m)." 2Obesity is defined as a condition in which the mean (weight) of obesity is 25 or higher. Obesity is primarily caused by an imbalance between energy intake and expenditure (an increase in the ratio of intake to expenditure) in physical activity and daily life. Therefore, obesity includes, for example, food-induced insulin resistance and / or weight gain.

[0043] As used herein, treatment and / or prevention of obesity includes preventing an increase in body weight and / or fat mass, as well as reducing fat mass and / or body weight.

[0044] Furthermore, the composition for use in the treatment and / or prevention of obesity according to this embodiment may also be effective in treating and / or preventing diseases, conditions, and complications caused by and / or associated with obesity. Diseases and conditions caused by and / or associated with overweight due to obesity include sleep apnea syndrome and orthopedic musculoskeletal disorders. Diseases, conditions, and complications caused by and / or associated with metabolic disorders due to obesity include impaired glucose tolerance / diabetes, dyslipidemia, fatty liver, hyperuricemia / gout, cardiovascular disease / cerebrovascular disease (atherosclerosis), non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity-related kidney disease, diabetic nephropathy, diabetic retinopathy, diabetic vasculopathy, diabetic neuropathy, hyperleptinemia, renal steatosis, pancreatic steatosis, cardiac steatosis, steatohepatitis, fibrosis, cirrhosis, chronic low-grade inflammation, hypertension, cardiovascular disease, and other obesity-related inflammatory conditions and diseases.

[0045] Pharmaceutical Composition One aspect of the present invention relates to a pharmaceutical composition for use in the treatment and / or prevention of triple-negative breast cancer, inflammatory diseases, or obesity, which comprises as an active ingredient an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene. That is, one aspect of the present invention relates to the use of an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene in the manufacture of a medicament for the treatment or prevention of triple-negative breast cancer, inflammatory diseases, or obesity. Furthermore, in some embodiments, a pharmaceutical composition for use in the treatment of triple-negative breast cancer, which comprises as an active ingredient an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene, can further comprise a BET inhibitor.

[0046] The content of the ZCCHC24 protein inhibitor or the agent that suppresses ZCCHC24 gene expression in a 100% by weight pharmaceutical composition can be appropriately set within the range of 0.001 to 99.99% by weight. The content of the BET inhibitor can also be appropriately set within the range of 0 to 99.99% by weight. Other components in the pharmaceutical composition of the present invention are not particularly limited and can be selected appropriately depending on the purpose, and include, for example, pharmaceutically acceptable carriers or additives. The carriers or additives are not particularly limited and can be selected appropriately depending on, for example, the dosage form, and may include any carrier, buffer, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener. The content in the pharmaceutical composition of the present invention is also not particularly limited and can be selected appropriately depending on the purpose. Therefore, in some embodiments, the pharmaceutical composition can comprise or consist of a ZCCHC24 protein inhibitor or an agent that suppresses ZCCHC24 gene expression and a carrier or buffer.

[0047] The dosage form of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the desired administration method. Examples include injections (solutions, suspensions, solid preparations for dissolution before use, etc.) and solid preparations (tablets, capsules, suppositories, powders, etc.). For example, injections for subcutaneous, intramuscular, or intravenous use can be prepared by adding a pH adjuster, buffer, stabilizer, isotonicity agent, local anesthetic, etc. to the composition and using conventional methods. Examples of pH adjusters and buffers include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of isotonicity agents include sodium chloride and glucose. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Solid preparations may be enteric-coated.

[0048] The method of administration of the pharmaceutical composition according to the present invention is not particularly limited, and either local administration or systemic administration can be selected depending on, for example, the dosage form of the pharmaceutical composition, the condition of the patient, etc. Administration can be performed, for example, by intravenous administration, subcutaneous administration, intramuscular administration, oral administration, enteral administration, enema administration, enteral nutrition, etc. Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The insertion site is not limited to the intestine, and examples include the esophagus, stomach, small intestine, large intestine, etc.

[0049] The recipient of the pharmaceutical composition of the present invention is not particularly limited and can be selected appropriately depending on the purpose. Examples include humans and non-human mammals, such as mice, rats, cows, pigs, monkeys, dogs, and cats. However, humans are preferred, particularly human patients with triple-negative breast cancer, inflammatory diseases, or obesity. The pharmaceutical composition of the present invention may also be administered for the purpose of preventing the onset of triple-negative breast cancer, inflammatory diseases, or obesity, particularly for the purpose of preventing recurrence. In some embodiments, the recipient may be a human with stem cell fraction Stem II, identified as NRP1(+)NCAM1(-).

[0050] The dosage of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the dosage form, the age and body weight of the subject, the degree of desired effect, etc. The dosage of the ZCCHC24 protein inhibitor or the agent that suppresses the expression of the ZCCHC24 gene can be, for example, 100 to 1,000,000 nmol, preferably 150 to 100,000 nmol per day, and the administration frequency can be, for example, 1 to 100 times per month.

[0051] The timing of administration of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the purpose, and for example, it may be administered prophylactically to patients susceptible to the above-mentioned diseases, or may be administered therapeutically to patients exhibiting symptoms. The number of administrations is also not particularly limited and can be appropriately selected depending on the age, body weight, desired degree of effect, etc. of the subject to be administered.

[0052] One aspect of the present invention relates to a method for treating triple-negative breast cancer, inflammatory disease, or obesity in a subject in need of such treatment and / or prevention, comprising administering to the subject an effective amount of an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene. The subject in need of such treatment and / or prevention is a mammal, for example, a human. The dosage can be determined appropriately depending on the type of agent used and the subject to which it is administered. The route of administration can also be determined appropriately depending on the type of agent used and the subject to which it is administered. A preferred route of administration is, for example, intravenous administration.

[0053] Some embodiments also relate to a method for treating triple-negative breast cancer, an inflammatory disease, or obesity in a subject in need thereof, comprising administering to the subject (i) an effective amount of an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene, and (ii) an effective amount of a BET inhibitor. The administration of the inhibitor of ZCCHC24 protein or the agent that suppresses expression of the ZCCHC24 gene and the administration of the BET inhibitor may be performed simultaneously or at different times. Furthermore, the number of administrations within a certain period of time may differ between the administration of the inhibitor of ZCCHC24 protein or the agent that suppresses expression of the ZCCHC24 gene and the administration of the BET inhibitor.

[0054] One aspect of the present invention relates to a method for detecting breast cancer. As described above, the present inventors have found that ZCCHC24 is an RNA-binding protein that is strongly expressed in cancer tissues and cells, particularly in breast cancer stem cells.

[0055] Thus, one embodiment of the present invention relates to a method for testing for breast cancer in a subject, comprising: a) measuring the amount of ZCCHC24 protein in a body fluid sample derived from the subject; and b) comparing the amount of ZCCHC24 protein with a predetermined standard value, wherein a higher amount of ZCCHC24 protein than the predetermined standard value indicates the possibility of breast cancer. That is, one aspect of the present invention relates to the use of ZCCHC24 protein as a blood marker for breast cancer. Another aspect of the present invention can also be said to relate to an in vitro method for testing for breast cancer, a method for measuring a blood marker for breast cancer, or a method for obtaining an indicator of the possibility of breast cancer.

[0056] Measurement of the amount of ZCCHC24 protein can be performed in vitro, for example, but not limited to, by ELISA. The body fluid sample can be, for example, an isolated blood sample, such as a plasma or serum sample.

[0057] The predetermined reference value to be compared with the amount of ZCCHC24 protein in the blood sample derived from the subject can be determined, for example, based on the average amount of ZCCHC24 protein in body fluid samples derived from multiple healthy individuals.

