SFRP4 biomarker as therapeutic target for hepatocellular carcinoma and uses thereof

Abstract

The present invention relates to an SFRP4 biomarker as a therapeutic target for hepatocellular carcinoma and uses thereof. The present invention aims to discover a hepatocellular carcinoma treatment method by elucidating the effect of CAF on hepatocellular carcinoma in a tumor microenvironment and identifying the role of a CAF-derived substance interacting with hepatocellular carcinoma through CAF genome analysis. That is, by analyzing secretomes specifically secreted from CAF, which is one of the components of the tumor microenvironment and is responsible for the stromal production in hepatocellular carcinoma and malignant tumors, the SFRP4 biomarker was discovered as a therapeutic target for hepatocellular carcinoma. As a result, the hepatocellular carcinoma CAF-derived SFRP4 is highly likely to be advantageously useful for hepatocellular carcinoma diagnosis, prediction of therapeutic response, and as a therapeutic target for hepatocellular carcinoma.

Description

SFRP4 biomarker and its use as a therapeutic target for hepatocellular carcinoma

[0001] The present invention relates to the SFRP4 biomarker and its use as a treatment target for hepatocellular carcinoma.

[0002] Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the sixth most common, according to statistics published by the National Cancer Center. However, it is a fatal disease with a survival rate of only 37%. Furthermore, it has a relatively high metastasis rate (13.1%) compared to all cancer types.

[0003] Recently, there has been a growing emphasis on the need for research aimed at understanding cancer cell characteristics from the perspective of the tumor microenvironment to suppress cancer progression and metastasis. In particular, HCC, which develops in an environment characterized by fibrosis due to recurrent inflammation, is a solid tumor. Among the cells within the tumor microenvironment, stromal cells, such as cancer-associated fibroblasts (CAFs), play a crucial role.

[0004] CAFs are known to secrete various activating factors in the tumor microenvironment, constantly communicating and interacting with tumor cells. This allows them to support tumor cell growth and extracellular matrix remodeling, promote tumor progression and metastasis, and evade immune responses. Therefore, research on CAFs, which secrete various activating factors to regulate tumor progression, metastasis, and immune responses in the tumor microenvironment, is an essential field.

[0005] The purpose of the present invention is to provide a biomarker composition for diagnosing hepatocellular carcinoma, predicting prognosis, or predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma-targeting anticancer drugs, including SFRP4 protein or a gene encoding the same.

[0006] In addition, another object of the present invention is to provide a composition for diagnosing hepatocellular carcinoma, predicting prognosis, or predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma-targeting anticancer drugs, comprising as an active ingredient a preparation capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0007] In addition, another object of the present invention is to provide a kit for diagnosing hepatocellular carcinoma, predicting prognosis, or predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma-targeting anticancer drugs, including the composition.

[0008] In addition, another object of the present invention is to provide a method for providing information necessary for diagnosing hepatocellular carcinoma, predicting prognosis, or predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma-targeting anticancer drugs, including a step of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0009] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatocellular carcinoma, which comprises an expression inhibitor of SFRP4 protein or a gene encoding the same as an active ingredient.

[0010] In addition, another object of the present invention is to provide a method for screening a hepatocellular carcinoma treatment agent, which includes a step of selecting a test substance having a reduced expression or activity level of SFRP4 protein or a gene encoding the same.

[0011] To achieve the above purpose, the present invention provides a biomarker composition for diagnosing hepatocellular carcinoma comprising SFRP4 protein or a gene encoding the same.

[0012] In addition, the present invention provides a biomarker composition for predicting the prognosis of hepatocellular carcinoma, comprising SFRP4 protein or a gene encoding the same.

[0013] In addition, the present invention provides a biomarker composition for predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, including SFRP4 protein or a gene encoding the same.

