KR101906657B1 - Biomarker composition for predicting prognosis of gastric cancer - Google Patents

Abstract

The present invention confirms the difference in the expression of PARP1 or FOXO3A gene in patients with poor prognosis and gastric cancer with benign prognosis. In patients with high expression of PARP1, the patient has poor prognosis such as tumor, invasion, lymph node metastasis and venous invasion , And patients with high FOXO3A expression had a good prognosis. In particular, patients with PARP + / FOXO3A-expression have poor prognosis, so PPAR1 or FOXO3A can be used independently or as a marker for predicting the prognosis of gastric cancer.

Description

TECHNICAL FIELD The present invention relates to a biomarker composition for predicting prognosis of gastric cancer,

The present invention relates to a biomarker composition for predicting gastric cancer prognosis, a kit, a method for providing information useful for predicting the prognosis of gastric cancer, or a pharmaceutical composition for inhibiting gastric cancer metastasis.

Gastric cancer is the second leading cause of cancer deaths worldwide and is one of the major causes of cancer death in Korea. The primary treatment of gastric cancer is surgical removal, but with the exception of early gastric cancer, recurrence occurs in many patients after surgical resection. The 5-year survival rate for stage 2 gastric cancer is only 30-70%, and for stage 3 gastric cancer only 5-30%. Predicting prognosis after a complete resection of the same stage of the disease is still in progress. Although there has been considerable research to develop a model for predicting the prognosis of gastric cancer using clinical and pathologic classification, there is a high probability of recurrence after surgery, and patients who need adjuvant chemotherapy after surgery are less likely to have recurrence. There is no established method for distinguishing patients with low need for Therefore, all patients with gastric cancer that can be operated at the present time are treated with complete remission, and most of them are treated with additional adjuvant chemotherapy except early gastric cancer.

However, even if the surgical stage is the same, the prognosis of the patient after surgery is variable. Even in the case of advanced gastric cancer, the prognosis is highly variable depending on the patient, so accurate prediction of the prognosis is urgently needed. The clinical diversity of these gastric cancers is attributed to the molecular biology of gastric cancer, which is due to changes in several genes. However, the present study is still limited and there is no approved biomarker that can predict the prognosis of gastric cancer.

1. Korean Patent No. 10-1026676.

Accordingly, it is an object of the present invention to provide a biomarker composition for prediction of stomach cancer prognosis.

The present invention also provides a kit for predicting the prognosis of stomach cancer.

Another object of the present invention is to provide a method for providing information useful for stomach cancer prognosis prediction.

It is another object of the present invention to provide a pharmaceutical composition for preventing or inhibiting gastric cancer metastasis.

In order to achieve the above object, the present invention provides a biomarker composition for predicting the prognosis of gastric cancer, comprising one or more genes selected from the group consisting of PARP1 and FOXO3A or a protein encoded by the gene from a biological sample.

In order to achieve the above another object, the present invention provides a primer or a probe specifically binding to at least one gene selected from the group consisting of PARP1 and FOXO3A, an antibody specifically binding to the protein encoded by the gene, The present invention provides a kit for predicting the prognosis of gastric cancer.

According to another aspect of the present invention, there is provided a method for screening a gastric cancer patient, comprising: measuring mRNA expression levels of at least one gene selected from the group consisting of PARP1 and FOXO3A or the expression level of a protein encoded by the gene; And comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with a control sample.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or inhibiting gastric cancer metastasis, which comprises an inhibitor for inhibiting the mRNA expression of the PARP1 gene or an inhibitor for inhibiting the expression or activity of the PARP1 protein Lt; / RTI >

The present invention also provides a pharmaceutical composition for preventing or inhibiting stomach cancer, which comprises as an active ingredient an agonist for activating mRNA expression of FOXO3A gene or an agonist for promoting expression or activity of FOXO3A protein.

The present invention confirms the difference in expression of PARP1 or FOXO3A gene in patients with poor prognosis and those with advanced prognosis. Patients with high expression of PARP1 have a poor prognosis such as tumor invasion, lymph node metastasis and venous invasion , And patients with high expression of FOXO3A showed good prognosis. In particular, patients with PARP + / FOXO3A-expression have a poor prognosis, so PPAR1 or FOXO3A can be used independently or as a marker for predicting the prognosis of gastric cancer.