[0058] The method for measuring the amount of ZCCHC24 protein is not particularly limited, but examples include immunostaining.

[0059] In some embodiments, the breast cancer being tested for may specifically be triple-negative breast cancer.

[0060] Furthermore, as described above, the present inventors discovered a new breast cancer stem cell fraction, Stem II, in which ZCCHC24 is specifically and highly expressed, and identified NRP1(+)NCAM1(-) as a surface antibody combination that can identify this Stem II.

[0061] Thus, one aspect of the present invention relates to a method for testing for breast cancer in a subject, which comprises determining whether a cell sample derived from the subject has an NRP1(+)NCAM1(-) cell fraction, wherein the presence of the NRP1(+)NCAM1(-) cell fraction indicates the possibility of breast cancer. That is, one aspect of the present invention relates to the use of the combination of cell surface markers NRP1(+)NCAM1(-) as a testing marker for breast cancer.

[0062] One aspect of the present invention relates to a method for examining the state of breast cancer in a subject, which comprises determining whether a cell sample derived from the subject has an NRP1(+)NCAM1(-) cell fraction, and determining that the breast cancer is highly malignant when the cell sample has the NRP1(+)NCAM1(-) cell fraction. That is, one aspect of the present invention relates to the use of the combination of cell surface markers NRP1(+)NCAM1(-) as an indicator of the malignancy of breast cancer.

[0063] Another aspect of the present invention is a method for examining the state of breast cancer in a subject, comprising determining whether a cell sample derived from the subject has an NRP1(+)NCAM1(-) cell fraction, and identifying the subject as a candidate for administration of a composition for use in treating triple-negative breast cancer if the cell sample has the cell fraction. In some embodiments, the composition for use in treating triple-negative breast cancer comprises, as an active ingredient, an inhibitor of ZCCHC24 protein or an agent that suppresses expression of the ZCCHC24 gene.

[0064] The cell sample may be a tumor-like site obtained from a subject.

[0065] Methods for determining whether a cell sample contains an NRP1(+)NCAM1(-) cell fraction include, for example, single-cell RNA sequencing analysis (scRNAseq), fluorescence-activated cell sorting (FACS), immunostaining, etc.

[0066] Another aspect of the present invention can be said to be an in vitro method for testing breast cancer or the state of breast cancer, a method for measuring a test marker for breast cancer, or a method for obtaining an index of the possibility or malignancy of breast cancer.

[0067] Screening Method One aspect of the present invention relates to a method for screening drugs for use in treating triple-negative breast cancer. In some embodiments, the screening method comprises the steps of (i) preparing cells that express ZCCHC24, (ii) contacting the cells with a test substance, (iii) measuring the expression level of ZCCHC24, and (iv) selecting a substance that reduces the expression level of ZCCHC24. Another aspect of the present invention relates to a method for screening drugs for use in treating inflammatory diseases. In some embodiments, the screening method comprises the steps of (i) preparing cells that express ZCCHC24, (ii) contacting the cells with a test substance, (iii) measuring the expression level of ZCCHC24, and (iv) selecting a substance that reduces the expression level of ZCCHC24. Another aspect of the present invention relates to a method for screening drugs for use in treating obesity. In some embodiments, the screening method includes the steps of (i) preparing cells that express ZCCHC24, (ii) contacting the cells with a test substance, (iii) measuring the expression level of ZCCHC24, and (iv) selecting a substance that reduces the expression level of ZCCHC24.

[0068] In some embodiments, the cells expressing ZCCHC24 may be cells modified to express labeled ZCCHC24. The label may be any label known to those skilled in the art, such as a fluorescent protein. The cells may be, for example, MDAMB231 cells.

[0069] The test substance may be, for example, a small molecule, peptide, protein, or biological extract contained in a compound library. Contact between the cell and the test substance can be achieved, for example, by adding the test substance to the cell culture medium. The expression level of ZCCHC24 can be measured, for example, by measuring the amount of ZCCHC24 mRNA or the amount of ZCCHC24 protein. These measurements can be performed by any method known to those skilled in the art, such as PCR, ELISA, Northern blot, or Southern blot. The decrease in the expression level of ZCCHC24 can be determined by comparison with a predetermined reference value. The reference value can be, for example, the expression level of ZCCHC24 measured under identical conditions except for the presence or absence of the test substance.

[0070] Substances identified by such screening methods could be used to treat triple-negative breast cancer, inflammatory diseases, or obesity.

[0071] The present invention will be specifically explained below by showing examples, but the present invention is not limited by these examples.

[0072] Materials and Methods: Single-cell RNA-Seq Reanalysis: Single-cell RNA-Seq data for five human breast cancer specimens from a previous report (Wu et al., EMBO J. 2020) was reanalyzed using Cell ranger (10x genomics) and Seurat (https: / / satijalab.org / seurat / articles / get_started.html) software.

[0073] RNA-Seq 2 x 10 5Human triple-negative breast cancer cell line MDAMB231 cells were seeded in 6-well plates. After 24 hours, cells were transfected with a negative control (Stealth RNAi™ siRNA Negative Control Hi GC (Table 1, *4), Thermofisher Scientific) or siRNA against ZCCHC24 (HSS137253, stealth RNA (Table 1, *5), Thermofisher Scientific) using RNA iMax (Thermofisher Scientific). After a further 48 hours, RNA was isolated using the Relia RNA miniprep system (Promega). 500 ng of RNA was used for RNA-Seq library preparation using the NEB Next rRNA depletion kit v2 (NEB) and the NEBNext Ultra II RNA library prep kit for Illumina (NEB). RNA-Seq was performed using Next-Seq (Illumina). Adapter processing of RNA-Seq was performed using Trim Galore (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ). Mapping was performed using STAR (https: / / github.com / alexdobin / STAR). Quantification was performed using RSEM (R package). Differentially expressed genes were identified using iDEP 91 (http: / / bioinformatics.sdstate.edu / idep / ).

[0074] qPCR analysis 2×10 5Human breast cancer patient-derived cells (PDX) were seeded in 6-well plates. 24 hours later, they were transfected with control or ZCCHC24 siRNA (*4 and 5 in Table 1). 48 hours later, RNA was collected using the Relia RNA Miniprep System (Promega). Reverse transcription was performed using dNTPs (Toyobo), random primers (Toyobo), and PrimeScript (Takara). qPCR was then performed using primer sets for CD44, ZCCHC24, ZEB1, and NRP1 (*6 to 9 in Table 1 (SEQ ID NOs: 1 to 8)).