[0014] In addition, the present invention provides a composition for diagnosing hepatocellular carcinoma, which comprises as an active ingredient an agent capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0015] In addition, the present invention provides a composition for predicting the prognosis of hepatocellular carcinoma, which comprises as an active ingredient an agent capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0016] In addition, the present invention provides a composition for predicting responsiveness to sorafenib or lenvatinib, which are anticancer drugs targeting hepatocellular carcinoma, comprising as an active ingredient a preparation capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0017] In addition, the present invention provides a kit for diagnosing hepatocellular carcinoma comprising the composition.

[0018] In addition, the present invention provides a kit for predicting the prognosis of hepatocellular carcinoma comprising the composition.

[0019] In addition, the present invention provides a kit for predicting the responsiveness of sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, including the composition.

[0020] In addition, the present invention provides a method for providing information necessary for diagnosing hepatocellular carcinoma, comprising the steps of: (1) measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) determining hepatocellular carcinoma if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

[0021] In addition, the present invention provides a method for providing information necessary for diagnosing hepatocellular carcinoma, comprising the steps of (1) measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) determining that the prognosis of hepatocellular carcinoma is poor if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

[0022] In addition, the present invention provides a method for providing information necessary for predicting responsiveness to sorafenib or lenvatinib, a hepatocellular carcinoma-targeting anticancer agent, comprising the steps of: (1) measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) comparing the expression level of the SFRP4 protein or the gene encoding it with that of a control sample; and (3) determining that the responsiveness to sorafenib or lenvatinib, a hepatocellular carcinoma-targeting anticancer agent, is low if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

[0023] In addition, the present invention provides a pharmaceutical composition for preventing or treating hepatocellular carcinoma, which comprises an expression inhibitor of SFRP4 protein or a gene encoding the same as an active ingredient.

[0024] In addition, the present invention provides a method for screening a hepatocellular carcinoma treatment agent, comprising the steps of (1) contacting a test substance with isolated hepatoma cells; (2) measuring the level of expression or activity of SFRP4 protein or a gene encoding it in the hepatoma cells contacted with the test substance; and (3) selecting a test substance in which the level of expression or activity of the SFRP4 protein or the gene encoding it is reduced compared to a control sample.

[0025] The present invention relates to the SFRP4 biomarker as a therapeutic target for hepatocellular carcinoma and its use. In order to elucidate the influence of CAFs in the tumor microenvironment on hepatocellular carcinoma, and to identify the role of CAF-derived substances that interact with hepatocellular carcinoma through CAF genome analysis, we aimed to discover a treatment method for hepatocellular carcinoma. In other words, we analyzed the secreted protein specifically secreted by CAFs, which are one of the components of the tumor microenvironment and are responsible for the formation of hepatocellular carcinoma and malignant tumor stroma, and discovered the SFRP4 biomarker as a therapeutic target for hepatocellular carcinoma. As a result, SFRP4 derived from hepatocellular carcinoma CAF has a very high possibility of being useful in the diagnosis of hepatocellular carcinoma, prediction of treatment response, and as a therapeutic target for hepatocellular carcinoma.

[0026] Figure 1 shows a schematic diagram of the identification of differentially expressed EMT core genes in HCC CAFs.

[0027] Figure 2 shows the results of verification of the association between CAF and EMT and confirmation of clinical validity.

[0028] Figure 3 shows the results of the prognostic evaluation of the selected genes.

[0029] Figure 4 shows the results of liver cancer and non-liver cancer diagnostic performance according to SFRP4 expression in hepatocellular carcinoma tissue.

[0030] Figure 5 shows the results of survival analysis of hepatocellular carcinoma patients according to SFRP4 expression.

[0031] Figure 6 shows the results of comparing the difference in SFRP4 expression according to the treatment response to sorafenib and lenvatinib and the difference in progression-free survival according to SFRP4 expression.

[0032] Figure 7 shows the results of confirming the association between SFRP4 and immune cells.

[0033] Figure 8 shows the results of characterization of CAF-derived SFRP4 in the tumor microenvironment through single-cell analysis.

[0034] Figure 9 shows the results of confirming the tumorigenicity of CAF-derived SFRP4 according to an orthotopic transplantation model that reproduces the tumor microenvironment.

