Abstract 1120: Wnt/β-catenin mediated enrichment of EpCAM positive cancer stem cells promote drug resistance in hepatocellular carcinoma

Abstract

Abstract Introduction: Hepatocellular carcinoma (HCC) attains resistance to anti-cancer drugs which accounts for most of the therapeutic failures, and is one of the major factors for poor prognosis and poor overall survival of HCC patients. Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation, drug resistance and recurrence in tumor microenvironment. We hypothesize that canonical Wnt/β-catenin mediated CSC enrichment is responsible for acquired drug resistance in HCC. We aimed to study HCC CSCs for aberrant Wnt/β-catenin signaling, possible dedifferentiation, and drug resistance property. Methods: Total n=26 human HCC specimens with adjacent controls were analyzed for EpCAM and β-catenin expression by histology and western blot. In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. CSC enrichment was confirmed by analyzing for established surface markers (EpCAM, CD44, CD133, CD90) and functional markers (Aldeflour assay and Hoechst-33342 efflux). For possible dedifferentiation, we analyzed gene expression profile during enrichment process using qRT-PCR for dedifferentiation and stem cell specific genes. Drug resistance property of CSCs was studied for Doxorubicin and Sorafenib by XTT assay. To investigate CSC activation, Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets (C-Myc,Cyclin-D1,TCF1) and findings were confirmed by β-catenin knockdown, inhibitors of Wnt pathways (LiCl, XAV939, FH535) and TOP flash reporter assay. To study in vivo tumorigenesis of HCC CSCs, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells for possible lineage tracking. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Human specimens showed concomitant EpCAM and beta-catenin expression. Spheroid forming HCC CSCs showed significant higher β-catenin and EpCAM expression, and acquired drug resistance compared with control HCC cells. Silencing β-catenin by knockdown abolish drug resistance property of CSCs for Doxorubicin and reverse spheroids to adherent phenotype. Our in vivo findings confirmed EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p<0.005). Conclusions: Our findings suggest that possible dedifferentiation by canonical β-catenin confer drug resistance and CSC properties to HCC cells. Sorted EpCAM+ CSCs showed very aggressive in vivo tumor initiation and growth in immunocompetent orthotopic mouse model. This EpCAM+ CSCs population could potentially responsible for HCC recurrence and therapeutic failure. Citation Format: Harshul Pandit, Yan Li, Gouzhen Cui, Qianqian Zheng, Suping Li, Robert Martin. Wnt/β-catenin mediated enrichment of EpCAM positive cancer stem cells promote drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1120.