The Mechanism Of Tax Protein Promotes Adult T-cell Leukemia Through Regulating Hippo/YAP Pathway

Human T-cell leukemia virus type 1(HTLV-1)is the pathogenic agent of adult T-cell leukemia(ATL).HTLV-1 virus codes structural genes gag,pol and env.Apart from that,HTLV-1 also encodes a series of regulatory genes containing Rex,p12,p13,p30,Tax,and HBZ.Tax is the most important protein,and it promotes tumorigenesis by regulating multiple signaling pathways,such as TGF-?/Smad,AP-1,NF-?B,AKT,etc.However,there is no clear molecular mechanism to elucidate how Tax regulates the malignant proliferation of ATL cells and induce the development of ATL.Hippo/YAP signaling pathway was first discovered in Drosophila.In mammals,the components of the Hippo/YAP signaling pathway are also highly conserved,and it mainly regulates embryonic development and organ size.In multiple cancers,YAP protein which is the downstream effectors of the Hippo/YAP signaling pathway was over-expressed through various regulatory mechanisms.Inhibition of the Hippo/YAP signaling pathway stimulates the malignant proliferation of cancer cells.Based on this,we speculated whether Tax could affect the development of adult T-cell leukemia by regulating the Hippo /YAP signaling pathway.In order to confirm this hypothesis,this study analyzed the regulation of Tax on Hippo/YAP signaling pathway,and further studied the potential molecular mechanism of this regulatory function and physiological significance.The main research contents are shown as follows:PART ?: Hippo/YAP signaling pathway is inhibited in ATL cell linesTo explore the activation of Hippo/YAP signaling pathway in ATL cell lines,we examined the expression level of YAP mRNA and YAP protein,intracellular localization of YAP protein,and the expression of YAP downstream target genes.Firstly,we detected the expression of YAP by RT-PCR and Western Blot.Compared with control cell lines(PBMC and PHA-stimulated PBMC cells),the levels of YAP mRNA and protein were highly expressed in ATL cell lines(ATL-T,ATL-2,MT-4,MT-2,MT-1,TL-Om1,and ED).Subsequently,we performed immunofluorescence analysis,and the results showed that the YAP protein was mainly localized in the nucleus of the ATL cell lines(ATL-T and ATL-2)comparing with the control cell lines(Jurkat and Hut-78).Finally,our results showed that the YAP downstream target genes,such as CTGF,cyr61,RASSF4,and TIMP1,which associated with cell proliferation were also highly expressed in ATL cell lines.Taken together,the above findings indicated that the Hippo /YAP signaling pathway was inhibited in ATL cell lines.PART ?: Tax promotes YAP expression and activates its regulatory functionTo investigate the regulation of HTLV-1 on the Hippo /YAP signaling pathway and on YAP protein,we examined the effects of HTLV-1 Tax and HBZ on the expression of YAP firstly.After transfected with HTLV-1 infectious cloning,Tax induce the expression of YAP,while HBZ had no such effect.Then,we studied the molecular mechanism by which Tax promotes YAP expression.We cloned the YAP promoter and its serial mutants.In the dual-luciferase reporter assay,we found that Tax can activate the YAP promoter activity,and-158 to +117 region of YAP promoter is the main target of Tax.Further studies revealed that Tax significantly activated the activity of YAP to induce the activity of 8×GTIIC-Luc.These results demonstrated that Tax can activate the expression and function of YAP,thereby inhibiting the Hippo/YAP signaling pathway.PART ?: Molecular mechanism of Tax activates YAP functionIn order to study the molecular mechanism of Tax regulates Hippo/YAP signaling pathway,we performed co-immunoprecipitation experiments.We found that Tax can bind to YAP protein.The results of immunofluorescence experiments confirmed that they are co-localized in the nucleus.To further analyze the binding sites of each protein,we used YAP and Tax mutants for the co-immunoprecipitation analysis.The results showed that YAP mainly interacted with Tax through YAP(1-174)or YAP(204-233)or YAP(263-292)domain.Tax binded with YAP through its(99-319)domains.Further Immunofluorescence and Co-IP analysis indicated that Tax can promote the entry of YAP into the nucleus.Tax did not inhibit the binding of YAP to 14-3-3.Immunoprecipitation experiments proved that Tax inhibited the binding of YAP to LATS1 kinase and decreased the phosphorylation level of YAP S127.In addition,we performed Western Blot to examine the effect of Tax on the half-life of YAP.CHX and ubiquitination experiments demonstrated that Tax can inhibit the ubiquitination and degradation of YAP,and maintain the stability of YAP.Taken together,these results indicated that Tax can inhibit the binding of YAP to LTAS1 and suppressed phosphorylation of YAP S127 through binding to YAP.It facilitates the nuclear retention of YAP.Moreover,Tax could induce the stability of YAP,and activate the transcriptional activation function of YAP.PART ?: YAP promotes HTLV-1 infection and tumor formation in mouse modelTo explore whether Tax can affect the development of ATL by regulating Hippo/YAP signaling pathway,we stable knocked down YAP and overexpressed YAP in ATL cells.The results showed that silencing of YAP significantly inhibit the proliferation of ATL-T cells.In a virus infection experiments,we found that the YAP significantly enhanced the infectivity of HTLV-1.In YAP overexpressing ATL-T cells,YAP downstream genes which are positively associated with cell growth and migration(CTGF,C-MYC,NRP-1,HSPG,FGF-1)were significantly up-regulated,and the genes which are negatively associated with cell growth and migration(RASSF4,TIMP1,BMP2)were significantly down-regulated.Finally,in the experiments using immunodeficient mice,we injected equivalent numbers of ED negative-control and ED YAP knockdown cells into mice and analyzed tumor growth.The tumors formed by ED-shYAP cells were smaller in both size and weight than ED control tumors.Real-time PCR revealed that tumor nodules originating from ED-shYAP cells had decreased expression of cyr61 and NRP1.In conclusion,the Hippo/YAP signaling pathway is inhibited in ATL cell lines.HTLV-1Tax activates YAP-induced transcriptional activation through interacting with YAP protein.The formation of YAP/Tax complex inhibits the binding between YAP and LATS1,leading to suppression of YAP phosphorylation on Ser127.It facilitates the nuclear retention of YAP in ATL cells.In addition,Tax could induce the expression of YAP through activating its promoter activity,and Tax maintain the stability of YAP through inhibiting the ubiquitination and degradation of YAP.Finally,we found that YAP activates the infection of HTLV-1 and the tumorigenicity of ATL cells.Overexpression or suppression of YAP in ATL cells influenced the expression of YAP target genes involved in migration and cell growth.This study found that Tax promotes the development of ATL through regulating Hippo/YAP signaling pathway.It disclosed the new molecular mechanism of HTLV-1 Tax promoting ATL,and it provides a new target for diagnosis and clinical treatment of ATL.