Blocking CDK1/PDK1/ß-Catenin signaling by CDK1 inhibitor RO3306 increased the efficacy of sorafenib treatment by targeting cancer stem cells in a preclinical model of hepatocellular carcinoma.

Rationale: Hepatocellular carcinoma (HCC) is an aggressive malignant solid tumor wherein CDK1/PDK1/ß-Catenin is activated, suggesting that inhibition of this pathway may have therapeutic potential. Methods: CDK1 overexpression and clinicopathological parameters were analyzed. HCC patient-derived xenograft (PDX) tumor models were treated with RO3306 (4 mg/kg) or sorafenib (30 mg/kg), alone or in combination. The relevant signaling of CDK1/PDK1/ß-Catenin was measured by western blot. Silencing of CDK1 with shRNA and corresponding inhibitors was performed for mechanism and functional studies. Results: We found that CDK1 was frequently augmented in up to 46% (18/39) of HCC tissues, which was significantly associated with poor overall survival (p=0.008). CDK1 inhibitor RO3306 in combination with sorafenib treatment significantly decreased tumor growth in PDX tumor models. Furthermore, the combinatorial treatment could overcome sorafenib resistance in the HCC case #10 PDX model. Western blot results demonstrated the combined administration resulted in synergistic down-regulation of CDK1, PDK1 and ß-Catenin as well as concurrent decreases of pluripotency proteins Oct4, Sox2 and Nanog. Decreased CDK1/PDK1/ß-Catenin was associated with suppression of epithelial mesenchymal transition (EMT). In addition, a low dose of RO3306 and sorafenib combination could inhibit 97H CSC growth via decreasing the S phase and promoting cells to enter into a Sub-G1 phase. Mechanistic and functional studies silencing CDK1 with shRNA and RO3306 combined with sorafenib abolished oncogenic function via downregulating CDK1, with downstream PDK1 and ß-Catenin inactivation. Conclusion: Anti-CDK1 treatment can boost sorafenib antitumor responses in PDX tumor models, providing a rational combined treatment to increase sorafenib efficacy in the clinic.

Efficacy of annexin A3 blockade in sensitizing hepatocellular carcinoma to sorafenib and regorafenib.

BACKGROUND & AIMS: Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Sorafenib is the only FDA-approved first-line targeted drug for advanced HCC, but its effect on patient survival is limited. Further, patients ultimately present with disease progression. A better understanding of the causes of sorafenib resistance, enhancing the efficacy of sorafenib and finding a reliable predictive biomarker are crucial to achieve efficient control of HCC. METHODS: The functional effects of ANXA3 in conferring sorafenib resistance to HCC cells were analyzed in apoptotic and tumorigenicity assays. The role of ANXA3/PKCδ-mediated p38 signaling, and subsequently altered autophagic and apoptotic events, was assessed by immunoprecipitation, immunoblotting, immunofluorescence and transmission electron microscopy assays. The prognostic value of ANXA3 in predicting response to sorafenib was evaluated by immunohistochemistry. The therapeutic value of targeting ANXA3 to combat HCC with anti-ANXA3 monoclonal antibody alone or in combination with sorafenib/regorafenib was investigated ex vivo and in vivo. RESULTS: ANXA3 conferred HCC cells with resistance to sorafenib. ANXA3 was found enriched in sorafenib-resistant HCC cells and patient-derived xenografts. Mechanistically, overexpression of ANXA3 in sorafenib-resistant HCC cells suppressed PKCδ/p38 associated apoptosis and activated autophagy for cell survival. Clinically, ANXA3 expression correlated positively with the autophagic marker LC3B in HCC and was associated with a worse overall survival in patients who went on to receive sorafenib treatment. Anti-ANXA3 monoclonal antibody therapy combined with sorafenib/regorafenib impaired tumor growth in vivo and significantly increased survival. CONCLUSION: Anti-ANXA3 therapy in combination with sorafenib/regorafenib represents a novel therapeutic strategy for HCC treatment. ANXA3 represents a useful predictive biomarker to stratify patients with HCC for sorafenib treatment. LAY SUMMARY: This study represents the most extensive pre-clinical characterization of anti-ANXA3 monoclonal antibodies for the treatment of hepatocellular carcinoma to date. These results support the clinical trial development of anti-ANXA3 antibodies in combination with sorafenib/regorafenib. Further studies will optimize patient target selection and identify the best treatment combinations.

