WO2021075559A1 - Cell growth inhibitor or cell death inducer for cancer-associated fibroblasts - Google Patents

Cell growth inhibitor or cell death inducer for cancer-associated fibroblasts Download PDF

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WO2021075559A1
WO2021075559A1 PCT/JP2020/039145 JP2020039145W WO2021075559A1 WO 2021075559 A1 WO2021075559 A1 WO 2021075559A1 JP 2020039145 W JP2020039145 W JP 2020039145W WO 2021075559 A1 WO2021075559 A1 WO 2021075559A1
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foxo1
cafs
cancer
inhibitor
antibody
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French (fr)
Japanese (ja)
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義弘 目澤
拓洋 小山
朗 片倉
彰 折茂
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学校法人順天堂
学校法人東京歯科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a cell growth inhibitor or cell death inducer of cancer-related fibroblasts (Carcinoma-Associated Fibroblasts; CAFs) or activated fibroblasts.
  • cancer-related fibroblasts Carcinoma-Associated Fibroblasts; CAFs
  • activated fibroblasts Carcinoma-Associated Fibroblasts
  • Cancer-related fibroblasts are cancer-promoting activated fibroblasts that are rich in ⁇ -smooth muscle actin ( ⁇ -SMA) -positive myofibroblasts.
  • ⁇ -SMA smooth muscle actin
  • the present inventor has previously shown that CAFs constitutively activate the autocline signals of TGF- ⁇ and SDF-1 during cancer progression and maintain their cancer-promoting activated fibroblast potential. (Non-Patent Document 1).
  • FOXO1 forkheadbox protein O1
  • PDX1 pancreatic and duodenal homeobox 1
  • neurogenin 3 the regulation of NeuroD and MafA.
  • Non-Patent Document 2 It has also been reported that FOXO1 is recruited to the TGF- ⁇ -Smad complex and contributes to transcriptional regulation.
  • AS1842856 is known to have a therapeutic effect on myelogenous leukemia, but AS1708727 is known not to have a therapeutic effect on myelogenous leukemia (Patent Document 1).
  • An object of the present invention is to elucidate how FOXO1 interacts with CAFs and to provide an inhibitor for CAFs.
  • FOXO1 inhibitors such as anti-FOX1 antibody, shRNA, and AS1842856 act on CAFs, a decrease in myofibroblast ability, a decrease in inflammatory cytokine production ability, and a decrease in tumor promoting ability in CAFs are observed, and at the same time, a decrease in tumor promoting ability is observed.
  • control human normal fibroblasts did not cause such growth inhibition or cell death.
  • the present invention provides the following [1] to [8].
  • [1] A CAFs cell growth inhibitor or a CAFs cell death inducer containing a FOXO1 inhibitor as an active ingredient.
  • FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  • FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  • the FOXO1 inhibitor according to [5] which is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  • a method for inhibiting cell growth of CAFs or a method for inducing cell death of CAFs which comprises administering an effective amount of a FOXO1 inhibitor.
  • the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  • the present invention it is possible to provide a new drug having a cell growth inhibitory action and a cell death inducing action on CAFs which are cancer-promoting activated fibroblasts.
  • FOXO1-positive myofibroblasts increased in patient breast cancer tissue a. Immunostaining of sections prepared from non-cancerous and cancerous parts of human breast cancer tissue using anti-FOXO1 antibody. FOXO1 is positively detected in Alpha-smooth muscle actin ( ⁇ -SMA) -positive myofibroblasts (arrow). Scale bar, 50 ⁇ m b. Comparison of the proportion of FOXO1-positive fibroblasts in non-cancerous and cancerous areas in 9 breast cancer patients. Immunohistochemistry with anti-FOXO1 antibody in the non-cancerous and cancerous areas resected from the same patient significantly increased the proportion of FOXO1-positive fibroblasts in the cancerous areas compared to the non-cancerous areas.
  • Scale bar 50 ⁇ m Decreased FOXO1 expression due to shRNA introduction in CAFs reduces myofibroblast ability.
  • B Western blot using myofibroblast markers in CAFs into which two different FOXO1-SHRNAs were introduced and antibodies specific for Smad2 / 3 activation. Suppression of FOXO1 expression by shRNA introduction in CAFs reduces the ability to produce inflammatory cytokines.
  • Suppression of FOXO1 activity in CAFs treated with AS1842856 reduces myofibroblast capacity.
  • An asterisk indicates that there is a significant difference in the student's t-test as compared with CAFs after DMSO treatment.
  • Suppression of FOXO1 activity in CAFs treated with AS1842856 reduces myofibroblast capacity and TGF-b-Smad2 / 3 signal activation over time.
  • Western blot analysis using antibodies against various genes in CAFs treated with AS1842856 Suppression of FOXO1 activity in CAFs treated with AS1842856 reduces the ability to produce inflammatory cytokines.
  • CAFs treated with DMSO and AS1842856 (1.0 ⁇ m) by Incute TM over time Asterisks indicate that there is a significant difference compared to DMSO-treated CAFs. Significant suppression of cell proliferation and increased apoptosis in CAFs treated with AS1842856.
  • Expression of FOXO1 is essential for the ability of CAFs to promote cancer cell growth in vivo.
  • Control fibroblasts and CAFs into which GFP-SHRNA or FOXO1-SHRNA was introduced were co-transplanted subcutaneously with DCIS cancer cells and immunodeficient mice. Then, the volume (a) and weight (b) of the formed cancer were measured over time. Compared with the control GFP-SHRNA, CAFs into which FOXO1-SHRNA was introduced significantly suppressed the volume and weight of DICS cancer formed in mice.
  • the CAFs cell growth inhibitor or CAFs cell death inducer of the present invention contains a FOXO1 inhibitor as an active ingredient.
  • FOXO1 is present in various organs, and in the liver, it controls systemic glucose metabolism through the regulation of gluconeogenic enzymes. In the pancreas, it regulates the new generation of ⁇ cells. Then, the FOXO1 inhibitor is known to suppress gluconeogenesis and lower the blood glucose level in a diabetic state. However, the association between FOXO1 inhibitors and CAFs has not been clearly shown.
  • FOXO1 inhibitor examples include anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, and the following AS1842856 and AS1708727.
  • AS1842856 and AS1708727 are already known compounds, can be produced by a known method, and commercially available products can also be used.
  • Anti-FOXO1 antibodies include monoclonal and polyclonal antibodies, as well as antibody variants and derivatives such as antibodies and T-cell receptor fragments that retain the ability to specifically bind antigenic determinants.
  • the type of anti-FOXO1 antibody is not particularly limited, and is artificial for the purpose of reducing heterologous antigenicity to humans, mouse antibody, human antibody, rat antibody, rabbit antibody, sheep antibody, camel antibody, triantibody, etc.
  • a recombinant antibody for example, a chimeric antibody, a humanized antibody, or the like, which is specifically modified, can be appropriately used. Recombinant antibodies can be produced using known methods.
  • a chimeric antibody is an antibody consisting of a heavy chain and a variable region of a light chain of a non-human mammal, for example, a mouse antibody and a constant region of a heavy chain and a light chain of a human antibody, and is a DNA encoding the variable region of a mouse antibody.
  • a humanized antibody also referred to as a restored human antibody, is obtained by transplanting a complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody, into a complementarity determining region of a human antibody.
  • CDR complementarity determining region
  • oligos prepared so as to have an overlapping portion at the end of a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody. It is synthesized from nucleotides by the PCR method. It is obtained by ligating the obtained DNA with the DNA encoding the human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. EP 239400, International Patent Application Publication No. See WO 96/02576).
  • the FR of the human antibody linked via the CDR is selected so that the complementarity determining region forms a good antigen-binding site.
  • amino acids in the framework regions of the variable region of the antibody may be substituted such that the complementarity determining regions of the reconstituted human antibody form the appropriate antigen binding site (Sato, K. et al., Cancer). Res, 1993, 53, 851-856.).
  • the siRNA of FOXO1 may be a low molecular weight double-stranded RNA consisting of 21-23 base pairs, which is involved in PNA interference (RNAi) and can suppress gene expression in a sequence-specific manner by disrupting the mRNA. ..
  • RNAi PNA interference
  • the shRNA of FOXO1 may be one that is transcribed from a plasmid, forms a hairpin structure, and is processed to become siRNA.
  • FOXO1 is highly expressed in myofibroblasts and cultured CAFs in patient breast cancer tissues, as shown in Examples below.
  • FOXO1 inhibitors such as anti-FOX1 antibody, shRNA, and AS1842856 act on CAFs
  • the myofibroblast characteristics of CAFs are attenuated, the ability of CAFs to produce inflammatory cytokines is attenuated, and the cell proliferation of CAFs is remarkable.
  • inhibition and cell death induction occur, and that the tumor-promoting ability of CAFs is attenuated, and that such growth inhibition and cell death do not occur in control human normal fibroblasts. Therefore, since the FOXO1 inhibitor is useful as a cell growth inhibitor and a cell death inducer for CAFs, it is suggested that it may be useful as a cancer progression inhibitor in which CAFs coexist.
  • the cancer to be treated by the medicament of the present invention is a cancer in which CAFs coexist, and specifically, epithelial cancer (respiratory cancer, digestive organ cancer, urogenital cancer, secretory system cancer, skin cancer, etc.). , Middle dermatoma, breast cancer, sarcoma, central nervous system tumor, peripheral nervous system tumor and the like.
  • respiratory cancer include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.).
  • Examples of gastrointestinal cancer include esophageal cancer, gastric cancer, duodenal cancer, liver cancer, biliary tract cancer (cholangiocarcinoma / cholangiocarcinoma, etc.), pancreatic cancer, colon cancer, colorectal cancer (colon cancer, rectal cancer, etc.) and the like. ..
  • Examples of urogenital cancer include ovarian cancer, uterine cancer (cervical cancer, endometrial cancer, etc.), renal cancer, bladder cancer, prostate cancer, testicular tumor and the like.
  • Examples of secretory cancers include neuroendocrine tumors.
  • mesothelioma examples include pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma.
  • sarcoma examples include gastrointestinal stromal tumors, bone / soft tissue tumors, and the like.
  • central nervous system tumors examples include brain tumors and the like.
  • Peripheral nervous system tumors include, for example, malignant schwannoma. Of these, gastrointestinal stromal tumor, breast cancer, lung cancer, gastric cancer, prostate cancer, ovarian cancer and colon cancer are preferable, and among lung cancers, non-small cell lung cancer is preferable.
  • the route of administration of the medicament of the present invention is not particularly limited, and the drug can be administered orally or parenterally.
  • Parenteral administration includes intravenous, intramuscular, subcutaneous or intradermal injection, inhalation, rectal, intranasal administration, external administration and the like.
