KR102325972B1 - SPP1 biomarker for predicting the response of anticancer drugs to liver cancer and use thereof - Google Patents

Abstract

The present invention relates to an SPP1 biomarker for predicting a response to a liver cancer treatment and a use thereof. An effect of cancer associated fibroblasts (CAF) on resistance to a liver cancer targeted therapeutics is examined to find a target substance for overcoming resistance to a liver cancer targeted therapeutics and a biomarker for predicting a treatment response thereof. That is, in case of the liver cancer, an efficacy is verified as a candidate for overcoming the resistance to the liver cancer targeted therapeutics by controlling liver cancer CAF-derived SPP1, and it is confirmed whether clinical significance is exhibited as a marker for predicting a treatment response. As a result, it is highly possible that liver cancer CAF-derived SPP1 is efficiently used as a biomarker for predicting a response to a liver cancer treatment.

Description

SPP1 biomarker for predicting the response of anticancer drugs to liver cancer and use thereof

The present invention relates to a SPP1 biomarker for predicting liver cancer treatment response and use thereof.

Liver cancer is one of the most common cancers in Koreans. In 2015, the incidence rate was 29.5 males and 8.2 females per 100,000, ranking 4th among males and 6th among females. It is a malignant tumor with a poor prognosis, ranking 2nd in women and 3rd in women.

Recently, the necessity of controlling the surrounding cells that form the tumor microenvironment together with cancer cells has been emphasized in order to suppress cancer progression and overcome treatment resistance. Studies have been reported that the tumor microenvironment plays an important role in liver cancer progression and drug resistance development in liver cancer, increasing the need to control the hepatocellular tumor microenvironment.

Cancer associated fibroblasts (CAFs) are primarily responsible for the formation of the matrix of malignant tumors. Recently, it has been reported that CAF plays an important role in tumor promotion and anticancer drug resistance induction by exchanging various signals with cancer cells as well as in matrix generation.

Liver cancer is a malignant tumor that occurs in the background of fibrosis due to chronic inflammation. It is assumed that the role of stromal cells such as CAF is more important in the development and progression of cancer than any other tumor, but related studies are lacking.

Meanwhile, in the treatment guidelines for advanced liver cancer, targeted therapies, sorafenib and lenvatinib, are recommended as the first-line treatment, but the objective response rate for tumor reduction is 3-4% sorafenib and 41% lenvatinib, and patients with advanced liver cancer Many of these patients are resistant to treatment and, despite treatment, have an average survival rate of 6 to 9 months. Therefore, in order to improve the survival rate of patients with advanced liver cancer, it is urgent to establish a strategy to overcome liver cancer resistance based on the analysis of the interaction of cells constituting the tumor microenvironment and to discover response markers for predictive treatment of liver cancer targeted therapy.

R Soc Open Sci. 2018 Dec 5;5(12):181006.(2018.12.05. Public)

It is an object of the present invention to provide a biomarker composition for predicting liver cancer-targeted anticancer drug reactivity comprising a secreted phosphoprotein 1 (SPP1) protein or a gene encoding the same.

In addition, another object of the present invention is to provide a composition for predicting liver cancer-targeted anticancer agent reactivity comprising an agent capable of measuring the expression level of SPP1 as an active ingredient, and a kit for predicting liver cancer-targeted anticancer agent reactivity including the same.

In addition, another object of the present invention is to provide a method for providing information necessary for predicting liver cancer-targeted anticancer drug reactivity, including measuring the expression level of SPP1.

In addition, another object of the present invention is to provide a pharmaceutical composition for enhancing the reactivity of a liver cancer target anticancer agent comprising an SPP1 expression or activity inhibitor as an active ingredient.

In addition, another object of the present invention is to provide a screening method for a liver cancer-targeted anticancer agent responsiveness enhancer comprising the step of selecting a test substance with reduced expression or activity level of SPP1 protein.

In order to achieve the above object, the present invention provides a biomarker composition for predicting liver cancer-targeted anticancer drug reactivity, which includes a secreted phosphoprotein 1 (SPP1) protein or a gene encoding the same.