[0035] Figure 10 shows the results of confirming changes in EMT-related genes in response to SFRP4 inhibition in an orthotopic transplantation model.

[0036] Figure 11 shows the results of single cell analysis confirming that SFRP4+ CAFs interact with Tregs and TAMs through CXCL12-CXCR4 signals.

[0037] Figure 12 shows the results of single cell analysis confirming that SFRP4+ CAFs interact with Tregs through NECTIN2-TIGIT signals.

[0038] Figure 13 shows the results of single cell analysis confirming that SFRP4+ CAFs interact with HCC cells through HGF-MET signaling.

[0039] Figure 14 is a schematic diagram showing the role of SFRP4+ CAFs in the hepatocellular carcinoma tumor microenvironment.

[0040] The present invention provides a biomarker composition for diagnosing hepatocellular carcinoma comprising SFRP4 protein or a gene encoding the same.

[0041] In addition, the present invention provides a biomarker composition for predicting the prognosis of hepatocellular carcinoma, comprising SFRP4 protein or a gene encoding the same.

[0042] In addition, the present invention provides a biomarker composition for predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, including SFRP4 protein or a gene encoding the same.

[0043] Preferably, the SFRP4 protein or the gene encoding it may be derived from hepatocellular carcinoma-cancer associated fibroblasts (HCC-CAF), but is not limited thereto.

[0044]

[0045] The "SFRP4" of the present invention may be NCBI Gene ID 6424, UniProtKB / Swiss-Prot: Q6FHJ7, but is not limited thereto.

[0046]

[0047] The term “diagnosis” as used herein includes determining the susceptibility of a subject to a particular disease or condition, determining whether a subject currently has a particular disease or condition, determining the prognosis of a subject having a particular disease or condition, or therametrics (e.g., monitoring the condition of a subject to provide information about the efficacy of a treatment).

[0048]

[0049] The term "prognosis" in this specification refers to the outlook for future symptoms or course of a disease, as determined by diagnosis. In cancer patients, prognosis typically refers to the presence or absence of metastasis or survival within a certain period following cancer onset or surgical treatment. Predicting prognosis is a crucial clinical task, as it provides clues to the future direction of hepatocellular carcinoma treatment, including the appropriateness of chemotherapy for hepatocellular carcinoma patients.

[0050]

[0051] The term "prediction" is used herein to refer to the likelihood that a subject patient will respond favorably or unfavorably to a drug or set of drugs. In one embodiment, the prediction relates to the extent of such response. For example, the prediction relates to whether and / or the probability that a patient will survive without cancer recurrence after treatment, such as treatment with a specific therapeutic agent and / or surgical removal of a primary tumor and / or chemotherapy for a specific period of time. The prediction of the present invention can be clinically used to determine treatment by selecting the most appropriate treatment modality for a patient with hepatocellular carcinoma. The prediction of the present invention is a useful tool for predicting whether a patient will respond favorably to a treatment, such as administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc., or whether a patient will likely survive long-term after a treatment.

[0052]

[0053] As used herein, the term "prediction of anticancer drug responsiveness" refers to predicting whether a patient will respond favorably or unfavorably to anticancer drug treatment, or predicting the risk of anticancer drug resistance. Therefore, if the present invention can predict patients who are expected to respond (responders) and those who are not expected to respond (non-responders) before treatment initiation, highly effective and safe chemotherapy can be realized. Furthermore, the prediction method of the present invention can be clinically applied to guide treatment decisions by selecting the most appropriate treatment modality for patients with hepatocellular carcinoma.

[0054]

[0055] In addition, the present invention provides a composition for diagnosing hepatocellular carcinoma, which comprises as an active ingredient an agent capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0056] In addition, the present invention provides a composition for predicting the prognosis of hepatocellular carcinoma, which comprises as an active ingredient an agent capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0057] In addition, the present invention provides a composition for predicting responsiveness to sorafenib or lenvatinib, which are anticancer drugs targeting hepatocellular carcinoma, comprising as an active ingredient a preparation capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

[0058] Specifically, the agent capable of measuring the expression level of the SFRP4 protein or the gene encoding it may be, but is not limited to, a primer or probe that specifically binds to the SFRP4 gene, an antibody, peptide, aptamer or compound that specifically binds to the SFRP4 protein.

