The Role Of SIRT1in Tumor Metastasis And The Molecular Mechanism Underlying Hypoxia Induced SIRT1Suppression

Objectives: Tumor metastasis refers to the spread of malignant cells from the site oforigin and the subsequent colonization of secondary organs，a pathological processthat includes reduced cell-cell connection, enhanced motility, invasion of surroundingtissues, intravasation, arrest and growth at a distant site. Though metastatic tumoraccounts for90%of cancer associated death, it remains the most enigmaticcomponent of tumorigenesis. Dissecting the molecular mechanism of metastaticcascade, therefore, sheds new light on the therapeutic intervention of metastaticdiseases.SIRT1is a type Ⅲ deacetylase which by deacetylating both histone andnumerous non-histone proteins regulates a vast array of cellular functions includinggene expression, metabolism homeostasis, cell proliferation and apoptosis. As theenzymatic activity of SIRT1depends on NAD+, SIRT1is considered both a sensorand a regulator of metabolism. Indeed, various in vivo and in vitro experimentsconsistently support a protective role for SIRT1in metabolic diseases such asdiabetes, obesity and atherosclerosis. Recently, accumulating evidence obtained fromgenetically modified mouse models suggests that SIRT1has strong tumor suppressiveactivity at least in certain types of cancer. However, the role of SIRT1in tumormetastasis has not been addressed thus far.SIRT1has been shown to protect from high fat diet induced fatty liver and livercancer though deacetylating and inhibiting NF-κB dependent inflammation. SIRT1isalso capable of deacetylating HIF1α and thereby inhibiting HIF1α dependent hypoxia associated responses. Since inflammation and hypoxia are both critical components ofthe tumor microenvironment that contributes to enhanced motility, invasiveness,stemness and drug resistance of tumor cells, these lines of evidence position SIRT1asa likely suppressor of metastasis. Consistently, reports have shown that hypoxiadownregulates both mRNA and protein levels of SIRT1alluding to a scenario inwhich SIRT1functions as a suppressor of metastasis under normoxia throughinhibiting HIF1α and NF-κB dependent responses, and its subsequent repression byhypoxia leads to tumor invasiveness and metastasis. The underlying mechanism,however, is not clearly defined. Therefore, we sought to determine whether SIRT1functions as a suppressor of metastasis and the molecular mechanism underlyinghypoxia induced SIRT1repression.Methods: Cells were cultured under1%oxygen using a Tri-Gas/CO2incubator tosimulate hypoxic condition. Real-time quantitative PCR (RT-qPCR) andimmunoblotting（IB）were used to examine the mRNA and protein levels of SIRT1inhuman liver cancer cell line HepG2, ovarian cancer cell line SKOV3，lung cancer cellline A549and breast cancer cell line MCF7. The sequence element within SIRT1gene promoter that mediates the hypoxia dependent suppression was indentifiedthough luciferase reporter assays. The recruitment of protein factors to SIRT1promoter was assessed by chromatin immunoprecipitation assay (ChIP). Enhancedexpression and activity of SIRT1were achived by transient transfection with SIRT1expression constructs and treatment with the SIRT1agonist resveratrol, respectively.The in vitro invasive potential of SKOV3was assessed by transwell invasion assay.The in vivo metastasis potential of SKOV3was assessed in nude mice. Smallinterfering RNA (siRNA) was used to knockdown PIASy expression and to monitorthe effect of PIASy knockdown on SKOV3invasion and metastasis. Oncomine database was used to analyze the expression of SIRT1in human ovarian cancertissues compared with normal ovarian tissues.Results: SIRT1over-expression by transient transfection or activation by resveratrolboth reduced hypoxia promoted invasiveness of SKOV3in vitro. Consistently，resveratrol administration also significantly impaired the metastasis potential ofSKOV3in nude mice. Moreover，SIRT1expression was significantly down-regulatedin tumor tissues compared with normal tissues in a cohort of human ovarian cancerpatients. These lines of evidence suggest that SIRT1is likely a metastasis suppressor,and hypoxia may contribute to metastasis though inhibiting SIRT1gene transcription,since our data showed that hypoxia downregulated both mRNA and protein levels ofSIRT1in HepG2，SKOV3，A549and MCF7cells. Hypoxia also reduced therecruitment of Sp1, a known SIRT1transcriptional activator, to the SIRT1promotervia PIASy dependent SUMOylation. PIASy may mediate hypoxia induced SIRT1suppression since our data showed that hypoxia stimulated PIASy expression andsubsequent recruitment to SIRT1promoter. In addition, siRNA mediated PIASyknockdown rescued Sp1binding to SIRT1promoter under hypoxia. Consistently,PIASy knockdown relieved SIRT1repression under hypoxia. PIASy knockdownantagonized both in vitro invasiveness and in vivo metastasis of SKOV3.