FIG. 1 shows the inhibition of growth of gastric cancer cells by PPAR1 inhibition,
FIG. 2 shows the results of induction of FOXO3A expression and G2 / M cell cycle arrest by inhibition of PARP1,
Figure 3 shows the results of determining the cutoffs of positive immunostaining for PARP1 and FOXO3A by performing a receiver operating characteristic curve (ROC) analysis on patient survival,
FIG. 4 is a result of comparing the clinico-pathological features and the expression status of PARP1 or FOXO3A in the cohort of gastric cancer patients,
FIG. 5 is a graph depicting the overall survival (OS) or recurrence-free survival (RFS) prediction of PARP1 and FOXO3A expression (FIG. 5A) and expression of PARP1 and FOXO3A Results,
FIG. 6 shows Kaplan-Meyer plot analysis of subgroups classified on the basis of the expression status of PARP1 or FOXO3A, showing that the expression of PARP1 or FOXO3A can independently predict the prognosis of gastric cancer patients ,
Figure 7 shows Kaplan Meier plot analysis for each subtype according to Lauren classification to compare the expression of PARP1 or FOXO3A with the prognosis of gastric cancer patients,
FIG. 8 shows the results of confirming the inhibitory effect of Olaparib on the growth of gastric cancer cells in subtypes according to Lauren classification,
FIG. 9 shows the results of the classification of patients based on the immunostaining strength of PARP1 or the immunostaining strength of FOXO3A,
FIG. 10 shows the results of confirming the correlation between immunostaining scores of PARP1 and FOXO3A,
Figure 11 shows the results of confirming that the combined expression status of PARP1 and FOXO3A has a higher reliability in predicting the clinical outcome of gastric cancer patients.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have studied the method for predicting the prognosis of gastric cancer, and confirmed the difference in expression of PARP1 or FOXO3A gene in patients with poor prognosis and patients with improved prognosis, thereby completing the present invention.

The PARP1 (poly (ADP-ribose) polymerase 1) refers to a large population of 17 proteins. PARP1 of the present invention is a 113 kDa protein and is located at the nucleus. The PARP1 gene is located on the long arm of chromosome 1 (1q42.12) and has four domains: catalytic, auto-modification, caspase-cleaved, (DNA-binding) domain.

PARP1 plays an important role in the base excision repair pathway by trapping DNA repair proteins at DNA break binding and damage sites. PARP1 is a polymerase that is a protein that activates ADP (adenosine diphosphate) in NAD ⁺ . One of its main functions is to repair single strand DNA breaks (SSB) and shows high expression in cancer.

The FOXO3A (Forkhead box O3A) is a member of the fork head class 'O' (FOXO) and is a transcription factor family. The FOXO3A gene is located in the long arm of chromosome 6 (6q21) and controls a broad spectrum of biological processes such as DNA repair, apoptosis, and cell cycle regulation.

FOXO3A is a tumor suppressor transcription factor gene that regulates expression of key genes such as p27, Bim, Fas ligands, and GADD45 alpha (GADD45 alpha), which are primarily responsible for cell cycle arrest and / or apoptosis .

Accordingly, the present invention provides a biomarker composition for predicting the prognosis of gastric cancer, comprising at least one gene selected from the group consisting of PARP1 and FOXO3A or a protein encoded by the gene from a biological sample.

In addition, the present invention relates to a primer or a probe specifically binding to at least one gene selected from the group consisting of PARP1 and FOXO3A, an antibody specifically binding to the protein encoded by the gene, or a peptide having a binding domain specific to the protein And a prognostic value of the prognosis of the gastric cancer.

The antibody is a polyclonal antibody or a monoclonal antibody.

The polyclonal antibody can be prepared by injecting an external host with a protein or fragment thereof expressed by the gene 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 intraperitoneally, intraperitoneally or subcutaneously, and is generally administered with an adjuvant to increase the antigenicity. Serum is collected periodically from an external host to collect the sera showing improved titer and specificity for the antigen or isolate and purify the antibody therefrom.

