The Role Of Hippo Pathway Effector Protein YAP In Regulating Proliferation Of Leukemic Cells And Its Molecular Mechanism

Adult T-cell leukemia is a kind of malignant lymphoid tumors caused by Human T-cell leukemia virus type 1(HTLV-1).HTLV-1 encodes a series of regulatory genes including Tax,Rex,p12,p21 and HBZ.Tax,HBZ and other viral proteins can regulate multiple signaling pathways in vivo,such as NF-?B,AP-1,TGF-?/Smad,etc.The activation of NF-?B signaling pathway plays a very important role in the carcinogenesis of ATL,which can promote the proliferation of ATL cells and induce the development of ATL.However,the molecular mechanism of HTLV-1 induced activation of NF-?B signaling is not clearly yet.The Hippo signaling pathway was initially found in Drosophila,and it mainly regulates organ development and regeneration in mammals.In addition,the Hippo signaling pathway inhibits cell proliferation and induces apoptosis by negatively regulating its downstream protein YAP.YAP is an oncogene,which is closely related to the malignant proliferation of cancer cells and poor prognosis of multiple cancers.Studies have shown that YAP is highly expressed in multiple tumors.At present,the function of YAP protein in ATL cells has not been studied.Therefore,our study mainly focus on studying the role of Hippo pathway key protein YAP in regulating the malignant proliferation of leukemia cells,and verifies its molecular mechanism.The main contents of this study are shown as follows:Part I:YAP is overexpressed in ATL cellsIn order to study the expression of YAP in ATL cells,the mRNA level and protein level of YAP were detected by RT-PCR and Western blot.Results showed that compared with the HTLV-1 negative cell lines(MOLT-4,Hut78,Jurkat),YAP genes was highly expressed in ATL cell lines(ATL-T,ATL-2,MT-1,MT-2,MT-4,TL-Om1).Western bolt results also showed that YAP protein was overexpressed in the ATL cell lines.Part II:YAP promotes malignant proliferation of ATL cells.In order to study whether YAP can regulate the malignant proliferation of ATL cells,we used lentivirus mediated gene silencing technique to knock down the expression of YAP gene in ATL-T and ATL-2 cells,and then selected stable knock-down cells.Subsequently,we preformed MTT assay,clone formation assay and flow cytometry to study the effect of YAP silencing on cell proliferation and apoptosis.The results showed that knock down of YAP inhibit the proliferation of ATL-T and ATL-2 cells,and cell apoptosis was significantly increased in YAP silencing cells.To confirm these results,we used a YAP specific inhibitor Verteporfin.The results showed that the proliferation of ATL-T and ATL-2 cells was also significantly inhibited after the treatment of YAP inhibitor.The above results indicate that YAP could promote the malignant proliferation of ATL cells and inhibit apoptosis.Part ?:Tax activates YAP through NF-?B signaling pathwayIn order to explore the regulatory effect of HTLV-1 on YAP protein and its molecular mechanism,we used dual luciferase reporter assay to test the activation of YAP in ATL cells.The results showed that the CTGF-Luc reporter was significant activated in ATL cells,indicating that the function of YAP in ATL was activated.To further confirm the result,we studied the effects of HTLV-1 on the function of YAP protein by using HTLV-1 infectious clone transfecting Jurkat cells method and by co-culturing Jurkat cells with HTLV-1 positive cells ATL-T.The results showed that HTLV-1 could significantly activate the function of YAP protein.Subsequently,we studied the role of viral protein Tax and HBZ on YAP activation.The reporter assay results showed that Tax could activate YAP,while HBZ has no obvious effect.Mutation assay showed that Tax-M22 mutants(lacking the ability to activate NF-?B)failed to activate YAP.To confirm this result,we used NF-?B inhibitory plasmid I?B(S32A/S36A),and found that Tax induced activation of YAP was inhibited when NF-T?B was blocked.Taken together,these results show that Tax can activate YAP,and the activation was depend on the NF-?B signaling pathway.Part IV:p65 induced activation of YAP and its molecular mechanismIn order to explore whether NF-?B/p65 could activate the function of YAP,we first study the effect of NF-?B/p65 on YAP by reporter assay.Results showed that p65 could significantly enhance the activity of 8×GTIIC-Luc(YAP downstream effector reporter),indicating that p65 can activate YAP.Subsequently,we studied the molecular mechanism of NF-?B/p65 mediated activation of YAP.The interaction of p65 and YAP protein was found by immunoprecipitation assay.Further study shown that p65 inhibiter the phosphorylation of YAP-S127,and blocked the binding of YAP with its inhibitory regulation protein 14-3-3.The result of fluorescence observation and western blot assay shown that p65 could induce the nuclear localization of YAP protein.Taken together,the above results show that NF-?B/p65 could interact with YAP can inhibit the phosphorylation of YAP,and then block the binding between YAP and 14-3-3 protein,thereby promoting YAP translocated intonucleus,leading to the activation of YAP function.In conclusion,we found that YAP,a key protein of Hippo signaling pathway,can promote the growth of adult T-cell leukemia cells and NF-?B/p65 could activate p65 through interacting with YAP.This study provides a basis for the mechanism of adult T cell leukemia,and also provides a new idea and strategy for the clinical treatment of adult T-cell leukemia.