Composition for treating gastric cancer containing SYT11 inhibitor as an active ingredient

Abstract

The present invention relates to a composition for treating gastric cancer containing a SYT11 (Synaptotagmin 11) inhibitor as an active ingredient, and a method for diagnosing esophageal carcinoma (ESC) including measuring SYT11 expression. The composition containing the SYT11 inhibitor of the present invention inhibits the migration and invasion of gastric cancer cells, inhibits their adhesion to extracellular matrix, suppresses the secretion of various cancer metastasis-related cytokines, and inhibits the proliferation of gastric cancer cells, thereby exhibiting excellent effects as a composition for inhibiting gastric cancer metastasis and preventing or treating gastric cancer. Furthermore, the present invention confirmed the correlation between SYT11 expression and esophageal carcinoma, and measuring the expression level of SYT11 demonstrated excellent effects in diagnosing esophageal carcinoma.

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2018-0125073, filed on October 19, 2018, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a pharmaceutical composition for preventing or treating gastric cancer comprising an SYT11 expression inhibitor as an active ingredient, a composition for diagnosing gastric cancer comprising a preparation for measuring the expression level of SYT11, a method for providing information for diagnosing gastric cancer comprising a step of measuring the expression level of SYT11, and a method for screening a preparation for treating gastric cancer. [Background technology]

[0003] Cancer has a high mortality rate worldwide, and in Western societies, it is the second most common cause of death after cardiovascular disease. In particular, the incidence of colon cancer, breast cancer, prostate cancer, and the like continues to increase due to the aging of the population, the generalization of high-fat diets due to the Westernization of dietary habits, the rapid increase in environmental pollutants, and the increase in alcohol consumption. In this situation, there is an urgent need to develop anticancer substances that can enable early prevention and treatment of cancer, and contribute to the promotion of human health, the improvement of the quality of healthy life, and the promotion of human health.

[0004] Among these, gastric cancer is particularly prevalent in Asia and is the leading cause of cancer-related deaths. In Korea, it is estimated that 16.2% of cancer patients (20.3% of male cancer patients and 11.2% of female cancer patients) are gastric cancer patients. Symptoms of gastric cancer range from no symptoms at all to severe pain, and do not have specific symptoms of gastric cancer, but rather show general digestive symptoms. In most cases, gastric cancer has no symptoms in the early stages, and even if symptoms are present, they are relatively minor, such as slight indigestion or discomfort in the upper abdomen, which is easily overlooked by most people and can be a cause of increased mortality from gastric cancer.

[0005] There are various standards for classifying gastric cancer tissue. For example, the type of gastric cancer can be classified by the Lauren classification. According to the Lauren classification, adenocarcinoma, which accounts for the majority of gastric cancers, is divided into intestinal type and diffuse type. Intestinal type gastric cancer occurs particularly frequently in cases of atrophic gastritis caused by long-term Helicobacter pylori infection, which often forms ulcers and has a characteristic tubular structure formed by adhesion of tumor cells. On the other hand, diffuse type is a type in which tumor cells have low adhesion and individual cells invade the stomach wall without forming a clear mass, and it occurs frequently in young people and has a poor prognosis. Most patients with diffuse type gastric cancer are those who become infected with Helicobacter for the first time as adults, and the cancer progresses to diffuse type gastric cancer due to a strong rejection reaction by the host. This reaction is more pronounced in young women who have less atrophic gastritis, and as a result, the acute infection cannot be cured, and the gastric mucosa continues to show edematous and nodular changes, progressing to diffuse gastric cancer, which has a very poor prognosis. Therefore, even if the patient is the same gastric cancer patient, the younger the adult who is diagnosed with diffuse gastric cancer, the lower the chance of a complete cure, and the patient will eventually die within a few years. As mentioned above, diffuse gastric cancer is difficult to confirm clinically and quickly, which delays treatment, or even if treatment is started, there is no treatment that shows sufficient therapeutic effects, and the above problem continues.

[0006] Recently, microarray analysis of gastric cancer tissues has confirmed that molecular subtypes can be classified into intestinal, stem-like, mixed stromal, and inflammatory types based on the characteristics of gene expression patterns. As mentioned above, intestinal type is known to be easier to treat than stem-like, mixed stromal, and inflammatory types, while stem-like type has been reported to belong to a group of gastric cancers that are difficult to treat and have a very poor prognosis [Nature Medicine 2015;21:449-456 Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes].

[0007] Under these circumstances, there is an urgent need for research and development into new technologies that can rapidly diagnose and treat specific cancers that are difficult to diagnose as described above. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the inventors made extensive efforts to develop an anticancer preparation that can exhibit excellent effects in treating gastric cancer without causing side effects to the human body, and as a result, they discovered that a SYT11 expression inhibitor exhibits excellent effects in treating gastric cancer within a non-toxic range and can also be useful for diagnostic purposes, thereby completing the present invention.

[0009] An object of the present invention is to provide a pharmaceutical composition for preventing or treating gastric cancer, which comprises an inhibitor of SYT11 (Synaptotagmin 11) expression as an active ingredient.

[0010] Another object of the present invention is to provide a composition for diagnosing truncal gastric cancer, comprising a preparation for measuring the expression level of SYT11 (Synaptotagmin 11).

[0011] Yet another object of the present invention is to provide a method for providing information for diagnosing stem-stage gastric cancer, comprising the steps of: (a) measuring the expression level of SYT11 (Synaptotagmin 11) from an isolated biological gastric tissue sample; (b) comparing the expression level with the expression level of SYT11 (Synaptotagmin 11) in a normal control sample; and (c) determining that the biological gastric tissue sample has stem-stage gastric cancer if the expression level of SYT11 (Synaptotagmin 11) in the biological gastric tissue sample is higher than the expression level of SYT11 (Synaptotagmin 11) in the normal control sample.

