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

Abstract

Transforming growth factor-β (TGF-β) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-β signaling can lead to breast cancer. TGF-β elicits its effects on proliferation and differentiation via specific cell surface TGF-β type I and type II receptors (i.e., TβRI and TβRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-β-induced heteromeric complex formation, activated TβRI 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-β acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-β signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-βinduced 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-β-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-β receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-β-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-

ST3GAL5‐catalyzed gangliosides inhibit TGF‐β‐induced epithelial‐mesenchymal transition via TβRI degradation

“…To investigate the TGF‐β‐induced changes in GSL‐glycans in cells undergoing EMT, we chose two established cell models with prominent TGF‐β‐induced EMT responses, i.e., NMuMG (Zhang et al , 2020a) and A549‐vimentin (VIM)‐red fluorescent protein (RFP) cell lines (Wang et al , 2020). We performed quantitative glycomic analysis of these cell lines using porous graphitized carbon (PGC) nano‐liquid chromatography‐electrospray ionization tandem mass spectrometry (nano‐LC‐ESI‐MS/MS) in negative ESI mode, allowing in‐depth determination of GSL profiles (Appendix Fig S1; Anugraham et al , 2015).…”

“…Activation of EMT can endow cells with an enhanced ability to migrate (Zhang et al , 2020a). We thus further investigated the basal migration rate upon Ugcg KO in NMuMG cells.…”

“…Activation of EMT can endow cells with an enhanced ability to migrate (Zhang et al, 2020a). We thus further investigated the basal Eliglustat, an inhibitor of UGCG activity, promotes TGF-b signaling, TGF-b-induced EMT, cell migration, extravasation, and early metastatic outgrowth…”