KR20200041722A - SGK494 gene involved in gastric cancer and use thereof - Google Patents

Abstract

The present invention relates to the expression of a certain gene associated with gastric cancer, a composition for prognostication of gastric cancer using the same, and a prognostication method using the same, and more specifically, to prognostication, diagnosis, and treatment of gastric cancer using sugene kinase 494 (SGK494) gene. The present invention has confirmed that the SGK494 gene associated with gastric cancer is highly expressed in various gastric cancer cells or tissues, and moreover, also confirmed, through experiments, changes of gastric cancer cells according to differences in SGK494 gene expression. In addition, the present invention has newly identified gastric cancer inhibitory effects due to SGK494 gene expression inhibition, and a binding protein, BAG2, and the SGK494 gene and BAG2 gene may be utilized in combination for prognostication, diagnosis, or treatment of gastric cancer.

Description

SGK494 gene involved in gastric cancer and use thereof

The present invention relates to a composition for predicting gastric cancer prognosis using the expression of a specific gene related to gastric cancer, and a method for predicting the prognosis using the same, and more particularly, to a prognosis, diagnosis and treatment of gastric cancer using the SGK494 gene.

The incidence of gastric cancer in Korea ranks first in male cancer. In other words, 19545 patients in 2015 and 9662 patients in women. In addition, the number of cancer patients also ranked second with 15.9% (as of 2015) after thyroid cancer.

In gastric cancer, 5-fluorouracil (5-FU) and cisplatin are the most widely used anti-cancer chemotherapy, but there are still no established globally recognized standard therapies, and clinical trials of new drugs alone or in combination I am trying through. However, in recent years, the treatment of gastric cancer is entering a new phase as new molecular target treatments are developed and used through understanding of the mechanism of solid cancer and the signaling system involved in it.

Although surgical excision is the best way to diagnose and treat gastric cancer early, it is best to perform surgery, but many gastric cancer patients are diagnosed in locally-advanced or metastatic stages. In Korea or Japan where active early diagnosis is performed, nearly half of patients are diagnosed at the stage of early gastric cancer, and the survival rate of early gastric cancer in the United States and Europe is higher than in the Western countries such as the United States and Europe. As a result, the 5-year survival rate is only 20%. However, despite the difference between the stage and the treatment method at the time of diagnosis, the treatment performance of gastric cancer patients with similar prognosis is not significantly different between western and oriental patients. In addition, even in patients who were able to complete surgical resection, more than half of them eventually recur, so the treatment of gastric cancer, such as chemotherapy or radiation therapy, plays an important role besides surgery.

The main disadvantage of this method is the side effects induced by systemic chemotherapy used to treat cancer. The side effects of chemotherapy have a significant effect on the patient's life and can increase the patient's anxiety about treatment. In addition, side effects associated with chemotherapeutic agents generally include dose limiting toxicity (DLT), which requires caution when administering these drugs. For example, mucositis has dose limiting toxicity (DLT) for several anti-cancer agents (anti-metabolite cytotoxic agent 5-fluorouracil, mesotrexate and anti-tumor antibiotic doxorubicin). Many of the side effects of these chemotherapy are severe, requiring hospitalization or requiring pain relievers to treat pain. As such, side effects of chemotherapeutic agents and radiation therapy are emerging as important issues in the treatment of cancer patients.

On the other hand, even in the case of the same surgical stage, the prognosis of the patient after surgery is diverse, so there is an urgent need for accurate prediction of the prognosis for each patient and personalized treatment. In the case of advanced gastric cancer, the prognosis is very diverse depending on the patient even though the same treatment is performed, so accurate prediction of the prognosis is urgently needed. This clinical diversity of gastric cancer can be said to be due to the molecular biological diversity of gastric cancer, which is due to changes in various genes. However, research on this is still insignificant and has been approved or used as a biomarker that can predict the prognosis of gastric cancer, and is currently in use or rare.

Accordingly, as an alternative for solving the problems of the conventional cancer treatment technology as described above, treatment targeting a cancer-related gene has been spotlighted. Particularly, studies on a single antibody against EGFR, VEGF, etc., or an inhibitor against tyrosine kinase as a therapeutic agent for gastric cancer, are being actively conducted, but have not yet achieved great success.

Accordingly, the present invention was devised to solve the above problems, and the present inventors confirmed the possibility of the expression of a specific gene among kinase as a predictor that can predict the prognosis of gastric cancer and complete the present invention based on this. Became.

