JP2020019749A - Therapeutic agent for controlling cancer microenvironment - Google Patents

Abstract

To provide a novel pharmaceutical composition targeting a cancer microenvironment, a cancer treatment method using the pharmaceutical composition, and the like.SOLUTION: The problem is solved by a pharmaceutical composition for treating tumor and a pharmaceutical composition for improving prognosis, containing an AEBP1 inhibitor, and a cancer treatment method and a prognosis improving method using the pharmaceutical composition.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition for treating cancer, comprising an inhibitor of AEBP1 (adipocyte enhancer binding protein 1), a method for treating cancer using the pharmaceutical composition, and the like. Further, the present invention provides a method for predicting the prognosis of cancer treatment, which comprises determining the expression level of AEBP1, a pharmaceutical composition for improving the prognosis of cancer treatment comprising an AEBP1 inhibitor, and a pharmaceutical composition comprising the same. It also relates to methods to improve the prognosis of the used cancer treatment.

  Recent studies have revealed that the surrounding environment of cancer, for example, stroma including blood vessels, ECM, fibroblasts, etc., plays an important role in the development and progression of cancer. Against this background, the importance of the surrounding environment of cancer has been highlighted, and therapeutic methods targeting not only the cancer cells themselves but also the surrounding environment have been studied. Among them, tumor angiogenesis is considered to be an important mechanism involved in proliferation, invasion and metastasis. Therefore, controlling angiogenesis in the cancer microenvironment is a promising strategy for treating and suppressing progression of cancer.

  However, in the development and progression of cancer, the molecular mechanism in the cancer microenvironment, especially the molecular mechanism of tumor angiogenesis, has not yet been elucidated in many parts. Treatment is not well established.

  Adipocyte Enhancer Binding Protein 1 (AEBP1) is a member of the carboxypeptidase A protein family and a protein identified as a transcriptional repressor that binds to the aP2 promoter. AEBP1 is thought to play an important role in adipogenesis, smooth muscle cell differentiation, abdominal wall development and wound healing. Recently, it has been reported that the gene is highly expressed in glioblastoma, and that it is involved in the acquisition of resistance to a BRAF inhibitor in melanoma (Non-Patent Documents 1 and 2). In addition, Non-Patent Literature 3 discloses a cancer candidate fibroblast (CAF) marker candidate gene in skin tumors and malignant melanoma.

Ladha et al., Mol Cancer Res; 10 (8); 1039-11; 51 Hu et al., Cell Death Dis., 2013 Nov 7; 4: e914 Sasaki et al., Human Pathology (2018) 79, 1-8

  An object of the present invention includes a novel pharmaceutical composition for treating cancer, comprising an AEBP1 inhibitor, a method for treating cancer using the pharmaceutical composition, and determining the expression level of AEBP1 The present invention provides a method for predicting the prognosis of cancer treatment, a pharmaceutical composition containing an AEBP1 inhibitor for improving the prognosis of cancer treatment, a method for improving the prognosis of cancer treatment using the pharmaceutical composition, and the like. It is in.

  As mentioned above, there is no established cure for cancer targeting the cancer microenvironment. For example, angiogenesis inhibitory therapy for colorectal cancer is currently being treated with anti-VEGF humanized monoclonal antibody (bevacizumab) or low-molecular-weight VEGFR inhibitor (sorafenib), but targeting VEGF-VEGFR It has been reported that these angiogenesis therapies have limited efficacy and have serious adverse events. Therefore, a new treatment method with a small patient burden is required.

  The present inventors have sought a new treatment method to solve the above-mentioned problems, and have found that AEBP1 is significantly higher expressed in tumor vascular endothelial cells (TEC) as compared to normal vascular endothelial cells (TEC). I got a new finding. Based on these findings, the present inventors continued intensive studies on the molecular mechanism of the AEBP1 gene in the cancer microenvironment, and found that suppression of AEBP1 expression significantly reduced angiogenesis and increase in tumor tissue in tumor tissue. As a result, the present invention has been completed.

That is, the present invention relates to the following:
[1] A pharmaceutical composition for treating a tumor, comprising an inhibitor of AEBP1 (adipocyte enhancer binding protein 1).
[2] The pharmaceutical composition of [1], wherein the AEBP1 inhibitor is siRNA, antisense nucleic acid, shRNA or miRNA against AEBP1 transcript RNA.
[3] The pharmaceutical composition of [2], wherein the AEBP1 inhibitor is an siRNA against transcript RNA of AEBP1.
[4] The pharmaceutical composition of [1] to [3], wherein the tumor is a solid tumor.
[5] The pharmaceutical composition of [4], wherein the tumor is a colon cancer.
[6] The pharmaceutical composition of [1] to [5], wherein the pharmaceutical composition is an angiogenesis inhibitor.
[7] A method for predicting the prognosis of tumor treatment in a subject, the method comprising determining the expression level of an expression product of AEBP1 in a test sample derived from the subject.
[8] The method of [7], wherein the tumor is a colon cancer.
[9] A pharmaceutical composition comprising an inhibitor of AEBP1 for improving the prognosis of tumor treatment.

  According to the present invention, since the therapeutic method targets a gene that is highly expressed specifically in tumor blood vessels, side effects can be significantly reduced as compared with conventional angiogenesis inhibition therapy. In addition, since the proliferation of cancer-associated fibroblasts (CAF) as well as tumor angiogenesis can be significantly reduced, a novel therapeutic method having a new mechanism of action targeting the cancer microenvironment can be provided. . Furthermore, since AEBP1 was also found to be a poor prognostic factor, it is possible to predict and improve the prognosis of cancer treatment, and is therefore useful in preventing metastasis and recurrence.

