The Molecular Mechanism Of YAP Supports Proliferation Of Adult T-cell Leukemia Cells Through Suppression Of TGF? Signaling

Human T-cell leukemia virus type 1(HTLV-1)is a carcinogenic retrovirus,whose infection can cause Adult T-cell Leukemia.In addition to expressing structural proteins such as gag,pol and env,HTLV-1 also encodes regulatory proteins including p12,p21,p30,p13,Tax and HBZ.Tax is the key protein of HTLV-1,which can promote tumorigenesis by regulating multiple signaling pathways.Many studies have shown that Tax can inhibit the negative function of the TGF?(Transforming Growth Factor ?)signaling pathway on cell growth,thus promoting the malignant proliferation of ATL cells.However,in the later stage of ATL development,the expression of Tax will gradually decline or even not be detected.Therefore,what mechanism can be used to inhibit TGF? signaling pathway and promote malignant proliferation of ATL cells? It has been reported that YAP is a key protein of Hippo signaling pathway.In mammals,Hippo/YAP pathway not only regulates organ size and cell regeneration,but also influences the growth of cells by regulating other signaling pathways.Moreover,studies show that there may be a interaction between TGF? signaling pathway and Hippo/YAP pathway.Therefore,we speculated whether YAP can promote the development of Adult T-cell leukemia by regulating TGF? signaling pathway in ATL cells? In order to prove this inference and elucidate its molecular mechanism,we has carried on these research from the following aspects:Part I: High expression of YAP in ATL cells and its mechanismWe firstly detected the expression of YAP mRNA in ATL cell lines by RT-PCR.We found that,compared with control cell lines(Hut78,MOLT-4,Jurkat),ATL cell lines(MT-4,TL-Om-1,ATL-T,ATL-2,MT-1,and MT-2)had high expression of YAP gene at different levels.In order to further investigate the molecular mechanism of high expression of YAP in ATL cell lines,we conducted a dual luciferase reporter assay.The result showed that the Tax protein encoded by HTLV-1 could activate the activity of YAP promoter.Meanwhile,we further confirmed that the overexpression of Tax protein in T cells could promote the expression of YAP protein by Western blot.Part II: YAP regulates TGF? signaling pathwayIn order to investigate whether YAP has a regulatory effect on TGF? signaling pathway,firstly,we conducted the dual luciferase reporter assay and found that YAP can significantly inhibit TGF? signaling pathway.Besides,all the different reporter plasmids of TGF? signaling pathway and various cell lines used in the experiments confirmed the inhibitory effect of YAP to TGF? signaling pathway.Subsequently,we constructed the ATL-T YAP knockdown cell line and the ATL-T YAP overexpressed cell line through lentivirus infection,and used reporter gene experiment to detect the activation of TGF? signaling pathway in those cells.The results showed that in the ATL-T YAP knockdown cells,the activation of TGF? signaling pathway was up-regulated with the down-regulation of YAP expression in ATL-T cells.However,the activation of TGF? signaling pathway was significantly inhibited in the ATL-T YAP overexpressed cells.Therefore,those experiments further proved that TGF? signaling pathway is negatively regulated by YAP in ATL-T cells.Part III: molecular mechanisms of YAP regulating TGF? signaling pathwayIn order to study the molecular mechanism of YAP regulating TGF? signaling pathway,we first used Western blot experiment to detect whether YAP has an effect on the half-life of Smad3,a key protein of TGF? signaling pathway.The results showed that YAP had no effect on Smad3 protein degradation.Subsequently,we found that YAP can bind to Smad3 protein by immunoprecipitation assay.We also conducted some immunoprecipitation assays on mutants of YAP and Smad3,showing that the WW1 domain and the partial TAD domain of YAP protein are involved in binding to Smad3,and YAP protein mainly acts on the MH2 domain of Smad3 protein.We also conducted in-depth studies on the molecular mechanism and found that YAP does not affect the binding of Smad3 to Smad4 and Smad7.The result of reporter gene assay showed that p300,the Smad3 cotranscriptional activator,could restore the TGF? signaling pathway inhibited by YAP,In addition,we found that YAP can inhibit the binding of Smad3 to p300,while strengthening the combination of the inhibitory Smad protein-Smad7 to the TGF? I receptor in the meantime through the immunoprecipitation assay.Part IV: YAP promotes ATL cell growth by inhibiting TGF signaling pathwayIn order to investigate whether YAP can affect the growth of ATL cells by inhibiting the TGF? signaling pathway,we first used the ED YAP knockdown cells and the ATL-T YAP overexpressed cells.Through MTT experiment,we found that overexpression of YAP can promote the growth of ATL-T cells.Flow cytometry result revealed that silencing YAP expression in ED cells affected the G1/S phase of cell cycle,which then affected cell growth.Subsequently,we constructed a Mv-1-Lu YAP overexpressed cell line.MTT assay showed that the death rate of Mv-1-Lu-control cells increased gradually with the increasing of TGF? concentration after TGF? treatment,while the death rate of Mv-1-Lu YAP overexpressed cells did not change significantly after TGF? treatment.It suggests that YAP antagonizes the negative function of TGF? on cell growth.Finally,qRT-PCR was processed to find that overexpression of YAP in ATL cell lines can significantly inhibit the expression of TGF? downstream target gene,p15 gene,the cell cycle suppressor,and promote the cell cycle transformation and the cell growth.To sum up,in this study,we found that YAP was highly expressed in ATL cells,and it was the viral protein Tax encoded by HTLV-1 that promoted the expression of YAP.YAP could bind to Smad3 protein and then inhibit the combination between the Smad3 and p300,the cotranscription factor.Meanwhile,studies have proved that YAP can enhance the binding of Smad7 to TGF? type I receptors,and inhibit the TGF? signaling pathway and the expression of its downstream target gene p15,thus ultimately promoting the growth of ATL cells.This study not only elucidated the molecular mechanism of YAP inhibiting TGF? signaling pathway and promoting cell growth when Tax expression is absent in the late ATL stage,but also provided new strategies and targets for clinical treatment of ATL.