Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/beta-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model. Methods: HCC-CSCs were enriched using established serum-free culture method. Wnt/beta-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of beta-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 x 10(6) Hepa1-6 CSC spheroids or control cells in upper left liver lobe. Results: The serum-free cultured Hepa1-6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/beta-catenin signaling. Wnt/beta-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3 beta serine-9 phosphorylation increased in Hepa1-6 spheroids. Silencing beta-catenin by siRNA reversed spheroid formation phenotype. Mice injected with Hepa1-6 CSC spheroids showed aggressive tumor initiation and growth compared with mice injected with control cells. Conclusions: Successfully induced Hepa1-6 spheroids were identified with CSC-like properties. Aberrant beta-catenin upregulation mediated by GSK-3 beta was observed in the Hepa1-6 spheroids. The beta-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Our study suggests plausible cell dedifferentiation mediated by beta-catenin contributes to CSC-initiated HCC tumor growth in vivo.