FIG. 1A is a diagram showing an outline of a test in which a CRC tissue was subjected to an enzyme treatment and cells were separated using a magnetic microbead method (IMS). B shows a stained image of a CRC tissue paraffin-embedded section stained with CD31 and CD146 (upper row) and the result of sorting CRC tissue cells by FACS using CD31 and CD146 (lower row). C is a diagram showing that the expression of 18 genes including ANTXR1 (TEM8) was enhanced in TECs by comparing the mRNA expression of TECs with NECs in three sets. FIG. 2 is a graph showing the results of evaluating the mRNA expression levels of candidate genes by quantitative RT-PCR in four groups of normal epithelium, tumor epithelium, NECs, and TECs of clinical specimens. FIG. 3A shows that the tumor blood vessels (upper row) and the non-cancerous normal blood vessels (lower row) of the human colon cancer tissue were stained with CD31 (left column), CD146 (center), and AEBP1 (right column). It is a stained image. B, C and D are stained images stained with stromal AEBP1 in the cancerous part of human colon cancer tissue. C and D are enlarged views of the dotted line portion of B, respectively. FIG. 4A shows AEBP1 using quantitative RT-PCR in colon cancer cell line cells (DLD1, SW480, RKO, WiDr, HT-29, HCT116, COLO320, LoVo), HUVEC, HMVEC, NHDF and CAF of normal cells. It is a graph showing the result of having evaluated the expression at the mRNA level of 7 genes containing and ANTXR1 (TEM8). B is a graph showing the results of evaluating the expression of vascular endothelial marker (CD31) and tumor vascular endothelial marker (CD146) at the mRNA level using the quantitative RT-PCR method for each of the above cells. FIG. 5A is a schematic diagram illustrating a procedure for collecting a tumor culture supernatant (TCM). B is a light micrograph of HUVEC cultured in TCM (serum free) and DMEM (serum free) derived from DLD1 and SW480. Images were taken 24h and 48h after the start of culture, respectively. C shows the results obtained by culturing HUVEC in TCM (serum-free) derived from eight types of colorectal cancer cell lines and successively evaluating changes in expression of AEBP1 at the mRNA level using a quantitative RT-PCR method. FIG. D represents the result of evaluating the change in expression of AEBP1 at the mRNA level when HUVEC was cultured with DLD1-derived TCM to which FBS was added (2.5%, 10%) using a quantitative RT-PCR method. It is a graph. For E, HUVEC and DLD1 were seeded on a 10 cm dish at a ratio of 4: 1 and co-cultured. By 10 ⁶ cells after culture initiation 24h and 96h were harvested, is a diagram illustrating a schematic of a test for separating and recovering HUVEC and DLD1 using IMS technique. F is a graph showing the results of evaluating the expression of HUVEC, DLD1 at the AEBP1 and CD31 mRNA levels at 24 h and 96 h after the start of co-culture using the quantitative RT-PCR method. G is an optical micrograph of HUVEC, DLD1, 24 h and 96 h after the start of co-culture. FIG. 6 shows survival curves of patients with AEBP1 high-expressing colorectal cancer and low-expressing colorectal cancer. FIGS. 7A and 7B are graphs in which two types of siAEBP1 or control siRNA were introduced into HUVEC and CAF, respectively, and expression at the mRNA level of AEBP1 was confirmed using a quantitative RT-PCR method to evaluate knockdown efficiency. . C and D are diagrams showing the results obtained by introducing two types of siAEBP1 or control siRNA into HUVEC or CAF, respectively, and examining cell growth over time immediately after the introduction. E is a graph showing the result of cell cycle analysis using FACS of HUVEC in which AEBP1 was knocked down. It can be seen that the number of cells having G1 arrest increased. F is a figure showing the result of a tube formation assay. It can be seen that the tube formation ability of HUVEC in which AEBP1 was knocked down was suppressed. G is a diagram showing the results of a wound healing assay. It can be seen that wound closure of HUVECs in which AEBP1 was knocked down was suppressed. H is a diagram showing the results of HUVEC expression array analysis 48 hours and 72 hours after introduction of two types of siAEBP1 or control siRNA. AQP1, POSTN1, etc. were identified as genes fluctuating in common between the two siRNAs, in addition to AEBP1. I and J are graphs showing the results of introducing two types of siAEBP1 or control siRNA into HUVEC and confirming the expression of AQP1 and POSTN1 at the mRNA level using a quantitative RT-PCR method. FIG. 8A shows a tumor growth curve when DLD1 or DLD1 and HUVEC were implanted in a nude mouse. B is a graph comparing tumor volumes (mm ³ ) on day 14 after transplantation. C is a graph showing the microvessel density of the tumor 12 days after transplantation. D is a stained image obtained by immunohistologically staining a paraffin-embedded section of the tumor with CD31. E shows a tumor growth curve when DLD1 was implanted in nude mice and siAebp1 was administered successively. F is a graph showing the microvessel density of the tumor 28 days after transplantation. G is a stained image obtained by immunohistologically staining a paraffin-embedded section of the tumor with CD31.

Hereinafter, the present invention will be described in detail.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referred to herein are hereby incorporated by reference in their entirety. In the case of inconsistencies between the publication referred to in this specification and the description of this specification, the description of this specification shall control.

  In the present disclosure, for example, when simply described by a gene name such as “AEBP1”, it means a gene having a known nucleic acid sequence represented by the gene name, unless otherwise specified, typically, cDNA or It represents an mRNA sequence, but is not limited thereto as long as a person skilled in the art can recognize it as a sequence of the gene. For example, one or more, preferably 1 to several, more preferably 1 to 10, 1 to 5 Base sequence including deletion, substitution, addition or insertion of 1, 3 or 1, or 2 nucleotides, 70% or more, 80% or more or 90% or more, preferably 95% or more of the cDNA or mRNA sequence And more preferably a base sequence having 98% or more or 99% or more sequence identity, and a degenerate sequence.

  Examples of a preferred gene of AEBP1 and its nucleic acid sequence in the present disclosure include a sequence represented by GenBank Accession No. NM_001129.4 and a homolog thereof. Further, a base sequence that specifically hybridizes with a nucleic acid molecule having such a sequence under stringent conditions, or about 60% or more, preferably about 70% or more, more preferably about 80% or more, more preferably the above sequence. A nucleic acid containing a nucleotide sequence having a homology of about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more is also encompassed in AEBP1 of the present disclosure.

  In the present disclosure, when a gene name such as "AEBP1 protein" is appended to "protein", it means the protein encoded by the gene, its isoform, and its homolog. Examples of the isoform include splicing variants and variants such as SNPs based on individual differences. Specifically, (1) a protein consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more homology in the protein encoded by the gene; One or more, preferably one to several, more preferably one to ten, one to five, one to three, one or two amino acids are substituted or deleted in the amino acid sequence of the encoded protein. , A protein consisting of an added or inserted amino acid sequence.