 An object of the present invention is to provide a biomarker composition for predicting gastric cancer prognosis through common expression of a specific gene and a protein binding thereto.

In addition, another object of the present invention is to provide a kit for predicting gastric cancer prognosis comprising a protein that specifically binds to a specific gene.

In addition, another object of the present invention is to find a method that provides useful information for predicting the prognosis of gastric cancer by measuring the expression level of a specific gene related to the prognosis of gastric cancer.

In addition, another object of the present invention is to find a pharmaceutical composition for preventing or inhibiting metastasis of gastric cancer by activating or inhibiting the expression of a specific gene related to the prognosis of gastric cancer.

In order to achieve the above object, the present invention provides a biomarker composition for predicting gastric cancer prognosis comprising at least one gene selected from the group consisting of SGK494 and BAG2 from a biological sample or a protein encoded with the gene.

In addition, the present invention is a primer or probe specifically binding to one or more genes selected from the group consisting of SGK494 and BAG2, an antibody specifically binding to a protein encoded with the gene, or a peptide having a binding domain specific to the protein. It provides a kit for predicting the prognosis of stomach cancer comprising a.

In addition, the present invention comprises the steps of measuring the mRNA expression level of one or more genes selected from the group consisting of SGK494 and BAG2 from the gastric cancer patient sample or the expression level of the protein encoded with the gene; And comparing the mRNA expression level of the gene or the expression level of the protein encoded with the gene with a control sample. It provides a method for providing useful information for predicting gastric cancer prognosis.

When the mRNA expression level of one or more genes selected from the group consisting of the SGK494 and BAG2 or the expression level of the protein encoded with the gene is measured as compared to the control sample, it is characterized by determining that the risk of metastasis is high.

The method for measuring the mRNA expression level is RT-PCR, competitive RT-PCR (RT-PCR), real-time RT-PCR (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting (Northern blotting) and characterized in that the measurement using any one selected from the group consisting of DNA chips.

Methods for measuring the protein expression level include Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and locating ( rocket) is characterized by measuring using any one selected from the group consisting of immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip.

In addition, the present invention comprises SGK494 and BAG2, gastric cancer metastasis characterized in that it comprises an inhibitor that inhibits the expression or activity of the protein encoded by the gene or an inhibitor that suppresses the mRNA expression of one or more genes selected from the group consisting of SGK494 and BAG2 Provided is a pharmaceutical composition for prevention or inhibition.

According to the present invention, it was confirmed that the SGK494 (Sugene kinase 494) gene related to gastric cancer has high expression in various gastric cancer cells or tissues, and experimentally confirmed the change in gastric cancer cells according to the difference in SGK494 gene expression. In addition, the effect of inhibiting the expression of the SGK494 gene and the inhibition effect of gastric cancer and the binding protein BAG2 were newly identified, and the SGK494 gene and the BAG2 gene may be used together to confirm, diagnose or treat the prognosis of gastric cancer.

1 is a KMPLOT result of measuring the difference in expression of SGK494 for various types of cancer.
Figure 2 is a result diagram showing the binding protein of SGK494 found in humans as a String.
Figure 3 is a result showing the binding protein of SGK494 found in the nematode nematode (C. elegans) in the form of a network.
Figure 4 is a result of KMPLOT obtained by simultaneously measuring and showing the expression of BAG2 and the expression of SGK494, the binding protein of SGK494, for the intestinal type / male / HER2 group, a group of patients with gastric cancer.
5 is a result of KMPLOT obtained by simultaneously measuring and showing the expression of BAG2 and SGK494, the binding protein of SGK494, for the intestinal type / male / HER2 / M0 group, a group of patients with gastric cancer.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a biomarker composition for predicting gastric cancer prognosis comprising at least one gene selected from the group consisting of SGK494 and BAG2 from a biological sample or a protein encoded with the gene.

In another aspect, the present invention provides a primer or probe specifically binding to one or more genes selected from the group consisting of SGK494 and BAG2, an antibody specifically binding to the protein encoded with the gene, or a binding domain specific to the protein. Provided is a kit for predicting gastric cancer prognosis that includes a peptide having.