[0075] eCLIP-Seq: MDAMB231 cells overexpressing ZCCHC24 with doxycycline were harvested and crosslinked with an Ultraviolet Crosslinker (UVP, CA, USA) at 300 mJ / cm on ice. 2The cells were subjected to 254 nm UV-crosslinking at 1000 rpm. Cells were harvested, centrifuged, and resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% sodium deoxycholate, and 1:100 protease inhibitor). After 15 minutes of incubation on ice, the cells were sonicated for 5 minutes at 4°C in a Bioruptor (Cosmo Bio, Tokyo, Japan) with a 30 sec on / 30 sec off setting. Then, the cells were thermomixed with 10 μl of 1:100 diluted RNase I (Thermo Fisher Scientific, MA, USA) and 2 μl of Turbo DNase (Thermo Fisher Scientific) per sample at 1200 rpm and 37°C for 5 minutes. Immediately after incubation, 11 μl of mouse RNase inhibitor was added and the cells were centrifuged at 4°C for 15 minutes. For immunoprecipitation, mouse anti-FLAG antibody (MBL) was coupled to Dynabeads Protein G (Thermo Fisher Scientific) according to the manufacturer's protocol. The coupled antibody was washed with lysis buffer. The whole lysate and the coupled antibody were mixed and rotated overnight at 4°C. The antibody-bound proteins and RNA-protein complexes were separated using a magnetic stand and washed with cold wash buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20), high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), and FastAP buffer (10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100). The bound RNA was treated with FastAP alkaline phosphatase (Thermo Fisher Scientific) for 30 min and T4 polynucleotide kinase (PNK) (NEB) for 45 min.The bound beads were then washed with cold wash buffer, high-salt wash buffer, and ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2). The bound RNA was subjected to 3' RNA linker ligation using high-concentration RNA ligase (NEB) and an RNA adapter (*11 (SEQ ID NO: 11) in Table 1) for 3 hours, followed by washing with cold wash buffer and high-salt wash buffer. The RNA-protein complexes were extracted with NuPAGE sample buffer (Invitrogen) and subjected to SDS-PAGE. They were then transferred to a nitrocellulose membrane. The target region of the membrane was excised using proteinase K (NEB), acid phenol / chloroform / isoamyl alcohol (Nippon Gene, Tokyo, Japan), and a Quick-RNA miniprep kit (Zymo Research, CA, USA), and RNA was extracted from the membrane. The purified RNA was reverse transcribed using a reverse transcription primer (*12 (SEQ ID NO: 12) in Table 1) and TGIRT-III enzyme (InGex, MO, USA). After treatment with ExoSAP-IT (Thermo Fisher Scientific), rand103Tr3 linker (*13 (SEQ ID NO: 13) in Table 1) was ligated to the 5' region of the cDNA using high-concentration RNA ligase (NEB) and incubated overnight at room temperature. The adapter-ligated cDNA was amplified by 15 cycles of PCR using Q5 PCR enzyme (NEB), purified with Ampure XP beads (Beckman Coulter, CA, USA), and gel-purified. The CLIP-Seq library was sequenced using Next-Seq 500 (Ilumina). Adapter sequences were removed from the sequence data using Cut-adapt. Adapter-removed reads were mapped to the GRCh38 genome data using STAR, and duplicate reads were removed using the UMI tool.

[0076] 100 μM 4SU (Sigma-Aldrich) was added to the human triple-negative breast cancer cell line MDAMB231 expressing PAR-CLIP ZCCHC24 for 24 hours. The cells were exposed to 365 nm and 500 mJ / cm. 2UV crosslinking was performed using a UV crosslinker (UVP) under the following conditions. Cells were harvested and placed on ice for 15 minutes in lysis buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitors (1:100)). Cells were then disrupted using a Bioruptor (Cosmo Bio) at low setting for 5 minutes at 4°C with on / off cycling. RNA was then fragmented using 10 μL of RNase I (1:100; Thermo Fisher Scientific) and 2 μL of Turbo DNase (Thermo Fisher Scientific) at 37°C and 1200 rpm for 5 minutes. After adding 11 μL of mouse RNase inhibitor (Thermofisher Scientific), the cells were centrifuged at 4°C for 15 minutes. Immunoprecipitation was performed overnight at 4°C using anti-FLAG antibody (MBL, Fla-1) conjugated to Dynabeads Protein G (Thermo Fisher Scientific) at a ratio of 1:1000. Antibody-bound proteins and RNA-protein complexes were purified using a magnetic stand (Invitrogen) and washed with wash buffer (20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.2% Tween-20), high-salt wash buffer (50 mM Tris-HCl (pH 7.4), 1 M NaCl, 1 mM ethylenediaminetetraacetic acid, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate), and FastAP buffer (10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 100 mM KCl, and 0.02% Triton X-100). The ligated RNA was treated with FastAP alkaline phosphatase (Thermo Fisher Scientific) for 30 minutes and T4 polynucleotide kinase (PNK) (NEB) for 45 minutes, followed by 3'-adapter ligation using a high concentration of RNA ligase (NEB) and an RNA adapter (*11 (SEQ ID NO: 11) in Table 1) for 3 hours.The RNA was then washed with wash buffer and high-salt wash buffer. RNA-protein complexes were extracted using NuPAGE sample buffer (Invitrogen) and subjected to SDS-PAGE. The digested samples were transferred to a nitrocellulose membrane (0.2 μm). The target protein region on the blot was excised, and RNA was extracted using proteinase K (NEB), acid phenol / chloroform / isoamyl alcohol (Nippon Gene), and a Quick-RNA miniprep kit (Zymo Research). The purified RNA was reverse transcribed using TGIRT-III enzyme (InGex) with a reverse transcription primer (*12 (SEQ ID NO: 12) in Table 1). The resulting cDNA was incubated with ExoSAP-IT (Thermo Fisher Scientific). The 5' end of the cDNA was then ligated to rand103Tr3 linker (*13 (SEQ ID NO: 13) in Table 1) overnight at room temperature using high-concentration RNA ligase (NEB). The adapter-ligated cDNA was amplified by PCR using Q5 PCR enzyme (NEB) for 15 cycles and purified with Ampure XP beads (Beckman Coulter). Samples were then recovered by gel purification. CLIP-Seq libraries were sequenced using Next-Seq 500 (Illumina).

[0077] PAR-CLIP analysis was performed as follows: Adapter sequences contained in the sequence reads were removed using Cutadapt (https: / / cutadapt.readthedocs.io / en / stable / guide.html). Reads were mapped to the GRCh38 genome using STAR (https: / / github.com / alexdobin / STAR), and duplicate reads were removed using UMI Tools (https: / / umi-tools.readthedocs.io / en / latest / reference / extract.html). Read data were annotated using RSeqC (http: / / rseqc.sourceforge.net / ).

[0078] BRIC-Seq 1 x 10 in a 15cm dish 6 MDAMB231 cells were seeded. 24 hours later, control or ZCCHC24 siRNA (*4, 5 in Table 1) was transfected using RNA iMax (Invitrogen). 24 hours later, 150 μM (final concentration) BrdU (Sigma-Aldrich) was added. After washing twice with PBS, the cells were harvested at 0 and 1 hour using TRIZOL (Thermofisher Scientific). To measure the rate of RNA degradation, immunoprecipitation with anti-BrdU antibody (MBL, 2B1) was performed as follows: 45 μg of the harvested RNA was immunoprecipitated with 5 μg of anti-BrdU antibody (MBL, 2B1) and IP buffer (1% Triton in PBS) and incubated with rotation at 4°C for 2 hours. After washing, RNA was harvested using the Monarch RNA cleanup kit (NEB). The recovered RNA library was prepared for next-generation sequencing using the NEBNext Ultra II RNA library prep kit for Illumina (NEB). RNA-Seq was performed on the library using HiSeq (Illumina). Adapters were then removed from the sequence data using Cutadapt (https: / / cutadapt.readthedocs.io / en / stable / guide.html). Reads were mapped to the GRCh38 genome using STAR (https: / / github.com / alexdobin / STAR). BridgeR (an R package) was used to measure RNA degradation rates.

[0079] Actinomycin D test: 5 × 10 MDAMB231 or HCC38 in a 24-well plate 4The cells were seeded at a ratio of 1 / well. 24 hours later, they were transfected with 0.5 μL of 20 μM siRNA (Thermofisher Scientific) and 3 μL of RNA iMax (Thermofisher Scientific). 48 hours after transfection, 10 μM Actinomycin D was added to the medium, and RNA was collected at 0, 1, and 2 hours. RNA collected using Relia Prep RNA mini prep (Promega) was reverse transcribed with Random Primer (Toyobo), dNTPs (NEB), and Prime Script (Takara), and changes in target gene expression were evaluated by qPCR.

