JP7205864B2 - Therapeutic agents that control the tumor microenvironment - Google Patents

Description

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

Recent studies have revealed that the surrounding environment of cancer, such as blood vessels, ECM, stroma containing fibroblasts, etc., plays an important role in the onset and progression of cancer. Against this background, the importance of the surrounding environment of cancer has been highlighted, and treatment methods that target the surrounding environment rather than the cancer cells themselves are being investigated. Among them, tumor angiogenesis is considered to be an important mechanism involved in growth, invasion and metastasis. Therefore, regulation of angiogenesis in the tumor microenvironment is a promising strategy for cancer treatment and inhibition of progression.

However, many aspects of the molecular mechanisms in the tumor microenvironment, especially the molecular mechanisms of tumor angiogenesis, are still unclear in the onset and progression of cancer. No cure has been established.

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

Objects of the present invention include a novel pharmaceutical composition for treating cancer containing an inhibitor of AEBP1, a method for treating cancer using the pharmaceutical composition, and determining the expression level of AEBP1. , a method for predicting the prognosis of cancer therapy, a pharmaceutical composition for improving the prognosis of cancer therapy containing an inhibitor of AEBP1, and a method for improving the prognosis of cancer therapy using the pharmaceutical composition. That's what it is.

As mentioned above, it cannot be said that a cancer treatment method targeting the cancer microenvironment has been established yet. For example, anti-angiogenic therapy in colorectal cancer is currently being treated with anti-VEGF humanized monoclonal antibody (bevacizumab) and low-molecular-weight VEGFR inhibitor (sorafenib). It has also been reported that the angiogenesis therapy has a limited effect and causes serious adverse events. Therefore, there is a need for a new treatment method that places less burden on the patient.

While seeking new therapeutic methods to solve the above problems, the present inventors found that AEBP1 was significantly highly expressed in tumor vascular endothelial cells (TEC) compared to normal vascular endothelial cells (NEC). I got the new knowledge that Based on these findings, we continued to study the molecular mechanism of the AEBP1 gene in the cancer microenvironment, and found that suppressing the expression of AEBP1 significantly reduced angiogenesis and tumor tissue growth in tumor tissue. As a result of the progress, the present invention was completed.

That is, the present invention relates to:
[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 siRNA against AEBP1 transcript RNA.
[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 colon cancer.
[6] The pharmaceutical composition of [1]-[5], wherein the pharmaceutical composition is an angiogenesis inhibitor.
[7] A method of predicting the prognosis of tumor treatment in a subject, said method comprising determining the expression level of the expression product of AEBP1 in a test sample from the subject.
[8] The method of [7], wherein the tumor is colon cancer.
[9] A pharmaceutical composition for improving the prognosis of tumor treatment, comprising an inhibitor of AEBP1.

INDUSTRIAL APPLICABILITY According to the present invention, a therapeutic method targeting a gene that is highly expressed in a tumor blood vessel-specific manner can significantly reduce side effects compared to conventional angiogenesis inhibitory therapy. In addition, since it can significantly reduce not only tumor angiogenesis but also cancer-associated fibroblast (CAF) proliferation, it is possible to provide a novel therapeutic method with a new mechanism of action that targets the cancer microenvironment. . Furthermore, since AEBP1 was found to be a poor prognostic factor, it is possible to predict and improve the prognosis of cancer treatment, so it is also useful in preventing metastasis and recurrence.