The antibody is a polyclonal antibody or a monoclonal antibody. The polyclonal antibody can be prepared by injecting a protein or fragment thereof expressed by the gene into an external host as an immunogen according to methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep or rabbits. The immunogen is injected by intramuscular, intraperitoneal or subcutaneous injection, and is usually administered with an adjuvant to increase antigenicity. Serum is collected from external hosts on a regular basis to collect serum showing improved titer and antigen specificity, or to separate and purify antibodies therefrom. The monoclonal antibody can be produced by an immortalized cell line production technique by fusion known to those skilled in the art. For example, a protein expressed by the gene is immunized with a mouse or a peptide is synthesized and bound to bovine serum albumin to immunize a mouse. The antigen-producing B lymphocytes isolated from mice are fused with myeloma of human or mouse to produce immortalized hybridomas, and the indirect enzyme-linked immunosorbent assay with the hybridoma cells (enzyme- Monoclonal antibodies can be prepared by determining whether monoclonal antibodies are generated using linked immunoabsorbent assays (ELISA), selecting positive clones, culturing them, and then separating and purifying the antibodies or injecting them into the abdominal cavity of rats, and then collecting ascites. have. The monoclonal antibody is generally quantified by coloring reaction using a secondary antibody bound with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and their substrate. It may be analyzed, or it may be quantitatively analyzed by directly using an AP or HRP enzyme coupled to the monoclonal antibody to the protein.

The reaction between the protein and the antibody is Western blot, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA) and immunostaining (Immunohistochemistry, IHC) It can be confirmed through, but is not limited to.

The kit containing the antibody may typically include a lyophilized antibody and a buffer, a stabilizer, an inactive protein, etc., wherein the antibody is prepared by radionuclides, fluorescors, enzymes, etc. Can be labeled.

In another aspect, the present invention comprises the steps of measuring the mRNA expression level of one or more genes selected from the group consisting of SGK494 and BAG2 from the gastric cancer patient sample or the expression level of the protein encoded with the gene; And comparing the mRNA expression level of the gene or the expression level of the protein encoded with the gene with a control sample. It provides a method for providing useful information for predicting gastric cancer prognosis.

When the mRNA expression level of one or more genes selected from the group consisting of the SGK494 and BAG2 or the expression level of the protein encoded with the gene is measured as compared to the control sample, it may be determined that the risk of metastasis is high.

The method for measuring the mRNA expression level is RT-PCR, competitive RT-PCR (RT-PCR), real-time RT-PCR (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting It can be measured using any one selected from the group consisting of (Northern blotting) and DNA chips.

Methods for measuring the protein expression level include Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and locating ( rocket) can be measured using any one selected from the group consisting of immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip.

In another aspect, the present invention is characterized in that it comprises an inhibitor that suppresses the mRNA expression of one or more genes selected from the group consisting of SGK494 and BAG2 or an inhibitor that inhibits the expression or activity of the protein encoded with the gene as an active ingredient. Provided is a pharmaceutical composition for preventing or inhibiting gastric cancer metastasis.

The inhibitor may be any one or more selected from the group consisting of Olaparib, Veliparib, Niraparib, Talazoparib, and Rucaparib, and preferably Olaparib. It is not limited thereto.

The pharmaceutical composition may further include a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

As a carrier, excipient or diluent usable in the present invention, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, And methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention is formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. You can.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc.These solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( sucrose) or lactose, gelatin, and the like.

Also, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

In addition, the dosage of the pharmaceutical composition according to the present invention can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, and age. Therefore, the above dosage does not limit the scope of the present invention in any way.

The present inventors confirmed the possibility of the expression of the specific gene SGK494 (Sugene kinase 494) gene among kinase as a predictor that can predict the prognosis of gastric cancer and completed the present invention based on this.

The SGK494 (Sugene kinase 494) gene, which has the potential as a target gene for the treatment of gastric cancer, is one of the serine / threonine protein kinase, and whether it can be applied to the diagnosis of gastric cancer through the expression of SGK494 as a kinase that is not yet known. It has not been reported as to whether or not the present invention has been reported.

The term "diagnosis" as used in the present invention means an act of confirming the pathological condition, that is, the presence or characteristic of gastric cancer by administration of the composition according to the present invention.

In the present invention, biological samples include blood and other liquid samples of biological origin, biopsy samples, solid tissue samples such as tissue culture, or cells derived therefrom. More specifically, tissue, extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, etc., the sample is an animal, preferably a mammal It can be obtained from animals, most preferably humans.

The sample may be pre-treated prior to use for detection, and may include, for example, filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, and the like. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the embodiments described below are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the embodiments described below.

Example 1 Analysis of the relationship between the expression of SGK494 and the survival rate of cancer patients (KMPLOT)

The expression of SGK494 and the prognosis of various cancers according to the analysis were analyzed using the KMPLOT method, and the effect of SGK494 expression on cancer prognosis in various cancers was investigated.