  • the FOXO1 inhibitor which is an active ingredient may be administered to a patient as it is, but it is preferably administered in the form of a pharmaceutical composition containing the active ingredient and a pharmaceutically acceptable additive. Should be.
  • Pharmaceutically acceptable additives include, for example, excipients, disintegrants or disintegrants, binders, lubricants, coatings, dyes, diluents, bases, solubilizers or solubilizers, etc. Tensioning agents, pH regulators, stabilizers, propellants, adhesives and the like can be used.
  • preparations suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, syrups and the like
  • preparations suitable for parenteral administration include, for example, injection.
  • Agents, drops, suppositories, inhalants or external preparations including patches, ointments, creams, gels, lotions, sprays, etc.
  • preparations suitable for parenteral administration include, for example, injection.
  • Agents, drops, suppositories, inhalants or external preparations including patches, ointments, creams, gels, lotions, sprays, etc.
  • Formulations suitable for oral administration include, as additives, excipients such as, for example, glucose, lactose, D-mannitol, starch, or crystalline cellulose; disintegrants or disintegrant aids such as carboxymethyl cellulose, starch, or carboxymethyl cellulose calcium.
  • Binders such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, or gelatin; Lubricants such as magnesium stearate or talc; Coating agents such as hydroxypropylmethyl cellulose, sucrose, polyethylene glycol or titanium oxide; Vaseline, liquid paraffin , Polyethylene glycol, gelatin, kaolin, glycerin, purified water, or a base such as hard fat can be used.
  • Formulations suitable for injection and infusion include solubilizers or solubilizers that may constitute aqueous or time-dissolving injectables such as distilled water for injection, saline, propylene glycol; glucose, sodium chloride, D- Isotonic agents such as mannitol and glycerin; pharmaceutical additives such as pH adjusters such as inorganic acids, organic acids, inorganic bases or organic bases can be used.
  • Suitable preparations for suppositories include, for example, bases such as polyethylene glycol, lanolin, cacao butter, fatty acid triglycerides, and optionally additives such as surfactants such as nonionic surfactants. ..
  • bases As a preparation suitable for an ointment, commonly used bases, stabilizers, wetting agents, preservatives and the like are used as necessary.
  • the base include liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like.
  • the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.
  • preparations suitable for patches include those obtained by applying the ointment, cream, gel, paste, or the like to a normal support by a conventional method.
  • a woven fabric made of cotton, rayon, or chemical fiber, a non-woven fabric, a film such as soft vinyl chloride, polyethylene, or polyurethane, or a foam sheet is suitable.
  • the other anticancer agent that can be used in combination is not particularly limited as long as it is an anticancer agent having an anticancer effect, but an anticancer agent having tumor cytotoxicity is particularly preferable in terms of obtaining a synergistic effect.
  • Examples of the other anticancer agent include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting agents, hormonal agents, biological preparations and the like.
  • Examples of the alkylating agent include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.
  • Antimetabolites include, for example, enocitabine, carmofur, capecitabine, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, cytarabine, cytarabine ocphosphat, neralabine, fluorouracil, fludalabine, pemetrexed
  • examples thereof include cladribine, doxiflulysin, hydroxycarbamide, mercaptopurine and the like.
  • the microtubule inhibitor include alkaloid anticancer agents such as vincristine and taxane anticancer agents such as docetaxel and paclitaxel.
  • Antibiotics Anticancer agents include, for example, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idarubicin, pyrarubicin, pepromycin, mitoxantrone, amurubicin, dinostatin stimalamar and the like.
  • the topoisomerase inhibitor include CPT-11 having a topoisomerase I inhibitory action, irinotecan, nogitecan, and etoposide and sobzoxane having a topoisomerase II inhibitory action.
  • platinum preparation examples include cisplatin, nedaplatin, oxaliplatin, carboplatin and the like.
  • Hormonal agents include, for example, dexamethasone, finasteride, tamoxifen, astrosol, exemestane, ethinyl estradiol, chlormaginone, goserelin, bicalutamide, flutamide, bredonizolone, leuprorelin, letrozole, estramustine, toremifene, phosfestol, mitotan, Examples thereof include methyltestosterone, medroxyprogesterone, and mepitiostane.
  • biologics include interferon ⁇ , ⁇ and ⁇ , interleukin 2, ubenimex, dried BCG and the like.
  • Molecular-targeted drugs include, for example, rituximab, alemtuzumab, trastuzumab, cetuximab, panitummab, imatinib, dasatinib, nilotinib, gefitinib, elrotinib, temsirolimus, bebashizumab, temshirolimus, bebashizumab, vEGF trap, , Ibritumomab tiuxetan, Tamibarotene, Tretinoin and the like.
  • human epithelial growth factor receptor 2 inhibitor epithelial growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epithelial growth factor tyrosine kinase inhibitor, mTOR inhibitor, vascular endothelial growth factor receptor 2 Inhibitors targeting angiogenesis such as inhibitors ( ⁇ -VEGFR-2 antibody), various tyrosine kinase inhibitors such as MAP kinase inhibitors, cytokine targeting inhibitors, proteasome inhibitors, antibodies-anticancer Molecular-targeted drugs such as drug formulations can also be used.
  • alkylating agents characterized by cytotoxic activity, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting agents and the like are particularly preferable.
  • gemcitabine 5-FU, CPT-11, etoposide, cisplatin, oxaliplatin, paclitaxel, docetaxel, dacarbazine, doxorubicin, bevacizumab, cetuximab, antivascular endothelial growth factor receptor 2 inhibitor antibody, epithelial growth factor tyrosine.
  • Kinase inhibitors and the like are particularly preferred.
  • the dose of the medicament of the present invention can be appropriately selected according to various conditions such as the progress of the disease or the degree of symptoms, the age and body weight of the patient, and for example, in the case of oral administration, 1 mg to 300 mg per day is 1 mg. It can be administered in divided doses of about 3 times.
  • Example 1 It was unclear whether FOXO1 was expressed in CAFs of human breast cancer. Therefore, we prepared sections from human breast cancer and performed immunohistochemical staining using anti-FOXO1 antibody. FOXO1-positive was significantly observed in ⁇ -SMA-positive CAFs in the cancerous region. However, no FOXO1-positive cells were found in the non-cancerous fibroblasts of the same patient (Fig. 1a). To generalize this observation, breast cancer sections of 9 more patients were checked (Table 1). Of note, the proportion of FOXO1-positive fibroblasts was significantly increased in CAFs of all breast cancer patients examined (Fig. 1b). Expression at the FOXO1 mRNA level was also increased 4-fold in CAFs compared to the control (Fig. 2a).
  • Example 2 The myofibroblast ability and inflammatory cytokine production ability of CAFs by suppressing FOXO1 expression were investigated. Two different shRNAs capable of significantly suppressing the expression of FOXO1 mRNA and protein were introduced into fibroblasts. CAF into which FOXO1-SHRNA was introduced showed a significant reduction in the expression of myofibroblast markers such as ⁇ -SMA, TGF-b1, SDF-1 mRNA, and pSmad2 protein (Fig. 3). Furthermore, we confirmed the expression level of inflammatory cytokines that contribute to the activation of CAFs and the recruitment of inflammatory immune cells into cancer.
  • Example 3 The specificity of AS1842856 was examined by the FOXO1 luciferase reporter assay using HEK293T cells into which the FOXO1 expression vector or control empty vector was introduced.
  • the cDNA3.11-FOXO1 cDNA vector was introduced into HEK293T cells, AS1842856 (concentration 1.0 ⁇ M) was added 24 hours later, and the transcriptional activity of FOXO1 was measured by Dual-Luciferase TM Reporter Assay System 24 hours later.
  • FIG. 5 it was shown that AS1842856 remarkably suppresses the transcriptional activity of FOXO1.
  • Example 4 It was investigated whether suppression of FOXO1 activity by AS1842856 treatment of CAFs reduced myofibroblast ability.
  • Real-time PCR using a marker gene-specific primer for myofibroblasts was performed using CAFs treated with AS1842856 for 12 hours.
  • CAFs treated with AS1842856 showed a significant decrease in myofibroblast markers.
  • Example 5 Western blot analysis using antibodies against various genes in CAFs treated with AS1842856 was performed. As a result, as shown in FIG. 7, in CAFs treated with AS1842856, the expression of FOXO1, phosphorylated FOXO1, ⁇ -SMA and phosphorylated Smad2 / 3 was observed to be attenuated with time. This result suggests that activation of FOXO1 is essential for the maintenance of CAFs myofibroblast ability and TGF- ⁇ -Smad2 / 3 signal.
  • Example 6 Real-time PCR analysis of CAFs treated with AS18428568 (concentration 1.0 ⁇ M) for 12 hours was performed. As a result, as shown in FIG. 8, the expression of inflammatory cytokines such as CXCL1, CXCL2, IL-1 ⁇ , IL1 ⁇ , IL-8, and LIF in CAFs was remarkably suppressed 24 hours after the treatment with AS1842856. From these results, it was found that treatment with AS18428568, which is a FOXO1 inhibitor, suppresses the myofibroblast ability and inflammatory cytokine production ability of CAFs.
  • AS18428568 which is a FOXO1 inhibitor
  • Example 7 Suppression of CAFs proliferation and induction of cell death by suppression of FOXO1 activity Since the phenotype of activated CAFs was attenuated when FOXO1-SHRNA was introduced, inhibition of FOXO1 activity affects the proliferation and viability of these cells. I checked whether to give it. Compared to the effect of DMSO treatment of the control, CAFs treated with AS1842856 for 72 hours markedly suppressed cell proliferation and increased cell death (FIGS. 9a, 9b), and also controlled human normal mammary fibroblasts. Almost no of these phenomena were observed in cells (Fig. 9b).
  • Example 8 Since FOXO1 is required for promotion of activated fibroblast ability, promotion of proliferation and suppression of cell death of CAFs, it was investigated whether expression of FOXO1 is also necessary for the ability of CAFs to promote migration and proliferation of tumor cells.
  • a Boyden chamber cell migration assay was performed using ductal carcinoma in situ (DCIS) cells from non-invasive human ductal carcinoma in situ. DCIS cells treated with culture supernatants of CAFs introduced with FOXO1-SHRNA showed a significant reduction in migration ability compared to treatment with culture supernatants of CAFs introduced with GFP-SHRNA (Fig.). 11).
  • FOXO1 expression in CAFs affects the in vivo growth of nearby cancer cells.
  • DCIS cells and human normal mammary fibroblasts or CAFs into which GFP-SHRNA or FOXO1-shRNA were introduced were subcutaneously transplanted into immunodeficient nude mice.
  • the volume and weight of tumors containing CAFs whose expression of FOXO1 was suppressed by the introduction of FOXO1-SHRNA was significantly suppressed as compared with the effect of control GFP-SHRNA (Fig. 12). From the above results, it was clarified that the expression of FOXO1 in CAFs is essential for the migration ability and cancer growth ability of nearby DCIS cells.