FIG. 1A is a schematic representation of a study in which CRC tissue was enzymatically treated and the cells were separated using the magnetic microbead method (IMS). B shows a stained image (upper) obtained by staining a paraffin-embedded section of CRC tissue with CD31 and CD146, and the result of sorting CRC tissue cells using CD31 and CD146 by FACS (lower). C is a diagram showing that the expression of 18 genes, including ANTXR1 (TEM8), was enhanced in TECs when the mRNA expression in 3 pairs of TECs and NECs was compared. FIG. 2 is a graph showing the results of evaluating the mRNA expression level of each candidate gene by quantitative RT-PCR in four groups of clinical specimens: normal epithelium, tumor epithelium, NECs, and TECs. FIG. 3A shows tumor blood vessels in cancerous parts of human colon cancer tissue (upper row) and normal blood vessels in non-cancerous parts (lower row) stained with CD31 (left column), CD146 (middle), and AEBP1 (right column). This is a stained image. B, C and D are images stained with stromal AEBP1 of cancerous parts of human colon cancer tissue. C and D are enlarged views of the dotted line portion of B, respectively. FIG. 4A shows AEBP1 in colon cancer cell lines (DLD1, SW480, RKO, WiDr, HT-29, HCT116, COLO320, LoVo), normal cells HUVEC, HMVEC, NHDF and CAF using quantitative RT-PCR method. and ANTXR1 (TEM8). B is a graph showing the results of quantitative RT-PCR evaluation of expression of a vascular endothelial marker (CD31) and a tumor vascular endothelial marker (CD146) at the mRNA level for each of the above cells. FIG. 5A is a schematic diagram showing the procedure for collecting tumor culture supernatant (TCM). B, Optical micrographs of HUVECs cultured in DLD1 and SW480-derived TCM (serum-free) and DMEM (serum-free). Photographs were taken 24 hours and 48 hours after the start of culture, respectively. C shows the results of culturing HUVECs with TCM (serum-free) derived from 8 types of colon cancer cell lines and evaluating expression changes at the mRNA level of AEBP1 over time using a quantitative RT-PCR method. It is a graph representing. D represents the result of evaluating expression changes at the mRNA level of AEBP1 when HUVEC was cultured in DLD1-derived TCM supplemented with FBS (2.5%, 10%) using a quantitative RT-PCR method. graph. In E, HUVEC and DLD1 were seeded on a 10 cm dish at a ratio of 4:1 and co-cultured. 10 ⁶ cells are collected 24 h and 96 h after initiation of culture, and HUVEC and DLD1 are separated and collected using the IMS method. FIG. F is a graph showing the results of evaluating the expression at the mRNA level of AEBP1 and CD31 of HUVEC and DLD1 at 24 h and 96 h after initiation of coculture using quantitative RT-PCR. G is an optical micrograph of HUVEC, DLD1, 24 h and 96 h after initiation of co-culture. FIG. 6 depicts survival curves for patients with AEBP1-high and low-expressing colorectal cancer. Figures 7A and B are graphs in which two types of siAEBP1 or control siRNA were introduced into HUVEC and CAF, respectively, and the knockdown efficiency was evaluated by confirming the expression of AEBP1 at the mRNA level using a quantitative RT-PCR method. . C and D are diagrams showing the results of introducing two types of siAEBP1 or control siRNA into HUVEC or CAF, respectively, and examining cell proliferation over time immediately after introduction. E is a graph showing the results of cell cycle analysis using FACS of HUVEC with knockdown of AEBP1. It can be seen that the number of cells with G1 arrest increased. F is a diagram representing the results of a tube formation assay. It can be seen that the tube formation ability of HUVECs with AEBP1 knocked down was suppressed. G depicts the results of the wound healing assay. It can be seen that the wound closure of HUVEC knocked down AEBP1 was suppressed. H is a diagram showing the results of expression array analysis of HUVEC 48 hours and 72 hours after introduction of two types of siAEBP1 or control siRNA. In addition to AEBP1, AQP1, POSTN1 and the like were identified as genes that fluctuate in common with the two types of siRNA. 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 tumor growth curves when DLD1 or DLD1 and HUVEC were implanted in nude mice. B is a graph comparing tumor volumes (mm ³ ) 14 days after transplantation. C is a graph depicting tumor microvessel density at 12 days post-implantation. D is a stained image obtained by immunohistological staining of a paraffin-embedded section of a tumor with CD31. E shows tumor growth curves when DLD1 was implanted in nude mice and siAebp1 was administered over time. F is a graph depicting tumor microvessel density at 28 days post-implantation. G is a stained image obtained by immunohistological staining of a paraffin-embedded section of a tumor with CD31.

The present invention will be described in detail below.
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 referenced herein are hereby incorporated by reference in their entirety. In addition, if there is any discrepancy between the publications referred to in this specification and the description in this specification, the description in this specification shall take precedence.

In the present disclosure, when simply referred to as a gene name such as "AEBP1", unless otherwise specified, it means a gene having a known nucleic acid sequence represented by the gene name, typically cDNA or Although it represents an mRNA sequence, it is not limited to this as long as a person skilled in the art can recognize it as the sequence of the gene. 70% or more, 80% or more, or 90% or more, preferably 95% or more of the nucleotide sequence containing deletion, substitution, addition or insertion of 1, 1 to 3, 1 or 2 nucleotides, the cDNA or mRNA sequence , more preferably a nucleotide sequence having a sequence identity of 98% or more or 99% or more, or a degenerate sequence.

Examples of preferred AEBP1 genes and nucleic acid sequences thereof in the present disclosure include the sequence represented by GenBank Accession No. NM_001129.4 and homologues thereof. Also, a nucleotide sequence that specifically hybridizes to a nucleic acid molecule having such a sequence under stringent conditions, or a nucleotide sequence that is about 60% or more, preferably about 70% or more, more preferably about 80% or more, more preferably about 80% or more of the above sequence. Also included in the AEBP1 of the present disclosure are nucleic acids containing nucleotide sequences having a homology of about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more.

In the present disclosure, when a gene name is appended with "protein", such as "AEBP1 protein", it means the protein encoded by the gene, isoforms thereof, and homologues thereof. Examples of such isoforms include splicing variants, variants such as SNPs based on individual differences, and the like. Specifically, (1) a protein consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more homology with the protein encoded by the gene, (2) the gene Substitution or deletion of one or more, preferably 1 to several, more preferably 1 to 10, 1 to 5, 1 to 3, 1 or 2 amino acids in the amino acid sequence of the encoded protein , proteins consisting of added or inserted amino acid sequences.