In addition, the present invention provides a composition for predicting liver cancer-targeted anticancer agent reactivity comprising an agent capable of measuring the expression level of SPP1 as an active ingredient.

In addition, the present invention provides a kit for predicting liver cancer-targeted anticancer agent reactivity comprising the composition.

In addition, the present invention comprises the steps of (1) measuring the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein from a sample isolated from a liver cancer patient; (2) comparing the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein with a control sample; and (3) determining that the reactivity to the liver cancer target anticancer agent is low when the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein is higher than that of the control sample. A method of providing information necessary for predicting liver cancer target anticancer agent reactivity to provide.

In addition, the present invention provides a pharmaceutical composition for enhancing liver cancer-targeted anticancer agent responsiveness comprising an SPP1 expression or activity inhibitor as an active ingredient.

In addition, the present invention comprises the steps of (1) contacting a test substance with liver cancer cells; (2) measuring the level of expression or activity of SPP1 protein in liver cancer cells contacted with the test substance; and (3) selecting a test substance having a reduced level of expression or activity of the SPP1 protein as compared to a control sample.

The present invention relates to a SPP1 biomarker for predicting liver cancer treatment response and its use, by elucidating the effect of cancer associated fibroblasts (CAF) on resistance to a targeted treatment for liver cancer, and overcoming resistance to a targeted treatment for liver cancer through CAF genome analysis We discovered target materials and biomarkers for predicting treatment response for That is, the efficacy of SPP1 derived from hepatocarcinoma CAF in liver cancer was verified as a candidate for overcoming target therapy resistance, and clinical significance was confirmed as a therapeutic response predictive marker. As a result, SPP1 derived from liver cancer CAF is highly likely to be usefully utilized as a biomarker for predicting liver cancer treatment response.

1 shows the results of observation under an optical microscope (200×). Morphological differences between NF, PAF and CAF were confirmed. CAF shows larger and broader morphological features than NF and PAF.
2 shows the results of confirming the liver cancer CAF marker. It was confirmed that FSP-1, an EMT-related fibroblast marker, was increased nearly 30-fold compared to PAF.
Figure 3 shows the results of processing Huh7, Hep3B NF / PAF / CAF CM. Cell growth was increased only upon CAF CM treatment.
Figure 4 shows the results of processing Huh7, Hep3B NF / PAF / CAF CM. Sorafenib/lenvatinib resistance was induced upon CAF CM treatment.
5A shows a study flow diagram. Figure 5B shows the results of comparing NF, PAF, CAF expression of genes that increase in the order of NF, PAF, CAF. 5C shows the results of GSEA analysis of CAF-specific overexpressed genes. It was confirmed that the correlation with EMT and KRAS signaling is high. 5D shows the CAF-derived candidate material selection process.
6 shows the Western blot results of CM SPP1.
7 is a result showing that the sorafenib/lenvatinib resistance acquired by CAF co-culture is overcome when the SPP1 blocking peptide is administered. A. Cell growth, B. Cell viability, C. BrdU cell proliferation, D. Caspase 3/7 activity.
FIG. 8 shows the results of comparing whether there is a difference in SPP1 expression depending on whether or not a sorafenib response is performed using the BIOSTORM study data. It was confirmed that the expression of SPP1 was statistically significantly higher in the sorafenib non-responder group (SOR_NR).

The present invention provides a biomarker composition for predicting liver cancer-targeted anticancer drug reactivity, comprising a secreted phosphoprotein 1 (SPP1) protein or a gene encoding the same.

Preferably, the SPP1 protein may be derived from cancer associated fibroblast (CAF), but is not limited thereto.

Preferably, the liver cancer-targeting anticancer agent may be sorafenib or lenvatinib, but is not limited thereto.

"SPP1 (secreted phosphoprotein 1)" of the present invention is NCBI accession no. It may be NM_001040058.2 or NP_001035147.1, but is not limited thereto.

