Exo70 Expression In Stage Ⅲ Gastric Cancer And Its Clinical Significance

Backgroud and ObjectionGastric cancer is one of the most common malignant tumors of the digestive tract.Gastric adenocarcinoma(GAC)is the main pathologic type.The characteristics of gastric cancer are easy invasion and metastasis,and a very low early diagnosis rate.Despite the gradual decline in incidence and mortality of gastric cancer around the world in recent years,the incidence of gastric cancer in East Asia,particularly in China and Japan,remains high.In China,gastric cancer is the second most common cancer,with approximately 300,000 deaths and 400,000 new cases each year.Despite significant progress in the diagnosis and treatment of gastric cancer in recent years,surgical operations are still the main method for eradicating tumor,and a large proportion of patients undergo recurrence and metastasis after surgery.In fact,the prognosis of patients with gastric cancer has not been significantly improved.Thus,to explore the molecular mechanisms related to tumor invasion and metastasis,and explore biomarkers that are helpful for the diagnosis and prognosis of gastric cancer would not only help clinicians predict prognosis,formulate individualized treatment plans,but also help clarify the mechanism of gastric carcinogenesis and development to seek potential target for treatment.The exocyst is a hetero octamer composed of Sec3,Sec5,Sec6,Sec8,Sec10,Sec15,Exo70 and Exo84.It mediates the tethering and fusion of secretory vesicles to the plasma membrane with its different subunits.The transport substances in vesicle such as phospholipids and proteins would fuse to the cell membrane or be secreted extracellularly,then implicating a variety of cellular processes,including establishment of epithelial polarity,cell growth,cell adhesion,cell migration and tumor invasion,etc.Exo70 is the critical component mediating the anchorage of the vesicle and the plasma membrane.Recent studies suggested that Exo70 is implicated in several key stages of tumorigenesis and cancer dissemination.Isoform switching of Exo70 plays an important role in the epithelial-mesenchymal transition(EMT)and promotes Invadopodia formation and cell invasion.Exo70 interacts with the actin-related Protein 2/3(Arp2/3)complex and stimulates actin dynamics,promotes invadopodia formation and secretion of matrix metalloproteinase(MMP),which in turn mediates in degradation of extracellular matrix(ECM)and tumor invasion.Exo70 acted downstream of hepatic nuclear factor 4a(HNF4a)to promote G2/M transition via increasing cell division cycle 2 protein(Cdc2)expression in HCC cells.In addition,It has been demonstrated that increased Exo70 expression is associated with poor survival in colon cancer patients.These studies suggest that Exo70 is implicated in tumor proliferation and migration.However,we know little about the role of Exo70 in tumorigenesis and development so far,and the expression and prognostic signifcance of Exo70 have not yet been reported in gastric cancer.The aim of this study was to investigate the expression level of Exo70 in patients with gastric cancer and to investigate the prognostic signifcance of Exo70.Oncomine and Kaplan-Meier Plotter database were used to predict the expression and prognostic value of Exo70 mRNA in gastric cancer patients.A total of ninety specimens of paraffin-embedded gastric cancer tissue and paired adjacent normal tissue were collected retrospectively to examine the Exo70 protein expression level by immunohistochemical(IHC)staining.Then,the correlation between Exo70 expression and clinicopathological parameters of gastric cancer patients and the prognostic significance of Exo70 were verified.Materials and Methods1.Materials1.1 Tissue specimens and PatientsA total of ninety specimens of paraffin-embedded gastric cancer tissue and paired adjacent normal tissue were collected from stage III gastric cancer patients between 2003 and 2006 at the Sun Yat-sen University Cancer Center.All the gastric cancer patients had a histologically confirmed diagnosis which was pathologically confirmed after surgery.The clinical data of gastric cancer patients contains gender,age,preoperative serum CEA level,tumor site,tumor size,Boarrmann classification,tumor differentiation,lymph node metastasis,recurrence,disease-free survival(DFS)and overall survival(OS).All of the patients underwent radical resection,received 5-Fluorouracil-based adjuvant chemotherapy post-operatively for 6 months,and none of all the patients had chemotherapy or radiotherapy before sample collection.1.2 ReagentsThe rabbit anti-Exo70 polyclonal antibody(12014-1-AP)was purchased from PROTEINTECH Corporation.Immunohistochemistry kit(KIT-9730),DAB solution(DAB0031)was