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Abstract

The present invention elucidates the interaction of FOXO1 in CAFs, and provides a novel therapeutic drug.　This cell growth inhibitor for CAFs or this cell death inducer for CAFs contains an FOXO1 inhibitor as an active ingredient.

Description

癌関連線維芽細胞の細胞増殖阻害剤又は細胞死誘導剤Cancer-related fibroblast cell growth inhibitor or cell death inducer
　本発明は、癌関連線維芽細胞（Ｃａｒｃｉｎｏｍａ－Ａｓｓｏｃｉａｔｅｄ　Ｆｉｂｒｏｂｌａｓｔｓ；ＣＡＦｓ）又は活性化線維芽細胞の細胞増殖阻害剤又は細胞死誘導剤に関する。 The present invention relates to a cell growth inhibitor or cell death inducer of cancer-related fibroblasts (Carcinoma-Associated Fibroblasts; CAFs) or activated fibroblasts.
　癌関連線維芽細胞は、α－ｓｍｏｏｔｈ ｍｕｓｃｌｅ ａｃｔｉｎ（α－ＳＭＡ）陽性の筋線維芽細胞を豊富に含んだ癌促進性の活性化線維芽細胞である。本発明者は、以前ＣＡＦｓが癌進展過程においてＴＧＦ－β及びＳＤＦ－１のオートクラインシグナルを恒常的に活性化し、これらの癌促進性の活性化線維芽細胞能を維持していることを明らかにした（非特許文献１）。 Cancer-related fibroblasts are cancer-promoting activated fibroblasts that are rich in α-smooth muscle actin (α-SMA) -positive myofibroblasts. The present inventor has previously shown that CAFs constitutively activate the autocline signals of TGF-β and SDF-1 during cancer progression and maintain their cancer-promoting activated fibroblast potential. (Non-Patent Document 1).
　一方、Ｃａｅｎｏｒｈａｂｄｉｔｉｓ　ｅｌｅｇａｎｓ　Ｄａｆ－１タンパク質の哺乳類ホモログであるフォークヘッドボックスタンパク質Ｏ１（ＦＯＸＯ１）は、ＰＥＰＣＫ（ホスホエノールピルビン酸カルボキシキナーゼ）及びｇ６Ｐａｓｅ（グルコース６－ホスファターゼ）を介して肝臓の糖新生を促進することが示されており、ＦＯＸＯ１阻害剤による治療は糖新生を抑制し、糖尿病状態の血糖値を低下させる。膵臓では、ＦＯＸＯ１はＰＤＸ１（膵臓及び十二指腸のホメオボックス１）とニューロゲニン３の転写、及びＮｅｕｒｏＤとＭａｆＡの調節を制御することにより、β細胞ストレス耐性を調節し、インスリン発現を調節する。また ＦＯＸＯ１はＴＧＦ－β－Ｓｍａｄ複合体にリクルートされ、転写調節に寄与すると報告されている（非特許文献２）。
　ＦＯＸＯ１阻害剤のうち、ＡＳ１８４２８５６は骨髄性白血病治療作用を有するが、ＡＳ１７０８７２７は骨髄性白血病治療作用を示さないことが知られている（特許文献１）。 On the other hand, forkheadbox protein O1 (FOXO1), which is a mammalian homologue of Caenorhabditis elegans Daf-1, promotes hepatic gluconeogenesis via PEPCK (phosphoenolpyruvate carboxykinase) and g6Pase (glucose 6-phosphatase). It has been shown that treatment with FOXO1 inhibitors suppresses gluconeogenesis and lowers blood glucose levels in diabetic conditions. In the pancreas, FOXO1 regulates β-cell stress tolerance and regulates insulin expression by controlling the transcription of PDX1 (pancreatic and duodenal homeobox 1) and neurogenin 3 and the regulation of NeuroD and MafA. It has also been reported that FOXO1 is recruited to the TGF-β-Smad complex and contributes to transcriptional regulation (Non-Patent Document 2).
Among the FOXO1 inhibitors, AS1842856 is known to have a therapeutic effect on myelogenous leukemia, but AS1708727 is known not to have a therapeutic effect on myelogenous leukemia (Patent Document 1).
特開２０１６－６５０５５号公報Japanese Unexamined Patent Publication No. 2016-65055
　しかしながら、ＣＡＦｓとＦＯＸＯ１との関係はまだ解明されていない。
　本発明の課題は、ＣＡＦｓにおいてＦＯＸＯ１がどのような相互作用をしているのかを解明し、ＣＡＦｓに対する阻害剤を提供することにある。 However, the relationship between CAFs and FOXO1 has not yet been elucidated.
An object of the present invention is to elucidate how FOXO1 interacts with CAFs and to provide an inhibitor for CAFs.
　そこで、本発明者は、ＣＡＦｓにおけるＦＯＸＯ１の役割を解明すべく種々検討したところ、ＦＯＸＯ１は癌関連組織中のＣＡＦｓに高発現していることを見出した。また、抗ＦＯＸＯ１抗体、ｓｈＲＮＡ、ＡＳ１８４２８５６などのＦＯＸＯ１阻害剤をＣＡＦｓに作用させると、ＣＡＦｓにおける筋線維芽細胞能の低下や炎症性サイトカイン産生能の低下や腫瘍促進能の低下が観察されると共に、顕著なＣＡＦｓの細胞増殖阻害及び細胞死誘導が生じた。重要な事に、コントロールのヒト正常線維芽細胞ではこのような増殖阻害や細胞死が生じないことを見出した。 Therefore, the present inventor conducted various studies to elucidate the role of FOXO1 in CAFs, and found that FOXO1 is highly expressed in CAFs in cancer-related tissues. In addition, when FOXO1 inhibitors such as anti-FOX1 antibody, shRNA, and AS1842856 act on CAFs, a decrease in myofibroblast ability, a decrease in inflammatory cytokine production ability, and a decrease in tumor promoting ability in CAFs are observed, and at the same time, a decrease in tumor promoting ability is observed. Significant inhibition of cell proliferation and induction of cell death of CAFs occurred. Importantly, we found that control human normal fibroblasts did not cause such growth inhibition or cell death.
　すなわち、本発明は次の［１］～［８］を提供するものである。
［１］ＦＯＸＯ１阻害剤を有効成分とするＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤。
［２］ＦＯＸＯ１阻害剤が、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである［１］記載のＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤。
［３］ＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤製造のための、ＦＯＸＯ１阻害剤の使用。
［４］ＦＯＸＯ１阻害剤が、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである［３］記載の使用。
［５］ＣＡＦｓの細胞増殖を阻害又はＣＡＦｓの細胞死を誘導するための、ＦＯＸＯ１阻害剤。
［６］抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである［５］記載のＦＯＸＯ１阻害剤。
［７］ＦＯＸＯ１阻害剤の有効量を投与することを特徴とするＣＡＦｓの細胞増殖阻害方法又はＣＡＦｓの細胞死誘導方法。
［８］ＦＯＸＯ１阻害剤が、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである［７］記載の方法。 That is, the present invention provides the following [1] to [8].
[1] A CAFs cell growth inhibitor or a CAFs cell death inducer containing a FOXO1 inhibitor as an active ingredient.
[2] The CAFs cell growth inhibitor or CAFs cell death inducer according to [1], wherein the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
[3] Use of a FOXO1 inhibitor for producing a cell growth inhibitor of CAFs or a cell death inducer of CAFs.
[4] The use according to [3], wherein the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
[5] A FOXO1 inhibitor for inhibiting cell proliferation of CAFs or inducing cell death of CAFs.
[6] The FOXO1 inhibitor according to [5], which is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
[7] A method for inhibiting cell growth of CAFs or a method for inducing cell death of CAFs, which comprises administering an effective amount of a FOXO1 inhibitor.
[8] The method according to [7], wherein the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
　本発明によれば、癌促進性の活性化線維芽細胞であるＣＡＦｓに対する細胞増殖抑制作用及び細胞死誘導作用を有する新たな医薬が提供できる。 According to the present invention, it is possible to provide a new drug having a cell growth inhibitory action and a cell death inducing action on CAFs which are cancer-promoting activated fibroblasts.
