Metformin Inhibits Pancreatic Cancer Cells Growth By Activating Hippo Signaling Pathway

Background and Objective: Pancreatic Cancer(PCa)is one of the most malignant cancers with increased morbidity and mortality in recent years.Currently,surgical resection is the best therapeutic method for this disease.Because pancreatic cancer has insidious onset and progresses rapidly with high metastasis rate,patients with pancreatic cancer usually missed the good opportunity of surgery when they were diagnosed.In addition,PCa is insensitive to radiotherapy and chemotherapy because of the low degree of differentiation.The PCa patients have poor prognosis and the 5-year overall survival rate is about 4%.It is a global difficult problem to conquer PCa because of its high malignancy,high mortality and poor treatment effect Therefore,specific diagnostic methods and novel therapeutic strategies are urgently required to treat this disease.Metformin is one of the most widely prescribed drugs for treatment of type-2 diabetes.In addition to inhibiting hepatic glucose output and promoting glucose utilization in peripheral tissues,metformin can also improve insulin resistance,reduce triglyceride and cholesterol levels and improve the obese state.Recently,a number of clinical studies have shown that type-2 diabetic patients treated with metformin for a long time had a lower incidence of PCa when compared with patients treated with other hypoglycemic drugs or insulin.In vitro experiments have also demonstrated that metformin significantly inhibited malignant biological behaviors of many pancreatic cancer cell lines including Aspc-1,Panc-1,Sw1990 and large doses of metformin can kill them.Some scientists believed that metformin suppressed the growth of pancreatic cancer partly by reducing the level of circulating insulin and blocking tumor-promoting effect of insulin /insulin-like growth factor-1(IGF-1).Recently several studies have shown that metformin induced AMP-activated protein kinase(AMPK)phosphorylation,which in turn inhibited or activated some tumor-promoting or tumor-suppressor genes.Currently,the precise mechanisms involved remain incompletely understood and further study is required.We treated pancreatic cancer cells with metformin and then verified the impact of metformin on the proliferation,apoptosis,cell cycle,migration and invasion of pancreatic cancer cells by varieties of methods.We used microarray to screen related gene of metformin inhibitory effect and analyzed the possible involved signaling pathways.Then real-time fluorescent quantitative PCR and western blot were used to further explore the regulations of metformin on these pathways.Previously,we found that MST1(the core protein of Hippo pathway)were both negative in tumor tissues and adjacent tissues.YAP was strongly expressed in tumor tissues,while in the adjacent tissues was weakly positive,indicating that the YAP activation of Hippo pathway is involved in the occurrence of pancreatic cancer.Hippo signaling is a pathway to inhibit cell growth and play a key regulatory role in mediating cell contact inhibition,maintaining organ size and on the balance between proliferation and apoptosis of cells.We thus hypothesized that metformin inhibited the growth of pancreatic cancer cells by relating with Hippo signaling pathway.In order to confirm the speculation,We focused on the expression of Hippo pathway factors in the microarray and explored the relationship between metformin’s tumor inhibitory effects and Hippo signaling pathway.Methods: 1.Pancreatic cancer PANC-1 cells were cultured in vitro and treated with different concentrations of metformin(5,10,15,20,40mmol/L)for 24 h,48h,60 h,72h,respectively.The PANC-1 cells treated with no metformin were set as the control cells.CCK-8 method was used to examine the proliferation inhibition of PANC-1 cells treated with metformin.Choosing half inhibitory concentration(IC50)for following experiments according to cell inhibition rate.2.Flow cytometry was used to detect cell cycle and apoptosis of PANC-1 cells treated with metformin.3.The migration abilities of PANC-1 cells treated with metformin were measured by wound healing test.4.Transwell invasion assay was used to examine the invasion abilities of PANC-1 cells.5.We used microarray to screen related gene of metformin inhibitory effect and analyzed the possible involved signaling pathways6.Real-time fluorescent quantitive PCR and Western bloting were used to detecte the effects of metformin on involved signaling pathways.Results: 1.Treatment with metformin could observably inhibit the proliferation of PANC-1 cells in a dose-dependent manner.The IC50 of metformin to inhibit PANC-1proliferation was about 20mmol/L.2.After treated with 20mmol/L metformin for 48 h,we found that cell cycle arrested in G0 / G1 phase and S phase shortened,and apoptosis levels increased significantly..3.The wound healing test showed that compared with the cells of control group(almost completely healed after 48h),the treated cells scratche widths healed more slowly,indicatingthat metformin could inhibit migration of PANC-1 cells.4.In Transwell invasion assay,we found that cell number of control group was significantly more than treatment group,which showed that metformin significantly inhibited the invasion abilities of PANC-1 cells in vitro.5.Microarray analysis results showed that the expressions of Hippo pathway factors(YAP1,MST1,TEAD3)were all significantly changed compared with control group.6.After the treatment of metformin,the relative expression levels of MST1,LATS1,YAP1 mRNA were increased;Western Blot results demonstrated that the expression levels of MST1,LATS1 and phosphorylated YAP1 were significantly increased compared with control group,while the overall expression level of YAP1 was not notably changed.Conclusion : Metformin significantly inhibited PANC-1 pancreatic cancer cells proliferation,migration and invasion and can also affect the cell cycle,promoting apoptosis.Metformin could activate Hippo signaling pathway and promote YAP(major downstream effector molecules)inactivation through phosphorylation cascade reaction,leading to tumor promoters negative expressed and thus inhibiting the growth of pancreatic cancer cells.