As used herein, "prediction" is used 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 a response. For example, the prediction relates to whether and/or the probability that a patient will survive cancer recurrence-free after treatment, e.g., after treatment with certain therapeutic agents and/or surgical removal of the primary tumor and/or chemotherapy for a certain period of time . The predictions of the present invention can be used clinically to determine treatment by selecting the most appropriate treatment modality for liver cancer patients. The prediction of the present invention is whether a patient will respond favorably to a therapeutic treatment, e.g., a given therapeutic treatment, e.g., administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc., or whether long-term survival of the patient is possible after the therapeutic treatment. It is a useful tool for predicting whether

In the present invention, "prediction of anticancer drug responsiveness" means predicting whether a patient will respond favorably or unfavorably to anticancer drug treatment, or predicting the risk of anticancer drug resistance. Therefore, according to the present invention, if the patient (responder) who can expect an effect and the patient who cannot expect the effect (non-responder) can be predicted before the start of treatment according to the present invention, chemotherapy with high efficacy and safety can be realized. In addition, the predictive method of the present invention can be used clinically to make a treatment decision by selecting the most appropriate treatment method for a patient with liver cancer.

In addition, the present invention provides a composition for predicting liver cancer-targeted anticancer agent reactivity comprising an agent capable of measuring the expression level of SPP1 as an active ingredient.

Specifically, the agent capable of measuring the expression level of SPP1 may be a primer or probe that specifically binds to the SPP1 gene, an antibody, peptide, aptamer or compound that specifically binds to the SPP1 protein, The present invention is not limited thereto.

Preferably, the SPP1 may be derived from liver cancer CAF, but is not limited thereto.

Preferably, the liver cancer-targeting anticancer agent may be sorafenib or lenvatinib, but is not limited thereto.

In addition, the present invention provides a kit for predicting liver cancer-targeted anticancer agent reactivity comprising the composition.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, capable of base pairing with a complementary template and serving as a starting point for template strand copying. nucleic acid sequence. The primer is capable of initiating DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases in length that can be specifically bound to mRNA, and is labeled to determine the presence or absence of a specific mRNA, expression quantity can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Suitable probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.

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 SPP1 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. Moreover, the said antibody also includes special antibodies, such as a humanized antibody.

As used herein, the term "peptide" has an advantage of high binding strength to a target material, and no denaturation occurs even during heat/chemical treatment. In addition, since the molecular size is small, it can be attached to other proteins and used as a fusion protein. Specifically, it can be used as a diagnostic kit and drug delivery material because it can be used by attaching it to a polymer protein chain.

As used herein, the term "aptamer" refers to a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and binding to a target molecule with high affinity and specificity. ) means a kind of polynucleotide composed of As described above, the aptamer can specifically bind to an antigenic substance in the same way as an antibody, but has higher stability than a protein, has a simple structure, and is composed of a polynucleotide that is easy to synthesize, so it can be used as an alternative to an antibody. can

In addition, the present invention comprises the steps of (1) measuring the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein from a sample isolated from a liver cancer patient; (2) comparing the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein with a control sample; and (3) determining that the reactivity to the liver cancer target anticancer agent is low when the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein is higher than that of the control sample. A method of providing information necessary for predicting liver cancer target anticancer agent reactivity to provide.

Preferably, the liver cancer-targeting anticancer agent may be sorafenib or lenvatinib, but is not limited thereto.

Specifically, the method for measuring the mRNA expression level is RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay) ), Northern blotting and DNA chips are used, but are not limited thereto.

Specifically, the method for measuring the protein expression level is Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS and protein chip are used, but are not limited thereto.

As used herein, the term "a sample isolated from a liver cancer patient" refers to a tissue, cell, whole blood, serum, plasma, saliva that is different from the control in the expression level of the SPP1 gene or SPP1 protein, which is a biomarker for predicting liver cancer target anticancer drug reactivity. , sputum, cerebrospinal fluid, or samples such as urine. Preferably, the sample may be liver cancer tissue.

In addition, the present invention provides a pharmaceutical composition for enhancing liver cancer-targeted anticancer agent responsiveness comprising an SPP1 expression or activity inhibitor as an active ingredient.