Such monoclonal antibodies can be produced by immortalized cell line generation techniques by fusion as known to those of skill 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 and immunized with a mouse. An antigen-producing B lymphocyte isolated from a mouse is fused with a myeloma of a human or a mouse to generate an immortalized hybridoma, and an enzyme-linked immunosorbent assay (ELISA) linked immunoabsorbent assays (ELISA) to identify monoclonal antibodies. After positive clones were selected, the antibodies were isolated and purified, or injected into the abdominal cavity of rats, and the ascites was collected to obtain monoclonal antibodies have.

The monoclonal antibody can be quantitatively assayed by using a secondary antibody to which an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) is conjugated, or a substrate thereof, Or may be quantitatively analyzed using a monoclonal antibody against the protein directly coupled with an AP or HRP enzyme or the like.

The reaction of the protein with the antibody can be carried out using a protein identification test such as western blot, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA) and immunohistochemistry (IHC) However, the present invention is not limited thereto.

The kit comprising the antibody may typically comprise a lyophilized form of the antibody and a buffer, a stabilizer, an inert protein, and the like, wherein the antibody is radionuclides, fluorescors, enzymes, etc. Lt; / RTI >

Also, the present invention provides a method for diagnosing cancer, comprising the steps of: measuring mRNA expression levels of at least one gene selected from the group consisting of PARP1 and FOXO3A from the gastric cancer patient sample or the expression level of the protein encoded by the gene; And comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with a control sample.

The biological sample may be any one or more selected from the group consisting of tissue, cells, whole blood, serum, and plasma.

The mRNA expression level of the PARP1 gene or the expression level of the protein encoded by the gene is high as compared with the control sample, or the mRNA expression level of the FOXO3A gene or the expression level of the protein encoded by the gene is higher than that of the control sample The risk of metastasis is high.

Methods for measuring the level of mRNA expression include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) Northern blotting, and DNA chips. However, the present invention is not limited thereto.

Methods for measuring the protein expression level include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket ) Can be measured using any one selected from the group consisting of immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, FACS and protein chip, but not limited thereto .

In addition, the present invention provides a pharmaceutical composition for preventing or inhibiting gastric cancer metastasis, which comprises an inhibitor that inhibits mRNA expression of the PARP1 gene or an inhibitor that inhibits the expression or activity of the PARP1 protein as an active ingredient.

The inhibitor may be any one or more selected from the group consisting of Olaparib, veliparib, Niraparib, talazoparib and Rucaparib, But is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or inhibiting stomach cancer, which comprises as an active ingredient an agonist for activating mRNA expression of FOXO3A gene or an agonist for promoting expression or activity of FOXO3A protein.

The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

Further, the dosage of the pharmaceutical composition according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Reference example  1> Reagent

Mouse anti-beta-actin antibody and dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, thiazolyl blue tetrazolium Thiazolyl Blue Tetrazolium Bromide, Trizma base and Tween 20 were purchased from Sigma (St. Louis, Mo.). Mouse anti-PARP1 and Rabbit anti-FOXO3A antibodies were purchased from Santa Cruz, CA, and Rabbit anti-caspase 3 and rabbit anti- The rabbit anti-Bax antibody was purchased from Cell Signaling (Danvers, MA). Goat anti-mouse and Goat anti-rabbit horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch, West Grove, Pa. . ECL (electrochemiluminescence) Western Blotting Detection Reagents were purchased from Genedepot (Barker, TX) and the PARP1 inhibitor Olaparib was purchased from Selleckchem.

< Example  1>

Ⅰ. Experimental Method

1. Cell culture

Human gastric cancer cell lines MKN28, MKN74, NCIN87, MKN45 and KATOIII were purchased from ATCC (American Type Culture Collection, Manassas, Va.) And cultured in RPMI 1640 supplemented with 10% FBS (Fetal Bovine Serum, Gibco, NY, US) and Penicillin / Streptomycin And cultured in a 5% CO ₂ incubator at 37 ° C using a PMI 1640 (Roswell Park Memorial Institute 1640, GlutaMAX ™, Gibco, NY, USA) medium containing P / S, Gibco,

2. siRNA Intermediate  Knock-down siRNA  mediated knock-down)

SiRNAs against non-targeting control siRNA, human PARP1 and FOXO3A were purchased from Santa Cruz and followed by guidelines using Lipofectamine 2000 (Invitrogen, CA) Cells were transfected.