  In the present disclosure, the term “inhibitor” of a certain gene includes an agent that inhibits or suppresses the expression of the gene, as well as an agent that inhibits the activity of a protein encoded by the gene. In the present disclosure, the “agent that inhibits or suppresses the expression of a gene” includes, for example, siRNA, antisense nucleic acid, shRNA, or miRNA against a transcript RNA of the gene, and also includes, for example, a transcription inhibitor of the gene. Examples of the agent that inhibits or suppresses the expression of AEBP1 of the present disclosure include siRNA (eg, a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2) or a precursor RNA thereof, which have an RNA interference (RNAi) effect. Or modified RNAs thereof, or vectors containing DNAs encoding siRNAs against transcript RNAs of the AEBP1 gene. In the present disclosure, the agent that inhibits or suppresses the expression of AEBP1 may be, for example, shRNA, miRNA, or the like. The vector may include antisense RNA or antisense DNA, DNA encoding the antisense RNA, or the antisense DNA.

  The siRNA is a sense RNA consisting of 18 to 25 nucleotides, preferably 20 to 24 nucleotides, more preferably 21 to 23 nucleotides complementary to a part of the transcript RNA of the AEBP1 gene, and having an RNAi (RNA interference) action. And an antisense RNA. Each of the 3 'ends of the sense RNA and the antisense RNA may have a protruding end of 2 to 5 nucleotides, preferably 2 nucleotides.

  In the present disclosure, the AEBP1 gene expression inhibitor may be, for example, a combination of an siRNA containing a sense strand and an antisense strand. For example, the nucleic acid of SEQ ID NO: 1 and the antisense strand may be used as the sense strand. Or a combination of the nucleic acid of SEQ ID NO: 2 as the sense strand and the nucleic acid of SEQ ID NO: 4 as the antisense strand. In addition, these nucleic acids may include any modified nucleic acids.

  In the present disclosure, the “agent that inhibits the activity of a protein encoded by a gene” may be any agent as long as the protein cannot exert its activity, and is not limited thereto. However, for example, a nucleic acid or protein that binds to the protein, a protein that converts an active form of the protein to an inactive form or a nucleotide containing a nucleic acid encoding such a protein, an agent that inhibits a protein that activates the protein, and the like Is mentioned. Examples of the agent that inhibits the activity of the AEBP1 protein of the present disclosure include, but are not limited to, an antibody that recognizes the AEBP1 protein, a compound that inhibits the AEBP1 protein, and the like.

The inhibitors of the present disclosure can be introduced into cells by any known introduction technique, including, but not limited to, the lipofectamine method, the lipofection method, the calcium phosphate method, the ultrasonic introduction method, the electroporation method, the particle gun method, and the virus. A method using a vector (for example, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or the like), a microinjection method, or the like can be used.
When a virus vector is used, the titer of the virus is 1 × 10 ³ to 1 × 10 ¹⁵ p. f. u. (Plaque forming unit), preferably 1 × 10 ⁵ to 1 × 10 ¹³ , more preferably 1 × 10 ⁷ to 1 × 10 ¹¹ , still more preferably 1 × 10 ⁸ to 1 × 10 ¹⁰ . be able to.

  When the inhibitors of the present disclosure are nucleic acids, such nucleic acid molecules may be used as naked nucleic acids or incorporated into various nucleic acid constructs or vectors. As the vector, any known vector such as a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, and a virus vector can be used. The nucleic acid construct or vector preferably contains at least appropriate transcription or translation control sequences derived from, for example, mammalian, microbial, viral, or insect genes. Such control sequences include sequences having a regulatory role in gene expression, such as transcription promoters or enhancers, operator sequences for regulating transcription, sequences encoding ribosome binding sites within messenger RNA, and transcription, translation initiation. Alternatively, a suitable sequence for regulating the termination of transcription is included.

<1> Pharmaceutical composition of the present disclosure One aspect of the present disclosure relates to a pharmaceutical composition containing an AEBP1 inhibitor as an active ingredient.
The pharmaceutical composition of the present disclosure is characterized in that it comprises an inhibitor of AEBP1 as an active ingredient, and further comprises one or more pharmaceutically acceptable surfactants, carriers, diluents and / or excipients. It may contain a shaping agent and the like. Pharmaceutically acceptable surfactants, carriers, diluents and / or excipients are well known in the pharmaceutical art, for example, see Remington's Pharmaceutical Sciences, 18th Ed., 18th Ed., Which is incorporated herein in its entirety. Mack Publishing Co., Easton, PA (1990). A person skilled in the art can select an appropriate pharmaceutically acceptable surfactant, carrier, diluent and / or excipient, etc., according to the administration form of the pharmaceutical composition and the like.

  The inhibitor of AEBP1 contained in the pharmaceutical composition of the present disclosure is not particularly limited as long as it is an agent capable of inhibiting or suppressing the activity of AEBP1 protein or the expression of AEBP1, and the activity of AEBP1 protein exemplified above is not limited. An agent that inhibits the expression of AEBP1 or an agent that inhibits or suppresses the expression of AEBP1 can be used. Typically, an agent that inhibits or suppresses the expression of AEBP1 is used. As described above, the agent for inhibiting or suppressing the expression of AEBP1 is typically an siRNA against the transcript RNA of AEBP1 (eg, a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2) or a precursor RNA thereof. Or modified RNAs, shRNAs and miRNAs thereof, or vectors containing DNAs encoding the RNAs. In a preferred embodiment, the inhibitor of AEBP1 is an siRNA against AEBP1 transcript RNA.

  The pharmaceutical composition of the present disclosure can be used, without limitation, for treating the above-mentioned diseases associated with high activity or high expression of AEBP1. Therefore, the present invention also relates to a pharmaceutical composition comprising an effective amount of the above-mentioned inhibitor of AEBP1 for the treatment of a disease in which high activity or high expression of AEBP1 is involved. The “disease associated with high activity or high expression of AEBP1” typically includes cell proliferative diseases such as tumors, fibrosis such as pulmonary fibrosis, and the like. Here, in the present disclosure, “tumor” includes a benign tumor and a malignant tumor (cancer, malignant neoplasm). Cancer includes hematopoietic tumors, epithelial malignancies (carcinoma) and non-epithelial malignancies (sarcoma, sarcoma).