[0012] Yet another object of the present invention is to provide a method for screening a pharmaceutical preparation for treating gastric cancer, comprising the steps of: (a) treating isolated gastric cancer cells expressing SYT11 (Synaptotagmin 11) with a candidate substance for treating gastric cancer; (b) measuring the expression level of SYT11 (Synaptotagmin 11) in the isolated gastric cancer cells treated with the candidate substance; and (c) determining that the candidate substance is usable as a pharmaceutical preparation for treating gastric cancer if the expression level of SYT11 (Synaptotagmin 11) measured in step (b) is lower than that in isolated gastric cancer cells not treated with the candidate substance.

[0013] It is still another object of the present invention to provide a method for preventing or treating gastric cancer, which comprises the step of administering to an individual a composition containing an inhibitor of the expression of SYT11 (Synaptotagmin 11).

[0014] It is still another object of the present invention to provide a method for preventing or treating gastric cancer, which comprises the step of administering to an individual a composition containing an inhibitor of the expression of SYT11 (Synaptotagmin 11). [Means for solving the problem]

[0015] To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating gastric cancer, comprising an inhibitor of SYT11 (Synaptotagmin 11) expression as an active ingredient.

[0016] Another aspect of the present invention provides a composition for diagnosing stem cell-type gastric cancer, comprising a preparation for measuring the expression level of SYT11 (Synaptotagmin 11).

[0017] In yet another aspect, the present invention provides a method for providing information for diagnosing stem-stage gastric cancer, comprising the steps of: (a) measuring the expression level of SYT11 (Synaptotagmin 11) from an isolated biological gastric tissue sample; (b) comparing the expression level with the expression level of SYT11 (Synaptotagmin 11) in a normal control sample; and (c) determining that the biological gastric tissue sample has stem-stage gastric cancer if the expression level of SYT11 (Synaptotagmin 11) in the biological gastric tissue sample is higher than the expression level of SYT11 (Synaptotagmin 11) in the normal control sample.

[0018] In yet another aspect, the present invention provides a method for screening a pharmaceutical preparation for treating gastric cancer, the method comprising the steps of: (a) treating isolated gastric cancer cells expressing SYT11 (Synaptotagmin 11) with a candidate substance for treating gastric cancer; (b) measuring the expression level of SYT11 (Synaptotagmin 11) in the isolated gastric cancer cells treated with the candidate substance; and (c) determining that the candidate substance is usable as a pharmaceutical preparation for treating gastric cancer if the expression level of SYT11 (Synaptotagmin 11) measured in step (b) is lower than that in isolated gastric cancer cells not treated with the candidate substance.

[0019] Furthermore, still another aspect of the present invention provides a method for preventing or treating gastric cancer, comprising the step of administering to an individual a composition containing an inhibitor of the expression of SYT11 (Synaptotagmin 11).

[0020] Furthermore, still another aspect of the present invention provides a preventive or therapeutic use of gastric cancer, comprising the step of administering to an individual a composition containing an inhibitor of SYT11 (Synaptotagmin 11) expression. Effect of the Invention

[0021] A composition containing the SYT11 inhibitor of the present invention inhibits the migration and invasion of gastric cancer cells, inhibits their adhesion to extracellular matrix, inhibits the secretion of various cytokines related to cancer metastasis, and inhibits the proliferation of gastric cancer cells, thereby exhibiting excellent effects as a composition for inhibiting cancer metastasis and preventing or treating cancer.

[0022] Furthermore, the present invention has confirmed the correlation between SYT11 expression and stem-type gastric cancer, and has demonstrated that measuring the expression level of SYT11 is highly effective in diagnosing stem-type gastric cancer. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the results of confirming enhanced SYT11 expression in stem-like gastric cancer cell lines among gastric cancer cell lines with intestinal, stem-like, mixed, and inflammatory subtypes. [Diagram 2] FIG. 1 shows the results of confirming inhibition of migration of gastric cancer cell lines by reduction of SYT11 expression in the gastric cancer cell lines. [Diagram 3] FIG. 1 shows the results of confirming that decreased SYT11 expression in gastric cancer cell lines inhibits the invasion of gastric cancer cell lines. [Figure 4] FIG. 1 shows the results of confirming the suppression of adhesion ability to extracellular matrix due to the decrease in SYT11 expression in gastric cancer cell lines. [Diagram 5] FIG. 1 shows the results of confirming the suppression of secretion of cancer metastasis-associated growth factors and cytokines by the reduction of SYT11 expression in gastric cancer cell lines. [Figure 6] FIG. 1 shows the results of confirming the tumor reduction effect of reduced SYT11 expression in a mouse animal model. [Figure 7] This figure shows the results of confirming the inhibition of secretion of cancer metastasis-associated growth factors and cytokines by reduced SYT11 expression in gastric cancer cell lines, and the results of confirming the inhibition of secretion of cancer metastasis-associated growth factors and cytokines by suppression of SYT11 in tumor tissue from a mouse animal model. [Figure 8]FIG. 1 shows the results of confirming the inhibition of cell proliferation by suppressing SYT11 expression using various siRNAs in gastric cancer cell lines. [Figure 9] FIG. 1 shows the results of confirming the inhibition of cancer cell proliferation in gastric cancer cell lines using SYT11 antisense oligonucleotides. [Figure 10] FIG. 1 shows the results of comparing cell growth inhibition by suppressing the expression of the SYT family in gastric cancer cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] To achieve the above object, one embodiment of the present invention is a pharmaceutical composition for preventing or treating gastric cancer, which comprises an inhibitor of SYT11 (Synaptotagmin 11) expression as an active ingredient.