3. Colony  Colony formation assay

0.5 × 10 ³ cells were placed in a 60Ø dish and cultured in a 5% CO ₂ incubator at 37 ° C. for 18 hours. After incubation, DMSO was treated as a control and Olaflex (2.5 μM) or PARP1 siRNA (100 nM) was treated for 14 h at the indicated concentration. The colonies were then washed twice with phosphate buffered saline (PBS), fixed with 3.7% paraformaldehyde, and washed with distilled water containing 1% crystal violet (Aldrich, USA) for 10 minutes Lt; / RTI &gt; After 10 minutes, the cells were washed with PBS until the color of the crystal violet was gone, and then dried and compared with the untreated group using the scanner.

4. Cell counting assay

Cells (1 x 10 ⁴ ) were dispensed into culture dishes and cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours. After incubation, the cells were plated on a membrane (2.5 μM) for 0, 24, 72 and 90 hours. At this time, DMSO was treated as a control. The number of cells was determined using a hemocytometer per hour.

5. Real time qPCR (Real-time qPCR )

Total RNA was prepared from a MI alba or ^TM miRNA extraction kit (mirVanaTM miRNA Isolation kit, Ambion) cells using FNF.

2 μg of the total RNA prepared was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystem).

Levels of gene expression were determined using the CF96 ^TM Optics Module (BIO-RAD) and the primers listed in Table 1 below. The expression of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was measured as a control.

gene order direction SEQ ID NO: PARP1
5'-CTA CTC GGT CCA AGA TCG-3 ' Forward One 5'-TTG AAA AAG CCC TAA AGG CTCA-3 ' Reverse 2 FOXO3A
5'-TCT ACG AGT GGA TGG TGC GTT-3 ' Forward 3 5'-CGA CTA TGC AGT GAC AGG TTG TG-3 ' Reverse 4 GAPDH
5'-ACC CAG AAG ACT GTG GAT GG-3 ' Forward 5 5'-TTC TAG ACG GCA GGT CAG GT-3 ' Reverse 6

All reactions were repeated twice and 2 - ^ΔΔCt was used for quantification.

6. Western Blotting (Western blotting)

Each well of 1 x 10 ⁴ cells / 6 well plate was washed once with PBS and then dissolved in a dissolution buffer containing protease and phosphorylation inhibitor for 30 minutes at 4 ° C . The lysates were centrifuged at 10,000 rpm for 10 minutes and then classified by protein size by 6%, 10% or 12% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis). It was then transferred electrically to nitrocellulose membranes (Bio-Rad).

Membranes were blocked with TBST (0.1% Tween 20 in Tris buffered saline) containing 3% BSA (bovine serum albumin, GenDEPOT, USA) for 1 hour and reacted with each primary antibody at 4 ° C for 24 hours. After the reaction, the cells were washed three times with TBST and reacted with each secondary antibody (1: 3000 or 1: 5000 dilution) for 1 hour. Protein expression was then visualized using ECL solution.

7. MTT (3- (4,5- dimethylthiazole -2- yl ) -2,5- 두henyltetrazolium  bromide) analysis

Cells (2 × 10 ³ ) were plated in 96-well plates and cultured overnight at 37 ° C in a 5% CO ₂ incubator. Olafibri and DMSO were treated for a fixed period of time and then 20 μL MTT solution (5 mg / mL in phosphate buffer) was added to each well and reacted for 2 hours. The resulting blue crystals were dissolved in DMSO (200 μL), and the absorbance was measured at 595 nm using a microplate reader.

Flow Cell Analysis cytometry  analysis)

Cells treated with Oraflavin or FOXO3A siRNA were harvested, washed twice with PBS and fixed overnight at 4 ° C with 70% cold ethanol. Before analysis, cells were washed twice with PBS, resuspended in 400 μL PBS, and treated with 100 μg / mL RNase A (Sigma-Aldrich) and 50 μg / mL PI (propidium iodide, Sigma-Aldrich).