患者乳癌組織で増加したＦＯＸＯ１陽性筋線維芽細胞　ａ．抗ＦＯＸＯ１抗体を用いたヒト乳がん組織の非癌部および癌部から調製された切片の免疫染色。Ａｌｐｈａ－ｓｍｏｏｔｈ　ｍｕｓｃｌｅ　ａｃｔｉｎ（α－ＳＭＡ）陽性の筋線維芽細胞にＦＯＸＯ１が陽性に検出されている（矢印）。スケールバー、５０μｍ　ｂ．９人の乳癌患者における非癌部および癌部におけるＦＯＸＯ１陽性の線維芽細胞の比率の比較。同一の患者から切除された非癌部および癌部領域の抗ＦＯＸＯ１抗体による免疫組織染色により、非癌部と比較し癌部においてはＦＯＸＯ１陽性の線維芽細胞の比率が有意に増加していることが明らかになった。アスタリスクは、ｓｔｕｄｅｎｔ’ｓ　ｔ検定にて両群で有意差があることを示す。FOXO1-positive myofibroblasts increased in patient breast cancer tissue a. Immunostaining of sections prepared from non-cancerous and cancerous parts of human breast cancer tissue using anti-FOXO1 antibody. FOXO1 is positively detected in Alpha-smooth muscle actin (α-SMA) -positive myofibroblasts (arrow). Scale bar, 50 μm b. Comparison of the proportion of FOXO1-positive fibroblasts in non-cancerous and cancerous areas in 9 breast cancer patients. Immunohistochemistry with anti-FOXO1 antibody in the non-cancerous and cancerous areas resected from the same patient significantly increased the proportion of FOXO1-positive fibroblasts in the cancerous areas compared to the non-cancerous areas. Became clear. An asterisk indicates that there is a significant difference between the two groups in the student's t-test. ＣＡＦｓにおけるＦＯＸＯ１ｍＲＮＡおよび蛋白の増加。　ａ．ＦＯＸＯ１遺伝子に特異的なプライマーを使用した、ヒト正常乳腺線維芽細胞（Ｃｏ　ｎｔ．　ｆ．）およびＣＡＦｓのリアルタイムＰＣＲ。アスタリスクは、ｓｔｕｄｅｎ　ｔ’ｓ　ｔ検定にてヒト正常乳腺線維芽細胞と比較して有意差があることを示す。　ｂ．線維芽細胞より抽出された全細胞エキストラクトおよび核エキストラクトにおける抗ＦＯＸＯ１抗体を使用したウエスタンブロット。　ｃ．抗ＦＯＸＯ１抗体を使用したヒト正常乳腺線維芽細胞とＣＡＦｓの蛍光免疫染色。スケールバー、５０μｍIncreased FOXO1 mRNA and protein in CAFs. A. Real-time PCR of human normal mammary fibroblasts (Cont. F.) and CAFs using primers specific for the FOXO1 gene. Asterisk indicates that there is a significant difference compared to human normal mammary fibroblasts in the student's t-test. B. Western blot using anti-FOXO1 antibody in whole cell extract and nuclear extract extracted from fibroblasts. C. Fluorescent immunostaining of human normal mammary fibroblasts and CAFs using anti-FOXO1 antibody. Scale bar, 50 μm ＣＡＦｓにおけるｓｈＲＮＡ導入によるＦＯＸＯ１発現低下は筋線維芽細胞能を低下させる。　ａ．２種類の異なるＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦｓにおける筋線維芽細胞のマーカ―遺伝子に特異的なプライマ―を用いたｒｅａｌ－ｔｉｍｅ　ＰＣＲ。アスタリスクは、ｓｔｕｄｅｎｔ'ｓ　ｔ検定にて対象のＧＦＰ－ｓｈＲＮＡが導入されたＣＡＦｓと比較して有意差があることを示す。　ｂ．２種類の異なるＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦｓにおける筋線維芽細胞のマーカ―およびＳｍａｄ２／３活性化に特異的な抗体を用いたウエスタンブロット。Decreased FOXO1 expression due to shRNA introduction in CAFs reduces myofibroblast ability. A. Real-time PCR using a marker gene-specific primer for myofibroblasts in CAFs into which two different FOXO1-SHRNAs have been introduced. The asterisk indicates that there is a significant difference in the student's t-test as compared with the CAFs into which the GFP-SHRNA of the subject was introduced. B. Western blot using myofibroblast markers in CAFs into which two different FOXO1-SHRNAs were introduced and antibodies specific for Smad2 / 3 activation. ＣＡＦｓにおけるｓｈＲＮＡ導入によるＦＯＸＯ１発現の抑制は炎症性サイトカインの産生能を低下させる。２種類の異なるＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦｓにおける種々の炎症性サイトカインの遺伝子に特異的なプライマ―を用いたｒｅａｌ－ｔｉｍｅ　ＰＣＲ。アスタリスクは、ｓｔｕｄｅｎｔ’ｓ　ｔ検定にて対象のＧＦＰ－ｓｈＲＮＡが導入されたＣＡＦｓと比較して有意差があることを示す。Suppression of FOXO1 expression by shRNA introduction in CAFs reduces the ability to produce inflammatory cytokines. Real-time PCR using gene-specific primers for various inflammatory cytokines in CAFs into which two different FOXO1-SHRNAs have been introduced. The asterisk indicates that there is a significant difference in the student's t-test as compared with the CAFs into which the GFP-SHRNA of the subject was introduced. ＦＯＸＯ１発現ベクターあるいはコントロールの空ベクターが導入されたＨＥＫ２９３Ｔ細胞を使用したＦＯＸＯ１ルシフェレースレポーターアッセイによるＡＳ１８４２８５６（ＡＳ）の特異性の検討結果。アスタリスクは、ＦＯＸＯ１ベクターの導入後にＤＭＳＯが処理されたグループと比較して、ｓｔｕｄｅｎｔ’ｓ　ｔ検定にて有意にレポーター活性が低下していることを示す。Results of examining the specificity of AS1842856 (AS) by the FOXO1 luciferase reporter assay using HEK293T cells into which the FOXO1 expression vector or control empty vector was introduced. An asterisk indicates that the reporter activity is significantly reduced in the student's t-test as compared to the group treated with DMSO after the introduction of the FOXO1 vector. ＡＳ１８４２８５６で処理されたＣＡＦｓにおけるＦＯＸＯ１活性の抑制は、筋線維芽細胞能を低下させる。ＡＳ１８４２８５６で１２時間処理したＣＡＦｓの筋線維芽細胞のマーカ―遺伝子に特異的なプライマ―を用いたｒｅａｌ－ｔｉｍｅ　ＰＣＲ。アスタリスクは、ｓｔｕｄｅｎｔ’ｓ　ｔ検定にてＤＭＳＯ処理後のＣＡＦｓと比較して有意差があることを示す。Suppression of FOXO1 activity in CAFs treated with AS1842856 reduces myofibroblast capacity. Real-time PCR using a marker gene-specific primer for CAFs myofibroblasts treated with AS1842856 for 12 hours. An asterisk indicates that there is a significant difference in the student's t-test as compared with CAFs after DMSO treatment. ＡＳ１８４２８５６で処理されたＣＡＦｓにおけるＦＯＸＯ１活性の抑制は、筋線維芽細胞能やＴＧＦ－ｂ－Ｓｍａｄ２／３シグナル活性化を経時的に低下させる。ＡＳ１８４２８５６で処理を行なったＣＡＦｓにおける種々の遺伝子に対する抗体を使用したウエスタンブロット解析。Suppression of FOXO1 activity in CAFs treated with AS1842856 reduces myofibroblast capacity and TGF-b-Smad2 / 3 signal activation over time. Western blot analysis using antibodies against various genes in CAFs treated with AS1842856. ＡＳ１８４２８５６で処理されたＣＡＦｓにおけるＦＯＸＯ１活性の抑制は、炎症性サイトカインの産生能を低下させる。ＡＳ１８４２８５６で１２時間処理したＣＡＦｓの炎症性サイトカインの遺伝子に特異的なプライマ―を用いたｒｅａｌ－ｔｉｍｅ　ＰＣＲ。アスタリスクは、ｓｔｕｄｅｎｔ’ｓ　ｔ検定にてＤＭＳＯ処理後のＣＡＦｓと比較して有意差があることを示す。Suppression of FOXO1 activity in CAFs treated with AS1842856 reduces the ability to produce inflammatory cytokines. Real-time PCR using a gene-specific primer for CAFs inflammatory cytokines treated with AS1842856 for 12 hours. An asterisk indicates that there is a significant difference in the student's t-test as compared with CAFs after DMSO treatment. ＡＳ１８４２８５６で処理されたＣＡＦｓにおける顕著な細胞増殖抑制。コントロールの正常乳腺線維芽細胞ではＡＳ１８４２８５６処理で有意な変化が観察されない。　ａ．ＤＭＳＯおよびＡＳ１８４２８５６（１．０μＭ）で７２時間処理した細胞の形状。ＡＳ１８４２８５６で処理されたＣＡＦｓにおける細胞死を矢印で示す。　ｂ．ＩｎｃｕｃｙｔｅＴＭによるＤＭＳＯおよびＡＳ１８４２８５６（１．０μｍ）で処理されたヒト正常乳腺線維芽細胞（Ｃｏｎｔ．　ｆ．）およびＣＡＦｓの経時的な細胞増殖能の測定。アスタリスクは、ＤＭＳＯ処理されたＣＡＦｓと比較して有意差があることを示す。Significant suppression of cell proliferation in CAFs treated with AS1842856. No significant changes were observed with AS1842856 treatment in control normal mammary fibroblasts. a. Cell shape treated with DMSO and AS1842856 (1.0 μM) for 72 hours. Cell death in CAFs treated with AS1842856 is indicated by arrows. b. Measurement of cell proliferation ability of human normal mammary fibroblasts (Cont. F.) and CAFs treated with DMSO and AS1842856 (1.0 μm) by Incute TM over time. Asterisks indicate that there is a significant difference compared to DMSO-treated CAFs. ＡＳ１８４２８５６で処理されたＣＡＦｓにおける顕著な細胞増殖抑制およびアポトーシスの増加。　ａ．還元型ニコチンアミドアデニンジヌクレオチド測定によるＤＭＳＯおよびＡＳ１８４２８５６（１．０μＭ）で７２時間処理された細胞の生細胞数の測定。　ｂ．乳酸デヒドロゲナーゼ活性の測定によるＤＭＳＯおよびＡＳ１８４２８５６（１．０μＭ）で７２時間処理された細胞の死細胞数の測定。　ｃ．ｃａｓｐａｓｅ　３／７　Ｇｌｏ　ａｓｓａｙによるＤＭＳＯおよびＡＳ１８４２８５６（１．０μＭ）で７２時間処理された細胞のアポトーシスの測定。アスタリスクは、ＤＭＳＯ処理後のＣＡＦｓと比較して有意差があることを示す。Significant suppression of cell proliferation and increased apoptosis in CAFs treated with AS1842856. A. Measurement of viable cell count of cells treated with DMSO and AS1842856 (1.0 μM) for 72 hours by reduced nicotinamide adenine dinucleotide measurement. B. Measurement of the number of dead cells in cells treated with DMSO and AS1842856 (1.0 μM) for 72 hours by measurement of lactate dehydrogenase activity. C. Measurement of apoptosis of cells treated with DMSO and AS1842856 (1.0 μM) for 72 hours by caspase 3/7 Glo assay. Asterisks indicate that there is a significant difference compared to CAFs after DMSO treatment. ＦＯＸＯ１の発現はＣＡＦｓによる癌細胞の遊走促進能に必須である。　ａ．Ｂｏｙｄｅｎチャンバー細胞遊走アッセイによるｄｕｃｔａｌ　ｃａｒｃｉｎｏｍａ　ｉｎ　ｓｉｔｕ（ＤＣＩＳ）由来ヒト乳癌細胞の遊走画像。異なるＦＯＸＯ１－ｓｈＲＮＡが導入されたｃｏｎｔｒｏｌ　ｆｉｂｒｏｂｌａｓｔｓおよびＣＡＦｓよりの培養上清がＤＣＩＳ癌細胞に６０時間処理後に、遊走した癌細胞がメイグリュンワルドギムザで染色されている。　b. 各群（ｎ=３）における腫瘍細胞の相対的な遊走能がグラフで示されている。アスタリスクは、ＧＦＰ－ｓｈＲＮＡが導入されたＣＡＦｓよりの培養上清が処理されたグループと比較して有意差があることを示す。Expression of FOXO1 is essential for the ability of CAFs to promote the migration of cancer cells. A. Migration images of human breast cancer cells derived from ductal carcinoma in situ (DCIS) by Boyden chamber cell migration assay. Culture supernatants from control fibroblasts and CAFs into which different FOXO1-SHRNAs have been introduced are treated with DCIS cancer cells for 60 hours, and then the migrating cancer cells are stained with Maygrünwald Giemsa. B. The relative migration ability of tumor cells in each group (n = 3) is shown in the graph. Asterisks indicate that there is a significant difference in culture supernatants from CAFs into which GFP-SHRNA has been introduced compared to the treated group. ＦＯＸＯ１の発現はＣＡＦｓのｉｎ ｖｉｖｏでの癌細胞増殖促進能に必須である。ＧＦＰ－ｓｈＲＮＡあるいはＦＯＸＯ１－ｓｈＲＮＡが導入されたｃｏｎｔｒｏｌ　ｆｉｂｒｏｂｌａｓｔｓやＣＡＦｓはＤＣＩＳ癌細胞と免疫不全マウスの皮下に共移植された。そして形成された癌の体積（ａ）や重量（ｂ）が経時的に測定された。コントロールのＧＦＰ－ｓｈＲＮＡと比較して、ＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦｓは、マウスに形成されたＤＩＣＳ癌の体積や重量を有意に抑制した。Expression of FOXO1 is essential for the ability of CAFs to promote cancer cell growth in vivo. Control fibroblasts and CAFs into which GFP-SHRNA or FOXO1-SHRNA was introduced were co-transplanted subcutaneously with DCIS cancer cells and immunodeficient mice. Then, the volume (a) and weight (b) of the formed cancer were measured over time. Compared with the control GFP-SHRNA, CAFs into which FOXO1-SHRNA was introduced significantly suppressed the volume and weight of DICS cancer formed in mice.
