ABSTRACT
The cooperation of Ras - extracellular signal-regulated kinase/mitogen-activated protein kinase and transforming growth factor (TGF)-beta signaling provokes an epithelial to mesenchymal transition (EMT) of differentiated p19(ARF) null hepatocytes, which is accompanied by a shift in malignancy and gain of metastatic properties. Upon EMT, TGF-beta induces the secretion and autocrine regulation of platelet-derived growth factor (PDGF) by upregulation of PDGF-A and both PDGF receptors. Here, we demonstrate by loss-of-function analyses that PDGF provides adhesive and migratory properties in vitro as well as proliferative stimuli during tumor formation. PDGF signaling resulted in the activation of phosphatidylinositol-3 kinase, and furthermore associated with nuclear beta-catenin accumulation upon EMT. Hepatocytes expressing constitutively active beta-catenin or its negative regulator Axin were employed to study the impact of nuclear beta-catenin. Unexpectedly, active beta-catenin failed to accelerate proliferation during tumor formation, but in contrast, correlated with growth arrest. Nuclear localization of beta-catenin was accompanied by strong expression of the Cdk inhibitor p16(INK4A) and the concomitant induction of the beta-catenin target genes cyclin D1 and c-myc. In addition, active beta-catenin revealed protection of malignant hepatocytes against anoikis, which provides a prerequisite for the dissemination of carcinoma. From these data, we conclude that TGF-beta acts tumor progressive by induction of PDGF signaling and subsequent activation of beta-catenin, which endows a subpopulation of neoplastic hepatocytes with features of cancer stem cells..
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Nucleus/metabolism , Platelet-Derived Growth Factor/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism , beta Catenin/metabolism , Anoikis , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Disease Progression , Epithelial Cells/metabolism , HumansABSTRACT
The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells. For both tumorigenesis and hepatic fibrogenesis, transforming growth factor (TGF)-beta signaling executes key roles and therefore is considered as a hallmark of these pathological events. By employing cellular transplantation we show that the interaction of neoplastic MIM-R hepatocytes with the tumor microenvironment, containing either activated hepatic stellate cells (M1-4HSCs) or myofibroblasts derived thereof (M-HTs), induces progression in malignancy. Cotransplantation of MIM-R hepatocytes with M-HTs yielded strongest MIM-R generated tumor formation accompanied by nuclear localization of Smad2/3 as well as of beta-catenin. Genetic interference with TGF-beta signaling by gain of antagonistic Smad7 in MIM-R hepatocytes diminished epithelial dedifferentiation and tumor progression upon interaction with M1-4HSCs or M-HTs. Further analysis showed that tumors harboring disrupted Smad signaling are devoid of nuclear beta-catenin accumulation, indicating a crosstalk between TGF-beta and beta-catenin signaling. Together, these data demonstrate that activated HSCs and myofibroblasts directly govern hepatocarcinogenesis in a TGF-beta dependent fashion by inducing autocrine TGF-beta signaling and nuclear beta-catenin accumulation in neoplastic hepatocytes. These results indicate that intervention with TGF-beta signaling is highly promising in liver cancer therapy.
Subject(s)
Hepatocytes/drug effects , Hepatocytes/pathology , Liver Neoplasms/pathology , Transforming Growth Factor beta/pharmacology , Animals , Cell Adhesion/drug effects , Cell Nucleus/metabolism , Cell Transplantation , Disease Progression , Fibroblasts/drug effects , Fibrosis , Humans , Mice , Models, Biological , Paracrine Communication/drug effects , Rats , Signal Transduction/drug effects , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Smad7 Protein/metabolism , beta Catenin/metabolismABSTRACT
Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.
