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

Harshul Pandit, Yan Li, Xuanyi Li, Weizhong Zhang, Suping Li, Robert C. G. Martin

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/β-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model. HCC-CSCs were enriched using established serum-free culture method. Wnt/β-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of β-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 × 106 Hepa1-6 CSC spheroids or control cells in upper left liver lobe. The serum-free cultured Hepa1-6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/β-catenin signaling. Wnt/β-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3β serine-9 phosphorylation increased in Hepa1-6 spheroids. Silencing β-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. Successfully induced Hepa1-6 spheroids were identified with CSC-like properties. Aberrant β-catenin upregulation mediated by GSK-3β was observed in the Hepa1-6 spheroids. The β-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 β-catenin contributes to CSC-initiated HCC tumor growth in vivo.