[0080] Sphere formation assay: Human breast cancer cell lines MDAMB231 and HCC38 were cultured at 2 × 10 5 The cells were seeded in a 6-well plate. After 24 hours, they were transfected with control or ZCCHC24 siRNA (*4, 5 in Table 1). After 24 hours, they were cultured in DMEM F-12 medium (Gibco) supplemented with 20 ng / mL EGF (R&D), 20 ng / mL FGF (Wako), and B27 supplement for one week, and images were taken from 10 random fields of view to calculate the number of spheres.

[0081] Extreme Dilution Assay (ELDA) Human breast cancer cell lines MDAMB231 and HCC38 were used at 2 × 10 5 spheres were seeded into a 6-well plate. 24 hours later, control or ZCCHC24 siRNA (*4, 5 in Table 1) was transfected. 24 hours later, 250, 125, 62.5, 31, and 15 spheres were seeded into each well (N = 8) and cultured for one week in DMEM F-12 medium (Gibco) supplemented with 20 ng / mL EGF (R&D), 20 ng / mL FGF (Wako), and B27 supplement, and the presence or absence of spheres was counted.

[0082] In vivo colony formation assay: 1 × 10 human breast cancer cell line MDAMB231 or breast cancer PDXs were transfected with control or siRNA against ZCCHC24 (*4, 5 in Table 1) 24 hours later. 4 , 10 3 , 10 2 The cells were subcutaneously implanted into 7-week-old female nude mice (NOG mice (Oriental Yeast)) at a rate of one cell per site along with 50% Matrigel (Corning). The number of tumors formed was counted one month later.

[0083] Single-cell RNA-Seq analysis of PDX xenograft models. PDX were transfected with negative control or ZCCHC24 siRNA (*4, 5 in Table 1) (Thermofisher Scientific). After 24 hours, 10 siRNA-transfected cells were 3 Each tumor was subcutaneously implanted into 7-week-old female NOG mice (Oriental Yeast) with 50% Matrigel (Corning). After 19 days, the grown tumors were harvested and treated with 3 μg / ml collagenase (Wako). Libraries were prepared from the treated tumors using the Chromium Next GEM Chip G Single Cell Kit (10x chromium). The prepared libraries were sequenced using Nova-Seq (Illumina). The sequencing data were analyzed using Seurat (https: / / satijalab.org / seurat / articles / get_started.html) to identify breast cancer stem cell fractions.

[0084] scRNAseq analysis of patient-derived breast cancer samples in subcutaneously transplanted NOG mice. Tumor samples from three triple-negative breast cancer patients were subcutaneously transplanted into NOG mice. When tumors reached an appropriate size, they were excised and treated with collagenase (Wako) to degrade the tumor mass. Libraries were prepared from the treated tumors using the Chromium Next GEM Chip G Single Cell Kit (10x chromium). The prepared libraries were sequenced using Nova-Seq (Illumina). Sequencing data was analyzed using Seurat (https: / / satijalab.org / seurat / articles / get_started.html) to identify breast cancer stem cell fractions.

[0085] Single-cell RNA-Seq analysis of reimplantation model 10 3 PDX were subcutaneously implanted into mice with Matrigel (Corning #354262) at a ratio of 1 / site. One month later, the formed tumors were excised, treated with collagenase, and sorted by FACS using antibodies against the surface antigens NRP1 and NCAM1 (NRP1-APC antibody: R&D, FAB3870A, NCAM1-APC-Cy7 antibody: BioLegend, BL318331). Stem I cell fractions were sorted according to the criteria of NRP1(+)NCAM1(+), and Stem II cells were sorted according to the criteria of NRP1(+)NCAM1(-).

[0086] The sorted cells were transfected into 7-week-old female NOG mice (CLEA Japan) at 10 dpi. The cells from the Stem I cell fraction, the Stem II cell fraction, and the mixed cells from the Stem I and Stem II cell fractions were transfected into 7-week-old female NOG mice (CLEA Japan). 4 , 10 3 , 10 2 The tumors were subcutaneously implanted with Matrigel (Corning, #354262) at a ratio of 1 / site, and tumors were sampled 6 weeks after implantation. Tumor formation ability was evaluated, and Stem II was incubated at 10x chromium (10x genomics). 2We performed scRNA-seq analysis on the reimplanted samples at each site and generated NGS libraries. The generated NGS libraries were sequenced using NovaSeq (Ilumina). The sequenced reads were mapped using cell ranger (https: / / support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / what-is-cell-ranger) and analyzed using Seurat (https: / / satijalab.org / seurat / ).

[0087] Compound screening using HiBiT-tagged knock-in cells. HiBiT-tagged MDAMB231 cells were generated as follows. First, crRNA (*14 (SEQ ID NO: 14) in Table 1) (IDT) resuspended in Nuclease-Free Duplex Buffer (IDT) to a final concentration of 100 μM was incubated with an equal amount of tracrRNA (IDT) at 95°C for 5 minutes. The oligo complex was then gradually cooled to room temperature and incubated with ALT-R Cas9 Nuclease V3 (61 μM) (Integrated DNA Technologies, IA, USA) at room temperature for 20 minutes to form a Cas9 complex. Next, MDAMB231 cells were co-transfected with a single-stranded DNA oligo (*15 (SEQ ID NO: 15) in Table 1) (containing sequences complementary to HiBiT and the C-terminal region of ZCCHC24) and the Cas9 complex by electroporation using a NEPA21 Superelectroporator (NEPAGENE). After single cell cloning, HiBiT-tagged cells were collected.

[0088] The procedure for compound screening was as follows: 1 × 10 HiBiT-tagged cells were cultured. 4The cells were plated in 96-well plates and cultured for 24 hours. A small molecule compound library (LOPAC; Sigma-Aldrich) was added at a final concentration of 1 μM for 24 hours. After discarding the culture medium, the cells were incubated with 12.5 μL PBS, 12.5 μL lysis buffer (Promega), 0.5 μL substrate (Promega), and 0.25 μL LgBiT (Promega) in the dark at room temperature for 10 minutes. Luminescence intensity was measured using an ARVO X3 (Perkin Elmer).

[0089] EC50 (Effective concentration 50) assay 1×10 in 96well plate 4 10 HiBiT-tagged cells were seeded on the cells. 24 hours later, 0.1% DMSO or 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 10 μM JQ1 (Selleck) or PD407824 (Sigma-Aldrich) were added. After 24 hours, the medium was discarded, and 12.5 μL of PBS, 12.5 μL of lytic buffer from the HiBiT kit (Promega), 0.5 μL of substrate, and 0.25 μL of LgBiT were added. After 10 minutes of incubation at room temperature, luminescence was measured using an ARVO X3 (Perkin Elmer).

[0090] IC50 (Inhibitory concentration 50) assay: 2 x 10 in a 96-well plate 3 10 HiBiT-tagged cells were seeded on the cells. 24 hours later, 0.1% DMSO or 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 1, 10 μM JQ1 (Selleck) or PD407824 (Sigma-Aldrich) was added. After 48 hours, 20 μL of Cell Titer Glo (Promega) was added. After 30 minutes of incubation at 37°C, the A490 absorbance was measured using an ARVO X3 (Perkin Elmer).

