Clinical Significance Of MACC1in Postoperative Recurrence Of Gastric Cancer And Exploration Of Its Possible Mechanism

Background and objectiveMetastasis-associated in colon cancer-1(MACC1), which was associated with the prognosis of colorectal cancer, was first found in the colon cancer as a oncogene. It’s now clear that MACC1plays an important role in regulating hepatocyte growth factor (hepatocyte growth factor, HGF)/hepatocyte growth factor receptor (Met tyrosine kinase receptor, c-Met) signal pathway. c-Met gene was found in the1980s as a proto-oncogene and HGF was the only c-Met receptor ligand. The abnormal of HGF/c-Met signal pathway activates the RAS-MARK (mitogen activated protein kinase), PI3K (phosphatidylinositol3-kinase)-Akt (serine/threonine kinase) and STAT signaling pathways, subsequently promoting proliferation, invasion, angiogenesis and anti-apoptosis. It associates with colon cancer, gastric cancer, hepatoma, lung cancer and other kinds of malignant tumors occurrence and prognosis.MACC1gene is located on chromosome7(7p21.1), containing seven exons and six introns, whose cDNA contains2559nucleotides, encoding protein with852amino acid residues. MACC1gene correlated with tumor metastasis was first discovered in colon cancer by Stein, etc. The metastasis-free survival (MFS) time in patients with higher expression of MACC1was shorter than patients with lower expression, suggesting that MACC1was an independent factor in predicting colon cancer metastasis and MFS. The expression of MACC1also has effect on the prognosis of patients with colon cancer.5year survival rate in patients with low expression of MACC1was as high as80%, compared to that of15%in patients with high expression. In addition, based on MACC1expression in primary tumor of colon cancer, MACC1in predicting patients with or without metachronous metastases was74%and80%, respectively. MACC1can predict metastasis of colon cancer effectively. In constructed colon cancer cell lines, the ability of motility and proliferation in the cells with higher expression of MACC1was significantly stronger than that in the inference cells. MACC1promoting tumor growth and metastasis in the xenograft mice model was also proved. Subsequently, more research supported the view of Stein, etc. MACC1was related with the TNM stage and peritoneal metastasis in colorectal cancer. Their data suggested that the expression of MACC1was much higher in stage IV patients than that of Ⅰ-Ⅲ stage patients and was significantly higher in patients with peritoneal metastasis than patients without peritoneal metastasis.However, little is known about its role in gastric cancer. According to a recent study the expression of MACC1was higher in patients with peritoneal metastasis of gastric carcinoma than that of patients without peritoneal metastasis. Currently, Gastric cancer is the second in the global mortality in malignant tumors. Surgery is still the unique way to cure gastric cancer. However, about half of patients after radical resection of tumor undergo recurrence, which always predict poor prognosis. In large part of postoperative recurrence in patients happened hematogenous or peritoneal metastasis for the reason of that probably tumor cells had happened to undetected micrometastases before operation. It is generally believed that the epithelial-to-mesenchymal transition (EMT) is an essential process for tumor cell invasion. During the invasion process, tumor cells must free themselves from cellular junctions and the surrounding matrix, and must breach these barriers by changing from an epithelial to a mesenchymal phenotype. EMT is induced by multiple signaling pathways which relationship is very complicated. Hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) signal via receptor tyrosine kinases (RTK) towards the central Ras-Raf-MAPK pathway or towards the PI3K pathway and the Src-STAT pathway. Transforming growth factor (TGF)-P signals via receptor serine/threonine kinases (RS/TK) towards the central R-Smad/Co-Smad pathway or towards the PI3K and MAPK pathways. Alternatively, the TGF-β receptor signals towards the polarity protein Par6, thus recruiting the ubiquitin ligase Smurf, which degrades Rho and leads to disassembly of tight junctions in epithelial cells. Jagged and Delta-like ligands signal via Notch receptors towards the transcription factor CSL. Wnt ligands signal via Frizzled receptors towards β-catenin and the transcription factors LEF-1/TCF. All these pathways modulate gene expression and lead to EMT and cell motility.HGF/c-Met signaling has been proved to be a downstream target of MACC1in colon cancer. Moreover, HGF/c-Met is known to be a tumorigenic and pro-metastatic factor for gastric cancer. Deregulation of HGF/c-Met has been reported to induce the EMT. Therefore, do MACC1is related with postoperative recurrence of gastric cancer? Do it change the malignant biological behavior of gastric cancer by the way of EMT? Our research will aim to solve these questions.In this study, we aimed to explore the roles of MACC1in gastric caner and its potential mechanisms by a retrospective analysis of264postoperative gastric caner patients categorized from stage I to stage III, and carrying