Recent data indicate that transforming growth factor-beta1 (TGF-beta1) can act to promote tumour progression in the late stages of carcinogenesis. The mechanism by which this occurs is unknown although a ligand-induced epithelial-mesenchymal transition (EMT) is thought to be important. In this study, we demonstrate that active Ras is required for TGF-beta1-induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF-beta1. Under conditions of TGF-beta1-induced EMT, cells were growth inhibited by the ligand resulting in G1 arrest. In cells containing normal Ras, TGF-beta1-activated ERK and p38 mitogen-activated protein kinases (MAPKs), and levels of activation were further increased by co-treatment with EGF. Inhibition of MAPK pathways and Smad2/3 signalling blocked the induction of EMT by TGF-beta1. Further, inhibition of the AP-1 transcriptional complex by [6]-Gingerol, or by the ectopic expression of JDP2, blocked TGF-beta1-induced EMT and conversely, stimulation of AP-1 by 12-O-tetradecanoylphorbol 13-acetate (TPA) substituted for EGF in the induction of EMT by TGF-beta1 in cells containing normal Ras. The presence of oncogenic Ras, the treatment of cells with EGF, or the treatment of cells with TPA to activate AP-1, potentiated TGF-beta1-induced Smad-dependent transcription, an effect that was attenuated by the inhibition of MAPKs and AP-1. The results demonstrate that active Ras and TGF-beta1 co-operate to reversibly induce EMT in human keratinocytes by mechanisms that involve MAPKs, Smad2/3 and AP-1. Further we demonstrate that MAPK/AP-1 signalling enhances Smad transcriptional activity under conditions associated with TGF-beta1-induced EMT.
This study examined the immunocytochemical expression of the transforming growth factor-beta (TGF-beta) isoforms TGF-beta1, TGF-beta2, and TGF-beta3, together with the TGF-beta cell surface receptors TbetaR-I and TbetaR-II, in patient-matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF-beta isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TbetaR-II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TbetaR-I, but not at a statistically significant level. This study also examined the structure of TbetaR-I and TbetaR-II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF-beta1. No structural abnormalities of TbetaR-II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TbetaR-II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709-718, codons 125-128), predicting a truncated receptor having no kinase domain. No defects were present in TbetaR-I. The structures of TbetaR-I and TbetaR-II did not correlate with growth inhibition by TGF-beta1. The data suggest that decreased expression of TGF-beta receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TbetaR-II expression, a dominant-negative TbetaR-II construct (dnTbetaR-II) was transfected into a human oral carcinoma cell line with a normal TGF-beta receptor profile and known to be markedly inhibited by TGF-beta1. In those clones that overexpressed the dnTbetaR-II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra-1 and collagenase-1 remained unchanged following treatment with TGF-beta1. The results demonstrate that a decrease in TbetaR-II relative to TbetaR-I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP-1-dependent genes that are involved in the regulation of the extracellular matrix.
This study examined the role of TGF-b1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGFb1-induced G 1 arrest, the presence of wild type TGF-b cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-b responsive cell line, and treatment of the cells with TGF-b1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-b1 resulted in a failure to induce transcription from an arti®cial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and ®bronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-b1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-b1 resulted in the attenuation of cell growth in vitro. To over-express TGF-b1, both cell lines were transfected with latent TGF-b1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that overexpressed endogenous TGF-b1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-b1 to the¯oor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-b type II receptor in cells that over-expressed endogenous TGF-b1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor eect of the ligand in vivo, indicating that the endogenous TGF-b1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-b1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-b1-induced G 1 arrest.
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