Dual Role Of PID1 In Regulating Apoptosis Induced By Anti-cancer Agents In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma（HCC）treatment efficiency is severely hampered due to its etiology and drug resistance,and increased understanding of genetic mechanisms underlying response of HCC to various anti-cancer agents is required.Phosphotyrosine interaction domain-containing protein 1（PID1）has been reported to be associated with cell proliferation,differentiation and lipids metabolism.However,little is known about the functions and underlying molecular mechanisms by which PID1regulates anti-cancer agents-induced apoptosis in HCC.Objective:The present study was aimed at identifying the role of PID1 in regulating response of HCC to different anti-cancer agents.Methods:（1）Apoptosis,ROS production and mitochondrial membrane potential were detected via flow cytometric analysis to determine the effects of PID1 on apoptosis induced by distinct anti-cancer agents.Xenograft model was constructed to further confirmed the effects of PID1 on efficacy of Doxorubicin and Sorafenib.（2）Bcl2 and Bcl-x L were inhibited to determine the role of BAD in PID1-mediated anti-apoptotic effects.Mitochondria isolation assay,Western blotting and immunofluorescence were performed to determine the effects of PID1 on mitochondrial BAD location and release of cytochrome c.AKT inhibition and Raf-1 knockdown were performed to determine the role of AKT/Raf-1 interaction in PID1-mediated anti-apoptotic effects.（3）Western blotting analysis was conducted to evaluate the effects of PID1 on AKT activation.Co-immunoprecipitation assay was conducted to determine the interaction between PID1 and PDPK1.Immunofluorescence was performed to detect the intracellular location of PID1during AKT activation.（4）Western blotting time-course analysis was conducted to evaluate the effects of PID1 on survival-related kinases upon Sorafenib treatment.BRAF inhibition and Raf-1 knockdown were performed to determine the mechanisms by which PID1 regulated survival-related kinases upon Sorafenib treatment.Results:（1）PID1 overexpression promoted resistance of hepatoma cells to conventional chemotherapeutic agents-induced mitochondrial dysfunction and apoptosis,but potentiated the efficacy of Sorafenib.Cell viability curves revealed that PID1 deficiency increased sensitivity of hepatoma cells to conventional chemotherapeutic agents,but declined efficacy of Sorafenib.（2）PID1 overexpression restrained recruitment of BAD to mitochondria and blocked release of cytochrome c induced by H₂O₂ and Doxorubicin.PID1 facilitated Raf-1 activation in an AKT-dependent manner.（3）PID1 did not affect insulin-induced AKT activation within 1 h,but subsequently restrained consistent AKT activation.PID1 translocated to cell membrane following growth factors stimulus and interacted with PDPK1 at phosphorylated tyrosine sites（Y9,Y373,Y376）to inhibits PDPK1.PID1 facilitates growth factors-mediated BAD inhibition,whereas Raf-1knockdown blocked this effect.（4）PID1 accelerated Sorafenib-induced Raf-1 activation and facilitated heterodimerization of Raf-1/BRAF,thus blocking AKT activation via Raf-1/BRAF/ERKMEK1 pathway.BRAF inhibition or Raf-1 knockdown could remove PID1-induced blockade in AKT activation upon Sorafenib treatment.Moreover,PID1 deficiency increased survival rates of HCC xenografts mice when treated with Doxorubicin,but declined efficacy of Sorafenib.Conclusions:PID1 regulates apoptosis induced by anticancer agents in an AKT/Raf-1-dependent manner.On the one hand,PID1 relieves AKT-induced Raf-1 inactivation and facilitates AKT or Raf-1-mediated BAD inhibition,thus restraining apoptosis induced by conventional chemotherapeutic agents.On the other hand,PID1 alters activity patterns of survival-related kinase and blocks AKT activation via Raf-1/BRAF/ERK/MEK1 pathway,thereby potentiating efficacy of Sorafenib.Our results suggest that PID1 may be a underlying biomarker of HCC and its upregulation predicts sensitivity to Sorafenib but resistance to conventional chemotherapeutic agents.