Introduction: Recurrence of Hepatocellular carcinoma (HCC) after definitive tumor treatment occurs in over 70% of patients within 14 months of initial treatment. The recurrent HCC cells demonstrate a more aggressive tumor growth pattern when compared with the initial HCC cells. Recurrent HCC attains resistance to chemotherapy which accounts for most of the therapeutic failures and is one of the major factors for poor overall survival (OS) in this disease. Underlying molecular mechanism(s) responsible for acquired chemotherapy resistance and tumor recurrence has not been well defined. Cancer stem cells (CSCs) are a sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. We hypothesize that in HCC, Wnt/β-catenin mediated CSC activation is responsible for acquired chemoresistance. Here, we aimed to study that enriched culture of EpCAM positive HCC cells with cancer stem-cell like properties could become resistant to anti-cancer drugs. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM, CD90, CD44, CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. Tumorigenic potential of enriched CSCs was studied in orthotopic HCC mouse models. Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets i.e. C-Myc, Cyclin-D1, TCF1. Lentivirus mediated overexpression and knockdown of Wnt/β-catenin signaling components confirmed effect of Wnt/β-catenin signaling on activation of CSCs and drug resistance. Total 24 paired human HCC specimens were obtained and analyzed to confirm in vitro and in vivo findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression (EpCAM+) and significant higher chemoresistance compared with control HCC cells. ABCG2 expression (and its efflux activity) was found to be increased in CSCs, which further supports acquired chemoresistance. EpCAM+ CSCs have shown significant higher tumor proliferation rate in the mouse models compared with control. Wnt/β-catenin signaling activity was found to be increased in EpCAM+ CSCs compared with control, and associated with chemoresistance. Human HCC specimens confirmed concomitant increase/decrease in EpCAM and β-catenin expression. Conclusions: Subpopulation of EpCAM+ CSCs, (1) contribute to acquired chemoresistance in HCC, and (2) showing increased Wnt/β-catenin signaling. We believe, this EpCAM+ CSCs population is primarily responsible for HCC recurrence and therapeutic failure.

Citation Format: Harshul Pandit, Yan Li, Guozhen Cui, Xuanyi Li, Suping Li, Yingbin Yang, Jack Rostas, Robert C.G. Martin. EpCAM-positive cancer stem cells acquired chemoresistance in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2535.