Specifically, the SPP1 expression inhibitor is any one selected from the group consisting of antisense nucleotides complementary to the mRNA of the SPP1 gene, small interfering RNA (siRNA) and short hairpin RNA (shRNA). may be, and the SPP1 activity inhibitor may be any one selected from the group consisting of a small molecule compound that specifically binds to the SPP1 protein, a peptide, a peptide mimetics, an aptamer, an antibody, and a natural product, but is not limited thereto.

Preferably, the liver cancer-targeting anticancer agent may be sorafenib or lenvatinib, but is not limited thereto.

The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisense, siRNA oligonucleotides, and natural extracts as active ingredients. The pharmaceutical composition or combination formulation of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , a solubilizing agent such as a lubricant or flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pill, capsule, granule or tablet.

The pharmaceutical formulation 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, sustained-release formulations of the active compound, etc. can The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered as The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for the prevention or treatment of diseases. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health status, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and drugs used at the same time. Although not limited thereto, for example, in the case of an adult, when administered once to several times a day, the composition of the present invention is administered once to several times a day, in the case of a compound, 0.1 ng/kg to 10 g/kg, polypeptide , in the case of protein or antibody, 0.1 ng/kg to 10 g/kg, antisense nucleotide, siRNA, shRNAi, or miRNA, it may be administered at a dose of 0.01 ng/kg to 10 g/kg.

In addition, the present invention comprises the steps of (1) contacting a test substance with liver cancer cells; (2) measuring the level of expression or activity of SPP1 protein in liver cancer cells contacted with the test substance; and (3) selecting a test substance having a reduced level of expression or activity of the SPP1 protein as compared to a control sample.

Preferably, the liver cancer-targeting anticancer agent may be sorafenib or lenvatinib, but is not limited thereto.

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

Hereinafter, the present invention will be described in detail according to non-limiting examples. Of course, the following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. Accordingly, what can be easily inferred by an expert in the technical field to which the present invention pertains from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention.

< Example 1> Liver cancer CAF's Characteristics and effects on liver cancer cells

The primary culture of a liver cancer CAF cell line/paracancer associated fibroblast (PAF) cell line and a normal liver tissue fibroblast (NF) cell line from a non-liver cancer patient was successful from liver cancer tissue of a liver cancer patient.

Accordingly, the basic characteristics of CAF/PAF/NF were confirmed. In morphological observation, liver cancer CAF showed a larger and broader shape than NF and PAF (FIG. 1). FSP-1, a fibroblast marker derived from epithelial-mesenchymal-transition (EMT), rather than a-SMA/FAP, an activated myofibroblast marker, in RT-PCR performed to confirm the CAF marker. It was confirmed that the CAF was overexpressed by about 30 times compared to the PAF (FIG. 2).

NF/PAF/CAF conditioned media (CM) was treated with hepatocellular carcinoma cell lines Hep3B and Huh-7. Tumor cell growth was significantly increased during CAF culture treatment (FIG. 3). Sorafenib and lenvatinib resistance was induced in liver cancer cell lines when the CAF culture medium was treated ( FIG. 4 ).

< Example 2> CAF Selection of genes that induce resistance to derived targeted therapeutics and verification in vitro

To confirm the CAF-specific gene expression pattern that affects the progression of liver cancer and the development of resistance to targeted drugs, 9 pairs of CAFs, PAFs (total 18 cell lines) and 2 NFs, 1 hepatic stellate (hepatic) cells that were successful in primary culture Whole transciptome sequencing (WTS) and analysis were performed on stellate cells (HSCs) ( FIG. 5A ).

790 gene clusters showing an increase in expression according to the progress of NF→PAF→CAF were identified (FIG. 5B).

Among the CAF-specific overexpressed genes, nine genes corresponding to growth factors or cytokines were selected ( FIG. 5D ).

Among the nine candidate genes, SPP1 showing CAF overexpression with the highest fold was selected, and SPP1 expression was confirmed in CAF/PAF/NF CM. It was confirmed that the highest expression of SPP1 in CAF CM (FIG. 6).

< Example 3> CAF origin of SPP1 In vitro verification of the effect of inducing resistance to targeted therapeutics

Upon co-culture of liver cancer cells/CAF, sorafenib/lenvatinib resistance was acquired. By confirming that the resistance acquired by CAF CM was overcome when the SPP1-blocking peptide was administered, it was demonstrated through an in vitro experiment that CAF-derived SPP1 plays an important role in acquiring sorafenib/lenvatinib resistance in liver cancer cells. (Fig. 7).