[0059] Preferably, the SFRP4 protein or the gene encoding it may be derived from HCC-CAF, but is not limited thereto.

[0060]

[0061] In addition, the present invention provides a kit for diagnosing hepatocellular carcinoma comprising the composition.

[0062] In addition, the present invention provides a kit for predicting the prognosis of hepatocellular carcinoma comprising the composition.

[0063] In addition, the present invention provides a kit for predicting the responsiveness of sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, including the composition.

[0064] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0065] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA, and is labeled so that the presence or absence of a specific mRNA and its expression level can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

[0066] As used herein, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes specialized antibodies such as humanized antibodies.

[0067] The term "peptide" used herein has the advantage of high binding affinity to target substances and resists denaturation even during heat and chemical treatments. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0068] As used herein, the term "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to an antigenic substance in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easy to synthesize, and therefore can be used as a substitute for antibodies.

[0069]

[0070] In addition, the present invention provides a method for providing information necessary for diagnosing hepatocellular carcinoma, comprising the steps of: (1) measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) determining hepatocellular carcinoma if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

[0071] In addition, the present invention provides a method for providing information necessary for diagnosing hepatocellular carcinoma, comprising the steps of (1) measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) determining that the prognosis of hepatocellular carcinoma is poor if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

[0072] In addition, the present invention provides a method for providing information necessary for predicting responsiveness to sorafenib or lenvatinib, a hepatocellular carcinoma-targeting anticancer agent, comprising the steps of: (1) measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) comparing the expression level of the SFRP4 protein or the gene encoding it with that of a control sample; and (3) determining that the responsiveness to sorafenib or lenvatinib, a hepatocellular carcinoma-targeting anticancer agent, is low if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

[0073]

[0074] Specifically, methods for measuring the gene expression level include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips.

[0075] Specifically, methods for measuring the protein expression level include, but are not limited to, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chips.

[0076]

[0077] As used herein, the term "sample isolated from a patient with hepatocellular carcinoma" includes, but is not limited to, a sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differs from a control group in the expression level of the SFRP4 gene or SFRP4 protein, which is a biomarker of the present invention. Preferably, the sample may be HCC-CAF.

[0078]

[0079] In addition, the present invention provides a pharmaceutical composition for preventing or treating hepatocellular carcinoma, which comprises an expression inhibitor of SFRP4 protein or a gene encoding the same as an active ingredient.

[0080] Specifically, the inhibitor of expression of the SFRP4 protein or the gene encoding it may be any one selected from the group consisting of an antisense nucleotide, a small interfering RNA (siRNA), and a short hairpin RNA (shRNA) that complementarily binds to the mRNA of the SFRP4 gene, but is not limited thereto.

[0081] Preferably, the composition can increase tumor immunity through regulation of Treg and TAM activation in a hepatocellular carcinoma tumor microenvironment, and inhibit the progression of hepatocellular carcinoma through HGF-MET signal transduction, but is not limited thereto.

[0082] The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or complex preparation of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients, and the adjuvants may include solubilizing agents such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredients for administration. In the composition to be formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0083] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. Although not limited thereto, for example, in the case of adults, when administered once to several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg in the case of a compound, 0.1 ng / kg to 10 g / kg in the case of a polypeptide, protein or antibody, and 0.01 ng / kg to 10 g / kg in the case of an antisense nucleotide, siRNA, shRNAi or miRNA, when administered once to several times a day.

[0084]

[0085] In addition, the present invention provides a method for screening a hepatocellular carcinoma treatment agent, comprising the steps of (1) contacting a test substance with isolated hepatoma cells; (2) measuring the level of expression or activity of SFRP4 protein or a gene encoding it in the hepatoma cells contacted with the test substance; and (3) selecting a test substance in which the level of expression or activity of the SFRP4 protein or the gene encoding it is reduced compared to a control sample.