As described above, the present inventors have newly found that the expression of AEBP1 is particularly enhanced in tumor vascular endothelial cells and cancer-related fibroblasts (CAF). Thus, in a preferred embodiment, the disease treated by the pharmaceutical composition of the present disclosure is a cancer, and in a more preferred embodiment, a solid tumor. Here, "solid tumor" means a tumor other than a hematopoietic tumor.
The cancer targeted by the pharmaceutical composition of the present disclosure is not limited as long as AEBP1 is expressed in tumor vascular endothelial cells and CAF, and examples thereof include fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and striated muscle. Tumors, leiomyosarcoma, hemangiosarcoma, Kaposi's sarcoma, lymphatic sarcoma, sarcoma such as synovial sarcoma, chondrosarcoma, osteosarcoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, Colorectal, rectal, liver, pancreatic, gallbladder, bile duct, anal, kidney, ureter, bladder, prostate, penis, testicular, uterine, ovarian, vulvar, vaginal, And carcinomas such as skin cancer. In one preferred aspect, the pharmaceutical composition of the present disclosure is for treating colorectal cancer.

  Whether or not AEBP1 is expressed in tumor vascular endothelial cells and CAF can be tested using methods known in the art, for example, a method for detecting the presence of AEBP1 protein, expression of AEBP1 transcript RNA Can be determined by a method of detecting at the gene level. Specifically, as a detection method at the protein level, for example, known protein expression analysis methods such as immunostaining, Western blotting, and ELISA using an antibody that specifically recognizes the AEBP1 protein, As a detection method, for example, detection can be performed by a known gene expression analysis method such as a Northern blotting method, a RNase protection assay, a PCR method such as RT-PCR, real-time PCR, an in situ hybridization method, and an in vitro transcription method. . In one embodiment of the present invention, the expression level of AEBP1 is increased by 2 times or more, preferably 3 times or more, more preferably 5 times or more, still more preferably 10 times or more, and particularly preferably 20 times or more compared to normal cells. I have.

  As described above, AEBP1 is highly expressed in tumor vascular endothelial cells, and by inhibiting or suppressing the activity of AEBP1 protein and / or the expression of AEBP1 in the cells, angiogenesis in tumor tissue can be inhibited; Thus, tumor growth can be suppressed and the tumor can be treated. Therefore, in a preferred embodiment, the pharmaceutical composition of the present invention can be used as an angiogenesis inhibitor, more preferably as a tumor angiogenesis inhibitor.

The pharmaceutical compositions of the present disclosure may further include other agents useful for treating the disease of interest, such as anti-tumor agents, anti-inflammatory agents, and the like.
Examples of the antitumor agent include, but are not limited to, ifosfamide, nimustine (eg, nimustine hydrochloride), cyclophosphamide, dacarbazine, melphalan, an alkylating agent such as ranimustine, gemcitabine (eg, gemcitabine hydrochloride), enositabine, Antimetabolites such as cytarabine ocphosphate, cytarabine preparations, tegafur uracil, tegafur gimeracil oteracil potassium combination (eg, TS-1), doxyfluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, idarubicin (eg, hydrochloric acid) Idarubicin), epirubicin (eg, epirubicin hydrochloride), daunorubicin (eg, daunorubicin hydrochloride, daunorubicin citrate), doxorubicin (eg, doxorubicin hydrochloride) ), Pirarubicin (eg, pirarubicin hydrochloride), bleomycin (eg, bleomycin hydrochloride), peplomycin (eg, peplomycin sulfate), mitoxantrone (eg, mitoxantrone hydrochloride), mitomycin C and other antitumor antibiotics, etoposide Irinotecan (eg, irinotecan hydrochloride), vinorelbine (eg, vinorelbine tartrate), docetaxel (eg, docetaxel hydrate), paclitaxel, vincristine (eg, vincristine sulfate), vindesine (eg, vindesine sulfate), vinblastine (eg, sulfate) Vinblastine), alkaloids, anastrozole, tamoxifen (eg, tamoxifen citrate), toremifene (eg, toremifene citrate), bicalutamide, flu Hormonal therapeutic agents such as mid, estramustine (for example, estramustine phosphate), platinum complexes such as carboplatin, cisplatin (CDDP) and nedaplatin, angiogenesis inhibitors such as thalidomide, neovastat, bevacizumab, L-asparaginase and the like Is mentioned.

  Examples of the anti-inflammatory agent include, but are not limited to, steroidal anti-inflammatory agents (prednisolone, beclomethasone, betamethasone, fluticasone, dexamethasone, hydrocortisone, etc.) and non-steroidal anti-inflammatory agents (acetylsalicylic acid, loxoprofen, acetaminophen, ketoprofen, thiaprofen Acids, suprofen, tolmetin, carprofen, benoxaprofen, piroxicam, benzidamine, naproxen, diclofenac, ibuprofen, diflunisal, azapropazone, etc.), RNAi molecules that suppress the expression of inflammatory cytokines (eg, siRNA, shRNA, ddRNA, miRNA, piRNA, rasRNA, etc.), substances such as antisense nucleic acids, and / or drugs that suppress the action of inflammatory cytokines, for example, And antibodies to the inflammatory cytokine, and the like receptor antagonists of inflammatory cytokines.

  The pharmaceutical compositions of the present disclosure can include the inhibitors of the present disclosure loaded on various drug delivery carriers. Such carriers include, but are not limited to, for example, polymer nanoparticles, polymer micelles, dendrimers, liposomes, virus nanoparticles, carbon nanotubes, and the like (Cho K. et al., Clin Cancer Res. 2008 Mar 1; 14). (5): see 1310-6).

  The inhibitors and pharmaceutical compositions of the present disclosure can be administered by a variety of routes, including both oral and parenteral, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, topical, rectum, intratumoral, intraarterial , Intraportal, intramedullary, intrapulp, sublingual, buccal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, etc. May be formulated in a suitable dosage form. As such a dosage form and formulation method, any known one can be appropriately adopted (for example, see Standard Pharmacology, edited by Yoshiteru Watanabe et al., Nankodo, 2003, Remington's Pharmaceutical Sciences, etc., above).

  For example, dosage forms suitable for oral administration include, without limitation, powders, granules, tablets, capsules, solutions, suspensions, emulsions, gels, syrups, and the like, and for parenteral administration. Suitable dosage forms include injections such as solution injections, suspension injections, emulsion injections, and ready-to-use injections. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.

  The present disclosure also provides kits for the preparation of compositions comprising one or more containers containing, alone or in combination, components that may be included in the various agents or compositions of the present disclosure, as well as in the form of such kits. As well as the necessary components of the various agents or compositions provided in. Such a kit may include, in addition to the above, instructions describing the method of preparing or administering the inhibitor or pharmaceutical composition of the present disclosure, such as instructions, an electronic recording medium such as a CD or DVD, and the like. .