[0025] In the present invention, the term "SYT11 (Synaptotagmin 11, NM_152280.4)" is a member of the Synaptotagmin gene family, which encodes a protein similar to other family members known as calcium sensors, and which mediates calcium-dependent regulation of membrane trafficking in synaptic transmission. The encoded protein is known to be a substrate for the ubiquitin-E3-ligase parkin. Said SYT11 has the amino acid sequence of SEQ ID NO:1.

[0026] In a specific embodiment of the present invention, human gastric cancer cell lines were analyzed to classify the molecular subtypes of cancer cell lines into intestinal, stem-like, mixed, and inflammatory, and it was confirmed that SYT11 expression was increased in stem-like subtypes among the classified human gastric cancer cell lines.

[0027] In another specific embodiment of the present invention, in order to confirm the correlation between the changes in cell metastasis due to inhibition of SYT11 expression, i.e., changes in migration and invasive abilities, it was confirmed that when SYT11 was inhibited in gastric cancer cells, the invasive and migratory abilities were significantly reduced.

[0028] In yet another specific embodiment of the present invention, in order to confirm the correlation between the inhibition of SYT11 expression and changes in the adhesion ability of cancer cells to extracellular matrix, it was confirmed that when SYT11 expression was inhibited in gastric cancer cells, the adhesion ability to extracellular matrix was significantly reduced.

[0029] In yet another specific embodiment of the present invention, in order to confirm the correlation between the inhibition of SYT11 expression and changes in growth factors or cytokines associated with cancer cell metastasis, it was confirmed that when SYT11 expression was inhibited in gastric cancer cells, PDGF-AA, VEGF, HGF, IGFBP-2, IL-17A, IL-8, angiopoietin-1, angiopoietin-2, etc., which are associated with cancer cell metastasis, were reduced.

[0030] In yet another specific embodiment of the present invention, tumor reduction due to inhibition of SYT11 expression was confirmed in a mouse animal model.

[0031] In yet another specific embodiment of the present invention, inhibition of tumor cell proliferation by inhibition of SYT11 was confirmed.

[0032] In the present invention, the term "SYT11 inhibitor" is used as a general term for all preparations that reduce the expression or activity of SYT11, and specifically includes all preparations that reduce the activity of SYT11 by reducing the expression level of SYT11 at the transcription, mRNA, or translation level, or by interfering with SYT11 activity, by affecting the reduction of SYT11 expression, acting directly on SYT11, or indirectly acting on its ligand.

[0033] The SYT11 inhibitor can be used in any form, including compounds, nucleic acids, peptides, viruses, or vectors containing the nucleic acids, that can inhibit the expression or activity of SYT11 by targeting SYT11. The SYT11 inhibitor can be, but is not limited to, siRNA or shRNA that degrades the mRNA of the SYT11 gene, or antisense oligonucleotides that reduce the expression of SYT11 protein. In addition, the SYT11 inhibitor that binds to the SYT11 protein to inhibit its function can be an aptamer or a low molecular weight compound.

[0034] In an exemplary embodiment, the siRNA may have a base sequence selected from SEQ ID NOs: 2, 3, 4 and 5.

[0035] [Table 1]

[0036] In an exemplary embodiment, the shRNA may be a synthetic or modified shRNA having a base sequence selected from SEQ ID NOs: 6, 15, 16 and 17, and its homologues, isoforms, mutants, derivatives, fragments, etc., and may be, for example, a base sequence selected from SEQ ID NOs: 6, 15 to 17 in which the loop sequence (underlined in Table 2 below) located in the center has been modified.

[0037] [Table 2]

[0038] In an exemplary embodiment, the antisense oligonucleotide may be any one or more antisense oligonucleotides selected from SEQ ID NOs: 18 and 19 and derivatives thereof, and the derivatives may have phosphorothioate modification and / or 2'-O-methylation modification in one or more oligonucleotides selected from SEQ ID NOs: 18 and 19.

[0039] [Table 3]

[0040] In the present invention, the term "treatment" refers to a clinical intervention to alter the natural processes of the individual or cells to be treated, which can be performed during the progression of a clinical pathological condition or to prevent it. The intended therapeutic effects include preventing the occurrence or recurrence of the disease, relieving symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily alleviating the disease state, and improving or improving the prognosis. Preferably, in the present invention, it includes all actions that improve the course of gastric cancer by administering a composition containing a substance that inhibits SYT11. Also, "prevention" refers to all actions that inhibit or delay the onset of said gastric cancer by administering a composition containing a substance that inhibits SYT11 according to the present invention.

[0041] In the present invention, the term "antisense oligonucleotide" refers to DNA, RNA or derivatives thereof that contain a nucleic acid sequence complementary to the sequence of a specific mRNA, and bind to the complementary sequence in the mRNA and inhibit the translation of the mRNA into a protein. The antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the SYT11 mRNA and can bind to the mRNA. This can inhibit the essential activity of the SYT11 mRNA for translation, translocation into the cytoplasm, maturation or any other overall biological function. The length of the antisense oligonucleotide can be 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. The antisense oligonucleotide can be synthesized in vitro by a conventional method and administered to a living body, or the antisense oligonucleotide can be synthesized in vivo. One example of synthesizing an antisense oligonucleotide in vitro is to use RNA polymerase I. One example of synthesizing an antisense RNA in vivo is to transcribe the antisense RNA using a vector with the origin of the multiple cloning site (MCS) in the reverse direction. The antisense RNA preferably has a translation termination codon in its sequence so that it is not translated into a peptide sequence. Antisense oligonucleotides that can be used in the present invention can be designed by referring to the nucleotide sequence of SYT11 and prepared according to methods known in the art.

[0042] Specifically, the antisense oligonucleotide of the present invention may be, but is not limited to, the oligonucleotide of SEQ ID NO:18 or SEQ ID NO:19.