After incubation at 37 ° C for 30 minutes, the DNA content of the cells was analyzed. PI fluorescence was analyzed using FACS Canto II (Becton Dickinson) and data were analyzed from at least 10,000 cells using BD FACSDIVA 7.0 (Becton Dickinson). The cell cycle distribution was calculated using ModiFit LT software.

9. Patients and specimens

Tissue microarray analysis was performed using a sample of 166 cases of gastric adenocarcinoma.

From January 1997 to December 2005, patients were treated with radical gastrectomy at Chonbuk National University Hospital.

The set of stomach cancer was classified according to sex, age (± 2 years), and staging system of the American Joint Committee on Cancer (AJCC), which were 50 cases originally for each stage I, II, III and IV It is a unified set for calendar year of surgery - (± 2 years).

After that, we re-staged 166 cases according to the guidelines of the AJCC Stage Classification System 7th edition and reviewed them according to the criteria of the World Health Organization (WHO).

This study was approved by the Institutional Review Board of Chonbuk National University Hospital and the agreement was provided in accordance with the Declaration of Helsinki.

Patients were divided into three groups according to their age, sex, preoperative serum carcinoembryonic (CEA) and tumor markers CA 19-9 (carbohydrate antigen 19-9), tumor stage (I and II vs III and IV) The presence of lymph node metastasis, presence of distant metastasis, presence of venous invasion, presence of histologic type, classification of WHO, histological grade tubular carcinoma and nipple papillary carcinoma, Lauren classifications, and tumor invasion [early gastric carcinoma (EGC) vs. early gastric carcinoma]. Advanced gastric carcinoma (AGC)]. A follow-up end point for the patient's overall survival (OS) and recurrence-free survival (RFS) was calculated using the last contact or date of death until December 2011 of death.

10. Organization - Microarray (tissue- microarray )

Immunohistochemistry (IHC) was performed using surgically removed paraffin-embedded tissue microarray blocks of cancer tissues from patients.

Tissue microarray analysis was performed with a size of about 3.0 mm core per case. Tissue sections were treated with a microwave antigen retrieval procedure of pH 6.0 sodium citrate buffer for 20 minutes and the markers were incubated with PARP1 (H-300) (1: 100, Santa Cruz Biotechnology, Santa Cruz, CA) and FOXO3A (D19A7) (1: 100, Cell Signaling Technology, Beverly, Mass.) Were used. Assessment of immunohistochemical staining for PARP1 and FOXO3 in human stomach samples was performed by agreement between two pathologists (Jang KY and Kim KM) without knowledge of clinical information.

Scoring for PARP1 and FOXO3A was performed following the Allred scoring system, which is generally used for the evaluation of nuclear expression.

The staining intensity of the nuclei was scored as 0 (no staining), 1 (weak staining), 2 (intermediate staining) or 3 (strong staining).

The staining area was evaluated according to the score: 0 (no stained cells), 1 (1% positive staining of cells), 2 (2-10% positive staining of cells), 3 (11-33% , 4 (34-66% positive staining of cells) and 5 (66-100% positive staining of cells).

The sum of strength scores and the proportion score were used for further analysis. The maximum total score was 8 and the minimum total score was 0.

Ⅱ. Experiment result

One. PPAR1  Inhibition of growth of gastric cancer cells by inhibition

In order to confirm the tumor suppressive activity of PARP1, Olafapil, a drug that inhibits PARP1, was treated with various concentrations of human gastric cancer cell lines NCIN87, MKN28 and MKN74 except for IC50 value (10 uM) to examine cell proliferation In MTT assay, cells Counting, and colony formation analysis.

The treatment of clumps showed inhibition of cancer cell growth in a dose-dependent manner (Fig. 1A, top). After 72 hours, the IC50 of Oraflavip was about 10 [mu] M in the three cell lines.

Time-dependent inhibition of growth was confirmed by up-fold processing in cell counting assays. When the number of cells was increased for 5 days, the collision ability (2.5 μM) was significantly reduced (FIG. 1A, middle) and the colony forming ability was also increased by the folding treatment (FIG. 1A, bottom).