  Also, as described below, the present inventors have compared subjects with tumors that express AEBP1 in tumor vascular endothelial cells and / or CAF with tumor controls that do not express AEBP1 in tumor vascular endothelial cells and / or CAF. It was found that the prognosis of tumor treatment was poor. This suggests that the inhibitors or pharmaceutical compositions of the present disclosure can be used to improve the prognosis of a subject receiving a tumor treatment. Accordingly, the pharmaceutical compositions of the present disclosure, in another aspect, include pharmaceutical compositions used to improve the prognosis of treating a tumor in a subject.

<2> Method for preventing and / or treating disease according to the present disclosure In another aspect, the present disclosure relates to a method for preventing and / or treating a disease associated with high activity or high expression of AEBP1 in a subject, the method comprising: (Hereinafter sometimes referred to as “the treatment method of the present disclosure”), which comprises the step of administering an effective amount of the inhibitor or the pharmaceutical composition to a subject in need thereof.

  The “subject” in the present disclosure may be any biological individual that can be afflicted with a disease associated with high activity or high expression of AEBP1, typically cancer, but is preferably human and Non-human mammals (eg, rodents such as mice, rats, guinea pigs, hamsters, primates such as chimpanzees, artiodactyla such as cattle, goats and sheep, equinoids such as horses, rabbits, dogs and cats) And more preferably a human individual. In the present disclosure, the subject may be healthy or suffering from any disease, but typically has cancer if, for example, prevention and / or treatment of cancer is contemplated. A subject is suffering or at risk of suffering. Prevention of cancer may also include prevention of cancer recurrence, and thus subjects at risk for having cancer may include subjects at risk for recurrence of cancer.

  Also, the term "treatment" as used herein encompasses all types of medically acceptable prophylactic and / or therapeutic interventions, such as for the cure, temporary remission or prevention of disease. Shall be. For example, the term “treatment” encompasses medically acceptable interventions for a variety of purposes, including slowing or stopping the progression of a disease, regressing or eliminating lesions, preventing the onset or preventing recurrence, and the like.

  Inhibitors or pharmaceutical compositions that can be used in the treatment methods of the present disclosure include any of those described herein. An effective amount in the present aspect is, for example, an amount that reduces the symptoms of cancer or delays or stops the progress thereof, and is preferably an amount that suppresses or cures cancer. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined by an in vitro test using cultured cells or the like, or a test using a model animal such as a mouse or a rat. Such a test method is well known to those skilled in the art. The specific dose of the active ingredient depends on various conditions related to the subject in need thereof, such as the severity of symptoms, general health of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, The determination can be made in consideration of the concomitant drug, response to treatment, dosage form, compliance with treatment, and the like. As for the administration method, any known and appropriate administration method as exemplified above can be used.

  One embodiment of the treatment method of the present disclosure may further include, before the administering step, selecting a subject having AEBP1-positive tumor vascular endothelial cells as a target for prevention / treatment. In such an embodiment, before the selecting step, the method may further include a step of collecting a tumor sample from the subject and a step of detecting AEBP1 expression in the tumor sample obtained from the subject. As a detection method used in such a step, the method exemplified for the method of detecting AEBP1 expression in the tumor vascular endothelial cells or CAF can be used.

<3> Prognostic prediction method of the present disclosure As described above, the present inventors have found that the higher the expression level of AEBP1 in a target tumor tissue, the worse the prognosis after tumor treatment. Accordingly, another aspect of the present disclosure includes a method of predicting the prognosis of a tumor treatment in a subject, comprising determining the expression level of an expression product of AEBP1 in a test sample (eg, tumor tissue from the subject) derived from the subject. (Hereinafter, may be referred to as “prognostic prediction method of the present disclosure”).

The prognostic method of the present disclosure includes the following steps:
(A) A step of determining the expression level of an AEBP1 expression product in a test sample derived from a subject.
In the prognosis prediction method of the present disclosure, the tumor is as described in detail in <1> above, and is preferably a colon cancer.

In the prognostic prediction method of the present disclosure, the “test sample” is not particularly limited as long as it is a sample containing tumor vascular endothelial cells and / or CAF derived from a subject, and is typically a tumor tissue sample derived from a subject.
In the prognostic method of the present disclosure, “expression product” means any substance produced in the expression process initiated by transcription of AEBP1, and typically, for example, transcript RNA of AEBP1 or AEBP1 protein, Or fragments thereof.

In the step (A), the expression level of the expression product of AEBP1 is determined. For determining the expression level of the expression product, a method for detecting an expression product as described in detail in <1> above can be used.
The prognosis prediction method of the present disclosure may optionally include the following steps after step (A):
(B) A step of comparing the expression level obtained in (A) with a reference value may be included.

As the reference value, any value may be used as long as the value is a reference for the amount of expression of AEBP1 in the sample. Typically, for example, the expression level of AEBP1 in the same tissue of a healthy subject and the prognosis are determined. The average value of the expression level of AEBP1 in the tumor tissue of a patient determined to be good, the average value of the expression level of ARBP1 of the tumor tissue in a patient determined to be poor in prognosis, and the like are given.
Herein, “poor prognosis” in the present disclosure typically refers to the period of remission after treatment of a subject treated with a tumor, the remission status after treatment in a control group treated with the same type of tumor. Is shorter than the average value of the period.

In one aspect, when the expression level of AEBP1 in the target sample is larger than the reference value, the prognosis is predicted to be poor. In another embodiment, if the expression level of AEBP1 in the control sample is small compared to the reference value, the prognosis is predicted to be good.
In a preferred embodiment, the reference value is 0. That is, when an AEBP1 expression product is detected in a target sample, the prognosis is predicted to be poor.

<4> Method for Improving Prognosis of the Present Disclosure As described above, a tumor that highly expresses AEBP1 in a tumor tissue, for example, a tumor microenvironment or tumor vascular endothelial cell, has a higher prognosis after treatment than a tumor that does not highly express it. Was found to be poor by the present inventors. AEBP1 is a factor involved in angiogenesis in tumor tissue, and inhibiting AEBP1 can inhibit angiogenesis in tumor tissue, thereby improving the prognosis of tumor treatment in a subject. Accordingly, one aspect of the present disclosure relates to a method for improving the prognosis of tumor treatment, comprising administering to a subject after tumor treatment an effective amount of an inhibitor or pharmaceutical composition of the present disclosure (see “Prognostic Improvement Methods of the Present Disclosure "May be referred to as").