[0043] The oligonucleotide of SEQ ID NO: 18 or 19 includes an oligonucleotide containing substantially the same base sequence as the oligonucleotide of SEQ ID NO: 18 or 19, and a derivative thereof. The oligonucleotide containing substantially the same base sequence means an oligonucleotide containing a base sequence having 75% or more, 80% or more, 90% or more, or 95% or more sequence homology with the base sequence of SEQ ID NO: 18 or 19, respectively. The derivative may be, but is not limited to, one or more oligonucleotides selected from SEQ ID NO: 18 and 19 that have phosphorothioate modification and / or 2'-O-methylation modification.

[0044] In the present invention, the term "aptamer" refers to a nucleic acid molecule that is a single-stranded oligonucleotide, has a size of about 20 to 60 nucleotides, and has binding activity to a specific target molecule. It has various three-dimensional structures depending on the sequence, and can have high affinity with a specific substance, such as an antigen-antibody reaction. An aptamer can inhibit the activity of a specific target molecule by binding to the specific target molecule. The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form. Preferably, the aptamer can bind to SYT11 and play a role in inhibiting the activity of SYT11. Such an aptamer can be produced from the sequence of SYT11 by a method known to those skilled in the art.

[0045] In the present invention, the terms "siRNA" and "shRNA" are used as an efficient gene knockdown method or gene therapy method, since they are nucleic acid molecules capable of mediating RNA interference or gene silencing and can suppress the expression of a target gene. shRNA is a single-stranded oligonucleotide that forms a hairpin structure by binding between complementary sequences, and in vivo, the shRNA is cleaved by dicer to become a double-stranded oligonucleotide siRNA, which is a small RNA fragment of 21 to 25 nucleotides in size, and can specifically bind to mRNA having a complementary sequence to suppress expression. Therefore, which of shRNA and siRNA is used can be determined by the selection of a person skilled in the art, and if the mRNA sequence targeted by them is the same, a similar effect of reducing expression can be expected. For the purpose of the present invention, the SYT11 can be suppressed by specifically acting on SYT11 to cleave the SYT11 mRNA molecule and inducing the phenomenon of RNA interference (RNAi). siRNA can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and any method known in the art can be used. For example, there are, but are not limited to, a method for directly chemically synthesizing siRNA, a method for synthesizing siRNA using in vitro transcription, a method for enzymatically cleaving long double-stranded RNA synthesized by in vitro transcription, an expression method by intracellular delivery of shRNA expression plasmid or viral vector, and an expression method by intracellular delivery of PCR (polymerase chain reaction)-induced siRNA expression cassette.

[0046] Specifically, the siRNA against SYT11 of the present invention may consist of a double strand of an siRNA having a base sequence selected from SEQ ID NOs: 2, 3, 4 and 5 and an siRNA having a complementary sequence thereto, but is not limited thereto.

[0047] The shRNA against SYT11 of the present invention may have a base sequence selected from SEQ ID NOs: 6, 15, 16 and 17, but is not limited thereto.

[0048] For the purposes of the present invention, the antibody may be an antibody capable of binding to SYT11 or a ligand protein of SYT11 and inhibiting the activity of SYT11.

[0049] In the present invention, the term "ligand" means a substance that forms a complex with a biomolecule to bring about a biological reaction, and a "ligand protein of SYT11" or a "ligand protein for SYT11" may be a protein that binds to SYT11 and activates or increases the activity of SYT11.

[0050] In the present invention, the term "gastric cancer" refers to a disease caused by the excessive proliferation of cells in the stomach. Such abnormally proliferating cells may invade surrounding tissues and organs to form a mass, destroying or deforming the normal structure of the stomach, and such a condition is called gastric cancer. Generally, a tumor refers to a mass that grows abnormally due to the autonomous excessive proliferation of body tissues, and can be classified into benign tumors and malignant tumors. Malignant tumors grow much faster than benign tumors, and invade surrounding tissues, causing metastasis and threatening life. In the present invention, the gastric cancer is preferably a stem cell subtype and / or a mixed subtype, more preferably a stem cell subtype.

[0051] In the present invention, the term "metastasis" refers to a state in which a malignant tumor spreads to other tissues distant from the organ where the tumor developed. As a malignant tumor that started in one organ progresses, it spreads from the organ where it first developed to other tissues, and this spread from the primary site to other tissues can be called metastasis. Metastasis can be said to be a phenomenon that accompanies the progression of a malignant tumor. As malignant tumor cells proliferate and cancer progresses, metastasis can occur when malignant tumor cells acquire new genetic traits. When tumor cells that have acquired new genetic traits invade blood vessels and lymph nodes, circulate through the blood and lymph, and settle and proliferate in other tissues, metastasis can occur.

[0052] Depending on the tissue in which metastasis occurs, various cancer diseases such as liver cancer, kidney cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, etc. The composition of the present invention can suppress metastasis and prevent and treat the spread of cancer.

[0053] In the present invention, the term "suppression" refers to any action of suppressing cancer metastasis by administering the composition according to the present invention.

[0054] Gastric cancer can be classified according to molecular subtypes. For example, whole genome level mRNA expression in gastric cancer samples can be investigated using microarray techniques, and intrinsic subtypes of gastric cancer can be identified by cluster analysis, after which genes specific to each subtype can be selected through statistical verification. Based on the gene expression levels selected in this way, subtypes with high expression of epithelial cell-specific genes can be classified into intestinal subtypes, subtypes with high expression of developmental signal transduction (EMT) and stroma-derived genes can be classified into stem-like subtypes, mixed stromal subtypes that express all the characteristics of intestinal and stem types, and inflammatory subtypes with high expression of immune regulation-related genes. Each subtype has been confirmed to have discriminatory characteristics associated with previously well-described clinical and histopathological findings.

[0055] Specifically, intestinal type is mainly located in the lower part of the stomach and is characterized by good histological differentiation. In the Lauren classification, intestinal type and indeterminate type are widely distributed.