[0086]

[0087] The term "test substance" used in referring to the screening method of the present invention refers to an unknown candidate substance used in screening to determine whether it affects the expression level of a gene or the expression or activity of a protein. The sample includes, but is not limited to, chemicals, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0088] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0089]

[0090] <Example>

[0091] We aimed to identify specific derived substances by analyzing secreted proteins specifically secreted by CAFs, which are one of the components of the tumor microenvironment and are responsible for the formation of the stroma of hepatocellular carcinoma and malignant tumors.

[0092] NGS RNA-seq was performed on samples that passed QC, including hepatocellular carcinoma tissues, para-cancer-associated fibroblasts (PAFs), and hepatocellular carcinoma-associated fibroblasts cultured from the tissues of eight hepatocellular carcinoma patients. Eighty-three signature genes of hepatocellular carcinoma-associated fibroblasts that were significantly differentially expressed more than fourfold in hepatocellular carcinoma CAFs compared to PAFs were selected, and GO and GSEA analyses were performed on these genes. Clinical efficacy was evaluated using the TCGA dataset for 60 genes specifically overexpressed in CAFs. Based on previous studies showing that fibroblasts significantly affect EMT, two key EMT genes were selected and verified through scRNA-seq analysis. Two key immune response genes were analyzed using the TIMER 2.0 database for immune infiltration analysis using the TCGA cohort (Fig. 1).

[0093] GO analysis confirmed that the HCC-CAF signature gene was highly correlated with EMT in gene groups related to cellular component, biological process, and molecular function (Fig. 2A). GSEA analysis also confirmed that the HCC-CAF signature was directly and closely related to EMT (Fig. 2B). To examine the clinical efficacy of this HCC-CAF signature, we divided it into high- and low-expression groups using clustering analysis from TCGA data (Fig. 2C). We confirmed that the proportion of the group with high expression of the HCC-CAF signature was higher in patients with macroscopic liver fibrosis compared to those without vascular invasion, as well as in patients with high Ishak score, AJCC stage, and histopathologic grade (Fig. 2D). Furthermore, we confirmed that the higher the proportion of the group with high expression of the HCC-CAF signature, the worse the overall survival (OS) and disease-specific survival (DSS) (Fig. 2E, left panel). The HCC-CAF signature was also confirmed to be a statistically significant risk factor in the Cox proportional model hazard measurement (Fig. 2E, right panel).

[0094] Two candidate genes belonging to the core EMT genes and differentially expressed and overexpressed in TCGA_LIHC were finally selected (Fig. 3A). Higher expression of the two selected candidates, SPP1 and SFRP4, was evaluated as being associated with a poorer patient prognosis in terms of OS, DFS, PFS, and DSS (Figs. 3B and 3C).

[0095] We analyzed SFRP4 expression between liver cancer and non-liver cancer in TCGA data. Results showed that SFRP4 expression was significantly increased in cancer tissues, and a high diagnostic performance of AUC 0.83 enabled differentiation between liver cancer and non-liver cancer (Fig. 4).

[0096] In the OS analysis based on the expression of SFRP4 in TCGA data, it was confirmed that patients with increased expression of SFRP4 had significantly poorer overall survival (Fig. 5).

[0097] Furthermore, the expression of SFRP4 in the patient's blood could predict the treatment response to sorafenib (800 mg / day, monotherapy) and lenvatinib (8-12 mg / day, monotherapy), which are multi-tyrosine kinase inhibitors (TKIs) that can be administered as first- or second-line treatments for hepatocellular carcinoma. SFRP4 expression was higher in the non-responder group, and a significant difference in the patient's PFS was observed according to plasma SFRP4 expression (Fig. 6).

