Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-beta1 and Ha-Ras: steps towards invasiveness.
ABSTRACT In hepatocarcinogenesis, it is an open question whether transforming growth factor (TGF)-beta1 provides a tumor-suppressive or a tumor-promoting role. To address this question, we employed immortalized murine hepatocytes, which display a high degree of differentiation and, expectedly, arrest in the G1 phase under exposure to TGF-beta1. These hepatocytes maintain epithelial polarization upon expression of oncogenic Ha-Ras. However, Ras-transformed hepatocytes rapidly convert to a spindle-shaped, fibroblastoid morphology upon treatment with TGF-beta1, which no longer inhibits proliferation. This epithelial to fibroblastoid conversion (EFC) is accompanied by disruption of intercellular contacts and remodeling of the cytoskeletal framework. Fibroblastoid derivatives form elongated branching cords in collagen gels and grow to severely vascularized tumors in vivo, indicating their increased malignancy and even invasive phenotype. Additionally, fibroblastoid cells secrete strongly enhanced levels of TGF-beta1, suggesting an autocrine regulation of TGF-beta signaling. Expression profiling further revealed that the loss of the adhesion component E-cadherin correlates with the upregulation of its transcriptional repressor Snail in fibroblastoid cells. Moreover, the phosphoinositide 3-OH (PI3) kinase pathway was required for the maintenance of EFC, as inhibition of PI3 kinase reverted fibroblastoid cells to an epithelial-like phenotype. Taken together, these data indicate a dual role of TGF-beta1 in hepatocytes: it induces proliferation arrest but provides a crucial function in promoting late malignant events in collaboration with activated Ha-Ras.
SourceAvailable from: Trina Das[Show abstract] [Hide abstract]
ABSTRACT: Aims: Recurrence is a major cause of mortality for patients with hepatocellular carcinoma (HCC) following liver transplantation. We previously identified RIOK3 as a prominently expressed gene in HCC tumors that recurred following transplantation. This is consistent with findings of increased RIOK3 expression in metastatic head, neck, and pancreatic cancers, which suggests RIOK3 is involved in cancer recurrence. In this study, we aimed to determine the pathway leading to HCC tumor invasion induced by RIOK3. Methods: Ectopic RIOK3 expression was created by introducing a RIOK3 expression plasmid construct in HepG2 cell line. We studied EMT related markers, E-cadherin, Snail1, and Twist1 by Western blot and immunofluorescence analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. Activation of Rac1 and WNT/β -catenin pathway were analyzed by western blot and quantitative PCR assays. Results: We demonstrated that RIOK3 transformed the epithelial HCC cell line, HepG2, into a mesenchymal phenotype via EMT induction. When transfected into HepG2 cells, RIOK3 suppressed E-cadherin expression and induced Snail1 and Twist1 expression. This correlated with metastasis-promoting, cell-specific morphological changes and increased cellular invasiveness. We also determined that RIOK3 overexpression promotes activation of the small G protein Rac1 and activation of Wnt/β -catenin signaling at multiple levels, including upregulation of Wnt6, Wnt11, FzD3 and FzD7 transcription, stabilization of β -catenin, and activation of WNT/β -catenin transcriptional activity compared to control cells. Conclusions: These findings suggest that RIOK3 influences HCC cell motility through regulation of EMT-associated genes and in cooperation with activated Rac1 and WNT/β -catenin signaling pathway activation.
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ABSTRACT: It is commonly assumed that somatic evolution drive the multi-step process that produces metastatic cancer. But it is difficult to reconcile the inexorable progression towards metastasis in virtually all carcinomas and the associated complex change of cancer cell phenotype characterized by an epithelial-to-mesenchymal transition with the random nature of gene mutations. Given their irreversible nature, it is also difficult to explain why certain metastatic carcinomas can reform normal tissue boundaries and remain dormant for years at distant sites. Here we propose an encompassing conceptual framework based on system-level dynamics of gene regulatory networks that may help reconcile these inconsistencies. The concepts of gene expression state space and attractors are introduced which provide a mathematical and molecular basis for an "epigenetic landscape". We then describe how cancer cells are trapped in "embryonic attractors" because of distortions of this landscape caused by mutational rewiring of the regulatory network. The implications of this concept for a new integrative understanding of tumor formation and metastatic progression are discussed. This formal framework of cancer progression unites mainstream genetic determinism with alternative ideas that emphasize non-genetic influences, including chronic growth stimulation, extracellular matrix remodeling, alteration of cell mechanics and disruption of tissue architecture.
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ABSTRACT: The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal-like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF-β) and high expression of the stem cell marker CD44 were refractory to sorafenib-induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial-like cells expressing the stem-related proteins EpCAM or CD133 were sensitive to sorafenib-induced apoptosis both in vitro and in vivo. A cross-talk between the TGF-β pathway and the acquisition of a mesenchymal-like phenotype with up-regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock-down in the mesenchymal-like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib-induced apoptosis. However, CD44 effect requires a TGF-β-induced mesenchymal background, since the only overexpression of CD44 in epithelial-like HCC cells is not sufficient to impair sorafenib-induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF-β pathway, may predict lack of response to sorafenib in HCC patients. © 2014 Wiley Periodicals, Inc.International Journal of Cancer 02/2015; 136(4). DOI:10.1002/ijc.29097 · 5.01 Impact Factor