BackgroundTumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.MethodsGene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.ConclusionTGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.

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Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality and the incidence of HCC is rising in the United States. Cancer stem cells (CSCs) are a subset of neoplastic cells that possess distinct survival mechanisms and plasticity crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by Transforming Growth Factor-β (TGF-β) has been linked to the acquisition of CSC characteristics in breast cancer. In patients with HCC, either a CSC or EMT phenotype correlates with a worse prognosis. In this report, we aim to link EMT to CSCs and to further elucidate the mechanism by which cells acquire CSC characteristics during EMT. We utilized human (Huh7, MHCC97-L) and murine (P2E, P2M; Ding et al Hepatology. 2010) liver cancer cell lines, and analysis included gene and protein expression, FACS, migration and invasion assays, and chemotherapy resistance assay. Human mesenchymal MHCC97 cells demonstrate decreased E-cadherin, increased Snail1, and enhanced migration and invasion compared to epithelial Huh7 cells. MHCC97 cells demonstrate significant upregulation of stem cell markers (Oct4, Nanog, BMI-1, and CD44; p<0.01), increased CD44high population (95% vs. 2% Huh7, p<0.01), resistance to chemotherapy, resistance to TRAIL-induced apoptosis, increased tumor-sphere formation and slower cell cycle compared to epithelial Huh7 cells. We recently reported EMT in murine epithelial liver cell lines, P2E and P2M (post EMT). Mesenchymal P2M cells demonstrate loss of E-cadherin, gain of Snail1, increased tumor-sphere formation, and increased expression of stem cell markers (Nanog, Oct4, CD44, and EpCam; p<0.001). As TGF-β induces EMT through Snail1, we hypothesize that Snail1 signaling plays a role in the induction of an EMT and CSC phenotype in HCC. TGF-β treatment, epithelial P2E cells demonstrate fibroblastic-like morphology, 14-fold increase in Snail1 (p<0.01) and 8 fold increase in Nanog expression (p<0.01), increased tumor-sphere formation and migration. siRNA knockdown of Snail1 in mesenchymal P2M cells resulted in significant decrease in EMT and CSC characteristics, with decreased tumor-sphere formation, decreased migration, and decreased stem cell gene expression (Nanog and CD44, p<0.01). In conclusion, both murine and human mesenchymal HCC cells possess multiple CSC characteristics compared to epithelial counterparts. The transient knockdown of Snail1 in mesenchymal HCC cells results in the reversal of EMT as well as loss of CSC characteristics. We propose that Snail1 may play an important role in the regulation of EMT and CSC phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3429. doi:10.1158/1538-7445.AM2011-3429