Interferon-gamma inducible protein 10 (IP10) induced cisplatin resistance of HCC after liver transplantation through ER stress signaling pathway.

Tumor recurrence remains an obstacle after liver surgery, especially in living donor liver transplantation (LDLT) for patients with hepatocellular carcinoma (HCC). The acute-phase liver graft injury might potentially induce poor response to chemotherapy in recurrent HCC after liver transplantation. We here intended to explore the mechanism and to identify a therapeutic target to overcome such chemoresistance. The associations among graft injury, overexpression of IP10 and multidrug resistant genes were investigated in a rat liver transplantation model, and further validated in clinical cohort. The role of IP10 on HCC cell proliferation and tumor growth under chemotherapy was studied both in vitro and in vivo. The underlying mechanism was revealed by detecting the activation of endoplasmic reticulum (ER) stress signaling pathways. Moreover, the effect of IP10 neutralizing antibody sensitizing cisplatin treatment was further explored. In rat liver transplantation model, significant up-regulation of IP10 associated with multidrug resistant genes was found in small-for-size liver graft. Clinically, high expression of circulating IP10 was significant correlated with tumor recurrence in HCC patients underwent LDLT. Overexpression of IP10 promoted HCC cell proliferation and tumor growth under cisplatin treatment by activation of ATF6/Grp78 signaling. IP10 neutralizing antibody sensitized cisplatin treatment in nude mice. The overexpression of IP10, which induced by liver graft injury, may lead to cisplatin resistance via ATF6/Grp78 ER stress signaling pathway. IP10 neutralizing antibody could be a potential adjuvant therapy to sensitize cisplatin treatment.

Rationale of personalized immunosuppressive medication for hepatocellular carcinoma patients after liver transplantation.

Liver transplantation is the only potentially curative treatment for hepatocellular carcinoma (HCC) that is not eligible for surgical resection. However, disease recurrence is the main challenge to the success of this treatment. Immunosuppressants that are universally used after transplantation to prevent graft rejection could potentially have a significant impact on HCC recurrence. Nevertheless, current research is exclusively focused on mammalian target of rapamycin inhibitors, which are thought to be the only class of immunosuppressive agents that can reduce HCC recurrence. In fact, substantial evidence from the bench to the bedside indicates that other classes of immunosuppressants may also exert diverse effects; for example, inosine monophosphate dehydrogenase inhibitors potentially have antitumor effects. In this article, we aim to provide a comprehensive overview of the potential effects of different types of immunosuppressants on HCC recurrence and their mechanisms of action from both experimental and clinical perspectives. To ultimately improve the outcomes of HCC patients after transplantation, we propose a concept and approaches for developing personalized immunosuppressive medication to be used either as immunosuppression maintenance or during the prevention/treatment of HCC recurrence.

A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.

F-actin reorganization and inactivation of rho signaling pathway involved in the inhibitory effect of Coptidis Rhizoma on hepatoma cell migration.

HYPOTHESIS: Hepatocellular carcinoma (HCC) is one of the most malignant human tumors and one of the risk factors is its highly metastatic property. Coptidis Rhizoma aqueous extract (CRAE) is able to suppress the migration and invasion of HCC cells, MHCC97-L, and F-actin reorganization and Rho signaling inhibition is involved. MAIN METHODS: CRAE was prepared and analyzed by high-performance liquid chromatography combined with mass spectrometry. The cytotoxicity and antimigration action of CRAE on MHCC97-L cells were evaluated; Immunofluorescence and immunoblotting were used to investigate the proposed mechanism of CRAE action. KEY FINDINGS: Chemical analysis reveals that the active components in CRAE are berberine and berberine-like alkaloids. CRAE exhibits a significant inhibitory effect on MHCC97-L cell migration as indicated by wound-healing and invasion-chamber assays. No significant alteration of matrix metalloproteinases and urokinase-type plasminogen activator (uPA) expression were observed in MHCC97-L cells exposed to CRAE. Reduction of F-actin polymerization and damage to cytoskeleton network in MHCC97-L cells were observed after CRAE treatment. Furthermore, it was found that CRAE significantly downregulated the Rho/ROCK signaling pathway. SIGNIFICANCE: These results indicate that CRAE may act as a Rho/ROCK signaling inhibitor to suppress MHCC97-L cell migration in vitro and suggested that total alkaloids in Coptidis Rhizoma may be a potential agent for suppressing liver cancer invasion.