Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma

•Co-activation of both Akt/mTOR and Wnt/ β-catenin signaling pathways was found in 14.4% of patients with HCC. •Co-activation of these pathways confers worse survival compared to Akt/mTOR or Wnt/β-catenin activation alone. •Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics. •MDR1 was the main driver of SP phenotype in the Akt/β-catenin-driven HCC. •Activated Stat3 regulates the self-renewing and tumorigenic population of SP/CD44+ cells in Akt/β-catenin tumors. Background & Aims Hepatic resection and liver transplantation with adjuvant chemo- and radiotherapy are the mainstay of hepatocellular carcinoma (HCC) treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Only sorafenib and lenvatinib are currently approved for the first-line treatment of advanced, unresected HCC, but they yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modalities. Methods The HCC tumor model was generated by hydrodynamic transfection of AKT1 and β-catenin (CTNNB1) oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors was analyzed in vitro by using tumorsphere culture and in vivo using an allograft mouse model. Results Co-activation of both Wnt/β-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/β-catenin or Akt/mTOR pathway activation alone, demonstrating the clinical relevance of our study. In addition, we observed that Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor self-renewal and drug resistance. Consequently, we identified small molecule inhibitors of the Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/β-catenin-driven HCC. Conclusions In conclusion, we have shown that Akt/β-catenin tumors contain a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics which can be effectively targeted with inhibitors of the Jak/Stat pathway, demonstrating that inhibition of the Jak/Stat pathway could be an alternative method to overcome drug resistance and effectively treat Akt/β-catenin-driven HCC tumors. Lay summary The prognosis for patients with hepatocellular carcinoma is poor, partly because of the lack of effective treatment options for those with more advanced disease. In this study, we identified a subpopulation of cancer cells with stem cell-like properties that were critical for tumor maintenance and growth in a mouse model of hepatocellular carcinoma. Through further experiments, we demonstrated that the Jak/Stat pathway is a promising therapeutic target in hepatocellular carcinoma. Hepatic resection and liver transplantation with adjuvant chemo- and radiotherapy are the mainstay of hepatocellular carcinoma (HCC) treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Only sorafenib and lenvatinib are currently approved for the first-line treatment of advanced, unresected HCC, but they yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modalities. The HCC tumor model was generated by hydrodynamic transfection of AKT1 and β-catenin (CTNNB1) oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors was analyzed in vitro by using tumorsphere culture and in vivo using an allograft mouse model. Co-activation of both Wnt/β-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/β-catenin or Akt/mTOR pathway activation alone, demonstrating the clinical relevance of our study. In addition, we observed that Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor self-renewal and drug resistance. Consequently, we identified small molecule inhibitors of the Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/β-catenin-driven HCC. In conclusion, we have shown that Akt/β-catenin tumors contain a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics which can be effectively targeted with inhibitors of the Jak/Stat pathway, demonstrating that inhibition of the Jak/Stat pathway could be an alternative method to overcome drug resistance and effectively treat Akt/β-catenin-driven HCC tumors.