Redox processes inform multivariate transdifferentiation trajectories associated with TGFβ-induced epithelial–mesenchymal transition

Abstract

Phenotype reprogramming during transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) is an extensive and dynamic process, orchestrated by the integration of biological signaling across multiple timescales. As part of the numerous transcriptional changes necessary for EMT, TGFβ-initiated Smad3 signaling results in remodeling of the redox environment and decreased nucleophilic tone. Because Smad3 itself is susceptible to attenuated activity through antioxidants, the possibility of a positive feedback loop exists, albeit the time scales on which these mechanisms operate are quite different. We hypothesized that the decreased nucleophilic tone acquired during EMT promotes Smad3 signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. Previous findings supporting such a mechanism were characterized independent of each other; we sought to investigate these relationships within a singular experimental context. In this study, we characterized multivariate representations of phenotype as they evolved over time, specifically measuring expression of epithelial/mesenchymal differentiation, redox regulators, and Smad transcription factors. In-cell western (ICW) assays were developed to evaluate multivariate phenotype states as they developed during EMT. Principal component analysis (PCA) extracted anti-correlations between phospho-Smad3 (pSmad3) and Smad2/Smad4, which reflected a compensatory up-regulation of Smad2 and Smad4 following cessation of TGFβ signaling. Measuring transcript expression following EMT, we identified down-regulation of numerous antioxidant genes concomitant with up-regulation of NADPH oxidase 4 (NOX4) and multiple mesenchymal phenotype markers. TGFβ treatment increased CM-H2DCF-DA oxidation, decreased H2O2 degradation rates, and increased glutathione redox potential. Our findings suggest that the decreased nucleophilic tone during EMT coincides with the acquisition of a mesenchymal phenotype over too long a time scale to enable enhanced Smad3 phosphorylation during initiation of EMT. We further challenged the mesenchymal phenotype following EMT through antioxidant and TGFβ inhibitor treatments, which failed to induce a mesenchymal-epithelial transition (MET). Our characterization of multivariate phenotype dynamics during EMT indicates that the decrease in nucleophilic tone occurs alongside EMT, however maintenance of the mesenchymal phenotype following EMT is independent of the both the nascent redox state and continuous TGFβ signaling.

Epithelial–mesenchymal transition of ovarian cancer cells is sustained by Rac1 through simultaneous activation of MEK1/2 and Src signaling pathways

“…EMT can be induced by diverse factors that include transforming growth factor β (TGFβ)/bone morphogenetic proteins (BMPs), receptor tyrosine kinases, Wnt and Notch signaling pathways ( 3 – 5 ). Recent studies have also established a strong connection between tumor microenvironment and EMT because hypoxia ( 6 , 7 ), inflammation ( 8 , 9 ) and oxidation stress ( 10 ), phenomenon commonly detected in tumor microenvironment, are potent EMT inducers. Signals triggered by these factors all converge on EMT-inducing transcriptional factors such as Snail, Slug, Twist, and Zeb1/2 that diminish the expression of epithelial-related genes such as E-cadherin and, at the same time, enhance the expression of mesenchymal-related genes such as vimentin ( 3 – 5 ).…”

IGFBP3 and MAPK/ERK signaling mediates melatonin‐induced antitumor activity in prostate cancer

“…It is well known that neuronal differentiation of rat PC12 cells is highly dependent on redox regulation by GSH and reactive oxygen species . In fact, redox balance directs the differentiation in neuroblastoma cells or in the well‐studied “epithelial‐to‐mesenchymal” (EMT) transdifferentiation . ROS, particularly H 2 O 2 , produced by SOD1/2 are regulators of the NED process in LNCaP cells and in the EMT in mammary carcinoma .…”

“…51 In fact, redox balance directs the differentiation in neuroblastoma cells 52 or in the well-studied "epithelial-to-mesenchymal" (EMT) transdifferentiation. 53 ROS, particularly H 2 O 2 , produced by SOD1/2 are regulators of the NED process in LNCaP cells 34 and in the EMT in mammary carcinoma. 54 Results shown here point…”