[0056] On the other hand, the trunk type often occurs in the relatively young age group under 60 years old, is located in the body and upper part of the stomach, and has a poor degree of histological differentiation. In particular, the signet ring cell type accounts for 20% of all histological types, and there is a high distribution of diffuse types in the Lauren classification. The trunk type has clinical characteristics that indicate a very poor prognosis.

[0057] The mixed type shares characteristics of both the intestinal and trunk types.

[0058] Compared to other subtypes, the inflammatory type is characteristically often located in the cardia, including the gastroesophageal junction, and is a type with a poor degree of histological differentiation. However, in the Lauren classification, the intestinal type and indeterminate type are common, and the characteristics are closer to the intestinal type than the truncal type. From a prognostic perspective, the intestinal and mixed types have an intermediate prognosis, while the inflammatory type has the best prognosis.

[0059] The pharmaceutical composition of the present invention may further include a suitable carrier, excipient, or diluent that is commonly used in the preparation of pharmaceutical compositions. The composition containing a pharma- ceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it may be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. that is commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquids, emulsions, syrups, etc., and may include various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried 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. Suppository bases may include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0060] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of, but not limited to, tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.

[0061] Another aspect of the present invention to achieve the above object is to provide a method for treating gastric cancer, comprising administering to an individual a pharmaceutical composition containing a SYT11 (Synaptotagmin 11) inhibitor as an active ingredient.

[0062] In the present invention, the term "individual" refers to any animal, including humans, that has or has developed the gastric cancer disease of the present invention, and may be an individual other than a human. By administering the pharmaceutical composition of the present invention to an individual, it is possible to show excellent effects in treating gastric cancer and inhibit metastasis of gastric cancer.

[0063] The pharmaceutical compositions of the present invention are administered in a pharma- ceutically effective amount.

[0064] In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into a subject in a suitable manner, and the administration route may be via various routes, such as oral or parenteral, as long as it can reach the target tissue.

[0065] The pharmaceutical composition can be appropriately administered to an individual according to the purpose or need according to the usual method, administration route, and dosage used in the art. Examples of administration routes include oral, parenteral, subcutaneous, intraperitoneal, pulmonary, and intranasal administration, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the appropriate dosage and administration frequency can be selected according to a method known in the art, and the amount and administration frequency of the pharmaceutical composition of the present invention actually administered can be appropriately determined according to various factors such as the type of symptoms to be treated, administration route, sex, health condition, diet, age and weight of the individual, and severity of the disease.

[0066] In the present invention, the term "pharmaceutical effective amount" means an amount sufficient to suppress or alleviate a disease at a reasonable rate applicable to medical use, and the effective dose level can be determined by factors including the type and severity of the individual, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, co-used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. For example, the pharmaceutical effective amount is 0.5 to 1000 mg / day / kg of body weight, preferably 0.5 to 500 mg / day / kg of body weight.

[0067] In still another aspect of the present invention to achieve the above object, there is provided a use of a composition comprising a SYT11 (Synaptotagmin 11) inhibitor in the manufacture of a medicament for treating gastric cancer.

[0068] In accordance with yet another aspect of the present invention to achieve the above object, there is provided a composition comprising a SYT11 (Synaptotagmin 11) inhibitor for use in treating gastric cancer.

[0069] Another aspect of the present invention to achieve the above object is to provide a composition for diagnosing stem cell-type gastric cancer, comprising a preparation for measuring the expression level of SYT11 (Synaptotagmin 11). For example, the base sequences of SEQ ID NO: 7 and SEQ ID NO: 8 can be used as preparations for measuring the expression level of SYT11 (Synaptotagmin 11).

[0070] Yet another object of the present invention is to provide a method for providing information for diagnosing stem-stage gastric cancer, comprising the steps of: (a) measuring the expression level of SYT11 (Synaptotagmin 11) from an isolated biological gastric tissue sample; (b) comparing the expression level with the expression level of SYT11 (Synaptotagmin 11) in a normal control sample; and (c) determining that the biological gastric tissue sample has stem-stage gastric cancer if the expression level of SYT11 (Synaptotagmin 11) in the biological gastric tissue sample is higher than the expression level of SYT11 (Synaptotagmin 11) in the normal control sample.

[0071] In the present invention, the term "diagnosis" means to confirm the presence or characteristics of stem-type gastric cancer disease by measuring the presence or absence of SYT11 of the present invention in a biological tissue specimen or tissue sample. In addition, the term "marker or diagnosis marker" refers to a substance that can distinguish and diagnose stem-type gastric cancer cells or individuals with stem-type gastric cancer disease from normal cells or normal individuals, and includes organic biomolecules such as polypeptides, proteins, or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that are increased or decreased in cells or individuals with stem-type gastric cancer compared to normal cells. For the purposes of the present invention, the stem-type gastric cancer diagnostic marker of the present invention is SYT11 that is specifically expressed at a high level in stem-type gastric cancer cells compared to cells of normal cells or tissues.

[0072] In the present invention, the term "isolated biological gastric cancer tissue sample" refers to a sample isolated from the tissue of the individual to be diagnosed, specifically gastric tissue.

[0073] In the present invention, measuring the expression level of SYT11 can be measuring the expression level of SYT11 mRNA or measuring the expression level of SYT11 protein.

[0074] The "measurement of mRNA expression level" refers to a process of confirming the presence and expression level of mRNA of a marker gene for stem cell gastric cancer in a biological tissue sample in order to diagnose stem cell gastric cancer, and can be determined by measuring the amount of mRNA. Analytical methods for this include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, etc.

[0075] The "protein expression level measurement" is a process of determining the presence and expression level of a protein expressed by a stem cell gastric cancer marker gene in a biological gastric tissue sample in order to diagnose stem cell gastric cancer, and the amount of the protein is determined using an antibody that specifically binds to the protein of the gene. Analytical methods for this include, but are not limited to, Western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip.

