Studies On The Mechanisms Of Choline Kinase Alpha-induced Tumor Progression And Drug Resistance In Hepatocellular Carcinoma

Part I Studies on The Mechanisms of Choline Kinase Alpha-induced Tumor Progression in Hepatocellular Carcinoma Background & Aims:Hepatocellular Carcinoma(HCC)ranks the sixth most common malignancies worldwide,and is one of the leading causes of cancer related deaths.Despite recent advancements in the diagnosis and treatment approaches,the prognosis of HCC patients remains dismal due to postsurgical recurrence and distant metastasis.Great efforts have been made to explore the mechanism underlying the pathogenesis of HCC during the past decades;however,the detailed molecular events contributing to HCC progression and metastasis are still not fully understood.Therefore,there remains and an urgent need to search for novel prognostic biomarkers and therapeutic targets for HCC therapy.Choline kinase alpha(CHKA)is the enzyme responsible for the biosynthesis of phosphocholine from choline and its expression has been reported to be higher in multiple human cancers and correlate with more aggressive phenotype.Activated choline metabolism,which is characterized by increased phosphocholine and total choline-containing compounds,is a fairly new metabolic hallmark of cancer.Previous studies from our research group uncovered CHKA to be increased in human HCCs,compared to non-tumor tissues,and increased expression to be associated with tumor aggressiveness and reduced survival times of patients.Overexpression of CHKA in HCC cell lines increased their metastasis,invasiveness,and ability to form metastatic tumors in mice by activating AKT signaling.However,the detailed mechanisms by which CHKA activates AKT signaling in HCC were still largely unknown.Methods:We used lentivirus system to construct CHKA overexpression and knockdown cell lines and their control counterparts.We used growth factors for signaling pathway stimulation.We used small interfering RNA-mediated gene knockdown and specific inhibitors for inhibition of selected genes.Western-blot analysis was employed to detect levels of signal transduction proteins.Cell metastasis and invasiveness were analyzed by transwell assays and Matrigel invasion assays.Protein interactions and colocaliazations were analyzed by Immunoprecipitation(IP)and Immunofluorescence(IF)assays.CHKA and p-AKT(Ser473)protein levels in HCC specimen were analyzed by Immunohistochemistry(IHC).Products of choline metabolism were analyzed by ELISA.Results:We found that CHKA physically interacts with EGFR,and promotes EGFR auto-phosphorylation and dimerization on plasma membrane.Moreover,CHKA promotes HCC cells invasion and metastasis mainly through integrating EGFR-mTORC2-AKT(Ser473)axis,acting as an adaptor molecule which mediates protein interactions between EGFR and mTORC2 to form EGFR/CHKA/mTORC2 complex.In addition,PI3 K,PDK1 and activated choline metabolism is not necessary for CHKA induced AKT(Ser473)activation and HCC progression.Combination of CHKA and p-AKT(Ser473)provides improved prognostic value for HCC.Conclusion:In this study,our results suggest a novel pathway driven by overexpressed CHKA.The detailed molecular mechanisms include its interaction with EGFR,which leads to enhanced dimerization of EGFR and membrane retention,resulting in enhanced EGF-mediated activation of AKT.In addition,CHKA serves as an adaptor protein that links EGFR with mTORC2,which activates AKT at Ser473.Finally,targeting this novel pathway rather than the classical PI3K-PDK1-AKT(Thr308)promotes tumor progression in HCC.CHKA protein itself may be more important than its catalytic role in this model.Among the aberrant genes located in chromosome 11 q.13,CHKA may serve as a cancer driver that promotes HCC progression with metastasis.Part II Studies on The Roles and Mechanisms of Choline Kinase Alpha in EGFR-targeted Therapy for HepatocellularCarcinoma Background & Aims:Epidermal Growth Factor Receptor(EGFR)has a vital role in tumorigenesis,promoting cell proliferation,survival,invasion and metastasis in cancers,and was a bona therapeutic target.The drugs targeting EGFR include the tyrosine kinase inhibitors such as Gefitinib and Erlotinib,and the monoclonal antibody Cetuximab.They have been widely used in lung cancer,breast cancer and colon cancer.EGFR overexpression occurs in about 40-70% of human HCC samples and is linked with cancer development,providing theoretical possibilities for application of anti-EGFR drugs for HCC treatment.However,only moderate effects and disappointing results of the drugs were found out during the clinical trials with unselected patients,with the reasons remaining obscure.In our previous studies,we found that CHKA was commonly overexpressed in Human HCC samples,and that CHKA acted as an adaptor protein to mediate interaction between EGFR and mTORC2,which was required for downstream AKT activation to promote tumor progression in HCC.Since CHKA is closely connected with EGFR-AKT signaling,its role in EGFR-targeted therapy for HCC remains largely unknown.Methods:We used short-term and long-term growth inhibition assay to analyzed the sensitivity of HCC cell lines to EGFR-targeted drugs Gefitinib and Erlotinib.We performed western-blot analysis to examine the levels of signal transduction proteins and apoptotic markers.We used small interfering RNA-mediated gene knockdown for selected gene silencing.We employed xenograft tumor formation experiment to examine the sensitivity of HCC cells to Erlotinib treatments in vivo.Results:We found that overexpression of CHKA rendered HCC cells to becoming resistant to anti-EGFR drugs Gefitinib and Erlotinib,and that knockdown of CHKA significantly increased HCC cells' sensitivity to Anti-EGFR drugs in vitro.Overexpression of CHKA also rendered HCC cells to becoming resistant to Erlotinib in vivo.Knockdown of RICTOR significantly reversed the resistance to anti-EGFR drugs of HCC cells induced by CHKA.Conclusion:Our results suggest an important role of CHKA in the resistance to EGFR-targeted therapy for HCC.Aberrant EGFR-mTORC2-AKT signaling driven by overexpressed CHKA is indispensable that it impairs HCC cells' sensitivity to EGFR inhibitors.In light of these results,CHKA may be not only used as a potential marker for the application of anti-EGFR drugs in HCC,but also a potential therapeutic target for EGFR-positive cancer prevention and treatment.