Studying TGF-beta Signaling and TGF-beta-induced Epithelial-to-mesenchymal Transition in Breast Cancer Cells

Transforming growth factor-beta (TGF-beta) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-beta signaling can lead to breast cancer. TGF-beta elicits its effects on proliferation and differentiation via specific cell surface TGF-beta type I and type II receptors (i.e., T beta RI and T beta RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-beta-induced heteromeric complex formation, activated T beta RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-beta acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-beta signaling and EMT responses using MCF10A-Ras and NMuMG breast cancer cell lines as examples. We describe methods to determine TGF-beta-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-beta-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-beta receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-beta-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-beta-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.Cancer Signaling networks and Molecular Therapeutic