Metastasis Suppressor1(MTSS1) Demonstrates Prognostic Value And Anti-metastatic Properties In Gastric Cancer

Given that high metastasis characteristic of gastric cancer is the major cause of death inpatients with gastric carcinoma and it results in a poor prognosis of the disease, to furtherinvestigate the field of the metastasis of gastric cancer will have significant meaning forimproving the survival of these patients. Metastasis suppressor1(MTSS1), as a potentialmetastasis suppressor gene, was considered to correlate with the invasion and metastasis oftumor through affecting the cystoskeleton migration in recent studies. The investigationabout this gene is new which has never been reported in gastric cancer both domestic andoverseas. In this study, we sought to determine the expression of MTSS1in resected gastriccancers, investigate the correlation of MTSS1expression and clinicopathologic featuresand survival and detect the LOH (loss of heterozygosity)of this gene in an attempt toidentify the potential mechanism of loss of MTSS1in the development of gastric cancer.Moreover, we will study the effect of MTSS1on biological behaviors such as invasion andmetastasis in gastric carcinoma cell lines and the molecular mechanism between MTSS1and probable pathway both in vitro and in vivo. With our study, MTSS1may serve as auseful biomarker for the prediction of metastasis and outcome of gastric cancer as well as apossible target to intercept the metastasis in this kind of carcinoma.Part I. Expression and clinicopathologic characteristics ofMTSS1in human gastric cancerObjective: To determine the expression of MTSS1in gastric cancer and to investigate theassociation of MTSS1with gastric cancer.Methods: Tissue microarray blocks containing primary gastric cancer, lymph nodemetastases, and adjacent normal mucosa specimens obtained from1,072patients wereconstructed. Expression of MTSS1in these specimens was analyzed usingimmunohistochemistry, Reverse transcription polymerase chain reaction (RT-PCR) andWestern blotting. Analysis implications between the expression of MTSS1and clinicalpathological characters was investigated.Results:(1) MTSS1expression was down-regulated in the tumor and metastases tissues ofgastric cancer compared with the adjacent normal tissues. Complete loss of MTSS1expression was observed in751cases (70.1%) of the1,072primary tumors and103(88%)of117nodal metastases. These results were in concordance with that in the Westernblotting study and Reverse transcription RT-PCR. (2) Significant differences were observed in the relationship between tumor size,depth of invasion, regional lymph node, TNM stage, differentiation and histological type.And from the well and moderate histological levels to the histological undifferentiated, theexpression of MTSS1was gradually decreased.(3) The GC patients with loss MTSS1expression had a significantly poor prognosiscompared to those with positive MTSS1expression (18months versus76months; P <0.001). MTSS1expression, just as the age, differentiation, nodal invasion, TNM stage wasan independent prognostic factor in gastric cancer(P<0.001).Conclusions: Loss of MTSS1expression was associated with poor outcome in patientswho underwent gastrectomy. MTSS1expression may serve as a useful biomarker for theprediction of outcome of gastric cancer.PartⅡ. The study on mechanism for loss expression of MTSS1in the progression and metastasis of late primary gastriccarcinomaObjective: To elucidate the role of the putative tumor metastasis suppressor gene MTSS1on tumorigenesis, progression and metastasis of gastric cancer.Methods: LOH (loss of heterozygosity) of four microsatellites loci(D8S1832,D8S2132,D8S1179and D8S1461) of the each thirty samples with the nomarltissue, primary tumor and metastasis tissue respectively were detected in the MTSS1gene site by using PCR—polyacrylamide gel electrophoresis-silver staining method.Results: A higher frequency of allelic loss was found in MTSS1-negative primary andmetastatic tumor samples.Conclusions:The frequent LOH of MTSS1gene and might play the important roles in thedevelopment, progression and metastasis of gastric cancer.Part III The biological effect of gene MTSS1in the metastasis ofgastric cancer cellsObjective: To construct a eukaryotic expression plasmid of MTSS1gene:pEGFP-N1-MTSS1, in order to study its effect on the biological behavior of gastriccarcinoma cell lines both in vitro and in vivo.Methods:1. The pEGFP-N1-MTSS1was constructed by use of recombinant DNA technique and was demonstrated by restriction endonuclease mapping and sequencing fromthe company of Takara in Dalian．The siRNA interference sequences for MTSS1have beendesigned by company. The pEGFP-N1-MTSS1/ShRNA431was transfected into humangastric cell line AGS/MGc80-3by using lipofectamine2000．The expression of MTSS1was detected by the Real time-PCR/Western blot assay.2. MTT growth test, transwell analysis were used in vitro to study the effects ofMTSS1expression on transfected cells proliferation. Tumor growth in nude mice was usedto access the tumorigenicity of gastric cancer cells. Change of cell cycles were alsodetected.Results:1,The level of protein expression of MTSS1in gastric cancer cell line AGStransfected with pEGFP-N1-MTSS1were significantly increased,and the assay of MTSS1activity showed the activity of AGS transfected with pEGFP-N1-MTSS1weresignificantly increased. MTSS1mRNA and protein expression was both assessed in thehuman gastric cancer cell lines which were transfected through the Real time PCR andWestern Blot analysis.And the overexpression and interference treatments were botheffective2,Transfected with MTSS1eukaryotic expression vector could significantly inhibitgrowth, cell migration and invasion compared with the untransfected cells in theMTT,colony formation ability assay and transwell tests compared with the controls butopposite in MTSS1ShRNA interference MGc80-3cells. Flow cytometry analysis showedthe proportion of G2/M phase cells in MTSS1-AGS group was significantly higher thanthe proportion in the control group.Conclusions: Our study indicates that MTSS1demonstrates the ability to modulatemetastatic ability, such as proliferation, invasion and migration of human gastric cancercell line in vitro and vivo. Therefore, metastasis suppressor1displays prognostic value forgastric cancer patients.