In the present disclosure, the term “inhibitor” of a gene includes agents that inhibit or suppress the expression of the gene, as well as agents that inhibit the activity of the protein encoded by the gene. In the present disclosure, "an agent that inhibits or suppresses gene expression" includes, for example, siRNA, antisense nucleic acid, shRNA, miRNA, etc. against the transcript RNA of the gene, as well as transcription repressors of the gene. Agents for inhibiting or suppressing the expression of AEBP1 of the present disclosure include, for example, siRNA (for example, a nucleic acid having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2) or its precursor RNA, which has an RNA interference (RNAi) effect, or modified RNAs thereof, or vectors containing DNAs encoding siRNAs against transcript RNAs of the AEBP1 gene. In addition, in the present disclosure, the agent that inhibits or suppresses the expression of AEBP1 may be, for example, shRNA, miRNA, and the like. The vector may contain antisense RNA or antisense DNA, DNA encoding said antisense RNA or said antisense DNA.

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

In the present disclosure, the inhibitor of AEBP1 gene expression may be, for example, a combination of siRNA containing a sense strand and an antisense strand. A combination of the nucleic acid set forth in SEQ ID NO: 3, or a combination of the nucleic acid set forth in SEQ ID NO: 2 as the sense strand and the nucleic acid set forth in SEQ ID NO: 4 as the antisense strand. These nucleic acids may also include optionally 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 it can prevent the protein from exerting its activity, and is limited to this. However, for example, a nucleic acid or protein that binds to the protein, a protein that converts the active form of the protein to an inactive form, or a nucleotide that encodes the protein, an agent that inhibits the protein that activates the protein, etc. is mentioned. Examples of agents that inhibit the activity of the AEBP1 protein of the present disclosure include, but are not limited to, antibodies that recognize the AEBP1 protein, compounds that inhibit the AEBP1 protein, and the like.

Introduction of the inhibitors of the present disclosure into cells can be performed by any known introduction technique, such as, but not limited to, lipofectamine, lipofection, calcium phosphate, sonophoresis, electroporation, particle gun, viral Methods using vectors (eg, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.) or microinjection methods can be used.
When using a viral vector, the virus titer is 1×10 ³ to 1×10 ¹⁵ p.o. f. u. (plaque-forming units), 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 this 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 a vector, any known vectors such as plasmid vectors, phage vectors, phagemid vectors, cosmid vectors and virus vectors can be used. Nucleic acid constructs or vectors preferably include at least suitable transcriptional or translational control sequences, eg, derived from mammalian, microbial, viral, or insect genes. Such regulatory sequences include sequences that have a regulatory role in gene expression, such as transcriptional promoters or enhancers, operator sequences for regulating transcription, sequences encoding ribosome binding sites within messenger RNAs, and transcription, translation initiation. or include appropriate sequences that regulate transcription termination.

<1> Pharmaceutical composition of the present disclosure One aspect of the present disclosure relates to a pharmaceutical composition comprising an AEBP1 inhibitor as an active ingredient.
The pharmaceutical composition of the present disclosure is characterized by comprising an AEBP1 inhibitor as an active ingredient, and further containing one or more pharmaceutically acceptable surfactants, carriers, diluents and/or excipients. Formulations and the like may also be included. Pharmaceutically acceptable surfactants, carriers, diluents and/or excipients, etc. are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990) and others. Those skilled in the art can select suitable pharmaceutically acceptable surfactants, carriers, diluents and/or excipients according to the dosage form of the pharmaceutical composition.

The AEBP1 inhibitor 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 the AEBP1 protein or the expression of AEBP1, and the activity of the AEBP1 protein exemplified above An agent that inhibits the expression of AEBP1 or an agent that inhibits or suppresses the expression of AEBP1 can be used, and typically includes an agent that inhibits or suppresses the expression of AEBP1. As described above, the agent for inhibiting or suppressing the expression of AEBP1 is typically an siRNA against transcript RNA of AEBP1 (for example, a nucleic acid having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2) or its precursor RNA. , or modified RNAs thereof, shRNAs, miRNAs, or vectors containing DNAs encoding the above RNAs. In one preferred embodiment, the inhibitor of AEBP1 is an siRNA against transcript RNA of AEBP1.

The pharmaceutical composition of the present disclosure can be used, without limitation, to treat diseases associated with high activity or high expression of AEBP1 described above. Accordingly, the present invention also relates to a pharmaceutical composition comprising an effective amount of the above-described inhibitor of AEBP1 for the treatment of diseases associated with high activity or expression of AEBP1. "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 benign and malignant tumors (cancer, malignant neoplasm). Cancer includes hematopoietic tumors, epithelial malignancies (carcinoma) and non-epithelial malignancies (sarcoma).

As described above, the present inventors newly found that the expression of AEBP1 is particularly enhanced in tumor vascular endothelial cells and cancer-associated fibroblasts (CAF). Accordingly, in preferred embodiments, the disease treated by the pharmaceutical compositions of the present disclosure is cancer, and in more preferred embodiments, solid tumors. As used herein, "solid tumor" means a tumor other than a hematopoietic tumor.
Cancers targeted by the pharmaceutical composition of the present disclosure are not limited as long as AEBP1 is expressed in tumor vascular endothelial cells and CAFs. leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendix cancer, Colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, Carcinomas such as skin cancer are included. In one preferred aspect, the pharmaceutical composition of the present disclosure is for treating colon cancer.