As used herein, "improving prognosis" refers to prolonging the expected duration of remission in a subject treated with a tumor. The prediction of the remission period may be predicted by the prognosis prediction method of the present disclosure, or may be predicted from, for example, the average value of the remission period of a subject group to which similar tumor treatment has been performed.
The “tumor treatment” in this aspect may be any treatment known in the art other than the treatment method of the present disclosure, such as surgical resection, drug therapy using an antitumor agent, radiation therapy, cancer It may be immunotherapy or the like.

  An effective amount in this aspect is, for example, an amount capable of inhibiting the activity or expression of AEBP1 in a subject, and preferably an amount capable of inhibiting neovascularization of tumor blood vessels. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined by an in vitro test using cultured cells or the like, or a test using a model animal such as a mouse or a rat. Such a test method is well known to those skilled in the art. The specific dose of the active ingredient depends on various conditions related to the subject in need thereof, such as the severity of symptoms, general health of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, The determination can be made in consideration of the concomitant drug, response to treatment, dosage form, compliance with treatment, and the like. As for the administration method, any known and appropriate administration method as exemplified above can be used.

  Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Isolation of vascular endothelial cells from human colon cancer tissue The human colon cancer tissue was cut into small fragments using scissors and a razor. To this, type I collagenase (Worthington) and DNase I (Worthington) were added, and an enzyme treatment was performed by shaking at 37 ° C. for 2 hours to obtain a tissue lysate. This was passed through a filter to collect the cells, and an ACK buffer (Thermo Fisher Scientific) was added to remove red blood cells. The obtained cell suspension was subjected to magnetic microbeads (immuno-magnetic separation, IMS) using Epihelial Enrich (invitrogen), anti-CD146 antibody (anti-MCAM antibody, EMD Millipore) and Dynabeads Pan mouse IgG (VERITAS). Epithelial cells and vascular endothelial cells were separated. Epithelial Enrich is obtained by binding an anti-EpCAM antibody to beads, and can directly bind to EpCAM-positive epithelial cells. The anti-CD146 antibody was shake-mixed with Dynabeads Pan mouse IgG beforehand to bind the antibody and the beads, and then bound to CD146-positive cells.

  As a preliminary experiment of cell separation by the IMS method (FIG. 1A), sorting by CD31 and CD146, which are vascular endothelial cell markers, was performed using FACS. As a result, 1.1% of CD31-positive cells and 7.7% of CD146-positive cells were obtained. (FIG. 1B). The 14 cases of colorectal cancer tissue were divided into cancerous and non-cancerous parts, and the epithelial cell markers were divided into EpiCAM-positive cells and CD146-positive cells, whereby tumor epithelial cells (n = 13), normal epithelial cells (n = 12), A total of 53 samples of TECs (n = 14) and NECs (n = 14) were obtained. Total RNA was extracted from the obtained sample using an RNeasy kit.

Example 2. RNA sequence analysis 14 cases of colorectal cancer tissue were divided into cancerous and non-cancerous parts and divided into epithelial cells and vascular endothelial cells, respectively, to obtain tumor epithelial cells (n = 13), normal epithelial cells (n = 12), and tumor blood vessels. A total of 53 samples of endothelial cells (TECs, n = 14) and normal endothelial cells (NECs, n = 14) were obtained. RNA was collected from these samples using TRI REAGENT (MRC) and quality checked using a 2100 Bioanalyzer-RNA Solution (Agilent, RNA6000 Nano Kit). RNA sequencing was performed on three samples each of TECs and NECs (all RIN values ≧ 8), and the obtained expression data was analyzed using a Volcano plot.

  The result of comprehensively examining the expression of mRNA in each of three samples of NECs and TECs using RNA-seq was analyzed using a Volcano plot, and the expression was statistically changed in NECs and TECs. Genes were recognized, and among them, 18 genes whose expression was predominantly enhanced in TECs were identified (FIG. 1C). Among these, ANTXR1 which was reported as a tumor endothelial maker 8 (TEM8) as a gene highly expressed in TECs was also included.

Example 3 Verification of Gene Expression in Clinical Specimens Expression of messenger RNA (mRNA) levels was confirmed for the genes listed as candidates by RNA sequence analysis using qRT-PCR. In addition, immunohistological staining of paraffin-embedded sections of clinical samples was performed to confirm the expression of candidate genes at the protein level.

  For all 53 samples, the expression at the mRNA level of 16 of the 18 genes except the non-coding genes was evaluated using quantitative RT-PCR (FIG. 2). AEBP1 was identified as the gene whose expression was most significantly enhanced in TECs, and immunohistochemical staining of paraffin-embedded sections of clinical samples confirmed AEBP1 expression in TEC (FIG. 3A). AEBP1 was found to stain not only in tumor blood vessels but also in tumor stroma including CAF (FIG. 3B).

Example 4. Verification of expression of candidate genes in colon cancer cell line cells and normal cells Eight types of colon cancer cell line cells (DLD1, SW480, RKO, WiDr, HT-29, HCT116, COLO320, LoVo), normal vascular endothelial cells Human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMVEC), normal human dermal fibroblasts (NHDF) (LONZA) and cancer-associated fibroblasts (CAF) (Vitro Biopharma) were prepared. . Eight types of colorectal cancer cell lines are in accordance with the ATCC protocol, DLD1, SW480, RKO, WiDr are DMEM / 10% FBS, HT-29, HCT116 are McCoy's 5A / 10% FBS, COLO320 is RPMI / 10%. FBS and LoVo were cultured using a F-12 / 10% FBS medium, and HUVECs and HMVECs were cultured using EGM-2 (LONZA). The expression of these cells at the mRNA level of the candidate gene was confirmed using qRT-PCR. Similarly, expression of vascular endothelial marker (CD31) and tumor vascular endothelial marker (CD146) at the mRNA level in these cells was also confirmed.