[0076] For example, the agent for measuring the mRNA level may be a primer pair, a probe, or an antisense nucleotide for the mRNA of the SYT11 of the present invention, and a person skilled in the art can easily design a primer, probe, or antisense nucleotide sequence based on the polynucleotide sequence of the SYT11 of the present invention. For another example, the agent for measuring the protein level may be an antibody.

[0077] In the present invention, the possibility of SYT11 as a diagnostic marker for gastric stem cell cancer was confirmed, and the effect of measuring the level of SYT11 and diagnosing gastric stem cell cancer was confirmed.

[0078] Yet another object of the present invention is to provide a method for screening a pharmaceutical preparation for treating gastric cancer, comprising the steps of: (a) treating isolated gastric cancer cells expressing SYT11 (Synaptotagmin 11) with a candidate substance for treating gastric cancer; (b) measuring the expression level of SYT11 (Synaptotagmin 11) in the isolated gastric cancer cells treated with the candidate substance; and (c) determining that the candidate substance is usable as a pharmaceutical preparation for treating gastric cancer if the expression level of SYT11 (Synaptotagmin 11) measured in step (b) is lower than that in isolated gastric cancer cells not treated with the candidate substance.

[0079] By measuring the expression level of the gene of the present invention or the level of the protein encoded by the gene in a cell in the absence of a candidate substance capable of treating gastric cancer, and also measuring the expression level of the gene of the present invention or the level of the protein encoded by the gene in the presence of the candidate substance, and comparing the two, a substance that reduces the expression level of the gene of the present invention or the level of the protein encoded by the gene in the presence of the candidate substance compared to the level in the absence of the candidate substance can be predicted as a preparation for treating gastric cancer.

[0080] The screening method can be performed in vivo or in vitro, and is not particularly limited. Candidate substances may be known or new substances, and can be screened on a large scale, for example, through plant extracts or chemical libraries. This can find a preparation that can suppress the expression or activity of SYT11 and suppress gastric cancer, particularly gastric cancer of the stem cell subtype.

[0081] In order to facilitate understanding of the present invention, preferred examples and formulation examples are presented below. However, the following examples and formulation examples are provided to facilitate understanding of the present invention, and the contents of the present invention are not limited by the examples or formulation examples.

[0082] <Example 1> Confirmation of changes in SYT11 expression in stem-like subtype gastric cancer cell lines Microarray analysis was used to classify the molecular subtypes of 25 human gastric cancer cell lines into intestinal, stem-like, mixed, and inflammatory. In addition, differences in SYT11 expression were confirmed using Western blot and RT-PCR techniques using 11 representative gastric cancer cell lines.

[0083] For Western blotting experiments, proteins were extracted from cell lines using RIPA buffer (Millipore), separated by electrophoresis on a polyacrylamide gel, and transferred from the gel to a polyvinylidene fluoride membrane. The transferred proteins were confirmed by reacting with the substrate using SYT11 antibody.

[0084] mRNA was isolated from the cell lines using an RNA extraction solution (Trizol, Invitrogen), and RT-PCR and microarray were performed. Complementary DNA was synthesized from the mRNA using an RT transcript (Enzynomics). PCR was performed using the SYT11 primer synthesized from the complementary DNA. In addition, the RPL13A primer was used as a control gene. The primer sequences used in the experiment are shown in Table 2. After PCR was completed, the samples were electrophoresed on an agarose gel containing ethidium bromide, and the bands were confirmed by UV irradiation.

[0085] [Table 4]

[0086] The expression level of each gene was measured using a microarray chip (Illumina, San Diego, CA, USA) containing oligonucleotides capable of complementary binding to the extracted RNA.

[0087] The results are shown in Figure 1A and Figure 1B.

[0088] FIG. 1A shows the results of microarray analysis of 25 human gastric cancer cell lines, confirming that SYT11 mRNA expression is significantly increased in the stem cell type gastric cancer subtype.

[0089] Figure 1B shows the results of confirming the expression of SYT11 by Western blot and RT-PCR in representative cell lines of each molecular subtype. As can be seen from Figure 1B, in all of the MKN1, SK4, SNU484, and SNU638 cell lines confirmed as stem-type gastric cancer subtype cell lines (corresponding to cell lines having characteristics of the stem-type gastric cancer subtype), the expression of SYT11 was confirmed to be significantly increased in all of the Western blot and RT-PCR results.

[0090] <Example 2> Confirmation of inhibition of migration / invasion by SYT11 inhibition in gastric cancer cell lines To confirm the inhibition of gastric cancer cell migration by siSYT11, an experiment was conducted using SNU484 cells, a gastric cell line. Specifically, SNU484 cells transfected with siSYT11 (SEQ ID NO: 2) or siSC (SEQ ID NO: 11) as a control were seeded in a 96 well-Image Loc plate and grown for 24 hours, after which they were scratched with a wound maker and cell migration was confirmed by a wound healing assay at 0, 20, and 40 hours using a real-time cell analysis system (Incucyte).

[0091] The results of the above experiment are shown in FIG.

[0092] FIG. 2A shows the results of confirming the suppression of SYT11 by Western blotting after applying siRNA (SYT11) (SEQ ID NO: 3, 4 or 5) or siSC (SEQ ID NO: 11) to SNU484 cells.

[0093] Figure 2B shows the results of a wound healing assay over time. As can be seen from Figure 2, treatment of gastric cancer cell lines with siSYT11 significantly reduced the migration ability of the gastric cancer cells.

[0094] To confirm the effect of siSYT11 in suppressing gastric cancer cell invasion, an invasion assay was performed. Specifically, an experiment was performed using a 24-well plate cell culture insert. SNU484 cells transfected with siSYT11 or siSC as described above were inoculated into the matrigel-coated insert. After 24 hours, the cells that had migrated under the insert were stained with sulforhodamine B (SRB) solution and the absorbance was measured.