In addition, an siRNA-mediated knock-down experiment was conducted to confirm the effect of Orafapl by PARP1 inhibition.

Similar to uptake treatment, knockdown of PARP1 by siRNA significantly inhibited colonization and proliferation of gastric cancer cells (Fig. 1B and Fig. 1C).

 However, since the treatment of clumps in the PARP1 siRNA-transfected cells had no effect on cell proliferation, it was found that the tumor suppression effect of clumps was due to inactivation of PARP1 (Fig. 1D).

2. PARP1  By inhibition FOXO3A  Expression induction and G2 / M cell cycle arrest

FOXO3A is considered to be one of the putative effector downstream targets of PARP1.

To confirm this theory, we confirmed the effect of Olafibril on the expression of FOXO3A.

As a result, Western blotting analysis up-regulated the expression of FOXO3A in a concentration-dependent manner in MKN28 and MKN74 cells (Fig. 2A).

In addition, when the clone was treated with FOXO3A knockdown cells, the knockdown of FOXO3A expression partially reduced the growth inhibitory effect of the clone (Fig. 2B).

 In contrast, knockdown of FOXO3A did not affect the expression levels of PARP1 mRNAs and protein (Fig. 2C and Fig. 2D).

These results indicate that FOXO3A is one of the downstream targets for the tumor suppression effect of PARP1 inhibitors.

In addition, the tumor suppressor effect of PARP1 inhibition was estimated to be mediated at least in part by FOXO3A activity, thus confirming the effect of PARP1 inhibition on tumor growth undergoing apoptotic procedures.

 As a result, the expression of pro-apoptotic proteins such as caspase 3 or box (Bax) by Olafrid treatment was not observed. Thus, it was confirmed that the effect of climbing did not go through the cell death process (Fig. 2E).

Because FOXO3A is known to be a DNA repair trigger through the activation of cell cycle arrest during DNA damage, the effect of clone on cell cycle system was confirmed by flow cytometry.

As a result, the ratio of G2 / M-phase cells (1 day, 2 days, 1 day, 2 days and 3 days, respectively) And 21%, 23%, and 28%, respectively, at 3 days) (Fig. 2F). In addition, when knockdown of FOXO3A expression using siRNAs, the ratio of arrested cells to G2 / M group due to Olafrid was significantly reduced (FIG. 2G).

Thus, PARP1 inhibition in gastric cancer cells induces G2 / M cell cycle arrest through FOXO3ADML activation.

3. PARP1  And FOXO3A  The relationship between expression and clinical outcome of gastric cancer patients

Receiver operating characteristic curves (ROC) analysis of patient survival were performed to determine the cutoff values of positive immunostaining for PARP1 and FOXO3A at the highest and lowest area under the curve (AUC) And determined to have a likelihood ratio.

Based on this analysis, the PARP1-positive group was determined with a cutoff score of 7 and the FOXO3A positive group with a cutoff score of 6 (FIG. 3).

Clinico-pathological features and expression status of PARP1 and FOXO3A in the cohort of gastric cancer patients are shown in FIG.

Overall, positive staining for the PARP1 and FOXO3A proteins was 54% (89 of 166) and 19% (32 of 166), respectively.

PARP1 expression was associated with high stage ( P <0.001), tumor invasion ( P <0.001), lymph node metastasis ( P <0.001) and venous invasion ( P = 0.017).

On the other hand, the group with characteristic expression of FOXO3A was significantly lower in the low stage ( P = 0.019) and EGC ( P = 0.042).

Thus, PARP1 expression is associated with aggressive phenotypes of gastric cancer and FOXO3A expression is associated with a less aggressive phenotype of gastric cancer.

To confirm the biological relevance of the expression of PARP1 and FOXO3A in gastric cancer cells, tissue microarrays were performed in 166 cases of gastric cancer patients.

Immunohistochemical staining revealed expression of PARP1 and FOXO3A, located in the nucleus of tumor cells, and weak expression in cytoplasm. Therefore, we only considered the expression of PARP1 and FOXO3A in the nucleus (Fig. 5A).