  AEBP1 and ANTXR1 (TEM8) using quantitative RT-PCR in colon cancer cell line cells (DLD1, SW480, RKO, WiDr, HT-29, HCT116, COLO320, LoVo), HUVEC, HMVEC, NHDF and CAF At the mRNA level of 7 genes, including, (FIG. 4A). The expression of AEBP1 in HUVEC and HMVEC was 100 times or more that of colon cancer cell line cells, and the expression in colon cancer cell line cells was extremely small. The specificity of AEBP1 expression in HUVEC, HMVEC, NHDF and CAF was higher than ANTXR1. It was also found that both AEBP1 and ANTXR1 are expressed not only in endothelial cells but also in NHDF and CAF. The expression of the vascular endothelial marker (CD31) was high in HUVEC and HMVEC and low in NHDF, CAF, and colon cancer cell line cells, but it was found that CD146 was also expressed to some extent in CAF (FIG. 4B).

Example 5 Changes in expression of candidate genes by tumor culture supernatant experiments and co-culture experiments Eight types of colon cancer cell line cells were cultured until they became semi-confluent, and the cells were cultured in a serum-free medium for 24 hours, and the culture supernatant was collected. The mixture was passed through a filter (MILLIPORE) having a pore size of 0.22 μm to obtain a tumor culture supernatant (TCM). The appearance of HUVEC cultured in TCM (serum-free) and control medium, which is a serum-free basal medium, was observed successively with an optical microscope. Furthermore, changes in the expression of AUVP1 in HUVEC cultured in TCM and control medium at the mRNA level were successively evaluated using qRT-PCR. HUVECs were cultured in TCM supplemented with FBS (2.5%), and changes in AEBP1 expression were evaluated over time. Further, DLD1 and HUVEC were inoculated in the same 10 cm dish at a ratio of 1: 4, and co-cultured using EGM-2. 10 ⁶ cell population recovered in was 24 hours and 96 hours, to separate the DLD1 and HUVEC using the IMS technique. For the antibody, DLD1 was recovered from the cell population by positive selection using only Epihelial Enrich, and HUVEC was recovered by negative selection. The recovered DLD1 and HUVEC were evaluated for their expression at the mRNA level of CD31 and AEBP1 using the qRT-PCR method.

  When HUVEC cultured in TCM (FIG. 5A) collected from colon cancer cell line cells were observed with an optical microscope, in HUVEC cultured in serum-free medium (DMEM), many cells came off the dish in 24 h and aggregated. , 48 h, these changes were observed in almost all cells (FIG. 5B). In HUVEC cultured in TCM, more cells aggregate over time, but less than in HUVEC cultured in serum-free medium. In addition, when cultured with DLD1-derived TCM, more HUVECs survived than when cultured with SW480-derived TCM, and differences in the survival and aggregation of HUVECs were observed depending on the type of cancer cells producing TCM (FIG. 5B). HUVECs were cultured in serum-free and TCM (serum-free) media derived from eight types of colorectal cancer cell lines, and the expression changes at the mRNA level of AEBP1 were evaluated successively using quantitative RT-PCR. did. In many cases, the expression of AEBP1 was enhanced in HUVEC cultured in TCM for 24 hours, whereas the expression was decreased in HUVEC cultured in serum-free medium. In particular, when the cells were cultured using DLD1 and TCM derived from HCT116, a significant difference was observed in the expression of AEBP1 at 12 hours and 24 hours after the start of the culture, as compared with the case where the cells were cultured in a serum-free medium (FIG. 5C). When cultured in TLD derived from DLD1 to which FBS was added (2.5%), the timing of the peak of AEBP1 expression was not changed in 24 hours, but the expression level increased compared to the case without serum. The expression level of AEBP1 at the peak was higher when FBS was added (2.5%). When the cells were cultured in a medium supplemented with FBS, the expression of AEBP1 was enhanced, but not so much as when the cells were cultured in TCM. When DLD1 and HUVEC were cultured in the same dish and observed with an optical microscope, DLD1: HUVEC = 1: 4 at the start of culture, but after 96 hours, the ratio was almost the same (FIG. 5G). At 24 hours and 96 hours after the start of the culture, DLD1 and HUVEC were separated using the IMS method, and the expression of AEBP1 and CD31 at the mRNA level was evaluated using the quantitative RT-PCR method. Even higher expression was seen in the HUVECs after 96 hours than in the TCM experiments (FIG. 5F).

Example 6. Published large-scale clinical data analysis
Analysis of mRNA expression data by RNA-seq and overall survival (OS) using large-scale clinical specimens published by The Cancer Genome Atlas (TCGA) revealed that subjects with colorectal cancer overexpressing AEBP1 Resulted in significantly lower survival rates. From this, it was found that high expression of AEBP1 was a poor prognostic factor for colorectal cancer (FIG. 6).

Example 7 Knockdown experiment of AEBP1 in HUVEC (1) Transfection of siAEBP1 Two kinds of siAEBP1 (siAEBP1_1 (SEQ ID NOs: 1 and 3) and siAEBP1_2 (SEQ ID NOs: 2 and 4)) adjusted to 20 μM (5 nmol / 250 μl) and control siRNA ( Ambion) was prepared and transfected into HUVEC by an electroporation method using a HUVEC Nucleofector Kit (Amaxa). HUVECs transfected with siAEBP1 and control siRNA were cultured in EGM-2, RNA was extracted after 48 hours, and expression of AEBP1 at the mRNA level was evaluated by RT-PCR to examine knockdown efficiency. . A similar experiment was performed using CAF into which two types of siAEBP1 and control siRNA had been introduced.

  SiAEBP1 was introduced into HUVEC and CAF using the electroporation method. RNA was extracted from HUVEC and CAF 48 hours after transfection, and the expression of AEBP1 at the mRNA level was evaluated using a quantitative RT-PCR method. The knockdown efficiencies were both around 90% (FIGS. 7A and B).

(2) Cell proliferation test Two types of siAEBP1 (siAEBP1_1 and siAEBP1_2) and HUVEC immediately after introduction of control siRNA were seeded on a 96-well plate, and a cell proliferation test was performed using Cell Counting Kit-8 (Dohin Chemical Laboratory). went. A similar experiment was performed using CAF into which two types of siAEBP1 and control siRNA had been introduced.
HUVEC and CAF knocked down by the two siRNAs were found to significantly suppress cell growth as compared to the control (FIGS. 7C and D).

(3) Cell cycle analysis Cell cycle analysis using a flow cytometer was performed using HUVECs after 48 hours of introduction of two types of siAEBP1 and control siRNA.
HUVEC knocked down by the two siRNAs was found to increase the number of cells undergoing G1 arrest compared to the control, which was a result consistent with the cell proliferation test (FIG. 7E).