[0098] When analyzing their expression and immune cell infiltration in the TCGA_LIHC dataset, SFRP4 showed a positive correlation with CAF, Treg, M2 macrophages, etc., and a negative correlation with CD8+ T cells, confirming that SFRP4 is related to tumor immune suppression in the tumor microenvironment (Figures 7A and 7B). Accordingly, when measuring the correlation between SFRP4 and Treg markers, it was confirmed that they showed a high correlation of 0.5 or higher (Figure 7C).

[0099] To determine whether SFRP4 expression is specifically expressed in hepatocellular carcinoma-associated fibroblasts, single-cell analysis was performed on public omics hepatocellular carcinoma data (n=25) (Fig. 8). We confirmed that SFRP4 expression was specifically increased in CAF (Figs. 8A and 8B). Subcluster analysis of CAF identified six CAF subclusters, and SFRP4 expression was particularly increased in C3 CAF (Figs. 8C-E). We confirmed that EMT gene expression was increased in the C3 CAF subtype with increased SFRP4 expression (Figs. 8F-H).

[0100] In order to confirm the tumorigenicity of SFRP4 derived from CAFs, we used an orthotopic mouse model that can reproduce the tumor microenvironment as an animal model. This experiment was conducted using HCC-CAF cells transfected with siSFRP4, which utilizes the inhibitory effect of siRNA. siRNA can inhibit the expression of target genes by complementary binding to the mRNA of the target gene, and by using an appropriate concentration and incubation time, the proportion of off-targets can be reduced and the target gene can be specifically inhibited. In this experiment, siSFRP4 (RNA Accession Number: NM_003014.3) was cultured for 48 hours at a concentration of 100 nM, and HCC-CAF transfected with siSFRP4 and hepatocellular carcinoma cell lines were co-cultured at a 1:1 ratio and used for animal experiments. A 1.5 cm skin and peritoneum were incised in the suprapubic wall of nude mice (BALB / c nude mice) to expose the left hepatic lobe to the outside of the body. Hepatocellular carcinoma cell lines co-cultured with HCC-CAF cells in which the SFRP4 gene was selectively suppressed were injected into the left lobe of the liver, and the tumorigenicity of each group was observed (Fig. 9).

[0101] When the tumor formation ability was observed in the HCC+CAF, HCC+CAF(siNC), and HCC+CAF(siSFRP4) groups in the orthotopic transplantation model, it was confirmed that tumor formation was significantly lower in the CAF(siSFRP4)+Huh-7 group compared to the HCC+CAF and HCC+CAF(siNC) groups (Fig. 10A and Fig. 10B). There was no significant difference in the body weights of the mice (Fig. 10C), and the tumor weights were confirmed to be higher in the HCC+CAF and HCC+CAF(siSFRP4) groups than in the HCC+CAF group (Fig. 10D). In addition, when IHC staining was performed on the obtained tumors, the expression of β-catenin, a key component of the Wnt signaling pathway involved in cell proliferation, and the expression of fibronectin, a mesenchymal marker, were observed to be low in the HCC+CAF (siSFRP4) group, and the expression of E-cadherin, an epithelial marker, was observed to be high, but the expression of Ki-67, a cell proliferation marker, was confirmed to be no different (Fig. 10E and Fig. 10F).

[0102] Single-cell analysis confirmed that SFRP4+ CAFs interact with Tregs and TAMs through CXCL12-CXCR4 signals (Fig. 11), and that SFRP4+ CAFs interact with Tregs through NECTIN2-TIGIT signals (Fig. 12). In addition, single-cell analysis confirmed that SFRP4+ CAFs interact with HCC cells through HGF-MET signals (Fig. 13).

[0103] Additional single cell analyses identified specific ligand-receptors through which SFRP4+ CAFs interact with Treg cells, HCC cells, and TAMs.

[0104] In summary, the above results confirmed that SFRP4+ CAFs suppress tumor immunity by regulating Treg and TAM activation and promote the progression of liver cancer through HGF-MET signaling (Fig. 14).

[0105]

[0106] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing hepatocellular carcinoma comprising SFRP4 protein or a gene encoding the same.

2. A biomarker composition for predicting the prognosis of hepatocellular carcinoma, comprising SFRP4 protein or a gene encoding the same.