Univariate Cox regression analysis results showed several clinical characteristics associated with shorter overall survival (OS) and / or no recurrence survival (RFS) (Table 2). This includes preoperative serum levels of CEA and CA 19-9, tumor stage, lymph node metastasis, tumor invasion classification and venous invasion.

Characteristic n OS RFS The hazard ratio (HR) (95% CI) P -value HR (95% CI) P -value Diurnal  analysis( Univariate  analysis) CEA, Upgraded (vs. Normal) 30/136 2.024 (1.230-3.330) 0.006 1.917 (1.170-3.144) 0.01 CA19-9, Upgraded (vs. Normal) 16/136 2.510 (1.376-4.580) 0.003 2.275 (1.251-4.138) 0.007 TNM steps, III and IV (vs. I and II) 93/166 4.660 (2.865-7.581) <0.001 4.488 (2.802-7.186) <0.001 Tumor metastasis, AGC
(vs. EGC) 135/166 3.539 (1.711-7.320) <0.001 3.767 (1.823-7.785) <0.001 LN transfer, presence
(none) 110/166 3.696 (2.178-6.271) <0.001 3.737 (2.230-6.261) <0.001 Vein invasion, presence
(none) 30/166 2.724 (1.705 - 4.352) <0.001 2.654 (1.666-4.229) <0.001 PARP1, positive
(vs. voice) 89/166 2.190 (1.424-3.370) <0.001 2.143 (1.406-3.265) <0.001 FOXO3A, voice
(vs. positive) 134/166 3.893 (1.800-8.416) <0.001 3.453 (1.673-7.127) <0.001 Multivariate analysis * TNM steps, III and IV (vs. I and II) 3.444 (1.922-6.171) <0.001 3.345
(1.918-5.836) <0.001 PARP1, positive
(vs. voice) 1.783 (1.090-2.915) 0.021 1.756 (1.089-2.830) 0.021 FOXO3A, voice
(vs. positive) 6.958 (2.163-22.383) 0.001 5.351 (1.929-14.845) 0.001

In addition, the PARP1-positive patient group showed significantly shorter OS (HR: 2.19, 95% CI: 1.424-3.370, P <0.001) and RFS (HR: 2.143, 95% CI: 1.406-3.265, P <0.001).

Conversely, the patient group of FOXO3A-negative showed short OS (HR: 3.893, 95% CI: 1.800-8.416, P <0.001) and RFS (HR: 3.453, 95% CI: 1.673-7.127, P <0.001).

Kaplan-Meier analyzes also showed prognostic predictions of OS and RES, respectively, through three features (tumor stage, PARP1 and FOXO3A) (log-rank test, P <0.001, Figure 5B).

Additionally, Kaplan-Meyer plot analysis was performed on subgroups classified on the basis of the expression status of PARP1 or FOXO3A to determine whether the prognostic significance of PARP1 and FOXO3A expression were independent of each other.

As a result, the expression level of PARP1 was able to predict the prognosis regardless of the expression level of FOXO3A in the subgroup (FIGS. 6A and 6B).

Also, the expression status of FOXO3A could predict the prognosis regardless of the PARP1 expression status of the subgroup (FIGS. 6C and 6D).

In addition, in Table 2, multivariate analysis showed that OS and RFS can be independently predicted through tumor stage and expression of PARP1 and FOXO3A. Thus, expression of PARP1 and FOXO3A can be usefully used for prediction of clinical prognosis in gastric cancer patients Could know.

In addition, the effect of clumping and / or the association of PARP1 and FOXO3A with the expression of PARP1 and FOXO3A proteins identified in Fig. 4 and the significant association of Lorene classification (P = 0.035 and P = 0.007, respectively) subtype).

Kaplan Meyer plot analysis was therefore performed for each subtype of Lauren class.

As a result, regardless of subtype, PARP1-positive tumors showed poor OS and RFS, while FOXO3A-positive tumors showed improved OS and RFS (Fig. 7).

We also confirmed that PARP1 inhibitors have different effects on tumor types. In addition to the diffuse type-derived gastric cancer cells (MKN45 and KATOIII) as well as the intestinal type-derived gastric cancer cells (MKN28 and MKN74) Of the growth rate.

