Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

Hypoxia inhibition of adipocytogenesis in human bone marrow stromal cells requires transforming growth factor-beta/Smad3 signaling

Although hypoxia and transforming growth factor-beta (TGF-beta) inhibit differentiation of adipocytes from preadipocytes and bone marrow-derived cells in several species, the relationship between hypoxia and TGF-beta signaling in adipocytogenesis is unknown. In this study, we evaluated the mechanisms of inhibition of adipocyte differentiation by hypoxia and TGF-beta in human and murine marrow stromal cells (MSCs) and the role of TGF-beta/Smad signaling in the inhibition of adipocytogenesis by hypoxia. Both hypoxia-mimetic deferoxamine mesylate (DFO) and TGF-beta1 inhibited adipocyte differentiation (1.0% versus the control at 15 microm DFO and 1.4% versus the control at 1 ng/ml TGF-beta1) and adipocyte gene expression (peroxisome proliferator-activated receptor-gamma2 and lipoprotein lipase) in human MSCs after 21 days of treatment. Hypoxia (2% O(2)) and DFO (but not TGF-beta1) increased hypoxia-inducible factor-1alpha as shown by Western blotting. Macroarrays and Western and Northern blot analyses showed that hypoxia activated the TGF-beta/Smad signaling pathway and that both hypoxia and TGF-beta1 modulated adipocyte differentiation pathways such as the insulin-, peroxisome proliferator-activated receptor-gamma-, phosphatidylinositol 3-kinase-, and MAPK-associated signaling pathways. Studies with mouse marrow stromal cell lines derived from Smad3(+/+) or Smad3(-/-) mice revealed that the TGF-beta type I receptor (ALK-5) and its intracellular signaling molecule Smad3 were necessary for the inhibition of adipocyte differentiation by both TGF-beta and hypoxia-mimetic DFO. Thus, the TGF-beta/Smad signaling pathway is required for hypoxia-mediated inhibition of adipocyte differentiation in MSCs.