Population heterogeneity in the epithelial to mesenchymal transition is controlled by NFAT and phosphorylated Sp1

Abstract

Epithelial to mesenchymal transition (EMT) is an essential differentiation program during tissue morphogenesis and remodeling. EMT is induced by soluble transforming growth factor {beta} (TGF-{beta}) family members, and restricted by vascular endothelial growth factor family members. While many downstream molecular regulators of EMT have been identified, these have been largely evaluated individually without considering potential crosstalk. In this study, we created an ensemble of dynamic mathematical models describing TGF-{beta} induced EMT to better understand the operational hierarchy of this complex molecular program. We used ordinary differential equations (ODEs) to describe the transcriptional and post-translational regulatory events driving EMT. Model parameters were estimated from multiple data sets using multiobjective optimization, in combination with cross-validation. TGF-{beta} exposure drove the model population toward a mesenchymal phenotype, while an epithelial phenotype was enhanced following vascular endothelial growth factor A (VEGF-A) exposure. Simulations predicted that the transcription factors phosphorylated SP1 and NFAT were master regulators promoting or inhibiting EMT, respectively. Surprisingly, simulations also predicted that a cellular population could exhibit phenotypic heterogeneity (characterized by a significant fraction of the population with both high epithelial and mesenchymal marker expression) if treated simultaneously with TGF-{beta} and VEGF-A. We tested this prediction experimentally in both MCF10A and DLD1 cells and found that upwards of 45% of the cellular population acquired this hybrid state in the presence of both TGF-{beta} and VEGF-A. We experimentally validated the predicted NFAT/Sp1 signaling axis for each phenotype response. Lastly, we found that cells in the hybrid state had significantly different functional behavior when compared to VEGF-A or TGF-{beta} treatment alone. Together, these results establish a predictive mechanistic model of EMT susceptibility, and potentially reveal a novel signaling axis which regulates carcinoma progression through an EMT versus tubulogenesis response.\n\nAuthor SummaryTissue formation and remodeling requires a complex and dynamic balance of interactions between epithelial cells, which reside on the surface, and mesenchymal cells that reside in the tissue interior. During embryonic development, wound healing, and cancer, epithelial cells transform into a mesenchymal cell to form new types of tissues. It is important to understand this process so that it can be controlled to generate beneficial effects and limit pathological differentiation. Much research over the past 20 years has identified many different molecular species that are relevant, but these have mainly been studied one at a time. In this study, we developed and implemented a novel computational strategy to interrogate the key players in this transformation process to identify which are the major bottlenecks. We determined that NFATc1 and pSP1 are essential for promoting epithelial or mesenchymal differentiation, respectively. We then predicted the existence of a partially transformed cell that exhibits both epithelial and mesenchymal characteristics. We found this partial cell type develops a network of invasive but stunted vascular structures that may be a unique cell target for understanding cancer progression and angiogenesis.