out cellular experiments to clarify the invasion and migration properties of MACC1in gastric cancer and the effect of MACC1on EMT.Research method1. Detection MACC1protein expression in patients with stage Ⅰ-Ⅲ gastric cancer after radical resection, and analyze association between MACC1expression and clinical features.A total of264patients who received gastric cancer radical resection were collected in Nanfang hospital from2004to2008. All patients had been histological diagnosed as gastric cancer, and all had been received radical resection. Tumor staging was defined according to the AJCC cancer staging manual (the7th edition,2010). Follow-up time was from surgery to the tumor recurrence, the follow-up deadline was March in2011. Paraffin-embedded cancerous tissue blocks were cut into4μm. Immunohistochemistry staining was used to detect MACC1protein content. Target protein expression level in cancerous tissue was scored by staining range and intensity. The relationship between MACC1expression with clinicopathological features.2. Establishment of stable MACC1overexpression and silence transfected gastric cancer cells for MACC1.According to MACC1gene sequences, we designed amplification or interference of MACC1expression plasmid and then transferred the plasmids into retroviruses. Human BGC-823and MKN-28gastric cancer cell lines were infected with the retroviruses. Therefore, MACC1overexpression (MACC1), no-load (vector), MACC1silence (shMACC1) and negative sequence (scramble) cells were established. Real-time fluorescent quantitative PCR and Western blot were used to detect the contents of MACC1mRNA and protein in these cell lines for purpose of identifying whether MACC1and shMACC1stable cell lines were successful established3. Detection of MACC1effects on gastric cancer cells invasion and migration.Transwell invasion assay and Wound closure assay were used to compare the invasion and migration activity of the four kinds of stably transfected cells respectively.4. Detection the effects of MACC1expression change for EMT-associated gene expression.Using RT-PCR and Western blot to detect expression of EMT-associated gene, involving E-Cadherin, α-Catenin, Fibronectin, MMP2, MMP9, Vimentin and CD44in established stable cells.5. Statistical analysisData were analyzed using the SPSS16.0software package.Kaplan-Meier survival analysis was used to analyze the relationship between MACC1and Disease free survival. The relationships between the expression of MACC1protein and their clinicalpathologic features were analyzed using χ2test. Hazard ratio (HR) was determined using Cox model. The difference significances were calculated using Student’s-T test for continuous variables. All p values less than0.05were considered statistically significant.Research results1. MACC1expression in gastric cancerous tissues.(1) The positive expression of MACC1is75.4%in264gastric cancerous tissues and24.6%was negative.(2) MACC1expression was not associated with gender (P=0.266) and age (P=0.446), but was correlated with clinical stage. In stage I patients the positive rate of MACC1expression was62.8%, but in the stage Ⅱ-Ⅲ patients the positive rate was77.8%(P=0.036).(3) The expression of MACC1was associated with the recurrence in gastric cancer patients, and the positive expression of MACC1was84.2%in patients who underwent recurrence versus60.6%in patients without recurrence (P<0.001).(4) DFS in the MACC1positive expression of patients was significantly shorter than the negative expression of patients, especially in the stage Ⅱ-Ⅲ patients. MACC1was an independent factor affecting DFS in patients of gastric cancer.2. The MACC1expression enhances the aggression and migration of gastric cancer cell lines.Invasive activity was further verified by a Transwell assay. The number of invading cells that migrated through the Matrigel was significantly greater for ectopic-MACC1cells versus vector controls, and was dramatically decreased for shMACC1cells versus scramble cells. In the wound closure migration assay, ectopic MACC1-transfected cells almost closed the wound after24h of culture, whereas the vector-transfected cells left a wide gap. In contrast, migration by shMACC1-transfected cells was significantly impaired, as evidenced by the fact that wound recovery was greatly suppressed in comparison with the scramble control.3.MACC1regulates EMT-associated genesOverexpression of MACC1decreased the expression of epithelial marker related gene significantly, such as E-Cadherin, a-Catenin. While the expression of mesenchymal transition associated gene, such as Fibronectin, Vimentin, MMP2, MMP9, CD44were up-regulated. While silencing MACC1gene had the opposite results. The difference was statistically significant between the vector group and the MACC1group, and the same result was obtained between the scramble group and the shMACC1group (P<0.05). Research conclusions1. MACC1which associated with clinic tumor stage and an increased postoperative recurrence is an independent risk factor predicting the recurrence of gastric cancer.2. MACC1promotes the migration and invasion of gastric cancer cells.3. MACC1affects the malignant biological behavior of gastric cancer through regulating EMT.