(4) In vitro tube formation assay
Matrigel (Corning), a soluble basement membrane preparation extracted from Engelbreth-Horm-Swarm (EHS) mouse sarcoma, was prepared. It is known that normal vascular endothelial cells cause angiogenesis on Matrigel. HUVEC into which two types of siAEBP1 and control siRNA were introduced were cultured in EGM-2, and after 48 hours, seeded on Matrigel, and the state of blood vessel formation was observed with an optical microscope. Furthermore, the effect of knockdown of AEBP1 on the formation of HUVEC lumens on Matrigel was quantitatively evaluated using image analysis software Image J.
HUVEC in which AEBP1 was knocked down had significantly reduced tube formation ability as compared to the control (FIG. 7F).

(5) Wound healing assay HUVEC into which siAEBP1 and control siRNA had been introduced were cultured in EGM-2 and seeded on a 6-well plate. Wounds were formed when they became semi-confluent, and wound closure was assessed at 12 and 24 hour time points thereafter.
HUVEC in which AEBP1 was knocked down significantly suppressed wound closure compared to the control (FIG. 7G).

(6) Gene expression microarray analysis Gene expression microarray analysis was performed to search for genes whose expression fluctuates due to knockdown of AEBP1. Microarray experiments using SurePrint G3 Human GE 8x60K V2 microarray (Agilent Technologies) after extracting RNA from HUVEC 48 hours and 72 hours after introducing two kinds of siAEBP1 (siAEBP1_1 and siAEBP1_2) and control siRNA and synthesizing cRNA Was done. For data analysis, GeneSpring GX version 13 (Agilent Technologies) was used. Gene Ontology (GO) analysis was performed on the identified gene.

  Two types of siAEBP1 or control siRNA were introduced into HUVEC, collected at 48 hours and 72 hours, and collected at two time points. The expression was fluctuated by comparing the sample and the control knocked down at each time point. Gene expression microarray analysis was performed to search for genes to be expressed. A plurality of genes whose expression levels decreased 2.0 times or more compared to the control were identified (FIG. 7H). Genes whose expression fluctuates due to knockdown of AEBP1 at a time point of 48 hours after siRNA introduction include POSTN, and genes that fluctuate in common at the 48 and 72 hour time points include AQP1 in addition to AEBP1. Had been. Furthermore, the expression level of mRNA was confirmed by quantitative RT-PCR for AQP1 and POSTN, which have already been reported such as tumor angiogenesis (FIGS. 7I and J).

Example 8 Mouse xenograft experiment (1) Co-transplantation of DLD1 and HUVEC Since it has been reported that tumors grow more when sarcoma and HUVEC are mixed and transplanted than when only sarcoma is transplanted, a similar experiment was performed on colon cancer cell line cells (DLD1) Was done. DLD1 or DLD1 and HUVEC were suspended in Matrigel (250 μl) diluted with PBS (250 μl) and transplanted to nude mice. The transplanted tumor was measured over time and the volume was roughly estimated (V = (W ² × L) / 2 (V: volume, W: minor axis, L: major axis), and a growth curve was determined.
It was confirmed that when DLD1 and HUVEC were transplanted, the number of transplanted tumors increased compared to when DLD1 was transplanted alone (FIGS. 8A and B).

(2) Co-transplantation of DLD1 and AEBP1 knockdown HUVEC HUVEC has been reported to be involved in angiogenesis of transplanted tumors in combination with mouse blood vessels. Therefore, in order to examine the effect of knocking down AEBP1 of HUVEC to be transplanted on angiogenesis in a transplanted tumor, HUVEC (5 × 10 ⁵ ) introduced with one kind of siAEBP1 or control siRNA, DLD1 (5 × 10 ⁵ ) were transplanted to nude mice. HUVEC and DLD1 into which siRNA had been introduced were suspended in Matrigel (250 μl) diluted with PBS (250 μl) and transplanted into mice. The transplanted tumor was excised on day 12 after transplantation in which no necrosis occurred inside the transplanted tumor, fixed in formalin, and paraffin-embedded sections were prepared. Tumor vessels were stained with a CD31 antibody, and the microvessel density (MVD) was measured.
It was found that when HUVEC in which AEBP1 was knocked down was co-transplanted with DLD1, MVD was smaller than that in the control, and tumor angiogenesis in the transplanted tumor was suppressed (FIGS. 8C and D).

(3) Knockdown of DLD1-transplanted tumor stroma In order to examine the effects of the knockdown of mouse Aebpl on tumor blood vessels, tumor stroma, and tumor growth invading the transplanted tumor, two types of siRNA against mouse Aebpl were prepared. DLD1 (1 × 10 ⁶ ) was suspended in Matrigel (250 μl) diluted with PBS (250 μl) containing siAebp1 and control siRNA and transplanted to nude mice. Further, two types of siAebp1 or control siRNA were administered every four days around the transplanted tumor. Transplanted tumors were measured over time and growth curves determined. In addition, a paraffin-embedded section of the transplanted tumor was immunohistologically stained with CD31, and MVD was objectively measured using Image J.

  In each of the groups to which two types of siAebp1 were administered around the transplanted tumor, the growth of the transplanted tumor was suppressed as compared with the control group (FIG. 8E). In addition, it was found that the group to which siAEBP1 was administered had less MVD than the control group, and that tumor angiogenesis in the transplanted tumor was also suppressed (FIGS. 8F and 8G).

Claims (9)

  A pharmaceutical composition for treating a tumor, comprising an inhibitor of AEBP1 (adipocyte enhancer binding protein 1).   The pharmaceutical composition according to claim 1, wherein the inhibitor of AEBP1 is an siRNA, antisense nucleic acid, shRNA or miRNA against transcript RNA of AEBP1.   The pharmaceutical composition according to claim 2, wherein the inhibitor of AEBP1 is an siRNA against a transcript RNA of AEBP1.   The pharmaceutical composition according to any one of claims 1 to 3, wherein the tumor is a solid tumor.   The pharmaceutical composition according to claim 4, wherein the tumor is a colon cancer.   The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is an angiogenesis inhibitor.   A method for predicting the prognosis of tumor treatment in a subject, comprising determining the expression level of an AEBP1 expression product in a test sample from the subject.   9. The method of claim 7, wherein the tumor is a colon cancer.   A pharmaceutical composition for improving the prognosis of tumor treatment, comprising an inhibitor of AEBP1.