The Role And Mechanism Of Cabozantinib And Cabozantinib Based Combination Therapy In Hepatocellular Carcinoma

【Background】Hepatocellular carcinoma（HCC）is the most common type of malignancy and the fourth leading cause of cancer-related deaths in the world.Patients with early stage HCC can be treated with curative strategies,including liver resection,interventional ablation and liver transplantation.However,the treatment options for advanced HCCs are restricted to systemic therapies.Despite advances in targeted therapies,the overall prognosis for advanced HCC remains dismal due to lacking the reliable biomarker for predict drug efficacy.Additionally,HCC showed limited benefit from single agent therapy and eventually developed drug resistance.Therefore,it is of great practical significance to clarify the molecular mechanism of these novel anti-HCC agents and discover potential combination therapeutic strategies for HCC.Cabozntinib is an orally bioavailable multi-kinase inhibitor which targets c-MET,VEGFR2 and AXL.Recent clinical trails have demonstrated that Cabozantinib effectively prolonged the overall survival（OS）and progression-free survival（PFS）of HCC patients who have been previously treated with Sorafenib.Therefore,Cabozantinib has been approved by EMA and FDA as second line therapy for advanced HCC.Despite the effecacy of Cabozantinib in the clinical practice of HCC,the knowledge of the underlying mechanism of how Cabozantinib exerts anti-HCC activity is still lacking.Hence,the main purpose of this study is to investigate the molecular mechanism of Cabozantinib in HCC treatment and evaluate the efficacy of Cabozantinib based combination therapy for the treatment of HCC accordingly.【Aim】1.To investigate the therapeutic efficacy as well as the molecular mechanism of Cabozantinib in HCC in vitro and in vivo.2.To explore the role of Cabozantinib in tumor microencironment and HCC metabolism.3.To study the therapeutic efficacy and molecular mechanism of combined Cabozantinib and MLN0128 treatment in HCC.4.To evaluate the therapeutic efficacy of Cabozantinib in combination with anti-PD-L1 in HCC.【Methods】1.Firstly,we assessed the efficacy of Cabozantinib on cell growth inhibiton by calculating the IC₅₀values of 14 HCC cell lines.Then we performed the Brdu cell proliferation assay,apoptosis assay as well as Brd U incorporation assay to determine the effect of Cabozantinib on cell proliferation,apoptosis and cell cycle of HCC.Next,western blot assays were conducted to illustrate the molecular targets of Cabozantinib in HCC inhibition in vitro.2.To investigate the therapeutic potential of Cabozantinib in vivo,hydrodynamic vein tail injection with plasmids encoding oncogene（s）was performed to generate 4different types of HCC mice models（c-Met/β-Catenin,Akt/c-Met,Akt/Ras,and c-Myc）.Cabozantinib was orally administered via gavage for 3 weeks in HCC mice to evaluate the therapeutic effect.Then,western blot assays as well as immunohistochemistry staining assays were conducted to reveal the molecular mechanism of Cabozantinib in HCC repression in vivo.3.The effect of Cabozantinib treatment on tumor microenvirenmont was evaluated.The status of tumor angiogenesis,tumor associated fibroblasts,and immune cells were assessed by performing the immunohistochemistry staining of the biomarkers respectively.Next,the glucose uptake,cholesterol levels,and triglycerides levels of HCC cells were assessed to investigate the effect of Cabozantinib treatment in HCC metabolism.The protein expression of glycolytic enzymes as well as lipometabolic enzymes was analyzed by western blot assays to clarify the poteintial targets of Cabozantinib in metabolic reprogramming of HCC.4.In vitro studies were performed to assess the therapeutic potential and the molecular mechanisms of combined Cabozantinib and MLN0128 treatment in HCC cells.The c-Met/β-Catenin HCC mouse model was used to determine the therapeutic efficacy of Cabozantinib/MLN0128 combination therapy in vivo.5.Different oncogene（s）induced HCC mouse models were recruited to test the therapeutic potential of combined Cabozantinib and anti-PD-L1 therapy in vivo.【Results】1.We demonstrated that Cabozantinib suppressed HCC cell growth in culture with variable IC₅₀ values.The sensitivities of Cabozantinib correlated with p-MET,AXL and p-ERK expression in HCC cell lines.Mechanistically,we demonstrate that Cabozantinib effectively suppressed HCC growth via inhibiting p-MET,p-ERK and p-AXL expression in vitro.2.We discovered that Cabozantinib treatment led to stable disease in c-Met/β-Catenin and Akt/c-Met mouse HCC,while possessing limited efficacy on Akt/Ras and c-Myc liver tumors.Mechanismly,Cabozantinib effectively inhibited p-MET/p-ERK activity,leading to increased p21 expression in c-Met/β-Catenin and Akt/c-Met HCC.However,Cabozantinib was ineffective in inhibiting the Akt/m TOR cascade.Cabozntinib induced apoptosis in all four HCC mouse models.3.We demonstrated that Cabozantinib effectively suppressed HCC glycolysis by inhibiting the activation of p-ERK/HK2 pathway.Cabozantinib also suppressed the p-VEGFR2,leading to decreased tumor angiogenesis in HCC mouse models regardless of the oncogenic drivers.Cabozantinib treatment did not affect the immune microenvironment or the cancer associated fibroblasts of mouse HCC.4.Combining Cabozantinib with AKT/m TOR cascade inhibitor MLN0128synergistically inhibited HCC cell growth in vitro.In vivo experiments showed that MLN0128 treatment didn’t affect HCC progression in c-Met/β-Catenin mice.However,Combining Cabozantinib with MLN0128 almost completely suppressed cell proliferation and led to tumor regression in c-Met/β-Catenin mice.5.We demonstrated that Cabozantinib did not affect PD-L1 expression of HCC in vitro and in vivo.Anti-PD-L1 treatment didn’t affect HCC progression in vivo.Combined treatment with Cabozantinib and the checkpoint inhibitor anti-PD-L1 antibody did not provide any additional therapeutic benefit in c-Met/β-Catenin and Akt/c-Met mouse HCC.【Conclusions】1.The p-MET/p-ERK/p21/PKM2 cascade and VEGFR2-induced angiogenesis are the primary targets of Cabozantinib in HCC treatment.p-MET levels could be a reliable biomarker for Cabozantinib efficacy.2.Combination therapies with Cabozantinib and m TOR inhibitor may be effective against HCC via inhibiting both p-MET/p-ERK pathway and AKT/m TOR cascade.3.Cabozantinib treatment didn’t significantly affect HCC immune microenvirenment.Combined Cabozantinib with anti-PD-L1 treatment didn’t lead tumor regression in c-Met/β-Catenin and Akt/c-Met mouse HCC.