[0091] Reanalysis of ChIP-Seq Data We reanalyzed the ChIP-Seq data for ZEB1, H3K2Ac, and H3K4Me1 from Rhie et al. BMC Genomics (2014) and Katsura et al., Mol Oncol. (2017). Rhie SK, Hazelett DJ, Coetzee SG, Yan C, Noushmehr H, Coetzee GA. Nucleosome positioning and histone modifications define relationships between regulatory elements and nearby gene expression in breast epithelial cells. BMC Genomics. 2014;15:331 Katsura A, Tamura Y, Hokari S, Harada M, Morikawa M, Sakurai T, et al. ZEB1-regulated inflammatory phenotype in breast cancer cells. Mol Oncol. 2017;11(9):1241-62

[0092] qPCR analysis of JQ1-treated MDAMB231 cells. 2 x 10 5 MDAMB231 cells were seeded on the plates. After 24 hours, 0.1% DMSO or 100 nM JQ1 was added. After 24 hours, RNA was extracted using Relia Prep RNA MiniPrep (Promega) and reverse transcribed using Random Primer (Takara), dNTP (NEB), and PrimeScript (Takara). The cDNA was used to quantify PSMB2 (*10 in Table 1 (SEQ ID NOs: 9 and 10)) and ZCCHC24 (*6 in Table 1 (SEQ ID NOs: 1 and 2)) by qPCR.

[0093] Evaluation of the effect of ZEB1 siRNA knockdown on JQ1 treatment. 1 × 10 HiBiT-tagged cells were plated onto a 96-well plate. 4After 24 hours, transfection was performed with 0.5 μL of control and ZCCHC24 siRNA and 3 μL of RNA iMax (Thermofisher). After 24 hours, 0.1% DMSO or 100 nM JQ1 was added. After 24 hours, 12.5 μL of PBS, 12.5 μL of HiBiT kit (Promega) Lytic Buffer, 0.5 μL of Substrate, and 0.25 μL of LgBiT were added. After 10 minutes of incubation at room temperature, luminescence was measured using an ARVO X3 (Perkin Elmer).

[0094] Analysis of breast cancer stem cell percentage after drug treatment by FACS. 2×10 cells were cultured in a 6-well plate. 5 MDAMB231 cells were seeded onto the plate. After 24 hours, 0.1% DMSO, 100 nM JQ1, 1 μM PD407824, or both were added. After 24 hours, the cells were harvested and stained with CD44-PE (IM7, Biolegend) and NRP1-APC (FAB3870A, R&D). FACS analysis was performed using a FACS Calibur (BD).

[0095] Cell Growth Assay 2 x 10 cells in a 96-well plate 3 MDAMB231 cells were seeded onto the plate. After 24 hours, 100 nM JQ1 (Selleck) or 1 μM PD407824 (Sigma-Aldrich) was added. Cell numbers were quantified at 0, 24, and 48 hours using Cell Titer Glo (Promega) and absorbance measurement using ARVO X3 (Perkin Elmer).

[0096] Generation of ZCCHC24 KO mice Synthesized gRNA (*16 (SEQ ID NO: 16) in Table 1) was injected together with Cas9 mRNA into fertilized eggs of BDF1 mice to generate KO mice using the CRISPR-Cas9 system.

[0097] LPS shock model: ZCCHC24KO mice and wild-type control mice were intraperitoneally injected with 15 mg / kg of lipopolysaccharide (LPS). After that, the survival of the mice was checked every 12 hours, and a survival curve was plotted.

[0098] High-fat diet model ZCCHC24KO mice and wild-type control mice were fed a high-fat diet, and their body weights were measured and recorded weekly.

[0099] Novel nucleic acid medicine experiments (miR-PBE1, 2) We designed mimic-miR-RNAs that bind complementary to the RNA sequence ("UGUAHAWA" (Table 1 *2)) to which ZCCHC24 binds. The seed target sequences used were "UGAUAUAU" (Table 1 *17) or "UGUACAU" (Table 1 *18). The sequences were miR-PBE1 (5'-AAUAUACAUCCUCCGGGAUCCA-3') (Table 1 *19 (SEQ ID NO: 17)) and miR-PBE2 (5'-AAUGUACAUCCUCCGGGAUCCA-3') (Table 1 *20 (SEQ ID NO: 18)) (Thermofisher miRNA mimics). These were transfected at a final concentration of 25 nM or 50 nM with Lipofectamine RNA iMax (Invitrogen) into the TNBC cell line MDAMB231, TNBC patient-derived cells (PDX), and the human chondrocyte cell line SW1353. Samples were harvested 48 hours later. SW1353 cells were stimulated with 10 nM human IL-1β starting 6 hours before harvest. After sample collection, RNA was purified using Relia-Prep RNA Miniprep Systems (Promega) and reverse transcribed with random primers (Takara), dNTP Mix (Takara), and PrimeScript (Takara). mRNA levels were then quantified by qPCR. The primer sets are shown in Table 1 (IL-1b: *21 in Table 1 (SEQ ID NOs: 19 and 20) and MMP13: *22 in Table 1 (SEQ ID NOs: 21 and 22)).

[0100] Results: Reanalysis of scRNA-seq data from human breast cancer samples By integrating reanalysis of single-cell RNA-seq data from human samples, we attempted to identify RNA-binding proteins specifically expressed in breast cancer stem cell fractions. We succeeded in identifying ZCCHC24 as one of the novel RNA-binding proteins with unknown function that was strongly expressed in cell fractions that showed breast cancer stem cell-like expression of NRP1(+)ZEB1(+)EPCAM(-) (Figure 1).

[0101] Genetic screening: We established the PC9-KI cell line by knocking in a HiBiT tag at the C-terminus of PDL1 in the lung adenocarcinoma cell line PC9. We then performed a genetic screening to quantitatively measure changes in PD-L1 protein expression by forcibly expressing 1030 RNA-binding proteins in this PC9-KI cell line using a lentiviral vector (Figure 2A). As a result, we identified ZCCHC24 as a gene that increases PDL1 expression (Figure 2B).

[0102] This upregulation of PDL1 expression by ZCCHC24 was confirmed by Western blotting (Fig. 2C). Furthermore, to identify the cells in which ZCCHC24 functions endogenously, we compared expression in multiple cell lines and found that high ZCCHC24 expression was observed in the triple-negative breast cancer cell line MDAMB231 (Fig. 2D).

[0103] Therefore, knockdown of ZCCHC24 by siRNA revealed that both PDL1 mRNA and protein expression were decreased (Fig. 2E, Fig. 2F). Based on these results, we analyzed the function of ZCCHC24 in triple-negative breast cancer cells.

[0104] Using the deposit data from a report (Wu et al., EMBO J. 2020) in which single-cell RNA-Seq analysis was performed on five triple-negative breast cancer samples, we attempted to identify fractions with strong ZCCHC24 expression. We divided the cell fractions using UMAP and found that they were separated into 23 clusters (Figure 3A). A violin plot revealed that ZCCHC24 was specifically expressed in cluster 9 (Figure 3B).

[0105] To further investigate the properties of the cells belonging to cluster 9, we identified marker genes and found strong expression of vimentin, which contributes significantly to epithelial-mesenchymal transition (EMT) and the maintenance of cancer stem cells (Figure 3C). We then compared the expression distribution of cancer stem cell markers and found that cluster 9 exhibited high CD44, negative CD24, positive NRP1, and positive ZEB1, suggesting that ZCCHC24 is strongly expressed in the cancer stem cell-like fraction. Furthermore, PDL1 expression was also observed, suggesting a correlation with ZCCHC24 expression (Figure 3D).