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

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

The pharmaceutical compositions of this disclosure may further comprise other agents useful in treating the disease of interest, such as anti-tumor agents, anti-inflammatory agents, and the like.
Antitumor agents include, but are not limited to, ifosfamide, nimustine (e.g., nimustine hydrochloride), cyclophosphamide, dacarbazine, melphalan, alkylating agents such as ranimustine, gemcitabine (e.g., gemcitabine hydrochloride), enocitabine, Cytarabine/Oxphosphate, Cytarabine preparations, Tegafur/Uracil, Tegafur/Gimeracil/Oteracil potassium combination (e.g., TS-1), antimetabolites such as doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, idarubicin (e.g., hydrochloric acid) idarubicin), epirubicin (e.g. epirubicin hydrochloride), daunorubicin (e.g. daunorubicin hydrochloride, daunorubicin citrate), doxorubicin (e.g. doxorubicin hydrochloride), pirarubicin (e.g. pirarubicin hydrochloride), bleomycin (e.g. bleomycin hydrochloride), peplomycin (e.g. , peplomycin sulfate), mitoxantrone (e.g. mitoxantrone hydrochloride), antineoplastic antibiotics such as mitomycin C, etoposide, irinotecan (e.g. irinotecan hydrochloride), vinorelbine (e.g. vinorelbine tartrate), docetaxel (e.g. docetaxel hydrate), alkaloids such as paclitaxel, vincristine (e.g. vincristine sulfate), vindesine (e.g. vindesine sulfate), vinblastine (e.g. vinblastine sulfate), anastrozole, tamoxifen (e.g. tamoxifen citrate), toremifene (e.g. , toremifene citrate), hormone therapy agents such as bicalutamide, flutamide, estramustine (e.g., estramustine phosphate), platinum complexes such as carboplatin, cisplatin (CDDP), nedaplatin, blood vessels such as thalidomide, neovastat, bevacizumab, etc. Neogenesis inhibitors, L-asparaginase and the like can be mentioned.

Anti-inflammatory agents 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, tiaprofen, etc.). acid, suprofen, tolmetin, carprofen, benoxaprofen, piroxicam, benzydamine, naproxen, diclofenac, ibuprofen, diflunisal, azapropazone, etc.), RNAi molecules that suppress the expression of inflammatory cytokines (e.g., siRNA, shRNA, ddRNA, miRNA, piRNA, rasiRNA, etc.), substances such as antisense nucleic acids, and/or drugs that suppress the action of inflammatory cytokines, such as antibodies to inflammatory cytokines and inflammatory cytokine receptor antagonists.

Pharmaceutical compositions of the present disclosure may include inhibitors of the present disclosure in the form of various drug delivery carriers. Examples of such carriers include, but are not limited to, polymeric nanoparticles, polymeric micelles, dendrimers, liposomes, viral nanoparticles, carbon nanotubes and the like (Cho K. et al., Clin Cancer Res. 2008 Mar 1;14 (5):1310-6, etc.).

The inhibitors and pharmaceutical compositions of this disclosure can be administered by a variety of routes including both oral and parenteral, including without limitation oral, intravenous, intramuscular, subcutaneous, topical, rectal, intratumoral, intraarterial. , intraportal vein, intramedullary, intramedullary, sublingual, intraoral, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, and the like. may be formulated into dosage forms suitable for Any known dosage form and formulation method can be appropriately employed (see, for example, Standard Pharmacology, Edited by Yoshiteru Watanabe, Nankodo, 2003, Remington's Pharmaceutical Sciences, etc.).

For example, dosage forms suitable for oral administration include, without limitation, powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, syrups and the like; Suitable dosage forms include injections such as solution injections, suspension injections, emulsion injections, and injections prepared just before use. Formulations for parenteral administration can 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, singly or in combination, the ingredients that can be included in various agents or compositions of the present disclosure, as well as forms of such kits. It also relates to necessary components of various agents or compositions provided in. In addition to the above, such kits may contain instructions describing methods for preparing and administering the inhibitors or pharmaceutical compositions of the present disclosure, such as instructions, electronic recording media such as CDs and DVDs, and the like. .

Also, as described below, the inventors have compared subjects with tumors that express AEBP1 on tumor vascular endothelial cells and/or CAFs to tumor controls that do not express AEBP1 on tumor vascular endothelial cells and/or CAFs. were found to have a poor prognosis for tumor therapy. Accordingly, it is believed that the inhibitors or pharmaceutical compositions of the present disclosure can be used to improve the prognosis of subjects undergoing tumor therapy. Accordingly, the pharmaceutical compositions of the present disclosure encompass, in another aspect, pharmaceutical compositions used to improve the prognosis of tumor therapy in a subject.

