REG? Deficiency Inhibits Metastasis Of Thyroid Cancer Through Regulation Of TGF-?/Smad Signaling

The proteasome activator REGy has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21,p16,and p19 in an ATP and ubiquitin-independent manner.The ability of REGy to degrade other oncogenic proteins,such as hepatitis C virus(HCV)core protein and pituitary tumor-transforming 1(PTTG1)provides good examples for its role in cancer development.Previous research has revealed that REGy plays special regulatory roles in several cancers such as colorectal,breast,liver,lung,skin and thyroid cancers.The process through which,epithelial morphology changes to mesenchymal phenotype is referred as epithelial-mesenchymal transition(EMT).Most carcinomas use this mechanism to evade into neighboring tissues,thus alternation of E-cadherin expression is a well-established hallmark of EMT.Increasing evidence suggests that transforming growth factor-beta(TGF-?)triggers EMT and facilitates cancer cell differentiation.However,the relationship between the expression level of REGy and TGF-P signaling for EMT progression in thyroid cancer cells remains elusive.In this study,we used shRNA-REGy to knockdown the REGy expression in thyroid cancer cells.The biological effects of REG?-knockdown on human thyroid cancer cells(SW1736,FTC,K18,and HTh-7)and lung cancer cells(A549)were investigated by examining the cell migration,EMT marker and MET marker genes regulation,cell morphological changes,cell proliferation,apoptosis and cell cycle arrest,and TGF-?/Smad signaling.Thus,this study was conducted to explore the role of REGy expression in EMT,as a regulator of metastasis in thyroid and lung cancer.We found that REGy siRNA knockdown in human thyroid cancer cells resulted in increased E-cadherin expression.In addition,morphological changes and potential inhibition of colony formation was revealed by siREGy cells.Furthermore,we demonstrated that shRNA knockdown of REGy led to promote E-cadherin and inhibited the cell migration and EMT.Likewise,in vivo study also demonstrated elevation of E-cadherin in REG?-/-mice tissues.Besides,overexpression of REGy indicated downregulation of E-cadherin expression in 293WT(REGy inducible)and 293T cells.REGy also influences the EMT transcription factors by regulating the gene expression.Moreover,REGy deficient cells inhibited cell migration and altered morphology of thyroid cancer cells.The cell phenotype was also altered by 2D and 3D cell culturing.2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay demonstrated statistically significant time-dependent inhibition of cell proliferation by REG?-knockdown in SW1736,FTC,K18,HTh-7,and A549 cells.Flow cytometric analysis of the anti-proliferative effect of REGy-knockdown was mediated by arresting cells in the G0/G1 phase,which was caused by increased expression of p53 and p21 followed by downregulation of c-Myc.TGF-? acted on REGy-knockdown(SW1736,FTC,K18,HTh-7 and A549)cells by changing cell morphology to a fibroblast-like shape.TGF-P dependent EMT changes were accompanied by the downregulation of E-cadherin protein and mRNA expression,upregulation of Zeb-1,Snail-1,and Vimentin.In contrast,co-treatment with TGF-P inhibitor(SB 431542)abolished the effects of TGF-P on the E-cadherin and transcription factors like,Zeb-1 and Snail-1 as well.Moreover,expression profiles of thyroid specific genes such as TTF-1,TTF-2,Pax-8,NIS,and Tg suggested that they are involved in TGF-?-mediated EMT in thyroid cancer cells.Moreover,TGF-? accentuated the cell migration ability in thyroid and lung cancer cells.REGy decreased Smurf2 on protein levels,while REGy depletion showed downregulation of Smad3 on protein levels.Therefore,we assume that REGy positively regulate TGF-?/Smad signaling.Consequently,REGy promotes EMT and cancer cell migration through regulation of TGF-?/Smad signaling.Collectively,our data indicated that REGy is a key mediator in EMT process via regulation of TGF-P signaling pathway and serve as a potential molecule for targeted cancer therapy,thereby leading to metastasis.