Abstract A52: c-Met and β-catenin independently regulate cancer stem cell self-renewal and maintenance in hepatocellular carcinoma cells.

Background: Physiologic activation of the hepatocyte growth factor receptor, c-Met, or Wnt/β-catenin has been shown to drive a variety of cellular processes including cell proliferation, motility and tissue repair. In hepatocellular carcinoma (HCC) overexpression and/or constitutive activation of c-Met or β-catenin is correlated with increased tumorigenesis and poor patient prognosis. Moreover, interactions between the c-Met or β-catenin signaling pathways have been described. Cancer stem cells (CSCs) are thought to play a key role in malignant tumor growth, as well as tumor recurrence and metastasis. Identification of the mechanisms that regulate CSC growth and survival is, therefore, of crucial importance for the development of HCC therapeutics. In this regard, we have recently shown that ionizing radiation or chemotherapy can induce stemness in HCC cells via upregulation of the pluripotency factors Oct3/4 and Sox2. Aim: The goal of this study was to determine whether c-Met plays a role in the self-renewal and maintenance of HCC CSCs, and whether crosstalk between the c-Met and Wnt signaling pathways participates in this process.Methods: To examine the role of c-Met or β-catenin HCC cells were transfected with asymmetric interfering RNAs (aiRNAs) against c-Met or β-catenin (100nM). Cell viability was assessed by MTT assay. Levels of phospho/total c-Met and phospho/total β-catenin protein were evaluated by Western blot. Levels of the c-Met signaling proteins Akt and Erk1/2 were also investigated. CSC self-renewal was assessed by spherogenesis assay. Single cell proliferation was analyzed by colony formation assay. Results: Huh7 and HepG2 cell viability was significantly inhibited by treatment with aiRNAs against c-Met or β-catenin. Moreover, aiRNA knockdown of c-Met inhibited c-Met phosphorylation and reduced HCC sphere formation by 50% compared to untreated cells, or cells treated with a control GFP aiRNA. Similarly, treatment with β-catenin aiRNA inhibited HCC self-renewal ability by ∼60% in both cell lines. aiRNA-mediated blockade of c-Met or β-catenin also reduced the colony formation ability of both Huh7 and HepG2 cells. Silencing of c-Met, however, had little effect on β-catenin expression suggesting c-Met and β-catenin independently regulate HCC CSC self-renewal. Similar results were obtained when cells were treated with the c-Met inhibitor Tivantinib. Conclusion: Our findings demonstrate that c-Met plays a key role in the self-renewal, growth and maintenance of HCC CSCs. Moreover, this appears to be independent of β-catenin signaling. These findings suggest that combined targeting c-Met and β-catenin may be an effective strategy for HCC therapeutics development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A52. Citation Format: Laura Ghisolfi, Andrew C. Keates, Dong-ki Lee, Chiang J. Li. c-Met and β-catenin independently regulate cancer stem cell self-renewal and maintenance in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A52.