　本発明のＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤は、ＦＯＸＯ１阻害剤を有効成分とする。
　ＦＯＸＯ１は、各種臓器に存在しており、肝臓では、糖新生酵素の調節を介して全身の糖代謝を制御している。膵臓では、β細胞の新生などを調節している。そして、ＦＯＸＯ１阻害剤は、糖新生を抑制し、糖尿病状態の血糖値を低下させることが知られている。
　しかし、ＦＯＸＯ１阻害剤とＣＡＦｓとの関連は明確に示されていない。 The CAFs cell growth inhibitor or CAFs cell death inducer of the present invention contains a FOXO1 inhibitor as an active ingredient.
FOXO1 is present in various organs, and in the liver, it controls systemic glucose metabolism through the regulation of gluconeogenic enzymes. In the pancreas, it regulates the new generation of β cells. Then, the FOXO1 inhibitor is known to suppress gluconeogenesis and lower the blood glucose level in a diabetic state.
However, the association between FOXO1 inhibitors and CAFs has not been clearly shown.
　ＦＯＸＯ１阻害剤としては、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、次のＡＳ１８４２８５６及びＡＳ１７０８７２７が挙げられる。 Examples of the FOXO1 inhibitor include anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, and the following AS1842856 and AS1708727.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　ＡＳ１８４２８５６及びＡＳ１７０８７２７は、すでに公知の化合物であり、公知の方法で製造でき、市販品を使用することもできる。 AS1842856 and AS1708727 are already known compounds, can be produced by a known method, and commercially available products can also be used.
　抗ＦＯＸＯ１抗体には、モノクローナル抗体及びポリクローナル抗体、並びに抗原決定基に特異的に結合する能力を保持している抗体及びＴ－細胞レセプターフラグメント等の、抗体の変種及び誘導体が含まれる。
　また、抗ＦＯＸＯ１抗体の種類は特に制限されず、マウス抗体、ヒト抗体、ラット抗体、ウサギ抗体、ヒツジ抗体、ラクダ抗体、トリ抗体等や、ヒトに対する異種抗原性を低下させること等を目的として人為的に改変した遺伝子組換え型抗体、例えば、キメラ抗体、ヒト化抗体等を適宜用いることができる。遺伝子組換え型抗体は、既知の方法を用いて製造することができる。キメラ抗体は、ヒト以外の哺乳動物、例えば、マウス抗体の重鎖、軽鎖の可変領域とヒト抗体の重鎖、軽鎖の定常領域からなる抗体であり、マウス抗体の可変領域をコードするＤＮＡをヒト抗体の定常領域をコードするＤＮＡと連結し、これを発現ベクターに組み込んで宿主に導入し産生させることにより得ることができる。ヒト化抗体は、再構成（ｒｅｓｈａｐｅｄ）ヒト抗体とも称され、ヒト以外の哺乳動物、たとえばマウス抗体の相補性決定領域（ＣＤＲ；ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ ｄｅｔｅｒｍｉｎｉｎｇ ｒｅｇｉｏｎ）をヒト抗体の相補性決定領域へ移植したものであり、その一般的な遺伝子組換え手法も知られている。具体的には、マウス抗体のＣＤＲとヒト抗体のフレームワーク領域（ｆｒａｍｅｗｏｒｋ　ｒｅｇｉｏｎ；ＦＲ）を連結するように設計したＤＮＡ配列を、末端部にオーバーラップする部分を有するように作製した数個のオリゴヌクレオチドからＰＣＲ法により合成する。得られたＤＮＡをヒト抗体定常領域をコードするＤＮＡと連結し、次いで発現ベクターに組み込んで、これを宿主に導入し産生させることにより得られる（欧州特許出願公開番号ＥＰ ２３９４００ 、国際特許出願公開番号ＷＯ　９６/０２５７６参照）。ＣＤＲを介して連結されるヒト抗体のＦＲは、相補性決定領域が良好な抗原結合部位を形成するものが選択される。必要に応じ、再構成ヒト抗体の相補性決定領域が適切な抗原結合部位を形成するように抗体の可変領域のフレームワーク領域のアミノ酸を置換してもよい（Ｓａｔｏ，　Ｋ．ｅｔ　ａｌ．，　Ｃａｎｃｅｒ　Ｒｅｓ，　１９９３，　５３，　８５１－８５６．）。 Anti-FOXO1 antibodies include monoclonal and polyclonal antibodies, as well as antibody variants and derivatives such as antibodies and T-cell receptor fragments that retain the ability to specifically bind antigenic determinants.
The type of anti-FOXO1 antibody is not particularly limited, and is artificial for the purpose of reducing heterologous antigenicity to humans, mouse antibody, human antibody, rat antibody, rabbit antibody, sheep antibody, camel antibody, triantibody, etc. A recombinant antibody, for example, a chimeric antibody, a humanized antibody, or the like, which is specifically modified, can be appropriately used. Recombinant antibodies can be produced using known methods. A chimeric antibody is an antibody consisting of a heavy chain and a variable region of a light chain of a non-human mammal, for example, a mouse antibody and a constant region of a heavy chain and a light chain of a human antibody, and is a DNA encoding the variable region of a mouse antibody. Can be obtained by ligating a DNA encoding a constant region of a human antibody, incorporating it into an expression vector, introducing it into a host, and producing it. A humanized antibody, also referred to as a restored human antibody, is obtained by transplanting a complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody, into a complementarity determining region of a human antibody. Yes, and the general gene recombination method is also known. Specifically, several oligos prepared so as to have an overlapping portion at the end of a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody. It is synthesized from nucleotides by the PCR method. It is obtained by ligating the obtained DNA with the DNA encoding the human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. EP 239400, International Patent Application Publication No. See WO 96/02576). The FR of the human antibody linked via the CDR is selected so that the complementarity determining region forms a good antigen-binding site. If desired, the amino acids in the framework regions of the variable region of the antibody may be substituted such that the complementarity determining regions of the reconstituted human antibody form the appropriate antigen binding site (Sato, K. et al., Cancer). Res, 1993, 53, 851-856.).
　ＦＯＸＯ１のｓｉＲＮＡとしては、２１－２３塩基対からなる低分子二本鎖ＲＮＡであり、ＰＮＡ干渉（ＲＮＡｉ）に関与し、ｍＲＮＡの破壊によって配列特異的に遺伝子の発現を抑制できるものであればよい。 The siRNA of FOXO1 may be a low molecular weight double-stranded RNA consisting of 21-23 base pairs, which is involved in PNA interference (RNAi) and can suppress gene expression in a sequence-specific manner by disrupting the mRNA. ..
　ＦＯＸＯ１のｓｈＲＮＡとしては、プラスミドから転写された後、ヘアピン構造を形成し、プロセシングを受けてｓｉＲＮＡとなるものであればよい。 The shRNA of FOXO1 may be one that is transcribed from a plasmid, forms a hairpin structure, and is processed to become siRNA.
　本発明者は、後記実施例に示すように、ＦＯＸＯ１が患者乳癌組織中の筋線維芽細胞や培養されたＣＡＦｓに高発現していることを見出した。また、抗ＦＯＸＯ１抗体、ｓｈＲＮＡ、ＡＳ１８４２８５６などのＦＯＸＯ１阻害剤をＣＡＦｓに作用させると、ＣＡＦｓの筋線維芽細胞特性の減弱、ＣＡＦｓにおける炎症性サイトカイン産生能の減弱が生じると共に、顕著なＣＡＦｓの細胞増殖阻害及び細胞死誘導が生じ、またＣＡＦｓの腫瘍促進能力を減弱させること、さらにコントロールのヒト正常線維芽細胞ではこのような増殖阻害や細胞死が生じないことを見出した。
　従って、ＦＯＸＯ１阻害剤が、ＣＡＦｓの細胞増殖阻害剤及び細胞死誘導剤として有用であることより、ＣＡＦｓが共存する癌の進展抑制剤としても有用である可能性が示唆された。 The present inventor has found that FOXO1 is highly expressed in myofibroblasts and cultured CAFs in patient breast cancer tissues, as shown in Examples below. In addition, when FOXO1 inhibitors such as anti-FOX1 antibody, shRNA, and AS1842856 act on CAFs, the myofibroblast characteristics of CAFs are attenuated, the ability of CAFs to produce inflammatory cytokines is attenuated, and the cell proliferation of CAFs is remarkable. It has been found that inhibition and cell death induction occur, and that the tumor-promoting ability of CAFs is attenuated, and that such growth inhibition and cell death do not occur in control human normal fibroblasts.
Therefore, since the FOXO1 inhibitor is useful as a cell growth inhibitor and a cell death inducer for CAFs, it is suggested that it may be useful as a cancer progression inhibitor in which CAFs coexist.
　本発明の医薬の治療の対象となる癌は、ＣＡＦｓが共存する癌であり、具体的には、上皮性癌（呼吸器癌、消化器癌、泌尿生殖器癌、分泌系癌、皮膚癌等）、中皮腫、乳癌、肉腫、中枢神経系腫瘍、末梢神経系腫瘍等が挙げられる。
　呼吸器癌としては、例えば、肺癌（非小細胞肺癌、小細胞肺癌等）等が挙げられる。消化器癌としては、例えば、食道癌、胃癌、十二指腸癌、肝臓癌、胆道癌（胆嚢・胆管癌等）、膵臓癌、大腸癌、結腸直腸癌（結腸癌、直腸癌等）等が挙げられる。泌尿生殖器癌としては、例えば、卵巣癌、子宮癌（子宮頚癌、子宮体癌等）、腎癌、膀胱癌、前立腺癌、精巣腫瘍等が挙げられる。分泌系癌としては、神経内分泌腫瘍等が挙げられる。中皮腫としては、例えば、胸膜中皮腫、腹膜中皮腫、心膜中皮腫、精巣中皮腫等が挙げられる。肉腫としては、例えば、消化管間質腫瘍、骨・軟部腫瘍等が挙げられる。中枢神経系腫瘍としては、例えば、脳腫瘍等が挙げられる。末梢神経系腫瘍としては、例えば、悪性神経鞘腫等が挙げられる。これらのうち、好ましくは、消化管間質腫瘍、乳癌、肺癌、胃癌、前立腺癌、卵巣癌及び大腸癌であり、肺癌のうち、非小細胞肺癌が好ましい。 The cancer to be treated by the medicament of the present invention is a cancer in which CAFs coexist, and specifically, epithelial cancer (respiratory cancer, digestive organ cancer, urogenital cancer, secretory system cancer, skin cancer, etc.). , Middle dermatoma, breast cancer, sarcoma, central nervous system tumor, peripheral nervous system tumor and the like.
