Downregulation Of Aif Potentiates Hypoxia-induced Emt Of Cancer Cells

Apoptosis-inducing Factor(AIF)is a mitochondrial oxidoreductase,which contains flavin adenine dinucleotide-binding domain and is NADH-dependent.AIF is a protein of dual roles.On the one hand,AIF induces caspase-independent cell death when it is released from mitochondria and subsequently translocated into nucleus under the action of certain stimulus.On the other hand,the oxidoreductase activity of AIF is essential for cell survival as it is required for mitochondrial energy production and optimal function of the respiratory chain.Numerous studies regarding the roles of these two aspects have been carried out.However,the relationship between AIF and cancer is largely unknown.In this study,by immunohistochemical(IHC)analysis,we found that AIF expression was inversely correlated with hypoxic microenvironment in colon cancer tissues.Hypoxia downregulated AIF mRNA and protein levels in cancer cells.Furthermore,by using multiple models,we demonstrated that hypoxia downregulated AIF expression through hypoxia-inducible factor 1?(HIF-1?).Subsequently,luciferase assay verified both hypoxic treatment and HIF-1? overexpression significantly inhibited the transcriptional activity of AIF promoter,which was mediated by the hypoxia-response element(HRE)in the first intron of AIF gene.Finally,chromatin immunoprecipitaion(CHIP)assay confirmed that both HIF-1? and HIF-1?,two subunits of HIF-1,directly bound to this HRE sequence.Next,we asked what are the functional consequences of AIF downregulation.Astonishingly,we found that AIF knockdown directly induced cancer cells to undergo epithelial-mesenchymal transition(EMT),as evidenced by cell morphological changes,and corresponding alterations of EMT markers including E-cadherin,Fibronectin and Vimentin.Hypoxic treatment has previously been reported to induce EMT.Interestingly,blocking the downregulation of AIF by AIF overexpression under hypoxia,significantly repressed hypoxia-induced EMT effects,whereas AIF knockdown accelerated hypoxia-induced EMT.In order to explore the mechanisms underlying AIF induced EMT,we employed proteomic strategies to look for AIF-interacting proteins.Interestingly,the tumor suppressor PTEN was identified as a new AIF-interacting protein.We found that AIF knockdown did not affect PTEN protein level,but increased its oxidative inactivation,with ensuing activation of AKT/GSK-3?/?-catenin signaling pathway.Hypoxic treatment also induced the activation of this signaling cascade,which was markedly inhibited by AIF overexpression.Taken together,our study identified AIF as a novel HIF-1 target gene,whose transcriptional activity was repressed by HIF-1.AIF downregulation potentiated hypoxia-induced EMT program by promoting oxidative inactivation of PTEN and activation of AKT/GSK-3?/?-catenin signaling cascade.