purchased from Fujian Maixin Biotechnology Campany.1.3 Cancer related public databasesOncomine database were used to predict the expression levels of Exo70 mRNA in gastric cancer patients.We entered the gene name "ExoC7" and choose the "Differential Analysis" module(Gastric Cancer vs.Normal Analysis).The Kaplan-Meier Plotter database was used to analyze relationship between Exo70 mRNA expression level and OS of gastric cancer patients.2.Experimental method2.1 ImmunohistochemistryImmunohistochemistry was performed using a standard streptavidin-perosidase(SP)method with operation according to the kit instructions.The paraffin sections were heated for 2h,deparafnized in xylene,hydrated in gradient concentrations of ethanol,then subjected to high-fire antigen retrieval in a microwave for 15 min in 0.01 mol/L sodium citrate buffer(pH 6.0).Endogenous peroxidase activity was blocked by incubation in 3%hydrogen peroxide for 30 min,and non-specifc staining was eliminated by incubating the sections with normal goat serum for 2h at room temperature.The sections were incubated with the primary antibody(1:100)at 4℃ overnight.After rewarming,sections were incubated with biotinylated goat anti-rat/rabbit secondary antibody(1:500)for 1h and then incubated with streptomycin antibiotic protein-peroxidase for 30 min.Staining was visualized using 3,3’-diaminobenzidine(DAB)solution.Then sections were counterstained with hematoxylin and dehydrated with xylene.2.2 Evaluation of immunohistochemical stainingThe results were blindly judged and evaluated by two experienced pathologists using an optical microscope.Ten visual fields from different areas were randomly selected at high magnification(400×).Based on a previous semi-quantitative method,an immunoreactive score(IRS)was calculated by percentage of positive cells(PP)(≤5%,0;5-25%,1;25-50%,2;50-75%,3;>75%,4)×staining intensity(SI)(negative,0;weak,1;moderate,2;strong,3)and the average of ten fields was obtained.Based on the IRS,the expression level of Exo70 was classifed into:0 point,negative(-);1～4 points,weakly positive(+);5～8 points,moderate positive(++);9～12 points,strongly positive(+++).3.Statistical methodsSPSS 18.0 software was performed to analyze the data.Quantitative data were expressed as mean ±standard deviation(SD).The differences between groups were analyzed by non-parametric test.The association between Exo70 expression and clinicopathological parameters was analyzed by chi-square test.Univariate survival analysis was done by the Kaplan-Meier method.Cox regression model was established for the multivariate survival analysis to determine independent risk factor.A p-value less than 0.05 was considered to be statistically significant.Results1.The Oncomine database predicted that Exo70 mRNA levels were higher in gastric cancer tissue compared to normal tissue(P<0.05).Survival analyses in Kaplan-Meier Plotter database showed that gastric cancer patients with high mRNA expression of Exo70 had signifcantly lower OS than those with low mRNA expression of Exo70(p<0.001).2.Subsequent immunohistochemical staining in 90 cases of gastric cancer tissues and paired adjacent normal tissue showed that Exo70 was predominantly located in the cytoplasm and membrane in both gastric cancer cells and normal gastric mucosal cells.Exo70 protein levels in gastric cancer tissues were highly expressed compared to normal tissues(P<0.001).3.Analysis of the the correlation between Exo70 expression and clinicopathological parameters of gastric cancer patients showed that overexpression of Exo70 was more common in male gastric cancer patients(P<0.001).No signifcant association was observed between Exo70 expression and patient’s age,tumor size,tumor site,tumor diferentiation,Boarrmann classification,preoperative serum CEA level and lymph node metastasis(P>0.05).4.Univariate survival analyses revealed that the gastric cancer patients with strongly positive Exo70 expression had remarkably decreased OS and DFS,compared to patients with slightly positive and moderately positive Exo70(P<0.05).Male gastric cancer patients had reduced OS compared to female patients(P<0.05).Multivariate Cox regression model analysis performed for expression of Exo70,gender,age,tumor size,tumor differentiation and lymph node metastasis confirmed that Exo70 high expression level was the only independent factor to predict the poor prognosis of gastric patients(P<0.05)。Conclusion1.The expression level of Exo70 in stage III gastric cancer tissue was higher than that in normal gastric tissue.2.Exo70 overexpression may be an independent risk factor for poor prognosis of gastric cancer patients.3.Exo70 is expected to be a new potential therapeutic target for the treatment of gastric cancer patients.