Dynamic Redox Signaling During TGF-Beta-Induced Epithelial-Mesenchymal Transition
Prasanphanich, Adam Franklin
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Individual biological processes operate within larger contexts and can participate in the emergence of complex phenotypes. The morphogen transforming growth factor β (TGFβ) can initiate diverse cellular responses, including down-regulation of numerous antioxidant species. TGFβ signaling itself has been shown to exhibit redox sensitivity and in the context of TGFβ-mediated epithelial-mesenchymal transition (EMT), there exists a possibility of a positive feedback loop operating over multiple temporal and biological scales to stabilize a mesenchymal phenotype. Additionally, drug resistant side populations (SP) arise in populations that exhibit heterogeneity of ABCG2 transporter activity, which is regulated within the same cellular program as antioxidants. Therefore, it is possible that SPs reflect heterogeneity in redox regulation within a population; however, how single-cell ABCG2 activity heterogeneity manifests at the population level is not known. The overall objective of this research was to investigate how redox regulated processes contribute to complex phenotypes that arise in the context of TGFβ-mediated EMT using multivariate and systems approaches. We investigated the dynamics of redox regulation in the context of EMT, hypothesizing that decreased nucleophilic tone acquired during EMT strengthens TGFβ signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. We demonstrated the sensitivity of TGFβ signaling to antioxidants and the down-regulation of antioxidants within a singular model. We developed in-cell western assays to evaluate multivariate phenotype states as they developed during EMT. TGFβ treatment decreased H2O2 degradation rates and increased glutathione redox potential, indicating decreased nucleophilic tone. Epithelial/mesenchymal differentiation and redox time course data were paired using principal component analysis (PCA) to construct a multivariate representation of phenotype over the time course of EMT. We found that decreased nucleophilic tone during EMT coincides with acquisition of a mesenchymal phenotype over too long a time scale to enable enhancement of EMT. In the second portion of this research, we investigated the role of heterogeneity of ABCG2 activity at the single cell level in the emergence of SPs at the population level and the means by which TGFβ signaling modulates heterogeneity to affect SP size. TGFβ was found to decrease the size of SPs as well as the magnitude of response. A multiscale ensemble model consisting of a heterogeneous population of individual cells was used to interrogate multiple kinetic schemas and identified a highly active subpopulation juxtaposed by an inactive main population, suggesting the SP cells may exhibit a distinct redox profile from main cells, the frequency of which was decreased in response to TGFβ. In summary, we developed an approach to investigate the dynamics of redox regulation during TGFβ-mediated EMT from the perspective of a multivariate phenotype, simultaneously accounting for changes in epithelial/mesenchymal differentiation and to the intracellular redox environment. Additionally, we developed a multiscale ensemble modeling approach to investigate the kinetic mechanisms by which heterogeneity of ABCG2 regulation at the single-cell level leads to emergence of a SP at the population level.