[0095] The results are shown in Figure 3.

[0096] As can be seen in Figure 3, Figure 3A shows the results of cell invasion confirmed by the invasion assay, and Figure 3B shows the diagrammatic results. As can be seen in Figure 3, it was confirmed that SYT11 inhibition reduced cancer cell invasion by about 40% compared to the control group.

[0097] <Example 3> Confirmation of inhibition of adhesion ability to extracellular matrix by SYT11 inhibition in gastric cancer cell lines The extracellular matrix (ECM) is a matrix consisting of proteins and polysaccharides that surrounds the outside of cells, providing an environment in which cells can perform normal functions. Integrins are present in the cell membrane and are involved in signal transduction related to focal adhesion through cell-cell or cell-matrix binding. In the process of cancer cell metastasis, integrins bind to fibronectin, collagen, laminin, etc. in the extracellular matrix, and act to induce cell adhesion during cancer cell migration through cell-matrix binding.

[0098] In order to confirm the effect of SYT11 on the adhesion of cancer cells, SNU484 cells transfected with siSYT11 or siSC were inoculated onto a 96-well plate coated with BSA, collagen, and fibronectin in the same manner as in Example 2. After 1 hour, the cells not attached to the plate were washed with PBS. The cells attached to the plate were stained with SRB solution and the absorbance was measured. In addition, 48 hours after transfection of SNU484 cells with siSYT11 or siSC, proteins were extracted, electrophoresed, and Western blot was performed to confirm the change in integrin protein expression.

[0099] The results are shown in Figure 4.

[0100] Figure 4A shows the results of the adhesion assay. As can be seen from Figure 4A, it was confirmed that the adhesion of cells bound to collagen or fibronectin was reduced by suppressing SYT11.

[0101] Figure 4B shows the results of confirming changes in integrin protein expression. As can be seen from Figure 4B, it was confirmed that the expression of various integrin proteins was inhibited by suppressing SYT11.

[0102] <Example 4> Confirmation of inhibition of secretion of cancer metastasis-related cytokines by SYT11 inhibition in gastric cancer cell lines To confirm whether SYT11 inhibition affects the secretion of growth factors and cytokines related to cancer cell metastasis, a cytokine array (R&D Systems, proteome profiler antibody arrays) was performed. Specifically, SNU484 cells were transfected with siSYT11 or siSC in the same manner as in Example 2, and 24 hours later, the medium was replaced with a serum-free medium. After culturing for 24 hours under hypoxic conditions (2% O2), the cytokines PDGF-AA, VEGF, HGF, IGFBP-2, IL-17A, IL-8, angiopoietin-1, and angiopoietin-2 were confirmed using the cell culture medium.

[0103] The cells were harvested, RNA was extracted, and complementary DNA was synthesized from the mRNA using RT transcriptome (Enzynomics). Next, qPCR was performed using real-time PCR pre mix (Solgent) and primers for VEGFA, HGF, IL-8, and angiopoietin-1 (Bioneer).

[0104] The experimental results are shown in FIG.

[0105] Figure 5A shows the results of a proteome profile-cytokine assay. As can be seen from Figure 5, siSYT11 treatment significantly reduced the secretion of growth factors and cytokines associated with cancer cell metastasis.

[0106] Also, Figure 5B shows the diagrammatic results of the qPCR results. As can be seen from Figure 5, it was confirmed that the expression of mRNA related to growth factors and cytokines associated with cancer cell metastasis was significantly decreased under both normoxia and hypoxia conditions.

[0107] <Example 5> Confirmation of the cancer treatment effect by SYT11 inhibition in animal models The gastric cancer cell line SNU484 was infected with shSYT11-Lentivirus and knocked down cells or shControl-Lentivirus-infected cells were injected into nude mice, and the tumor size was measured at intervals of 2 to 3 days to confirm changes in mass and volume.

[0108] Here, the shRNA indicated as shSTY11 (Sigma) has the base sequence of SEQ ID NO: 6, and the shRNA indicated as shControl (CTRL) (Sigma) has the base sequence of SEQ ID NO: 12.

[0109] The above results are shown in Figure 6. Figure 6A shows the change in tumor size over time, Figure 6B shows the results of measuring the tumor weight after sacrificing the mice on day 16, and Figure 6C shows a photograph of the tumors that were formed. As can be seen from the results in Figure 6, when shSYT11 was inhibited, tumor formation was suppressed compared to shControl.

[0110] In addition, tissues from the above animal models were taken and qPCR was used to confirm changes in expression of growth factors and cytokines associated with cancer cell metastasis, changes in expression of integrins, and ANTXR1, a tumor-specific endothelial marker, in the same manner as in Example 4.

[0111] In addition, SNU484 cells were transfected with siSYT11 or siSC in the same manner as in Example 2, and changes in gene expression were confirmed by RT-PCR.

[0112] The above results are shown in FIG.

[0113] As seen from SNU484 cells in FIG. 7A, it was confirmed that siSYT11 treatment reduced all of angiopoietin-1, angiopoietin-2, Intergrin-β1, and ANTXR1.

[0114] Furthermore, the results confirmed from the in vivo model in FIG. 7B also confirmed that angiopoietin-1, angiopoietin-2, Intergrin-β1, and ANTXR1 were all decreased by SYT11 treatment.

[0115] <Example 6> Confirmation of cancer cell proliferation inhibition by SYT11 inhibition <6-1> Confirmation of cancer cell proliferation inhibition using siRNA sequences SNU484 cells were transfected with siSYT11 or siSC in the same manner as in Example 2, and cell proliferation was monitored for 4 days using a real-time cell analysis system (InCucyte).