[0106] To further identify genes whose expression was altered by ZCCHC24 through whole-transcriptome analysis, we performed RNA-Seq on control and ZCCHC24-knockdown (human breast cancer cell line MDAMB231) cells. As a result, 797 genes were identified as differentially expressed genes (DEGs). In particular, we found significant decreases in the expression of genes important for characterizing cancer stemness in breast cancer, such as CD44, NRP1, ZEB1, and ZEB2 (Figure 4, left, Figure 5A). Furthermore, we found significant decreases in the expression of cytokines and chemokines important for cancer survival, such as PDL1, IL6, IL8, and CXCL1 (Figure 5A). Similarly, qPCR confirmed that ZCCHC24 knockdown also reduced the expression of CD44, NRP1, and ZEB1 in human breast cancer patient-derived cell lines (PDX) (Figure 4, right).

[0107] Therefore, we actually measured the change in the percentage of breast cancer stem cells, defined as CD44 positive and NRP1 positive, in MDAMB231 cells in which ZCCHC24 was knocked down using FACS, and found that knockdown of ZCCHC24 reduced the percentage of the cancer stem cell fraction (Figure 5B, Figure 5C).

[0108] Furthermore, to demonstrate that these events are not specific to MDAMB231, knockdown of ZCCHC24 in another triple-negative breast cancer cell line, HCC38, also resulted in a decrease in the expression of cancer stem cell-like cell surface markers, such as CD44 and NRP1, at the mRNA level (Fig. 5D).

[0109] Furthermore, analysis of surface antigens by FACS revealed that the CD44-positive and NRP1-positive cell fractions were significantly reduced by ZCCHC24 knockdown (Fig. 5E, 5F). These results suggest that ZCCHC24 is an important gene for maintaining the expression of genes that characterize cancer stemness.

[0110] Furthermore, we performed eCLIP-Seq on ZCCHC24-overexpressing cells to identify mRNA regions directly bound by ZCCHC24 as an RNA-binding protein through whole-transcriptome analysis. The results revealed that ZCCHC24 targets 4,821 genes. Merging these results with the DEGs from RNA-Seq identified 364 genes as target genes whose expression is regulated by direct binding of ZCCHC24 (Figure 6A). Classification of the binding regions in these target genes by RSeqC revealed that they bind to 3'UTR regions and CDS (Figure 6B).

[0111] In fact, analysis of the peak patterns of these target genes revealed that ZCCHC24 directly binds to the 3'UTRs of genes such as PDL1, IL6, IL8, CXCL1, CD44, and NRP1 (Figure 6C). Furthermore, GO term analysis and pathway analysis of these target genes using DAVID revealed that ZCCHC24 regulates genes that negatively regulate apoptosis and cell proliferation, as well as pathways that regulate focal adhesion and the actin cytoskeleton (Figure 6D, Figure 6E). These results strongly suggest that ZCCHC24 is an RNA-binding protein that directly binds to the CDS and 3'UTRs of genes important for regulating cancer stemness.

[0112] PAR-CLIP Furthermore, to identify the mRNAs directly targeted by ZCCHC24 and their binding motifs through whole-transcriptome analysis, PAR-CLIP analysis was performed on MDAMB231. We found that ZCCHC24 recognizes a novel, unique motif sequence, (A / U)GU(A / U)U(A / U)U, and binds to the 3'UTRs of the mRNAs of genes that characterize cancer stemness, such as ZEB1, CD44, and NRP1 (Figure 7).

[0113] BRIC-Seq To understand the level at which expression control by ZCCHC24 occurs, we performed BRIC-Seq, which measures the rate of RNA degradation, on MDAMB231 cells in which ZCCHC24 and the control were knocked down with siRNA. We found that knockdown of ZCCHC24 tended to significantly reduce the RNA stability of genes that characterize breast cancer stemness, such as ZEB1, CD44, and NRP1 (Figure 8).

[0114] To investigate the contribution of ZCCHC24 to target gene mRNA stability, we performed an actinomycin D test to measure mRNA stability by halting transcription with actinomycin D. In MDAMB231, we found that knockdown of ZCCHC24 reduced mRNA stability for target genes such as CD274 (PDL1), CXCL8 (IL8), IL6, CD44, NRP1, and ZEB1 (Figure 9A). Similarly, knockdown of ZCCHC24 also reduced mRNA stability for CD44 and NRP1 in HCC38 (Figure 9B). These results strongly suggest that ZCCHC24 stabilizes target gene mRNAs by directly binding to them.

[0115] To investigate the effect of ZCCHC24 on cancer stemness, we performed a sphere formation assay after knocking down ZCCHC24 in MDAMB231 with siRNA. We found that knockdown of ZCCHC24 significantly reduced the sphere-forming ability (Fig. 10A, Fig. 10B).

[0116] Extreme Dilution Assay (ELDA) ELDA was performed on the triple-negative breast cancer cell lines MDAMB231 and HCC38 to quantify changes in the number of cancer stem-like cells present per cell count. It was found that knockdown of ZCCHC24 with siRNA tended to reduce the number of breast cancer stem cells per cell count compared to the control (Figure 11).

[0117] In vivo colony formation assay: To evaluate tumorigenicity at the in vivo level, we performed in vivo tumorigenicity assays using the triple-negative breast cancer cell line MDAMB231 and a subcutaneously transplanted PDX model. We observed a trend toward a significant decrease in tumorigenicity in both cases with siRNA knockdown of ZCCHC24 compared to the control (Figure 12). These results demonstrated that ZCCHC24 characterizes cancer stemness both in vitro and in vivo.

[0118] Single-cell RNA-seq analysis of PDX subcutaneous transplantation model: To analyze the changes in cancer-forming cells due to ZCCHC24 knockdown in more detail, we performed single-cell RNA-seq analysis on samples subcutaneously transplanted into NOG mice with cells knocked down by siRNA for ZCCHC24 and control. We found that the stem cell-like population, which exhibited cancer stem cell-like expression of NRP1+, ZEB1+, CD24-, and EPCAM-, was significantly reduced. In particular, we found that the stem cell-like population was divided into two major populations: a cell population characterized by NRP1+ and NCAM1+ (referred to as "Stem I") and a cell population characterized by NRP1+ and NCAM1- (referred to as "Stem II"), and that the Stem II population in particular was significantly reduced by ZCCHC24 knockdown. This significantly reduced Stem II is a cell fraction positive for genes such as ZEB1 and NRP1, as well as ZCCHC24, and also expresses specific proteoglycans such as POSTN and DCN (Fig. 13A).

[0119] scRNAseq analysis of patient-derived breast cancer specimens in subcutaneous transplant models in NOG mice. Three triple-negative breast cancer patient specimens were subcutaneously transplanted into NOG mice, and scRNAseq analysis was performed on these specimens. The results showed that the NRP1(+)ZEB1(+)CD24(-)EPCAM(-) fraction, which has been identified as a breast cancer stem cell fraction in specimens from other breast cancer patients, can be broadly divided into two subpopulations. ZCCHC24 was identified as an RNA-binding protein specifically expressed in the NRP1(+)ZEB1(+)NCAM1(-) fraction (defined as Stem II) (Figure 13B).

[0120] Single-cell RNA-Seq analysis of the reimplantation model - Analysis of tumors formed by subcutaneous PDX transplantation Analysis of tumor sites formed by subcutaneous PDX transplantation revealed that ZCCHC24 was specifically expressed in the Stem II fraction of NRP1(+)NCAM1(-) (Figure 14).

[0121] Tumorigenicity Assay The tumorigenicity of sorted tumor cells was examined, and the tumorigenicity of Stem II was found to be significantly higher than that of Stem I and the mixture of Stem I and Stem II (Figure 15).