Therefore, Orafapi inhibited the growth of gastric cancer cells regardless of the subtypes of Lauren classifications (Fig. 8).

Next, the relationship between the intensity score of PARP1 or FOXO3A expression and clinical outcome was confirmed.

For this, the intensity of immunochemical staining was measured by Mean Allred Score ± standard error (4.8 ± 0.2) for PARP1 or the Allred Score ± standard error (3.8 ± 0.2) for FOXO3A.

Patients were subdivided based on immunostaining intensity (score 0-3, 4-5, 6-8) or immunostaining intensity (score 0-2, 3-6, 7-8) of FOXO3A on PARP1.

As a result, a group of patients with high scores on PARP1 showed poor prognosis of OS and RFS (P <0.001, Fig. 9A) and a group of patients with high score of FOXO3A showed improved prognosis of OS and RFS (P < 0.001, Fig. 9B).

Also in Fig. 4, the allele scores of PARP1 and FOXO3A were significantly positive, similar to that of PARP1-positive tumors (23 of 30, P = 0.021, Chi-square test) that were expressed as FOXO3A protein Spearman's rho 0.382, P &lt; 0.001, Fig. 10). Therefore, it could be predicted that there is a functional relation between PARP1 and FOXO3A.

However, in Fig. 4, PARP1-positive tumors showed aggressive phenotypes while FOXO3A-positive tumors showed less aggressive phenotypes.

These contradictory results suggest that oncogenic expression of PARP1 may be accompanied by expression of FOXO3A as a compensatory mechanism in suppressing tumor progression and may be thought to be counter-balanced by removing the expression of FOXO3A from PARP1 in tumors, Expression (FOXO3A- / PARP1 +) could not regulate tumor growth and was expected to stimulate an aggressive pattern.

Considering the functional linkage of PARP1 and FOXO3A, we confirmed that the combined expression status of PARP1 and FOXO3A is more helpful in predicting the clinical outcome of gastric cancer patients.

PARP1 / FOXO3A + patient group (n = 66) showed poor prognosis, whereas PARP- / FOXO3A + patient group (n = 9) showed poor prognosis when patients were classified based on the combined expression status of PARP1 and FOXO3A ( P = 6.0 × 10 ^-9 ) and FRS ( P = 2.2 × 10 ^-8 ) (FIG. 11).

The group of PARP + / FOXO3A + (n = 23) or PARP1- / FOXO3A- (n = 23) showed intermediate status prognosis.

Therefore, we concluded that the combined expression of PARP1 and FOXO3A is more reliable than the expression of each in predicting the clinical outcome of patients with gastric cancer.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

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Claims (8)

Wherein the expression of the PARP1 gene or the protein encoded by the gene is increased and the expression of the FOXO3A gene or the protein encoded by the gene is decreased, the expression of PARP1 and FOXO3A genes, Wherein the biomarker composition comprises a biomarker composition for predicting the risk of gastric cancer metastasis. A primer or a probe specifically binding to the PARP1 and FOXO3A genes, an antibody that specifically binds to the protein encoded by the gene, or a peptide having a binding domain specific to the protein. Measuring the mRNA expression level of the PARP1 and FOXO3A genes from the gastric cancer patient sample or the expression level of the protein encoded by the gene; And
And comparing the mRNA expression level of the gene or the expression level of the protein encoded by the gene with a control sample. The method of claim 3,
The mRNA expression level of the PARP1 gene or the expression level of the protein encoded by the gene is measured to be higher than that of the control sample and the mRNA expression level of the FOXO3A gene or the expression level of the protein encoded by the gene is compared with the control sample The risk of metastasis is high. &Lt; Desc / Clms Page number 15 &gt; The method according to claim 3 or 4,
Methods for measuring the level of mRNA expression include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) Wherein the measurement is performed using any one selected from the group consisting of Northern blotting and DNA chip. The method according to claim 3 or 4,
Methods for measuring the protein expression level include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket ) Is assessed using either immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, Complement Fixation Assay, FACS, and protein chips. To provide useful information. delete delete

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