<2> A method for preventing and/or treating a disease of the present disclosure In another aspect, the present disclosure is a method for preventing and/or treating a disease associated with high activity or high expression of AEBP1 in a subject, the present disclosure It also relates to a method comprising administering an effective amount of an inhibitor of or a pharmaceutical composition to a subject in need thereof (hereinafter sometimes referred to as a "method of treatment of the present disclosure").

The “subject” in the present disclosure may be any biological individual as long as it can be affected by a disease associated with high activity or high expression of AEBP1, typically cancer, but preferably humans and Non-human mammals (e.g., rodents such as mice, rats, guinea pigs and hamsters, primates such as chimpanzees, artiodactyls such as cows, goats and sheep, perissodacty 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 when prevention and/or treatment of cancer is intended, the subject typically has cancer. It means a subject who is afflicted or at risk of being afflicted. Prevention of cancer may also include prevention of recurrence of cancer, and thus subjects at risk of developing cancer may include subjects at risk of recurrence of cancer.

Also, the term "treatment" as used herein includes all types of medically acceptable prophylactic and/or therapeutic interventions aimed at cure, temporary remission or prevention of disease. shall be For example, the term "treatment" encompasses medically acceptable interventions for various purposes, including slowing or arresting disease progression, regression or disappearance of lesions, preventing onset or recurrence, and the like.

Inhibitors or pharmaceutical compositions that may be used in the treatment methods of the present disclosure include any described herein. An effective amount in this aspect is, for example, an amount that reduces symptoms of cancer or delays or stops the progression thereof, preferably an amount that suppresses or cures cancer. Also, an amount that does not cause adverse effects that exceed the benefits of administration is preferred. Such an amount can be appropriately determined by in vitro tests using cultured cells and the like, and tests using model animals such as mice and rats, and such test methods are well known to those skilled in the art. The specific dose of the active ingredient depends on various conditions related to the subject requiring it, such as severity of symptoms, general health condition of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, It can be determined in consideration of concomitant drugs, responsiveness to treatment, dosage form, compliance to treatment, and the like. As for the administration method, any known appropriate administration method as exemplified above can be used.

One aspect of the treatment method of the present disclosure may further comprise, prior to the administering step, selecting a subject having AEBP1-positive tumor vascular endothelial cells as a subject for prevention/treatment. Such embodiments may further comprise the steps of obtaining a tumor sample from the subject and detecting expression of AEBP1 in the tumor sample obtained from the subject prior to the selecting step. As a detection method used in such a step, the method exemplified for detecting the expression of AEBP1 in tumor vascular endothelial cells or CAFs can be used.

<3> Prognostic Prediction Method of the Present Disclosure As described above, the present inventors found that the higher the expression level of AEBP1 in the tumor tissue of a subject, the worse the prognosis after tumor treatment. Accordingly, another aspect of the present disclosure is a method of predicting the prognosis of tumor therapy in a subject, comprising determining the expression level of an expression product of AEBP1 in a test sample from the subject (e.g., tumor tissue from the subject) (hereinafter sometimes referred to as "the prognostic prediction method of the present disclosure").

The prognostic prediction method of the present disclosure includes the steps of:
(A) Determining the expression level of the AEBP1 expression product in a subject-derived test sample.
In the prognostic prediction method of the present disclosure, the tumor is as detailed in <1> above, preferably colon cancer.

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

In step (A), the expression level of the AEBP1 expression product is determined. The expression level of the expression product can be determined using the detection method of the expression product as described in detail in <1> above.
The prognostic prediction method of the present disclosure optionally comprises, after step (A), the following steps:
(B) may include a step of comparing the expression level obtained in (A) with a reference value.

The reference value may be any value as long as it is a reference value for the amount of expression of AEBP1 in a sample. Examples include the average AEBP1 expression level in tumor tissues of patients judged to have a good prognosis, and the average ARBP1 expression level of tumor tissues in patients judged to have a poor prognosis.
Here, "poor prognosis" in the present disclosure typically means that the period of post-treatment remission in subjects treated with a tumor is less than that in a control group treated with the same type of tumor. shorter than the average value for the period of

In one aspect, a higher level of AEBP1 expression in a subject's sample compared to a reference value is predictive of a poor prognosis. In another aspect, a lower level of AEBP1 expression in a control sample compared to a reference value is predictive of a good prognosis.
In preferred embodiments, the reference value is zero. That is, detection of an AEBP1 expression product in a subject's sample predicts a poor prognosis.

<4> Method for Improving Prognosis of the Present Disclosure As described above, tumors that highly express AEBP1 in tumor tissues, such as tumor microenvironments and tumor vascular endothelial cells, have better prognosis after treatment than tumors that do not express highly. was found by the inventors to be bad. AEBP1 is a factor involved in angiogenesis in tumor tissue, and inhibition of AEBP1 can inhibit angiogenesis in tumor tissue, thereby improving the prognosis of tumor treatment in a subject. Accordingly, one aspect of the present disclosure is a method for improving the prognosis of tumor treatment, comprising administering an effective amount of an inhibitor or pharmaceutical composition of the present disclosure to a subject after tumor treatment (“Method for improving prognosis of the present disclosure ”) is also related.

