FoxMl Regulates ADAM17in Promting The Development Of Gastric Cancer

Despite some decline in the incidence of gastric cancer worldwide in recent years,especially in western countries, the incidence and cancer-related mortality of gastriccancer are still highest amongst all cancers in developing countries. Each year, it isreported that more than300,000new cases is diagnosed in China; this is nearly aquarter of newly diagnosed cases worldwide. It is also demonstrated that more than60%of gastric cancer patients presents with lymph node metastasis at first diagnosis,5%of patients with liver metastasis and other organ metastases. Tumor invasion andmetastasis and cancer metastasis-related complications are the major causes of deathfor gastric cancer patients. As such, it is very important to study special genes andpathays of gastric cancer development, invasion, and metastasis in China to studybiological characteristics of gastric cancer and improve disease treatment and overallsurvival.Objective:1) To explore the expression of ADAMs in gastric cancer profile, themechanism of ADAMs mediated by FoxM1. In order to provide possibly new targetsfor the diagnosis, treatment and prognosis prediction of gastric cancer, the associationbetween ADAMs or FoxM1expression and the prognosis of patients with gastriccancer is also investigated.2) To analysis the influence of the FoxM1-ADAM17axismediated by melbine so as to provide experimental evidence for melbine in thetreatment of gastric cancer patients.Methods:1) The expression of soluble ADAMs in gastric cancer cells and tissuewere confirmed by real-time quantitative RT-PCR and Western blot. High expressionlevels of the ADAM family were extracted.2) The association between FoxM1andADAMs in gastric cancer cells and tissue were also confirmed by real-timequantitative RT-PCR and Western blot. The expression levels of ADAM mRNA andprotein were detected through upregulation or downregulation of FoxM1gene.3)Different concentrations of FoxM1were transfected into gastric cancer cells by usingLuciferase. The change of promoter region activation of ADAM was assessed.4)FoxM1and ADAM were analysized by immunochemistry in gastric cancer tissue.The association between FoxM1and ADAM and patients’ clinicopathological data and prognosis was also analysized.5) The change of FoxM1and ADAM protein withdifferent concentration of metformin was detected through western blot. MTTdetected the change of tumor cell proliferation and implantation experimentanalysized the influence of metformin in tumor formation.Results:1) The family members of soluble ADAM mRNA, including ADAM9、ADAM10、ADAM15、ADAM17and ADAM28, were highly expressed in gastriccancer tissue, while the ADAM12mRNA was down-regulated. ADAM10, ADAM17and ADAM28gene expression in tumor tissue samples were higher than those inmatched normal gastric mucosa. The effect of down-regulation of ADAM10,ADAM17and ADAM28by sh-RNA can obviously inhibited the tumor cellproliferation.2) There exists positive association between FoxM1and ADAM17in gastriccancer cells and tissue. The up-regulation of FoxM1in gastric cancer MKN45cells by3*flag-FoxM1could significantly elevate ADAM17mRNA and protein expressionlevels, thus promote MKN45cell proliferation. Down-regulation of FoxM1in gastriccancer SGC7901cells by sh-RNA transfection could significantly inhibit ADAM17mRNA and protein expression levels, thus inhibited the tumor cell proliferation. Invitro, after transfecting FoxM1shRNA into SGC7901cells, the tumorigenic ability ofSGC7901mice was decreased, with FoxM1and ADAM17protein expression levelsdown-regulating. The tumorigenic ability of MKN45mice was enhanced throughup-regulating FoxM1expression levels in MKN45cells by3*flag-FoxM1. However,The tumorigenic ability of MKN45mice through both FoxM1and ADAM17sh-RNAtransfection was more decreased than MKN45mice through either FoxM1orADAM17sh-RNA transfection.3) The higher the concentration of FoxM1plasmids in MKN45and293T celllines, the higher activity of ADAM17promoter. The activity of ADAM17promoterwith5ng FoxM1plasmid transfection was higher than those with2.5ng FoxM1plasmid transfection or without FoxM1plasmid transfection (p<0.05).Down-regulation of FoxM1in SGC7901and293T cell lines through sh-RNA inducedthe activity of ADAM17promoter decreasing. Furthermore, the activity of ADAM17promoter was more deceased in FoxM1down-regulation group through5ng sh-RNAthan that in non-transfection group (p<0.05). All of these suggested FoxM1could mediate the transcription of ADAM17.4) The expression levels of FoxM1and ADAM17in gastric cancer tissueswere significantly higher than those in matched normal gastric mucosa, with FoxM1expression reaching58.7%(121/206) and ADAM17expression reaching75.7%(156/206). The quantity of FoxM1protein expression was increased as the gastriccancer cells’ differentiation increased, the extent of gastric cancer invasion expanded,the lymph node metastases increased and the TNM stage increased (p<0.05). Theexpression of ADAM17in the gastric cancer tissues was significantly associated withthe patients’ age, the TNM stage and lymph node metastasis (p<0.001). The overallsurvival time in ADAM17and FoxM1-positive patients were significantly longer thanin ADAM17and FoxM1-negative patients (both P<0.001). Moreover, COXmultivariate analysis suggested high expressions of ADAM17and FoxM1wereindependent prognostic factors for gastric patients.5) In vitro, the tumor cell proliferation of gastric N87and MKN45cells waspositively associated with metformin dose. A subcutaneous tumor model wasestablished in mice. After tumor formation, the size of transplanted tumor was lowerin metformin-treated group than the control group (p<0.01). The transplanted tumorsafter treatment were more weigh in the control group than metformin-treated group(p<0.01), further suggesting metformin can inhibit tumor growth and tumor formation.In the mechanism study of metformin, it is found that the higher the concentration ofmetformin, the higher expression levels of pAMPKαin gastric cancer cell N87andMKN45. In addition, pAMPKαexpression was analyzed by immunohistochemistry ingastric cases. The expression levels of pAMPKαprotein in metformin-treated groupwas significantly higher than those in the control group. The expression levels ofp-mTOR, pS6, p4EBP1and cyclinD1in metformin-treated group was lower thanthose in the control group. Meanwhile, it is demonstrated that there exists negativeassociation between the expression levels of ADAM17and FoxM1and metformindose.Conclusion:1) The expression of ADAM17and FoxM1was higher in gastriccancer cells and tissues. The inhibitory effect of ADAM17or FoxM1can inhibitgastric cancer cell proliferation and tumor growth.2) FoxM1can combine withADAM17promoter region and mediate ADAM17directly.3) The expression levelsof ADAM17and FoxM1in gastric cancer tissue were higher than those in matchednormal gastric mucosa. ADAM17and FoxM1may be independent molecular markers for predicting the prognosis of gastric cancer.4) Metformin can inhibit gastric cancercell proliferation in vitro and vivo, which is potentially mediated through inhibition ofFoxM1-ADAM17axis.