Examples of respiratory cancer include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.). Examples of gastrointestinal cancer include esophageal cancer, gastric cancer, duodenal cancer, liver cancer, biliary tract cancer (cholangiocarcinoma / cholangiocarcinoma, etc.), pancreatic cancer, colon cancer, colorectal cancer (colon cancer, rectal cancer, etc.) and the like. .. Examples of urogenital cancer include ovarian cancer, uterine cancer (cervical cancer, endometrial cancer, etc.), renal cancer, bladder cancer, prostate cancer, testicular tumor and the like. Examples of secretory cancers include neuroendocrine tumors. Examples of mesothelioma include pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma. Examples of sarcoma include gastrointestinal stromal tumors, bone / soft tissue tumors, and the like. Examples of central nervous system tumors include brain tumors and the like. Peripheral nervous system tumors include, for example, malignant schwannoma. Of these, gastrointestinal stromal tumor, breast cancer, lung cancer, gastric cancer, prostate cancer, ovarian cancer and colon cancer are preferable, and among lung cancers, non-small cell lung cancer is preferable.
　本発明の医薬の投与経路は特に限定されず、経口的又は非経口的に投与することができる。非経口投与としては静脈内、筋肉内、皮下又は皮内等への注射、吸入、直腸内、鼻腔内投与、外用投与等が挙げられる。
　本発明の医薬としては、有効成分であるＦＯＸＯ１阻害剤をそのまま患者に投与してもよいが、好ましくは、有効成分と薬学的に許容し得る添加剤とを含む医薬組成物の形態として投与すべきである。薬学的に許容し得る添加剤としては、例えば、賦形剤、崩壊剤ないし崩壊補助剤、結合剤、滑沢剤、コーティング剤、色素、希釈剤、基剤、溶解剤ないし溶解補助剤、等張化剤、ｐＨ調節剤、安定化剤、噴射剤、及び粘着剤等を用いることができる。 The route of administration of the medicament of the present invention is not particularly limited, and the drug can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, subcutaneous or intradermal injection, inhalation, rectal, intranasal administration, external administration and the like.
As the medicament of the present invention, the FOXO1 inhibitor which is an active ingredient may be administered to a patient as it is, but it is preferably administered in the form of a pharmaceutical composition containing the active ingredient and a pharmaceutically acceptable additive. Should be. Pharmaceutically acceptable additives include, for example, excipients, disintegrants or disintegrants, binders, lubricants, coatings, dyes, diluents, bases, solubilizers or solubilizers, etc. Tensioning agents, pH regulators, stabilizers, propellants, adhesives and the like can be used.
　経口投与に適する製剤の例としては、例えば、錠剤、カプセル剤、散剤、細粒剤、顆粒剤、液剤、又はシロップ剤等を挙げることができ、非経口投与に適する製剤としては、例えば、注射剤、点滴剤、坐剤、吸入剤又は外用剤（貼付、軟膏、クリーム、ゲル、ローション、スプレーなどを含む）などを挙げることができる。 Examples of preparations suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, syrups and the like, and preparations suitable for parenteral administration include, for example, injection. Agents, drops, suppositories, inhalants or external preparations (including patches, ointments, creams, gels, lotions, sprays, etc.) and the like can be mentioned.
　経口投与に適する製剤には、添加物として、例えば、ブドウ糖、乳糖、Ｄ－マンニトール、デンプン、又は結晶セルロース等の賦形剤；カルボキシメチルセルロース、デンプン、又はカルボキシメチルセルロースカルシウム等の崩壊剤又は崩壊補助剤；ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、又はゼラチン等の結合剤；ステアリン酸マグネシウム又はタルク等の滑沢剤；ヒドロキシプロピルメチルセルロース、白糖、ポリエチレングリコール又は酸化チタン等のコーティング剤；ワセリン、流動パラフィン、ポリエチレングリコール、ゼラチン、カオリン、グリセリン、精製水、又はハードファット等の基剤を用いることができる。注射、点滴用剤に適する製剤には、注射用蒸留水、生理食塩水、プロピレングリコール等の水性あるいは用時溶解型注射剤を構成し得る溶解剤又は溶解補助剤；ブドウ糖、塩化ナトリウム、Ｄ－マンニトール、グリセリン等の等張化剤；無機酸、有機酸、無機塩基又は有機塩基等のｐＨ調節剤等の製剤用添加物を用いることができる。坐剤に適する製剤には、例えば、ポリエチレングリコール、ラノリン、カカオ脂、脂肪酸トリグリセリド等の基剤、及び必要に応じて非イオン界面活性剤のような界面活性剤等の添加物を用いることができる。
　軟膏剤に適する製剤には、通常使用される基剤、安定剤、湿潤剤、保存剤等が必要に応じて用いられる。基剤としては、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィン等が挙げられる。保存剤としては、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピル等が挙げられる。 Formulations suitable for oral administration include, as additives, excipients such as, for example, glucose, lactose, D-mannitol, starch, or crystalline cellulose; disintegrants or disintegrant aids such as carboxymethyl cellulose, starch, or carboxymethyl cellulose calcium. Binders such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, or gelatin; Lubricants such as magnesium stearate or talc; Coating agents such as hydroxypropylmethyl cellulose, sucrose, polyethylene glycol or titanium oxide; Vaseline, liquid paraffin , Polyethylene glycol, gelatin, kaolin, glycerin, purified water, or a base such as hard fat can be used. Formulations suitable for injection and infusion include solubilizers or solubilizers that may constitute aqueous or time-dissolving injectables such as distilled water for injection, saline, propylene glycol; glucose, sodium chloride, D- Isotonic agents such as mannitol and glycerin; pharmaceutical additives such as pH adjusters such as inorganic acids, organic acids, inorganic bases or organic bases can be used. Suitable preparations for suppositories include, for example, bases such as polyethylene glycol, lanolin, cacao butter, fatty acid triglycerides, and optionally additives such as surfactants such as nonionic surfactants. ..
As a preparation suitable for an ointment, commonly used bases, stabilizers, wetting agents, preservatives and the like are used as necessary. Examples of the base include liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.
　貼付剤に適する製剤としては、通常の支持体に前記軟膏、クリーム、ゲル、ペースト等を常法により塗布したものが挙げられる。支持体としては、綿、スフ、化学繊維からなる織布、不織布や軟質塩化ビニル、ポリエチレン、ポリウレタン等のフィルムあるいは発泡体シートが適当である。 Examples of preparations suitable for patches include those obtained by applying the ointment, cream, gel, paste, or the like to a normal support by a conventional method. As the support, a woven fabric made of cotton, rayon, or chemical fiber, a non-woven fabric, a film such as soft vinyl chloride, polyethylene, or polyurethane, or a foam sheet is suitable.
　また、本発明の医薬は、癌細胞に直接作用する他の抗癌剤と併用することも有用である。そのような併用できる他の抗癌剤としては、抗癌効果を有する抗癌剤であれば特に限定されないが、腫瘍細胞傷害性を有する抗癌剤であることが、相乗効果を得る点で特に好ましい。 It is also useful to use the medicament of the present invention in combination with other anticancer agents that act directly on cancer cells. The other anticancer agent that can be used in combination is not particularly limited as long as it is an anticancer agent having an anticancer effect, but an anticancer agent having tumor cytotoxicity is particularly preferable in terms of obtaining a synergistic effect.
　当該他の抗癌剤としては、アルキル化剤、代謝拮抗剤、微小管阻害剤、抗生物質抗癌剤、トポイソメラーゼ阻害剤、白金製剤、分子標的薬、ホルモン剤、生物製剤などが挙げられる。
　アルキル化剤としては、例えば、シクロホスファミド、イホスファミド、ニトロソウレア、ダカルバジン、テモゾロミド、ニムスチン、ブスルファン、メルファラン、プロカルバジン、ラニムスチン等が挙げられる。
　代謝拮抗剤としては、例えば、エノシタビン、カルモフール、カペシタビン、テガフール、テガフール・ウラシル、テガフール・ギメラシル・オテラシルカリウム、ゲムシタビン、シタラビン、シタラビンオクホスファート、ネララビン、フルオロウラシル、フルダラビン、ペメトレキセド、ペントスタチン、メトトレキサート、クラドリビン、ドキシフルリジン、ヒドロキシカルバミド、メルカプトプリン等が挙げられる。
　微小管阻害剤としては、例えば、ビンクリスチン等のアルカロイド系抗がん剤、ドセタキセル、パクリタキセル等のタキサン系抗がん剤が挙げられる。
　抗生物質抗がん剤としては、例えば、マイトマイシンＣ、ドキソルビシン、エピルビシン、ダウノルビシン、ブレオマイシン、アクチノマイシンＤ、アクラルビシン、イダルビシン、ピラルビシン、ペプロマイシン、ミトキサントロン、アムルビシン、ジノスタチンスチマラマー等が挙げられる。
　トポイソメラーゼ阻害剤としてはトポイソメラーゼＩ阻害作用を有するＣＰＴ－１１、イリノテカン、ノギテカン、トポイソメラーゼＩＩ阻害作用をもつエトポシド、ソブゾキサンが挙げられる。
　白金製剤としては、例えば、シスプラチン、ネダプラチン、オキサリプラチン、カルボプラチン等が挙げられる。
　ホルモン剤としては、例えば、デキサメタゾン、フィナステリド、タモキシフェン、アストロゾール、エキセメスタン、エチニルエストラジオール、クロルマジノン、ゴセレリン、ビカルタミド、フルタミド、ブレドニゾロン、リュープロレリン、レトロゾール、エストラムスチン、トレミフェン、ホスフェストロール、ミトタン、メチルテストステロン、メドロキシプロゲステロン、メピチオスタン等が挙げられる。
　生物製剤としては、例えば、インターフェロンα、β及びγ、インターロイキン２、ウベニメクス、乾燥ＢＣＧ等が挙げられる。
　分子標的薬としては、例えば、リツキシマブ、アレムツズマブ、トラスツズマブ、セツキシマブ、パニツムマブ、イマチニブ、ダサチニブ、ニロチニブ、ゲフィチニブ、エルロチニブ、テムシロリムス、ベバシズマブ、ＶＥＧＦ　ｔｒａｐ、スニチニブ、ソラフェニブ、トシツズマブ、ボルテゾミブ、ゲムツズマブ・オゾガマイシン、イブリツモマブ・オゾガマイシン、イブリツモマブチウキセタン、タミバロテン、トレチノイン等が挙げられる。
　さらに、ヒト上皮性増殖因子受容体２阻害剤、上皮性増殖因子受容体阻害剤、Ｂｃｒ－Ａｂｌチロシンキナーゼ阻害剤、上皮性増殖因子チロシンキナーゼ阻害剤、ｍＴＯＲ阻害剤、血管内皮増殖因子受容体２阻害剤（α－ＶＥＧＦＲ－２抗体）等の血管新生を標的にした阻害剤、ＭＡＰキナーゼ阻害剤などの各種チロシンキナーゼ阻害剤、サイトカインを標的とした阻害剤、プロテアソーム阻害剤、抗体―抗がん剤配合体等の分子標的薬なども使用できる。
　これらの他の抗癌剤のうち、細胞傷害活性を特徴とするアルキル化剤、代謝拮抗剤、微小管阻害剤、抗生物質抗がん剤、トポイソメラーゼ阻害剤、白金製剤、分子標的薬等が特に好ましい。具体的には、ゲムシタビン、５－ＦＵ、ＣＰＴ－１１、エトポシド、シスプラチン、オキサリプラチン、パクリタキセル、ドセタキセル、ダカルバジン、ドキソルビシン、ベバシズマブ、セツキシマブ、抗血管内皮増殖因子受容体２阻害抗体、上皮性増殖因子チロシンキナーゼ阻害剤等が特に好ましい。 Examples of the other anticancer agent include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting agents, hormonal agents, biological preparations and the like.