In the present disclosure, "improving prognosis" refers to prolonging the expected duration of remission in a subject treated with a tumor. Prediction of the duration of remission may be predicted by the prognostic prediction method of the present disclosure, or may be predicted, for example, from the average duration of remission of a group of subjects who underwent similar tumor treatments.
"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 anti-tumor agents, radiation therapy, cancer It can be immunotherapy and 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, preferably an amount that inhibits tumor angiogenesis. Also, an amount that does not cause adverse effects that exceed the benefits of administration is preferred. Such an amount can be appropriately determined by in vitro tests using cultured cells and the like, and tests using model animals such as mice and rats, and such test methods are well known to those skilled in the art. The specific dose of the active ingredient depends on various conditions related to the subject requiring it, such as severity of symptoms, general health condition of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, It can be determined in consideration of concomitant drugs, responsiveness to treatment, dosage form, compliance to treatment, and the like. As for the administration method, any known appropriate administration method as exemplified above can be used.

EXAMPLES The present invention will be specifically described below 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 Human colon cancer tissue was cut into small pieces using scissors and a razor. To this, type I collagenase (Worthington) and DNase I (Worthington) were added, followed by enzymatic treatment with shaking at 37°C for 2 hours to prepare a tissue lysate. This was passed through a filter to collect cells, and ACK buffer (Thermo Fisher Scientific) was added to remove erythrocytes. The obtained cell suspension was subjected to magnetic microbead method (immuno-magnetic separation, IMS method) using Epithelial 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 a bead conjugated with anti-EpCAM antibody, which can directly bind to EpCAM-positive epithelial cells. The anti-CD146 antibody was previously mixed with Dynabeads Pan mouse IgG by shaking to bind the antibody and the beads, and then bound to the CD146-positive cells.

As a preliminary experiment of cell separation by the IMS method (Fig. 1A), sorting by vascular endothelial cell markers CD31 and CD146 was performed using FACS. CD31-positive cells were 1.1% and CD146-positive cells were 7.7%. (Fig. 1B). 14 cases of colorectal cancer tissues were divided into cancerous and non-cancerous areas, and each was divided into EpiCAM-positive cells and CD146-positive cells, which are epithelial cell markers, to obtain 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 resulting sample using an RNeasy kit.

Example 2. RNA sequence analysis 14 cases of colorectal cancer tissues were divided into cancerous and non-cancerous parts, and each of them was divided into epithelial cells and vascular endothelial cells. A total of 53 samples of endothelial cells (TECs, n=14) and normal endothelial cells (NECs, n=14) were obtained. RNA was recovered from these samples using TRI REAGENT (MRC) and quality checked using the 2100 Bioanalyzer-RNA Solution (Agilent, RNA6000 nanokit). Three samples each of TECs and NECs (both RIN values ≥ 8) were subjected to RNA sequencing, and the resulting expression data were analyzed using Volcano plots.

A volcano plot analysis of the results of comprehensive investigation of mRNA expression in three samples each of NECs and TECs using RNA-seq revealed statistically significant changes in expression between NECs and TECs26. Among them, 18 genes whose expression was predominantly enhanced in TECs were identified (Fig. 1C). Among these genes, ANTXR1 reported as a tumor endothelial marker (tumor endothelial maker8, TEM8) was included as a gene with predominantly high expression in TECs.

Example 3. Verification of Gene Expression in Clinical Specimens Expression at the messenger RNA (mRNA) level was confirmed using the qRT-PCR method for genes nominated by RNA sequence analysis. Immunohistochemical staining of paraffin-embedded sections of clinical specimens confirmed the expression of candidate genes at the protein level.

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

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

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

Example 5. Candidate gene expression changes in tumor culture supernatant experiments and co-culture experiments Eight types of colon cancer cell lines were cultured to semi-confluence and cultured in serum-free medium for 24 hours, and the culture supernatant was collected. It was passed through a filter (MILLIPORE) with a pore size of 0.22 μm and used as a tumor culture supernatant (TCM). HUVECs cultured in TCM (serum-free) and a control medium, which is a serum-free basal medium, were successively observed under an optical microscope. Furthermore, expression changes at the AEBP1 mRNA level in HUVECs cultured in TCM and control medium were evaluated over time using the qRT-PCR method. In addition, HUVECs were cultured in TCM supplemented with FBS (2.5%), and changes in AEBP1 expression were evaluated over time. Furthermore, DLD1 and HUVEC were seeded in the same 10 cm dish at a ratio of 1:4 and co-cultured with EGM-2. After 24 hours and 96 hours, 10 ⁶ cell populations were collected, and DLD1 and HUVEC were separated using the IMS method. DLD1 was recovered from the cell population by positive selection using only Epithelial Enrich antibody, and HUVEC was recovered by negative selection. Collected DLD1 and HUVEC were evaluated for CD31 and AEBP1 mRNA level expression using qRT-PCR method.

