Metformin Inhibits SMAD4-deficiency Upregulated HNF4G Caused Pancreatic Ductal Adenocarcinoma Metastasis

BACKGROUND&AIMS:Pancreatic ductal adenocarcinoma(PDAC)is a highly lethal malignancy because of high invasion and metastasis.However,the development of effective therapies for PDAC remains lag behind,largely due to limited knowledge on the mechanism underlying PDAC invasiveness and metastasis.Genome-wide association study(GWAS)is a powerful tool to identify genetic variants associated with risk and phenotypes of diseases.Some GWAS-identified disease risk variants may also contribute to the disease progression or even outcomes through their molecular functions on disease phenotypes including the response to therapies.Genome-wide association studies(GWAS)have identified several genetic regions associated with risk of pancreatic ductal adenocarcinoma(PDAC)while the underlying mechanisms remain largely unknown.This study investigated the functions of genes in these genetic regions and their roles as therapeutic targets for PDAC.METHODS:We select 36 genes significantly associated with PDAC from GWAS reanalysis and the malignant phenotypes after silencing these genes were examined by using a high content screen method.Invasiveness and migration in cells were analyzed in transwell assay or in nude mice with orthotopic pancreas transplantation.Metastasis of xenograft tumors was quantified by live animal imaging.Genes expression were quantified by quantitative real-time PCR Analysis(qPCR)in RNA level and immunoblot or immunohistochemistry(IHC)analyses in protein level.We detected the interaction between protein and DNA using chromatin immunoprecipitation combined quantitative PCR(ChIP-qPCR)and dual-luciferase reporter gene assay.The binding site and phosphorylating site were determined through vectors constructed with site-directed mutagenesis.Clinical information was collected and over-all survival time was measured from the date of diagnosis to the date of last follow-up or death.Patients survival were compared using Kaplan-Meier methods and hazard ratios were calculated by Cox models.RESULTS:We identify HNF4G was the top one who's silencing significantly repressed PDAC cell invasiveness in 36 candidate genes(P<0.0001).We report for the first time that deficiency of SMAD4,the tumor suppressor gene frequently deleted or mutated in PDAC,induces HNF4G upregulation that causes PDAC invasion and metastasis and poor survival in patients(hazard ratio=1.60,95%confidential interval=1.03-2.47,P=0.036).We demonstrate that Metformin is able to significantly repress invasion and metastasis of PDAC cells in vitro and in vivo by suppressing HNF4G via AMPK-mediated phosphorylation-coupled ubiquitination degradation.Metfomin significantly repress invasion and metastasis of PDAC cells with SMAD4 deficiency in vitro(P<0.0001)and in vivo(P=0.0039).In our PDAC cohort,Metformin treatment could significantly improve clinical outcomes in patients with SMAD4-deficient PDAC(hazard ratio=0.3 1,95%confidential interval=0.14-0.68,P=0.022)but not in patients with SMAD4-efficient PDAC.CONCLUSIONS:HNF4G functions as an oncogene that plays a pivotal role in invasiveness and metastasis in PDAC,whose overexpression may be caused by SMAD4 deficiency.Overexpression of HNF4G evokes PDAC progression and invasiveness,but can be suppressed by the panacea Metformin.These findings hold the potential for clinical trials to test HNF4G pathway inhibitors including Metformin in SMAD4-deficient/HNF4G-overexpressing PDAC.