Examples of the alkylating agent include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.
Antimetabolites include, for example, enocitabine, carmofur, capecitabine, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, cytarabine, cytarabine ocphosphat, neralabine, fluorouracil, fludalabine, pemetrexed Examples thereof include cladribine, doxiflulysin, hydroxycarbamide, mercaptopurine and the like.
Examples of the microtubule inhibitor include alkaloid anticancer agents such as vincristine and taxane anticancer agents such as docetaxel and paclitaxel.
Antibiotics Anticancer agents include, for example, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idarubicin, pyrarubicin, pepromycin, mitoxantrone, amurubicin, dinostatin stimalamar and the like.
Examples of the topoisomerase inhibitor include CPT-11 having a topoisomerase I inhibitory action, irinotecan, nogitecan, and etoposide and sobzoxane having a topoisomerase II inhibitory action.
Examples of the platinum preparation include cisplatin, nedaplatin, oxaliplatin, carboplatin and the like.
Hormonal agents include, for example, dexamethasone, finasteride, tamoxifen, astrosol, exemestane, ethinyl estradiol, chlormaginone, goserelin, bicalutamide, flutamide, bredonizolone, leuprorelin, letrozole, estramustine, toremifene, phosfestol, mitotan, Examples thereof include methyltestosterone, medroxyprogesterone, and mepitiostane.
Examples of biologics include interferon α, β and γ, interleukin 2, ubenimex, dried BCG and the like.
Molecular-targeted drugs include, for example, rituximab, alemtuzumab, trastuzumab, cetuximab, panitummab, imatinib, dasatinib, nilotinib, gefitinib, elrotinib, temsirolimus, bebashizumab, temshirolimus, bebashizumab, vEGF trap, , Ibritumomab tiuxetan, Tamibarotene, Tretinoin and the like.
In addition, human epithelial growth factor receptor 2 inhibitor, epithelial growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epithelial growth factor tyrosine kinase inhibitor, mTOR inhibitor, vascular endothelial growth factor receptor 2 Inhibitors targeting angiogenesis such as inhibitors (α-VEGFR-2 antibody), various tyrosine kinase inhibitors such as MAP kinase inhibitors, cytokine targeting inhibitors, proteasome inhibitors, antibodies-anticancer Molecular-targeted drugs such as drug formulations can also be used.
Among these other anticancer agents, alkylating agents characterized by cytotoxic activity, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting agents and the like are particularly preferable. Specifically, gemcitabine, 5-FU, CPT-11, etoposide, cisplatin, oxaliplatin, paclitaxel, docetaxel, dacarbazine, doxorubicin, bevacizumab, cetuximab, antivascular endothelial growth factor receptor 2 inhibitor antibody, epithelial growth factor tyrosine. Kinase inhibitors and the like are particularly preferred.
　本発明の医薬の投与量は、疾患の進行状況又は症状の程度、患者の年齢や体重などの諸条件に応じて適宜選択可能であるが、例えば経口投与の場合、一日当たり１ｍｇから３００ｍｇを１回から３回程度に分けて投与することができる。 The dose of the medicament of the present invention can be appropriately selected according to various conditions such as the progress of the disease or the degree of symptoms, the age and body weight of the patient, and for example, in the case of oral administration, 1 mg to 300 mg per day is 1 mg. It can be administered in divided doses of about 3 times.
　次に、実施例を挙げて本発明をより詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
実施例１
　ヒト乳癌のＣＡＦｓにおいてＦＯＸＯ１が発現しているかどうかは不明であった。そこで、我々はヒト乳癌から切片を調製し、抗ＦＯＸＯ１抗体を使用して免疫組織化学染色を行った。癌部領域のα－ＳＭＡ陽性のＣＡＦｓにおいてＦＯＸＯ１陽性が有意に観察された。しかしながら、同じ患者の非癌部領域の線維芽細胞ではＦＯＸＯ１陽性細胞は認められなかった（図１ａ）。
　この観察事項を一般化するために、さらに９人の患者の乳癌切片をチェックした（表１）。注目すべきは、ＦＯＸＯ１陽性の線維芽細胞の割合が、調べたすべての乳癌患者のＣＡＦｓで有意に増加したことである（図１ｂ）。ＦＯＸＯ１ ｍＲＮＡレベルでの発現も、コントロールと比較して、ＣＡＦｓで４倍に増加していた（図２ａ）。さらに、線維芽細胞よりの全細胞抽出液および核抽出液を用いたＦＯＸＯ１のウエスタンブロットおよび免疫蛍光染色により、ＣＡＦｓの核および細胞質におけるＦＯＸＯ１の発現上昇が検出された（図２ｂ、ｃ）。これらのデータから、正常な乳腺線維芽細胞と比較してＣＡＦｓにおいてはＦＯＸＯ１の発現が増加していることが明らかになった。 Example 1
It was unclear whether FOXO1 was expressed in CAFs of human breast cancer. Therefore, we prepared sections from human breast cancer and performed immunohistochemical staining using anti-FOXO1 antibody. FOXO1-positive was significantly observed in α-SMA-positive CAFs in the cancerous region. However, no FOXO1-positive cells were found in the non-cancerous fibroblasts of the same patient (Fig. 1a).
To generalize this observation, breast cancer sections of 9 more patients were checked (Table 1). Of note, the proportion of FOXO1-positive fibroblasts was significantly increased in CAFs of all breast cancer patients examined (Fig. 1b). Expression at the FOXO1 mRNA level was also increased 4-fold in CAFs compared to the control (Fig. 2a). Furthermore, Western blot and immunofluorescent staining of FOXO1 using whole cell extract from fibroblasts and nuclear extract detected increased expression of FOXO1 in the nucleus and cytoplasm of CAFs (FIGS. 2b, c). From these data, it was revealed that the expression of FOXO1 was increased in CAFs as compared with normal mammary fibroblasts.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
実施例２
　ＦＯＸＯ１発現抑制によるＣＡＦｓの筋線維芽細胞能や炎症性サイトカイン産生能を調べた。ＦＯＸＯ１　ｍＲＮＡおよびタンパク質の発現を有意に抑制することが可能な２つの異なるｓｈＲＮＡが線維芽細胞に導入された。ＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦは、α－ＳＭＡ、ＴＧＦ－ｂ１、ＳＤＦ－１　ｍＲＮＡ、ｐＳｍａｄ２蛋白などの筋線維芽細胞性マーカーの発現の有意な低下を示した（図３）。さらに、ＣＡＦｓの活性化や炎症免疫細胞の癌内へのリクルートに寄与している炎症性サイトカインの発現レベルを確認した。ＣＸＣＬ１、ＣＸＣＬ２、ＩＬ－１ａ、ＩＬ－１ｂ、ＩＬ－８やＬＩＦなどの炎症性サイトカインのｍＲＮＡの発現は、ＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦｓでは顕著に抑制されていた（図４）。これらのデータから、ＣＡＦｓにおけるＦＯＸＯ１の発現抑制は、ＣＡＦｓの筋線維芽細胞能と炎症性サイトカイン産生能を抑制することが明らかになった。 Example 2
The myofibroblast ability and inflammatory cytokine production ability of CAFs by suppressing FOXO1 expression were investigated. Two different shRNAs capable of significantly suppressing the expression of FOXO1 mRNA and protein were introduced into fibroblasts. CAF into which FOXO1-SHRNA was introduced showed a significant reduction in the expression of myofibroblast markers such as α-SMA, TGF-b1, SDF-1 mRNA, and pSmad2 protein (Fig. 3). Furthermore, we confirmed the expression level of inflammatory cytokines that contribute to the activation of CAFs and the recruitment of inflammatory immune cells into cancer. Expression of mRNA of inflammatory cytokines such as CXCL1, CXCL2, IL-1a, IL-1b, IL-8 and LIF was significantly suppressed in CAFs into which FOXO1-SHRNA was introduced (Fig. 4). From these data, it was clarified that suppression of FOXO1 expression in CAFs suppresses myofibroblast ability and inflammatory cytokine production ability of CAFs.
実施例３
　ＦＯＸＯ１発現ベクターあるいはコントロールの空ベクターが導入されたＨＥＫ２９３Ｔ細胞を使用したＦＯＸＯ１ルシフェレースレポーターアッセイによるＡＳ１８４２８５６の特異性を検討した。
　ｐｃＤＮＡ３．１－ＦＯＸＯ１　ｃＤＮＡベクターをＨＥＫ２９３Ｔ細胞へ導入し、２４時間後にＡＳ１８４２８５６（濃度１．０μＭ）添加し、さらに２４時間後にＤｕａｌ－ＬｕｃｉｆｅｒａｓｅTM　Ｒｅｐｏｒｔｅｒ　Ａｓｓａｙ　ＳｙｓｔｅｍでＦＯＸＯ１の転写活性を測定した。その結果、図５に示すように、ＡＳ１８４２８５６は顕著にＦＯＸＯ１の転写活性を抑制することが示された。 Example 3
The specificity of AS1842856 was examined by the FOXO1 luciferase reporter assay using HEK293T cells into which the FOXO1 expression vector or control empty vector was introduced.
