TGF-ß-regulated genes implicated in invasion that might participate in the control of apoptosis in liver tumor cells

Resumen

In the last years our research has been focused on analyzing the signaling pathways induced by TGF-? in liver tumor cells, to understand the molecular mechanisms that confer resistance to its suppressor effects. TGF-? induces apoptosis in fetal and neonatal murine hepatocytes, as well as in liver tumor cells, and chronic exposure of these cells to TGF-? i?induces a process of Epithelial to Mesenchymal Transition (EMT). In the present work we wanted to identify TGF-?-regulated genes that being involved in EMT and cell invasion could also participate in the control of growth, apoptosis and/or differentiation. Firstly we analyzed the role of genes regulated by TGF-? and implicated in EMT that might participate in apoptosis control in hepatocytes, focusing on Snail and SPARC. Inhibition of Snail, through targeting knock-down with specific siRNA, impairs TGF-?-induced EMT in murine hepatocytes and significantly enhances their apoptotic response, which indicates that Snail plays a relevant role in conferring resistance to TGF-?-induced cell death. TGF-? also induces anti-apoptotic signals, mediated by the activation of the epidermal growth factor receptor (EGFR). Snail downregulation impairs the TGF-?-induced EGFR ligands expression and inhibits the phosphorylation of Akt, Erks and c-Src family, which is coincident with activation of mitochondrial-dependent apoptotic events and an earlier Smad3 phosphorylation in TGF-?-treated cells. We also demonstrate a role for Snail in sensitizing murine hepatocytes to cell death by anoikis, which is a relevant phenomenon in metastatic processes. Snail1 downregulation in human hepatocellular carcinoma (HCC) cells, which are partially or fully resistant to TGF-? ?suppressor effects, restores the apoptotic response to TGF-?. TGF-? induces SPARC expression in FaO rat liver tumor cells but not in neonatal murine untransformed hepatocytes. SPARC inhibition, through targeting knock-down with specific siRNA, reveals a role for SPARC in mediating TGF-?-induced EMT in liver tumor cells. Furthermore, SPARC knock-down significantly enhances the TGF-?-induced apoptotic response. Interestingly, SPARC effects might be mediated by Snail, since SPARC silencing impairs Snai1 up-regulation by TGF-? in FaO cells. We next wanted to study the tumorigenesis of FaO cells after in vitro chronic treatment with TGF-? for 4 weeks (T?T-FaO). For this, we injected these cells through both subcutaneous and intrasplenic procedures. Liver tumor formation derived from intrasplenic injection of FaO cells induced a multifocal highly proliferative hepatocarcinoma in all mice, whereas parallel inoculation of T?T-FaO cells promoted low proliferative unifocal and heterogeneous hepatic lesions which showed higher staining for phospho-Smad2. Detailed analysis of tumors revealed lesions with bile duct characteristics and lesions with a dedifferentiated (hepatoblast) phenotype. Primary culture of tumor cells from both FaO- and T?T-FaO-induced intrasplenic lesions indicated that only cells obtained from FaO-induced tumors undergo apoptosis in response to TGF-?, whereas T?T-FaO-derived tumors contain cells that are fully resistant. Analysis of the phenotype of tumors and their derived cells showed that intrasplenic injection of T?T-FaO cells may produce cholangiocarcinoma-like and hepatoblastoma-like tumors. In summary, chronic in vitro TGF- ? treatment of FaO cells changed their tumorigenic potential. Tumor growth was slower but cells are resistant to apoptosis. Futhermore, phenotype of lesions reflected a stem-like phenotype which provokes the appearance of less differentiated tumors (hepatoblastomas) or transdifferentiation to a different liver tumor lineage (cholangiocarcinomas).