< Example 4> Target therapy as a predictor of therapeutic response SPP1 valence

The present inventors performed an analysis using public omics data in order to determine whether the response to a target therapeutic agent is different depending on whether SPP1 is overexpressed or not.

The subjects of analysis are as follows. In the STORM phase 3 study to see the effect of sorafenib and placebo as adjuvant therapy, 67 patients enrolled in the BIOSTORM study conducted together as a biomarker study were analyzed. 67 patients were obtained from liver cancer before sorafenib and were subjected to tissue microarray.

The analysis method is as follows. Comparison of SPP1 expression between two groups divided into a sorafenib responder (Sor_R, n=21) and a sorafenib non-responder (Sor_NR, n=46) according to the response to sorafenib. did.

The analysis results are as follows. Analysis was performed on two SPP1 probes. It was confirmed that the expression of SPP1 was statistically significantly increased in Sor_NR in both probes. Through this analysis, it was confirmed that SPP1 not only serves as a resistance overcoming target, but also shows clinical significance as a biomarker for predicting response to sorafenib treatment ( FIG. 8 ).

Claims (17)

A biomarker composition for predicting responsiveness of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer drug, comprising a secreted phosphoprotein 1 (SPP1) protein or a gene encoding the protein. The biomarker composition according to claim 1, wherein the SPP1 protein is derived from cancer associated fibroblasts (CAF). delete A composition for predicting the reactivity of sorafenib or lenvatinib, a liver cancer-targeted anticancer agent, comprising an agent capable of measuring the expression level of SPP1 as an active ingredient. The method of claim 4, wherein the agent capable of measuring the expression level of SPP1 is a primer or probe that specifically binds to the SPP1 gene, an antibody, peptide, aptamer or compound that specifically binds to the SPP1 protein. A composition for predicting the reactivity of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer agent. The composition for predicting the reactivity of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer agent according to claim 4, wherein the SPP1 is derived from liver cancer CAF. delete A kit for predicting responsiveness of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer agent, comprising the composition of any one of claims 4 to 6. (1) measuring the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein from a sample isolated from a liver cancer patient;
(2) comparing the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein with a control sample; and
(3) when the mRNA expression level of the SPP1 gene or the expression level of the SPP1 protein is higher than that of the control sample, sorafenib, a liver cancer target anticancer agent comprising the step of determining that the reactivity to the liver cancer target anticancer agent sorafenib or lenvatinib is low; A method of providing the information needed to predict lenvatinib reactivity. 10. The method of claim 9, wherein the sample is liver cancer tissue, the method for providing information necessary for predicting the reactivity of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer agent. delete A pharmaceutical composition for enhancing the responsiveness of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer agent, comprising an SPP1 expression or activity inhibitor as an active ingredient. The method of claim 12, wherein the SPP1 expression inhibitor is selected from the group consisting of antisense nucleotides complementary to the mRNA of the SPP1 gene, small interfering RNA (siRNA) and short hairpin RNA (shRNA). A pharmaceutical composition for enhancing responsiveness of sorafenib or lenvatinib, which is a liver cancer-targeted anticancer agent, characterized in that it is any one. The liver cancer-targeted anticancer agent according to claim 12, wherein the SPP1 activity inhibitor is any one selected from the group consisting of a small molecule compound that specifically binds to the SPP1 protein, a peptide, a peptide mimetics, an aptamer, an antibody, and a natural product. A pharmaceutical composition for enhancing the responsiveness of phosphorus sorafenib or lenvatinib. delete (1) contacting the test substance with liver cancer cells;
(2) measuring the level of expression or activity of SPP1 protein in liver cancer cells contacted with the test substance; and
(3) Sorafenib or lenvatinib reactivity enhancing agent screening method, which is a liver cancer target anticancer agent, comprising the step of selecting a test substance having a reduced level of expression or activity of the SPP1 protein compared to a control sample.
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