The cDNA3.11-FOXO1 cDNA vector was introduced into HEK293T cells, AS1842856 (concentration 1.0 μM) was added 24 hours later, and the transcriptional activity of FOXO1 was measured by Dual-Luciferase TM Reporter Assay System 24 hours later. As a result, as shown in FIG. 5, it was shown that AS1842856 remarkably suppresses the transcriptional activity of FOXO1.
実施例４
　ＣＡＦｓのＡＳ１８４２８５６処理によるＦＯＸＯ１活性の抑制が、筋線維芽細胞能を低下させるか否か検討した。ＡＳ１８４２８５６で１２時間処理したＣＡＦｓを用いて、筋線維芽細胞のマーカ―遺伝子に特異的なプライマ―を使用したｒｅａｌ－ｔｉｍｅ　ＰＣＲを施行した。その結果、図６に示すように、ＡＳ１８４２８５６で処理されたＣＡＦｓでは、有意な筋線維芽細胞マーカーの低下が見られた。 Example 4
It was investigated whether suppression of FOXO1 activity by AS1842856 treatment of CAFs reduced myofibroblast ability. Real-time PCR using a marker gene-specific primer for myofibroblasts was performed using CAFs treated with AS1842856 for 12 hours. As a result, as shown in FIG. 6, CAFs treated with AS1842856 showed a significant decrease in myofibroblast markers.
実施例５
　ＡＳ１８４２８５６が処理されたＣＡＦｓにおける種々の遺伝子に対する抗体を使用したウエスタンブロット解析を行った。その結果、図７に示すように、ＡＳ１８４２８５６が処理されたＣＡＦｓにおいて、ＦＯＸＯ１、リン酸化ＦＯＸＯ１、α－ＳＭＡやリン酸化Ｓｍａｄ２／３の経時的な発現減弱を認めた。この結果より、ＦＯＸＯ１の活性化が、ＣＡＦｓの筋線維芽細胞能およびＴＧＦ－β－Ｓｍａｄ２／３シグナルの維持に必須であることが示唆された。 Example 5
Western blot analysis using antibodies against various genes in CAFs treated with AS1842856 was performed. As a result, as shown in FIG. 7, in CAFs treated with AS1842856, the expression of FOXO1, phosphorylated FOXO1, α-SMA and phosphorylated Smad2 / 3 was observed to be attenuated with time. This result suggests that activation of FOXO1 is essential for the maintenance of CAFs myofibroblast ability and TGF-β-Smad2 / 3 signal.
実施例６
　ＡＳ１８４２８５６８(濃度１．０μＭ)で１２時間処理されたＣＡＦｓのリアルタイムＰＣＲ解析を行った。その結果、図８に示すように、ＡＳ１８４２８５６処理後２４時間でＣＡＦｓにおけるＣＸＣＬ１，ＣＸＣＬ２，ＩＬ－１α，ＩＬ１β，ＩＬ－８，ＬＩＦなどの炎症性サイトカインの発現が顕著に抑制された。
　これらの結果から、ＦＯＸＯ１阻害剤であるＡＳ１８４２８５６８処理により、ＣＡＦｓの筋線維芽細胞能と炎症性サイトカイン産生能が抑制されることがわかった。 Example 6
Real-time PCR analysis of CAFs treated with AS18428568 (concentration 1.0 μM) for 12 hours was performed. As a result, as shown in FIG. 8, the expression of inflammatory cytokines such as CXCL1, CXCL2, IL-1α, IL1β, IL-8, and LIF in CAFs was remarkably suppressed 24 hours after the treatment with AS1842856.
From these results, it was found that treatment with AS18428568, which is a FOXO1 inhibitor, suppresses the myofibroblast ability and inflammatory cytokine production ability of CAFs.
実施例７
　ＦＯＸＯ１活性の抑制によるＣＡＦｓの増殖抑制および細胞死の誘導
　活性化されたＣＡＦｓのフェノタイプがＦＯＸＯ１－ｓｈＲＮＡを導入した際に減衰されたため、ＦＯＸＯ１活性阻害がこれらの細胞の増殖と生存率に影響を与えるかどうかを調べた。コントロールのＤＭＳＯ処理の効果と比較して、ＡＳ１８４２８５６で７２時間処理したＣＡＦｓでは、細胞増殖能が顕著に抑制され、細胞死も増加した（図９ａ、ｂ）、またコントロールであるヒト正常乳腺線維芽細胞ではこれらの現象はほとんど観察されなかった（図９ｂ）。これらの知見をさらに確認するために、生細胞のマーカーである還元型ニコチンアミドアデニンジヌクレオチドの測定、および細胞膜の損傷のマーカーである乳酸デヒドロゲナーゼ活性やｃａｓｐａｓｅ　３／７活性を測定した。結果として、ＡＳ１８４２８５６で処理されたＣＡＦｓにおける細胞増殖の抑制と細胞死（アポトーシス）の増加が観察された（図１０ａ，ｂ，ｃ）。 Example 7
Suppression of CAFs proliferation and induction of cell death by suppression of FOXO1 activity Since the phenotype of activated CAFs was attenuated when FOXO1-SHRNA was introduced, inhibition of FOXO1 activity affects the proliferation and viability of these cells. I checked whether to give it. Compared to the effect of DMSO treatment of the control, CAFs treated with AS1842856 for 72 hours markedly suppressed cell proliferation and increased cell death (FIGS. 9a, 9b), and also controlled human normal mammary fibroblasts. Almost no of these phenomena were observed in cells (Fig. 9b). In order to further confirm these findings, the measurement of reduced nicotinamide adenine dinucleotide, which is a marker of living cells, and the lactate dehydrogenase activity and caspase 3/7 activity, which are markers of cell membrane damage, were measured. As a result, suppression of cell proliferation and increased cell death (apoptosis) were observed in CAFs treated with AS1842856 (FIGS. 10a, b, c).
実施例８
　ＦＯＸＯ１はＣＡＦｓの活性化線維芽細胞能促進、増殖能促進および細胞死抑制に必要であることより、ＦＯＸＯ１の発現がＣＡＦｓによる腫瘍細胞の遊走能や増殖促進能にも必要かどうかを調べた。非侵襲性のヒト乳管癌由来乳癌（ＤＣＩＳ）細胞を使用してボイデンチャンバー細胞遊走アッセイを行った。
　ＦＯＸＯ１－ｓｈＲＮＡが導入されたＣＡＦｓの培養上清で処理されたＤＣＩＳ細胞は、ＧＦＰ－ｓｈＲＮＡが導入されたＣＡＦｓの培養上清による処理と比較して、有意な遊走能の減少を示した（図１１）。次に、ＣＡＦｓでのＦＯＸＯ１発現が近傍の癌細胞のｉｎ　ｖｉｖｏでの増殖に影響を与えるかどうかを調査した。これを確認するために、ＤＣＩＳ細胞とＧＦＰ―ｓｈＲＮＡあるいはＦＯＸＯ１－ｓｈＲＮＡが導入されたヒト正常乳腺線維芽細胞やＣＡＦｓが免疫不全ヌードマウスに皮下移植された。結果として、対照ＧＦＰ－ｓｈＲＮＡの効果と比較して、ＦＯＸＯ１－ｓｈＲＮＡの導入によりＦＯＸＯ１の発現が抑制されたＣＡＦｓを含む腫瘍ではその体積および重量が顕著に抑制された（図１２）。
　以上の結果より、ＣＡＦｓにおけるＦＯＸＯ１の発現が近傍のＤＣＩＳ細胞の遊走能や癌増殖能に必須であることが明らかになった。 Example 8
Since FOXO1 is required for promotion of activated fibroblast ability, promotion of proliferation and suppression of cell death of CAFs, it was investigated whether expression of FOXO1 is also necessary for the ability of CAFs to promote migration and proliferation of tumor cells. A Boyden chamber cell migration assay was performed using ductal carcinoma in situ (DCIS) cells from non-invasive human ductal carcinoma in situ.
DCIS cells treated with culture supernatants of CAFs introduced with FOXO1-SHRNA showed a significant reduction in migration ability compared to treatment with culture supernatants of CAFs introduced with GFP-SHRNA (Fig.). 11). Next, it was investigated whether FOXO1 expression in CAFs affects the in vivo growth of nearby cancer cells. To confirm this, DCIS cells and human normal mammary fibroblasts or CAFs into which GFP-SHRNA or FOXO1-shRNA were introduced were subcutaneously transplanted into immunodeficient nude mice. As a result, the volume and weight of tumors containing CAFs whose expression of FOXO1 was suppressed by the introduction of FOXO1-SHRNA was significantly suppressed as compared with the effect of control GFP-SHRNA (Fig. 12).
From the above results, it was clarified that the expression of FOXO1 in CAFs is essential for the migration ability and cancer growth ability of nearby DCIS cells.

Claims (8)

  1. 　ＦＯＸＯ１阻害剤を有効成分とするＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤。 A CAFs cell growth inhibitor or a CAFs cell death inducer containing a FOXO1 inhibitor as an active ingredient.
  2. 　ＦＯＸＯ１阻害剤が、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである請求項１記載のＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤。 The CAFs cell growth inhibitor or CAFs cell death inducer according to claim 1, wherein the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  3. 　ＣＡＦｓの細胞増殖阻害剤又はＣＡＦｓの細胞死誘導剤製造のための、ＦＯＸＯ１阻害剤の使用。 Use of FOXO1 inhibitors for the production of CAFs cell growth inhibitors or CAFs cell death inducers.
  4. 　ＦＯＸＯ１阻害剤が、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである請求項３記載の使用。 The use according to claim 3, wherein the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  5. 　ＣＡＦｓの細胞増殖を阻害又はＣＡＦｓの細胞死を誘導するための、ＦＯＸＯ１阻害剤。 A FOXO1 inhibitor for inhibiting cell proliferation of CAFs or inducing cell death of CAFs.
  6. 　抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである請求項５記載のＦＯＸＯ１阻害剤。 The FOXO1 inhibitor according to claim 5, which is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
  7. 　ＦＯＸＯ１阻害剤の有効量を投与することを特徴とするＣＡＦｓの細胞増殖阻害方法又はＣＡＦｓの細胞死誘導方法。 A method for inhibiting cell growth of CAFs or a method for inducing cell death of CAFs, which comprises administering an effective amount of a FOXO1 inhibitor.
  8. 　ＦＯＸＯ１阻害剤が、抗ＦＯＸＯ１抗体、ＦＯＸＯ１のｓｉＲＮＡ、ＦＯＸＯ１のｓｈＲＮＡ、ＡＳ１８４２８５６及びＡＳ１７０８７２７から選ばれるものである請求項７記載の方法。 The method according to claim 7, wherein the FOXO1 inhibitor is selected from anti-FOXO1 antibody, FOXO1 siRNA, FOXO1 shRNA, AS1842856 and AS1708727.
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