[0116] Similarly, siSYT11 or siSC was transfected with siSYT11 having other siRNA sequences (sequence numbers 2, 3, 4, or 5) in the same manner as in Example 2 above, and after 72 hours, the cells were stained with Sulforhodamine B (SRB) solution, the absorbance was measured, and the cell viability was confirmed.

[0117] The results are shown in Figure 8.

[0118] FIG. 8A shows the results of confirming the decrease in SYT11 expression by Western blotting, and FIG. 8B shows the results of confirming the change in cancer cell proliferation over time.

[0119] As can be seen from Figures 8A and 8B, it was confirmed that the proliferation of cancer cells was inhibited with a decrease in SYT11 expression.

[0120] In addition, Figure 8C shows the results of confirming the inhibition of SYT11 by Western blotting after applying siRNA (SYT11) (sequence number 3, 4 or 5) or siSC (sequence number 11) to SNU484 cells, and Figure 8D shows the results of confirming the cell proliferation rate, confirming that cell proliferation was inhibited by all siRNAs having sequence numbers 2, 3, 4 or 5.

[0121] <6-2> Confirmation of inhibition of cancer cell proliferation using antisense oligonucleotides SNU484 cells were transfected with the antisense oligonucleotide AS-SYT11 (sequence number 18 or 19) or the negative control AS-NC (sequence number 20) as in Example 6-1, and cell proliferation was monitored for 3 days using a real-time cell analysis system (InCucyte).

[0122] In addition, similar to Example 6-1 above, antisense oligonucleotide AS-SYT11 (sequence number 18 or 19) or negative control group AS-NC (sequence number 20) was transfected, and after 72 hours, the cells were stained with sulforhodamine B (SRB) solution, the absorbance was measured, and cell viability was confirmed.

[0123] The results are shown in Figure 9.

[0124] As can be seen from Figure 9, when antisense oligonucleotides were used as SYT11 inhibitors, it was confirmed that cancer cell proliferation was inhibited by suppressing SYT11, as in the experiment using siRNA in Example <6-1>. On the other hand, when the negative control group (SEQ ID NO: 20) was treated, there was no effect on cell proliferation.

[0125] From the above results, it was confirmed that SYT11 can be used as a diagnostic factor for stem cell type gastric cancer. In addition, it was confirmed that SYT11 inhibitors suppress the migration and invasion of gastric cancer cells, suppress the adhesion ability to extracellular matrix, suppress the secretion of various cancer metastasis-related cytokines, and suppress the proliferation of gastric cancer cells, and therefore, it was confirmed that they have excellent effects as a composition for treating gastric cancer.

[0126] <Example 7> Confirmation of SYT11-selective inhibition of cancer cell proliferation As in Example 2, SNU484 cells were transfected with siSC, siSYT11, siSYT4 (sequence number 13) which inhibits SYT4 using another gene of the SYT family, or siSYT7 (sequence number 14) which inhibits SYT7. After 72 hours, the cells were stained with sulforhodamine B (SRB) solution and the absorbance was measured to confirm the cell viability.

[0127] The results are shown in FIG.

[0128] As can be seen from FIG. 10, SN U484 cell proliferation was selectively suppressed by SYT11 knockdown, and was not affected by other SYT family members such as SYT4 and SYT7.

[0129] From the above results, it was confirmed that only SYT11 has a specific therapeutic effect on gastric cancer, particularly on truncal subtype gastric cancer.

Claims

1. A pharmaceutical composition for preventing or treating gastric cancer, comprising an SYT11 (Synaptotagmin 11) inhibitor as an active ingredient, The SYT11 inhibitor is selected from the group consisting of siRNA that specifically binds to SYT11 mRNA, shRNA that specifically binds to SYT11 mRNA, and antisense oligonucleotide that specifically binds to SYT11 mRNA; The gastric cancer is a gastric cancer having a stem cell subtype; A pharmaceutical composition for preventing or treating gastric cancer.

2. The pharmaceutical composition for preventing or treating gastric cancer according to claim 1 , wherein the siRNA has a base sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

3. The pharmaceutical composition for preventing or treating gastric cancer according to claim 1 , wherein the shRNA has a base sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

4. The pharmaceutical composition for preventing or treating gastric cancer according to claim 1 , wherein the antisense oligonucleotide has a base sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO:

19.

5. A diagnostic composition for gastric carcinoma, comprising a preparation for measuring the expression level of SYT11 (Synaptotagmin 11), The composition for diagnosing stem-stage gastric cancer, wherein the preparation for measuring the expression level of SYT11 (Synaptotagmin 11) is i) a preparation for measuring the SYT11 mRNA level selected from the group consisting of a primer pair for SYT11 mRNA, a probe for SYT11 mRNA, and an antisense oligonucleotide for SYT11 mRNA, or ii) a preparation for measuring the SYT11 protein level, which is an antibody that binds to SYT11 protein.

6. (a) measuring SYT11 (Synaptotagmin 11) expression levels from an isolated biological gastric tissue sample; (b) comparing said expression level with SYT11 (Synaptotagmin 11) expression levels in normal control samples; (c) determining that the separated biological gastric tissue sample is a gastric stem cell cancer if the expression level of SYT11 (Synaptotagmin 11) in the separated biological gastric tissue sample is higher than the expression level of SYT11 (Synaptotagmin 11) in a normal control sample.

7. (a) treating isolated gastric cancer cells expressing SYT11 (Synaptotagmin 11) with a candidate substance for treating stem-type gastric cancer; (b) measuring the expression level of Synaptotagmin 11 (SYT11) in the isolated gastric cancer cells treated with the candidate substance; (c) determining that the candidate substance can be used as a preparation for treating stem-type gastric cancer when the expression level of SYT11 (Synaptotagmin 11) measured in step (b) is lower than that of isolated gastric cancer cells not treated with the candidate substance.

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