Studies On The Function And Mechanism Of MTORC1-mediated Downregulation Of COX2 In Tuberous Sclerosis Complex

Mammalian target of rapamycin protein(mTOR),a highly conserved serine/threonine protein kinase,which plays a critical role in cell growth,proliferation,metabolism,and autophagy.mTOR mainly exist in two different forms of complex: mTOR complex 1(mammalian target of rapamycin complex 1,mTORCl)and mTOR complex 2(mammalian target of rapamycin complex 2,mTORC2).mTORC1 is under the control of energy and nutrition signals,and sensitive to rapamycin,while the upstream signals of mTORC2 remains unclear,and it is insensitive to rapamycin.Accumulating evidences indicate that mTORC1 exerts a crucial role in tumorigenesis.However,the downstream effectors of mTORC1 still remain incompletely characaterized.Tuberous sclerosis complex(TSC)is an autosomal dominant disease with benign tumors in multiple organs.This disorder is caused by inactivating mutations in either of the two tumor suppressor genes: TSC1 or TSC2.TSC1 and TSC2 protein form a functional complex which negatively regulates a small GTPase,Ras homologue enriched in brain(Rheb),through the GTPase-activating(GAP)activity of TSC2.Disruption of TSC1/TSC2 complex by inactivating mutations in either of TSC1 or TSC2 leads to the accumulation of GTP-bound Rheb,which in turn activates mammalian target of rapamycin complex 1(mTORC1).Hyperactivated mTORC1 signaling leads to uncontrolled cell growth and tumorigenesis,and thus is considered to be responsible for the tumor development in TSC.Interestingly,TSC patients are rarely seen with malignant lesions.Although it is believed that the negative feedback inhibition of AKT by deregulated mTORC1 is the major reason of benign nature of TSC tumors,whether additional signaling molecules contribute to restrict the tumor development of TSC remain less clear.Cyclooxygenases(COXs)are a family of myeloperoxidases which catalyze the biosynthesis of the prostaglandins(PGs)from arachidonic acid.So far,three different COX isoforms have been identified.COX2 is an inducible isoform that produces PGs during inflammatory and tumorigenic settings.Overexpression of COX2 has been reported in many types of tumors.COX2 has been shown to play a crucial role in carcinogenesis by promoting growth,survival,and metastasis of tumor cells.However,the exact role of COX2 in TSC tumors remains not understood.In this research,by analyzing Tsc2-null mouse embryonic fibroblasts(MEFs)and rat uterine leiomyoma-derived Tsc2-null ELT3 cells,here we present evidences for the involvement of Cyclooxygenase 2(COX2),as a novel downstream target of mTORC1,in the development of TSC.We showed that loss of TSC2 led to decreased expression of COX2 through activation of mTORC1.Moreover,signal transducer and activator of transcription 3(STAT3)inhibited the transcription of COX2 by acting as a downstream effector of TSC/mTORC1 signaling pathway.Overexpression of COX2 promoted the proliferation and tumor growth of Tsc2-null cells,while knockdown of COX2 inhibited the proliferation of the control cells.Furthermore,we demonstrated that interleukin-6(IL-6)mediated the effect of COX2 on the growth of TSC2-null cells.In addition,rapamycin in combination with celecoxib,a COX2 inhibitor,exhibited a strong inhibitory effect on the growth of Tsc2-deficient cells.We conclude that the downregulated COX2 may serve as a protective mechanism against hyperactivated mTORC1-mediated tumorigenesis due to loss of TSC2 and the combination of rapamycin with celecoxib may be a new strategy to treat TSC.