When HUVECs cultured in TCM (Fig. 5A) collected from colon cancer cell lines were observed under an optical microscope, many of the HUVECs cultured in serum-free medium (DMEM) detached from the dish and aggregated after 24 hours. , 48 h, these changes were observed in almost all cells (Fig. 5B). HUVEC cultured in TCM increased the number of cells that aggregated over time, but less than HUVEC cultured in serum-free medium. In addition, more HUVECs survived when cultured with DLD1-derived TCM than when cultured with SW480-derived TCM, and differences in HUVEC survival and aggregation were observed depending on the type of cancer cell that produces the TCM (Fig. 5B). HUVECs were cultured in TCM (serum-free) and serum-free media derived from 8 colon cancer cell lines, and quantitative RT-PCR was used to sequentially evaluate changes in expression of AEBP1 at the mRNA level. bottom. In many cases, HUVEC cultured in TCM for 24 hours showed enhanced expression of AEBP1, whereas HUVEC cultured in serum-free medium tended to decrease expression. In particular, when cultured using DLD1 and HCT116-derived TCM, there was a significant difference in the expression of AEBP1 at 12 hours and 24 hours after the start of culture compared to culture in a serum-free medium (Fig. 5C). When cultured in DLD1-derived TCM supplemented with FBS (2.5%), the timing of the peak expression of AEBP1 did not change at 24 hours, but the expression level increased compared to serum-free. In addition, the expression level of AEBP1 at the peak was higher when FBS was added (2.5%). The expression of AEBP1 was enhanced when cultured in a medium supplemented with FBS, but not as much as when cultured in TCM. When DLD1 and HUVEC were cultured in the same dish and observed with an optical microscope, the ratio of DLD1:HUVEC was 1:4 at the start of culture, but after 96 hours, the ratio was almost the same (Fig. 5G). After 24 hours and 96 hours from the initiation of culture, DLD1 and HUVEC were separated using the IMS method, and expression of AEBP1 and CD31 at the mRNA level was evaluated using the quantitative RT-PCR method. Even higher expression was seen in HUVECs after 96 hours than in experiments with TCM (Fig. 5F).

Example 6. Large-scale publicly available clinical data analysis
Analysis of RNA-seq mRNA expression data and overall survival (OS) using large-scale clinical specimens published by The Cancer Genome Atlas (TCGA) revealed that subjects with colorectal cancer with high AEBP1 expression resulted in a significantly lower survival rate. From this, high expression of AEBP1 was found to be a poor prognostic factor for colorectal cancer (Fig. 6).

Example 7. AEBP1 Knockdown Experiment in HUVEC (1) Transfection of siAEBP1 Ambion) was prepared and transfected into HUVEC by electroporation using HUVEC Nucleofector Kit (Amaxa). HUVEC transfected with siAEBP1 and control siRNA were cultured in EGM-2, RNA was extracted after 48 hours, and knockdown efficiency was examined by evaluating expression of AEBP1 at the mRNA level using RT-PCR. . A similar experiment was performed using CAFs into which two types of siAEBP1 and control siRNA were introduced.

siAEBP1 was introduced into HUVECs and CAFs using the electroporation method. RNA was extracted from HUVECs and CAFs 48 hours after transduction, and the expression of AEBP1 at the mRNA level was evaluated using a quantitative RT-PCR method. Both knockdown efficiencies were approximately 90% (FIGS. 7A and B).

(2) Cell proliferation test Two types of siAEBP1 (siAEBP1_1 and siAEBP1_2) and HUVECs immediately after introduction of control siRNA were seeded in a 96-well plate, and a cell proliferation test was performed using Cell Counting Kit-8 (Dojindo Laboratories). went. A similar experiment was performed using CAFs into which two types of siAEBP1 and control siRNA were introduced.
It was found that HUVECs and CAFs knocked down with two types of siRNA were significantly inhibited in cell growth compared to controls (Figs. 7C and D).

(3) Cell Cycle Analysis Cell cycle analysis was performed using a flow cytometer using HUVECs introduced with two types of siAEBP1 and control siRNA for 48 hours.
HUVECs knocked down with two types of siRNA were found to have more cells undergoing G1 phase arrest compared to controls, 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 provided. Normal vascular endothelial cells are known to undergo angiogenesis on Matrigel. HUVEC transfected with two types of siAEBP1 and control siRNA were cultured in EGM-2, seeded on matrigel after 48 hours, and angiogenesis was observed under an optical microscope. Furthermore, the effect of AEBP1 knockdown on HUVEC tube formation on Matrigel was quantitatively evaluated using image analysis software Image J.
AEBP1-knocked down HUVEC had significantly suppressed tube formation ability compared to the control (Fig. 7F).

(5) Wound Healing Assay HUVEC transfected with siAEBP1 and control siRNA were cultured with EGM-2 and seeded in a 6-well plate. Wounds were formed when semi-confluent, and wound closure was assessed at subsequent 12- and 24-hour time points.
AEBP1-knocked down HUVEC showed significantly suppressed wound closure compared to control (Fig. 7G).

