PDE4D Binds And Interacts With YAP To Cooperatively Promote HCC Progression

Objective: Hepatocellular carcinoma(HCC)is one of the common primary malignant tumors.Due to the insidious onset and the difficulty of early diagnosis,the prognosis is poor.Recently,the modulation of cyclic adenosine monophosphate(cAMP)has been shown to be of great interest in regulation of cell proliferation in different cell lines,including HCC cells.The expression level of the main cAMP-hydrolyzing enzyme phosphodiesterase 4D(PDE4D)is also different in various cancer tissues.However,the role and mechanism of PDE4 D in HCC progression is still unclear.Herein,we elucidated the functional role and mechanism of PDE4 D and the effects of pharmacological interventions in the progression of HCC.Methods: The correlation between the expression of PDE4 D and the survival probability of HCC patients in TCGA database was analyzed.RT-PCR and western blot was used to detect the m RNA and protein level of PDE4 D and Yes-associated protein(YAP)in HCC tissues and adjacent tissues.The mechanism of PDE4 D was further investigated by gain-and lossof-function assays in Hep G2 and MHCC-97 H cells.The functional contribution of PDE4 D to HCC progression was investigated in Hep G2 and Hep G2-stable expression of PDE4 D xenograft mouse model.In vitro and in vivo studies with pharmacological intervention were applied to address the therapeutic potential of targeting PDE4 D for HCC treatment.Results: PDE4 D was significantly upregulated in human HCC lesions and was associated with worse survival.Furthermore,PDE4 D bound to YAP and was positively correlated with YAP in HCC.PDE4 D gene silencing decreased cell proliferation,migration,and invasion in Hep G2 and MHCC-97 H cells with reduced YAP expression and increasing YAP phosphorylation.In turn,overexpression of PDE4 D promotes HCC cells proliferation with upregulated YAP expression and reducing YAP phosphorylation.Treatment with YAP inhibitor or YAP sh RNA in Hep G2 significantly reduced cell viability and the increase in YAP target genes induced by overexpression of PDE4 D.In vivo,overexpression of PDE4 D promotes tumor growth accompanied by increased YAP expression,which was attenuated by YAP inhibitor verteporfin.Of note,forced expression of YAP promoted PDE4 D and YAP target genes expression and cell growth,which were abrogated by PDE4 inhibitor roflumilast.Mechanistic insights further revealed that silencing of YAP induced PDE4 D downregulation and liver cancer cell apoptosis through activation of extracellular signalregulated kinase(ERK).The pharmacological inhibition of PDE4 D with roflumilast induced cAMP-PKA-dependent YAP phosphorylation at serine 127,restrained nuclear transfer of YAP,reduced total YAP and critical YAP target genes expression,resulting suppression of HCC cell growth and tumor progression in mice.The inhibition effects of roflumilast on YAP expression and cell growth were lost in cells expressing YAP S127 A mutant.Conclusion: Our findings reveal that PDE4 D is upregulated and binds to YAP in HCC.Targeting PDE4D-YAP signaling with selective PDE4 D inhibitor roflumilast may be an effective strategy for HCC treatment.