3. A biomarker composition for predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, including SFRP4 protein or a gene encoding the same.

4. A biomarker composition according to any one of claims 1 to 3, characterized in that the SFRP4 protein or the gene encoding it is derived from hepatocellular carcinoma-cancer associated fibroblasts (HCC-CAF).

5. A composition for diagnosing hepatocellular carcinoma, comprising as an active ingredient a preparation capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

6. A composition for predicting the prognosis of hepatocellular carcinoma, comprising as an active ingredient a preparation capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

7. A composition for predicting responsiveness to sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, comprising as an active ingredient a preparation capable of measuring the expression level of SFRP4 protein or a gene encoding the same.

8. A composition according to any one of claims 5 to 7, wherein the agent capable of measuring the expression level of the SFRP4 protein or the gene encoding it is a primer or probe that specifically binds to the SFRP4 gene, or an antibody, peptide, aptamer, or compound that specifically binds to the SFRP4 protein.

9. A composition according to any one of claims 5 to 7, characterized in that the SFRP4 protein or the gene encoding it is derived from HCC-CAF.

10. A kit for diagnosing hepatocellular carcinoma comprising the composition of Article 5.

11. A kit for predicting the prognosis of hepatocellular carcinoma comprising the composition of Article 6.

12. A kit for predicting the responsiveness of sorafenib or lenvatinib, which are hepatocellular carcinoma targeting anticancer drugs, comprising the composition of Article 7. 13.(1) A step of measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) a step of comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) A method for providing information necessary for diagnosing hepatocellular carcinoma, including a step of determining hepatocellular carcinoma if the expression level of the SFRP4 protein or the gene encoding it is higher than that of a control sample. 14.(1) A step of measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) a step of comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) A method for providing information necessary for diagnosing hepatocellular carcinoma, including a step of determining that the prognosis of hepatocellular carcinoma is poor if the expression level of the SFRP4 protein or the gene encoding it is higher than that of a control sample. 15.(1) A step of measuring the expression level of SFRP4 protein or a gene encoding it from a sample isolated from a hepatocellular carcinoma patient; (2) a step of comparing the expression level of the SFRP4 protein or the gene encoding it with a control sample; and (3) A method for providing information necessary for predicting responsiveness to sorafenib or lenvatinib, a hepatocellular carcinoma-targeting anticancer drug, including a step of determining that responsiveness to sorafenib or lenvatinib, a hepatocellular carcinoma-targeting anticancer drug, is low if the expression level of the SFRP4 protein or the gene encoding it is higher than that of the control sample.

16. A method according to any one of claims 13 to 15, wherein the sample isolated from the hepatocellular carcinoma patient is HCC-CAF.

17. A pharmaceutical composition for preventing or treating hepatocellular carcinoma, comprising an inhibitor of the expression of SFRP4 protein or a gene encoding the same as an active ingredient.

18. A pharmaceutical composition for preventing or treating hepatocellular carcinoma, characterized in that in claim 17, the expression inhibitor of the SFRP4 protein or the gene encoding it is any one selected from the group consisting of an antisense nucleotide, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily binds to SFRP4 mRNA.

19. A pharmaceutical composition for preventing or treating hepatocellular carcinoma, characterized in that the SFRP4 protein or the gene encoding it in claim 17 is derived from HCC-CAF.

20. A pharmaceutical composition for preventing or treating hepatocellular carcinoma, characterized in that the composition increases tumor immunity by regulating Treg and TAM activation in a hepatocellular carcinoma tumor microenvironment and inhibits the progression of hepatocellular carcinoma through HGF-MET signal transduction. 21.(1) Step of contacting the test substance with the separated liver cancer cells; (2) A step of measuring the level of expression or activity of SFRP4 protein or a gene encoding it in liver cancer cells that have come into contact with the test substance; and (3) A method for screening a hepatocellular carcinoma treatment agent, comprising a step of selecting a test substance in which the expression or activity level of the SFRP4 protein or the gene encoding it is reduced compared to a control sample.

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