(6) Gene Expression Microarray Analysis Gene expression microarray analysis was performed to search for genes whose expression changes due to AEBP1 knockdown. Two types of siAEBP1 (siAEBP1_1 and siAEBP1_2) and control siRNA were introduced, RNA was extracted from HUVEC after 48 hours and 72 hours, cRNA was synthesized, and microarray experiments were performed using SurePrint G3 Human GE 8x60K V2 microarray (Agilent Technologies). did For data analysis, GeneSpring GX version 13 (Agilent Technologies) was used. Gene Ontology (GO) analysis was also performed on the identified genes.

Two types of siAEBP1 or control siRNA were introduced into HUVECs and collected at two time points, 48 hours and 72 hours later. Expression changed by comparing knockdown samples and controls at each time point. Gene expression microarray analysis was performed to search for genes that A plurality of genes whose expression levels were decreased by 2.0-fold or more compared to the control were identified (Fig. 7H). Genes whose expression varies due to AEBP1 knockdown at the 48-hour time point after siRNA introduction include POSTN, and genes that vary commonly at the 48-hour and 72-hour time points include AQP1 and the like in addition to AEBP1. It was In addition, quantitative RT-PCR was used to confirm the expression levels of mRNAs of AQP1 and POSTN, for which tumor angiogenesis was previously reported (FIGS. 7I and J).

Example 8. Mouse xenograft experiment (1) Co-implantation of DLD1 and HUVEC There is a report that when sarcoma and HUVEC are implanted together, the tumor grows more than when only sarcoma is implanted. did 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 estimated (V=(W ² ×L)/2 (V: volume, W: minor axis, L: major axis) to obtain a growth curve.
It was confirmed that when DLD1 and HUVEC were co-transplanted, the transplanted tumor increased more than when DLD1 was transplanted alone (FIGS. 8A and B).

(2) Co-implantation of DLD1 and AEBP1-knockdown HUVECs It has been reported that HUVECs are involved in angiogenesis of transplanted tumors together with mouse blood vessels. Therefore, in order to investigate how knocking down AEBP1 in HUVECs to be transplanted affects angiogenesis in the transplanted tumor, HUVECs (5 × 10 ⁵ ) introduced with one type of siAEBP1 or control siRNA, DLD1 (5×10 ⁵ ) were transplanted to nude mice. siRNA-introduced HUVEC and DLD1 were suspended in Matrigel (250 μl) diluted with PBS (250 μl) and transplanted to mice. On the 12th day after transplantation when necrosis did not occur inside the transplanted tumor, the transplanted tumor was excised, formalin-fixed, and a paraffin-embedded section was prepared. Tumor vessels were stained with CD31 antibody and microvessel density (MVD) was measured.
Co-implantation of AEBP1-knockdown HUVECs with DLD1 resulted in fewer MVDs and suppressed tumor angiogenesis within the implanted tumors compared to controls (FIGS. 8C and D).

(3) Knockdown of DLD1-implanted tumor stroma In order to examine the effects of knockdown of mouse Aebp1 on tumor blood vessels invading the implanted tumor, tumor stroma, and tumor growth, two types of siRNA against mouse Aebp1 were prepared. DLD1 (1×10 ⁶ cells) was suspended in matrigel (250 μl) diluted with PBS (250 μl) containing siAebp1 and control siRNA and transplanted to nude mice. Furthermore, two types of siAebp1 or control siRNA were administered around the transplanted tumor every 4 days. The implanted tumors were measured over time to obtain a growth curve. In addition, paraffin-embedded sections of the transplanted tumor were immunohistochemically stained with CD31, and MVD was objectively measured using Image J.

In both groups in which two kinds of siAebp1 were administered around the transplanted tumor, growth of the transplanted tumor was suppressed more than in the control group (Fig. 8E). It was also found that the siAEBP1-administered group had less MVD than the control group, and tumor angiogenesis within the transplanted tumor was also suppressed (FIGS. 8F and G).

Claims (5)

A pharmaceutical composition for treating colorectal cancer, comprising an AEBP1 (adipocyte enhancer binding protein 1) inhibitor, wherein the AEBP1 inhibitor is siRNA, antisense nucleic acid, shRNA or miRNA against AEBP1 transcript RNA , or an antibody that recognizes AEBP1. 2. The pharmaceutical composition according to claim 1, wherein the AEBP1 inhibitor is an siRNA against AEBP1 transcript RNA. The pharmaceutical composition according to claim 1, wherein the AEBP1 inhibitor is an antibody that recognizes AEBP1. The pharmaceutical composition according to any one of claims 1 to 3, which is an angiogenesis inhibitor. A pharmaceutical composition for improving the prognosis of colorectal cancer treatment comprising an inhibitor of AEBP1, wherein the inhibitor of AEBP1 is an siRNA, antisense nucleic acid, shRNA or miRNA against transcript RNA of AEBP1, or AEBP1. Said pharmaceutical composition, which is a recognizing antibody.

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