As can be seen in (Fig. 1), when the expression of SGK494 is high, in the stomach cancer (HR = 1.24, p = 0.047) and (Fig. 1 (B)) can be seen in ((A) of Fig. 1) It was observed that the survival rate was lowered in the visible ovarian cancer (HR = 1.32, p = 0.0034).

Expression of SGK494 in breast cancer (HR = 0.63, p = 0) as seen in ((C) of FIG. 1) and lung cancer (HR = 0.73, p = 0.00017) as seen in (D) of FIG. 1 It was observed that the higher the survival rate, the higher the survival rate.

From these results, it can be seen that unlike other cancers, the prognosis of gastric cancer and ovarian cancer and the expression of SGK494 are closely related.

Example 2. Exploration of SGK494 binding protein

In order to find a partner that binds the SGK494 protein, human genes predicted to bind SGK494 in a bioinformatics approach are illustrated using a string as shown in FIG. 2 (FIG. 2). On the other hand, F31E3.2 corresponding to the SGK494 gene from the pretty little nematode (C. elegans) was identified by binding or predicting a partner (FIG. 3).

As a result, a gene called BAG2 was found as a result of searching and matching genes among human genes predicted to interact with SGK494 and genes of only nematodes.

Example 3. Prediction of gastric cancer prognosis using SGK494 and BAG3

In the case of gastric cancer patients whose prognosis was not predicted by expression of SGK494 alone, that is, intestinal type / male / HER2 group (HR = 0.91, p = 0.8) (see Fig. 4 (A)), the values of SGK494 and BAG2 co-expression When the expression of BAG2 was examined together when it was greater than the average value (see FIG. 4 (B)), the inverse survival rate of gastric cancer patients was poor in the intestinal type / male / HER2 group (HR = 3.44, p = 0.00086). Showed correlation.

In case of gastric cancer patient group not predicted by expression of SGK494, that is, intestinal type / male / HER2 / M0 group (HR = 1.18, p = 0.67) (see FIG. 5 (A)), and

When both the expressions of SGK494 and BAG2 were higher than the mean value (see median) (see FIG. 5 (B)), the expression of BAG2 was examined as a result, and the expression of SGK494 / BAG2 was high intestinal type / male / HER2 / M0 group (HR = 3.81, p = 0.001) showed a negative correlation with gastric cancer survival.

From these results, it is judged that the prognosis of a specific group of gastric cancer patients can be well indicated by examining expression including both SGK494 and BAG2.

As described above, specific parts of the present invention have been described in detail. For those skilled in the art, this specific technique is only a preferred embodiment, and it is obvious that the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

Biomarker composition for predicting gastric cancer prognosis comprising at least one gene selected from the group consisting of SGK494 and BAG2 from a biological sample or a protein encoded with the gene.
Gastric cancer comprising a primer or probe specifically binding to one or more genes selected from the group consisting of SGK494 and BAG2, an antibody specifically binding to the protein encoded with the gene, or a peptide having a binding domain specific to the protein. Prognosis prediction kit.
Measuring mRNA expression levels of one or more genes selected from the group consisting of SGK494 and BAG2 from gastric cancer patient samples or expression levels of proteins encoded with the genes; And
Comprising the step of comparing the mRNA expression level of the gene or the expression level of the protein encoded with the gene with a control sample; Method for providing useful information for predicting the prognosis of gastric cancer comprising a.
According to claim 3,
The mRNA expression level of one or more genes selected from the group consisting of the SGK494 and BAG2 or the expression level of the protein encoded with the gene,
A method that provides useful information for predicting the prognosis of gastric cancer, characterized by determining that the risk of metastasis is high when compared to a control sample.
The method of claim 4,
Method for measuring the mRNA expression level,
RT-PCR, Competitive RT-PCR (Competitive RT-PCR), Real-time RT-PCR (RT-PCR), RNase protection assay (RPA), Northern blotting and DNA chip group Method for providing useful information for predicting the prognosis of gastric cancer, characterized in that measured using any one selected from.
The method of claim 4,
Method for measuring the protein expression level,
Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, Method for providing useful information for predicting gastric cancer prognosis, characterized by measuring using any one selected from the group consisting of immunoprecipitation assay, complement fixation assay, FACS, and protein chip.
SGK494 and BAG2 is selected from the group consisting of inhibitors that inhibit the mRNA expression of one or more genes or inhibitors that inhibit the expression or activity of the protein encoded with the gene as an active ingredient for preventing or inhibiting metastasis of gastric cancer. Pharmaceutical composition.

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