[0122] Single-cell RNA-Seq analysis Furthermore, when scRNA-seq analysis was performed on the sample in which Stem II had been re-implanted, it was observed that the tumor was again separated into a Stem II fraction characterized by NRP1(+)NCAM1(-), as well as a Stem I fraction characterized by NRP1(+)NCAM1(+), and an epithelial fraction characterized by NRP1(-), showing fractions similar to those of the tumor before sorting (Figure 16).

[0123] Thus, single-cell RNA-seq analysis using patient-derived xenografts revealed that ZCCHC24 is specifically and highly expressed in breast cancer stem cells, which have been defined as CD44 positive, CD24 negative, NRP1 positive, and ZEB1 positive. Furthermore, single-cell RNA-seq analysis using PDX revealed that ZCCHC24 expression is particularly high in cells of the Stem II population, which are characterized as NRP1 positive and NCAM1 negative (Figure 3D). Knockdown of ZCCHC24 reduced the number of Stem II cells (Figure 13A) and reduced tumorigenicity (Figure 12), demonstrating the importance of ZCCHC24 in maintaining the Stem II population. Furthermore, tumorigenesis assays demonstrated that sorting and retransplantation of this Stem II population demonstrated high tumorigenicity (Figure 15), and single-cell RNA-Seq analysis revealed that the tumor population established by retransplantation of this Stem II population again differentiated into each tumor cell fraction (Figure 16). These results demonstrate that the Stem II population, centered on ZCCHC24, has high breast cancer-forming potential, and that in addition to ZCCHC24, the cancer stem cell fraction expressing ZCCHC24 itself may also be a therapeutic target.

[0124] As described above, ZCCHC24 is highly expressed in cancer stem-like cells and maintains the expression of genes important for maintaining cancer stemness by directly binding to their mRNA. To clarify the mechanism by which ZCCHC24 expression is induced, we created reporter cells labeled with a HiBiT tag at the C-terminus of ZCCHC24. We then administered 1,280 compounds from LOPAC (Sigma-Aldrich) at a concentration of 1 μM for 24 hours and performed compound screening (Figure 17A).

[0125] As a result, 11 up-regulators and 9 down-regulators were identified, defining compounds with a Z score >3.0 as up-regulators and compounds with a Z score <-3.0 as down-regulators (Figure 17B).

[0126] It is noteworthy that upregulators specifically identified included BET inhibitors, which are expected to be clinically effective against refractory breast cancers, including triple-negative breast cancer, as well as topoisomerase inhibitors such as idarubicin, nitidine, and camptothecin (Figure 17C). Meanwhile, downregulators identified included microtubule polymerization inhibitors and Wee1 / Chk1 inhibitors, which are important in the DNA damage response pathway (Figure 17D). Therefore, considering the possibility that the DNA damage response pathway may be particularly important in regulating ZCCHC24 expression, we decided to focus on BET inhibitors as upregulators and Wee1 / Chk1 inhibitors as downregulators.

[0127] Therefore, we used JQ1, a typical BET inhibitor, to measure the EC 50 The concentration was measured and found to be approximately 50 nM, demonstrating that ZCCHC24 expression was indeed increased (Fig. 18A). 50 The concentration was approximately 0.1 μM (FIG. 18B).

[0128] We focused on the transcription factor ZEB1 as the mechanism underlying the dramatic increase in ZCCHC24 expression by BET inhibitors. ZEB1 is a transcription factor whose importance in EMT and drug resistance has been strongly suggested in breast cancer, lung cancer, and other cancers. It has also been identified as a target gene of ZCCHC24, as shown in Figures 6 and 7. By comparing a histone marker ChIP database and previous ChIP-Seq data for ZEB1, we found that there was a region between the first and second exons where the histone markers H3K4Me1 and H3K27Ac peaked, along with a ZEB1 peak (Figure 18C). Furthermore, administration of JQ1 dramatically increased ZEB1 expression (Figure 18D).

[0129] Therefore, we examined the dependency of ZCCHC24 on ZEB1 by JQ1 using siRNA knockdown, and found that the increase in ZCCHC24 expression caused by JQ1 was significantly reduced by ZEB1 knockdown (Figure 18E).

[0130] These results strongly suggest that BET inhibitors increase ZCCHC24 expression through increased ZEB1 expression. Furthermore, compounds that regulate the ZEB1-ZCCHC24 axis are of great significance in reducing drug resistance caused by BET inhibitors. Therefore, we focused on Wee1 / Chk1 inhibitors in our compound screening. The EC 50 The concentration was measured and found to be approximately 50 nM (Fig. 19A). On the other hand, although PD407824 inhibited cell activity to a certain extent, the IC 50 However, the inhibition of cell activity was not observed to the extent that calculation of the β-amyloid ratio was possible (Fig. 19B).

[0131] Furthermore, when JQ1 and PD407824 were administered separately, no reduction in the cancer stem cell-like population was observed with JQ1 administration, whereas a tendency for this cell population to decrease with PD407824 administration was observed. Furthermore, a reduction in the cancer stem cell-like population was also observed with the combined administration of JQ1 and PD407824 (Fig. 19C, Fig. 19D).

[0132] Based on these trends, we investigated the inhibitory effect of combined use of 100 nM JQ1 and 1 μM PD407824 on cell proliferation, and found that the combined use did indeed have an additive effect (FIG. 19E).

[0133] ZCCHC24 knockout mice are resistant to LPS shock. ZCCHC24 knockout mice were established using the CRISPR-Cas9 system. When ZCCHC24 knockout mice were intraperitoneally injected with 15 mg / kg LPS, they showed a longer survival time after LPS injection compared to wild-type mice. This suggests that ZCCHC24 is an important RNA-binding protein that regulates inflammation (Figure 20).

[0134] ZCCHC24 knockout mice were fed a high-fat diet (HFD). Approximately three months after the start of the diet, weight gain was significantly suppressed in ZCCHC24 knockout mice compared to wild-type mice, suggesting that ZCCHC24 may also play an important role in obesity (Figure 21).

[0135] We attempted to simultaneously broadly regulate breast cancer stemness and inflammatory genes targeted by ZCCHC24 using a novel nucleic acid drug designed by us. When mimic-1 (miR-PBE1), mimic-2 (miR-PBE2), or a mixture of mimics 1 and 2 (miR-mix) was transfected into the MDAMB231 cell line, the expression of genes required for breast cancer stemness and cancer cell invasion, including ZEB1, IL-6, ITGB1, and THBS1, was suppressed (Figures 22 and 23).

[0136] Furthermore, when mimic-1 (miR-PBE1), mimic-2 (miR-PBE2), and a mixture of mimic 1 and 2 (miR-mix) were introduced into patient-derived cells (PDX) derived from breast cancer TNBC, the expression of genes necessary for breast cancer stemness and cancer cell invasion, such as IL-6, CD44, ITGB1, and NRP1, was similarly suppressed (Figure 24).

[0137] Furthermore, when mimic1 (miR-PBE1) was introduced into the human chondrocyte cell line SW1353, the expression of MMP13 and IL1-β, which are important effector molecules in knee osteoarthritis, was suppressed (Figure 25).

[0138] These results demonstrated in vitro that a novel mimicRNA formulation targeting the RNA sequence to which ZCCHC24 binds may have therapeutic effects against cancer and osteoarthritis of the knee.

[0139] Table 1 below shows the sequence symbols, sequence names, sequences, and sequence numbers in the attached sequence listing used in the examples.

[0140]

[0141] While preferred embodiments of the present invention are described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only, and that various modifications, changes, and substitutions may be made by those skilled in the art without departing from the invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the invention. Furthermore, the contents of all publications, including patents and patent applications, referenced in this specification should be construed as being incorporated by reference as if expressly set forth herein.

[0142] Many molecularly targeted drugs, including CDK4 / 6 inhibitors, have been used to treat refractory breast cancer, including triple-negative breast cancer. However, effective treatments have yet to be fully established. In these refractory breast cancers, ZCCHC24 upregulates cancer-promoting genes through post-transcriptional regulation, making it an important gene for explaining treatment resistance and a useful therapeutic target. Furthermore, the inventors have successfully identified a new breast cancer stem cell fraction that highly expresses ZCCHC24 and its corresponding cell surface markers. This ZCCHC24-expressing cancer stem cell fraction itself is considered a potential therapeutic target. Furthermore, the inventors have successfully identified compounds that downregulate ZCCHC24 expression, suggesting that the use of these compounds may alleviate drug resistance to existing refractory breast cancer therapies. Furthermore, ZCCHC24 is a useful therapeutic target not only for triple-negative breast cancer and other breast cancers, but also for inflammatory diseases and obesity.

Claims

1. A composition for reducing the NRP1(+)NCAM1(-) cell fraction in triple-negative breast cancer patients, the composition comprising as an active ingredient an inhibitor of the ZCCHC24 protein or a drug that suppresses the expression of the ZCCHC24 gene, for patients with triple-negative breast cancer who have been determined to have an NRP1(+)NCAM1(-) cell fraction.

2. The composition of claim 1, wherein the drug that suppresses the expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor.

3. 3. The composition of claim 1 or 2 for use in combination with a BET inhibitor.

4. The composition of claim 1, wherein the inhibitor of ZCCHC24 protein is selected from the group consisting of a neutralizing antibody, a nucleic acid drug, and a small molecule compound.

5. Wee1 / Chk1 inhibitors include PD407824, AZD-1775, ZN-c3, Debio-0123, IMP-7068, GDC-0575, ESP-01, PNT-737, BEBT-260, AZD7762, LY2603618, MK-8776, CHIR-124, PF-477736, rosovitine, SNS-032, and ginasin. The composition of claim 2, wherein the compound is selected from the group consisting of milciclib, flavopiridol, AT7519, purvalanol A, RO-3306, SU9516, XL413, NU6027, P276-00, AZD5438, PHA-793887, JNJ-7706621, BMS-265246, milciclib, R547, and adavosertib.

6. 10. The composition of claim 1, further comprising a BET inhibitor.

7. 7. The composition of claim 6, wherein the BET inhibitor is selected from the group consisting of JQ1, perabresive, BMS-986158, INCB-057643, ODM-207, PLX-2853, ABBV-744, BI-894999, BPI-23314, CC-90010, FT-1101, JAB-8263, mibebrexit, SF-1126, and SYHA-1801.

8. The composition of claim 5, wherein the Wee1 / Chk1 inhibitor is selected from the group consisting of PD407824, AZD-1775, ZN-c3, Debio-0123, IMP-7068, GDC-0575, ESP-01, PNT-737, BEBT-260, AZD7762, LY2603618, MK-8776, CHIR-124, PF-477736, rosovitine, SNS-032, dinaciclib, flavopiridol, AT7519, purvalanol A, RO-3306, SU9516, XL413, NU6027, P276-00, AZD5438, PHA-793887, JNJ-7706621, BMS-265246, and R547.

9. The composition according to claim 1, wherein the drug that suppresses the expression of the ZCCHC24 gene is a nucleic acid drug.

10. ZCCHC24 protein inhibitors (i) a seed sequence complementary to at least a portion of WGUWHWWA (ii) an adenine or uracil base 5' to the seed sequence, and (iii) a scrambled sequence 3' to the seed sequence The composition of claim 1, comprising a miRNA of 19 to 25 bases in length having a full-length GC content of 40 to 60%.

11. A composition for use in the treatment of triple-negative breast cancer, containing as an active ingredient an inhibitor of ZCCHC24 protein or a drug that suppresses the expression of the ZCCHC24 gene (excluding Wee1 inhibitors).

12. A composition for use in treating triple-negative breast cancer, comprising as an active ingredient a nucleic acid drug that inhibits ZCCHC24 protein or suppresses expression of the ZCCHC24 gene, wherein the nucleic acid drug is a microRNA-mimic that targets the RNA sequence targeted by ZCCHC24.

13. A composition for the treatment of triple-negative breast cancer, which contains as an active ingredient an inhibitor of the ZCCHC24 protein or a drug that suppresses the expression of the ZCCHC24 gene (excluding AZD-1775), and is to be used in combination with a BET inhibitor.

14. A method for testing a subject for breast cancer, comprising: a) measuring the expression level of ZCCHC24 in a sample derived from the subject; b) comparing the expression level of ZCCHC24 with a predetermined reference value; wherein the possibility of breast cancer is indicated when the expression level of ZCCHC24 is higher than a predetermined standard value.

15. A composition for use in treating an inflammatory disease, comprising as an active ingredient an inhibitor of ZCCHC24 protein or a drug that suppresses the expression of the ZCCHC24 gene.

16. A composition for use in treating an inflammatory disease according to claim 15, wherein the drug that suppresses the expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor.

17. The composition for use in treating an inflammatory disease according to claim 15, wherein the drug that suppresses the expression of the ZCCHC24 gene is a nucleic acid drug.

18. ZCCHC24 protein inhibitors (i) a seed sequence complementary to at least a portion of WGUWHWWA (ii) an adenine or uracil base 5' to the seed sequence, and (iii) a scrambled sequence 3' to the seed sequence The composition for use in treating an inflammatory disease according to claim 15, comprising a 19-25 base long miRNA having a sequence consisting of the above and a full-length GC content of 40-60%.

19. A composition for use in treating obesity, comprising as an active ingredient an inhibitor of ZCCHC24 protein or a drug that suppresses the expression of the ZCCHC24 gene.

20. A composition for use in treating obesity as described in claim 19, wherein the drug that suppresses the expression of the ZCCHC24 gene is a Wee1 / Chk1 inhibitor.

21. 20. A composition for use in treating obesity according to claim 19, wherein the drug that suppresses the expression of the ZCCHC24 gene is a nucleic acid drug.

22. ZCCHC24 protein inhibitors (i) a seed sequence complementary to at least a portion of WGUWHWWA (ii) an adenine or uracil base 5' to the seed sequence, and (iii) a scrambled sequence 3' to the seed sequence 20. The composition for use in treating obesity according to claim 19, comprising a 19-25 base miRNA having a sequence consisting of the above and a full-length GC content of 40-60%.

23. A method for screening drugs for use in treating triple-negative breast cancer, inflammatory diseases, or obesity, comprising: (i) providing cells expressing ZCCHC24; (ii) contacting the cells with a test substance; (iii) measuring the expression level of ZCCHC24; and (iv) selecting a substance that reduces the expression level of ZCCHC24 A method comprising:

24. Use of an inhibitor of ZCCHC24 protein or a drug that suppresses expression of the ZCCHC24 gene in the manufacture of a pharmaceutical for the treatment and / or prevention of inflammatory diseases or obesity.

25. Use of an inhibitor of ZCCHC24 protein or a drug that suppresses expression of the ZCCHC24 gene (excluding Wee1 inhibitors) in the manufacture of a pharmaceutical for the treatment and / or prevention of triple-negative breast cancer.