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 β (TGF-β) 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-β 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-β 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-β 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-β 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-β 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. Funding:The project described was supported by Award Number #U54CA143876 from the National Cancer Institute to JDV, National Science Foundation CBET-0955172 to JB, National Science Foundation Graduate research fellowship to DMB, Author 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 implement...

Hypoxia, partial EMT and collective migration: Emerging culprits in metastasis

“…Moreover, cells isolated from in vivo tumors contain hybrid E/M cells [ 39 , 103 , 163 ]. Thus, hybrid E/M state can be a stable phenotype, especially in the presence of phenotypic stability factors (PSFs) such as OVOL, NRF2, GRHL2, NUMB, ∆NP63α, and NFATc that can promote and stabilize hybrid E/M state and increase the mean residence times of cells in hybrid E/M states [ 63 , 83 , 85 , 87 , 162 , [164] , [165] , [166] ]. Knockdown of these PSFs in H1975 lung cancer cell line impaired a stable hybrid E/M phenotype and collective migration and drove the cells towards a complete EMT [ 63 , 165 ].…”

NFATc acts as a non-canonical phenotypic stability factor for a hybrid epithelial/mesenchymal phenotype

“…In presence of NFATc, cells undergo a delayed or stalled EMT; thus maintaining cells in hybrid E/M phenotypes; knockdown of NFATc in H1975 NSCLC cells drove the progression towards a complete EMT phenotype, reminiscent of observations made for other PSFs -GRHL2, OVOL2, NUMB and NRF2 (17)(18)(19)(20)(21)(22). Similar effects of NFATc knockdown were also seen in MCF10A and DLD1 cells where treatment with VIVIT, a soluble inhibitor of NFATc transcriptional activity, significantly reduced E-cadherin expression and protein level, and increased Slug and Vimentin levels (28), thus driving EMT. NFATc transcriptional activity was shown to be capable of maintaining Ecadherin levels even in the presence of TGFβ induced EMT (28), suggesting that NFATc acted as a "molecular brake" or "guardian" of epithelial traits, preventing a complete EMT (39).…”

“…Similar effects of NFATc knockdown were also seen in MCF10A and DLD1 cells where treatment with VIVIT, a soluble inhibitor of NFATc transcriptional activity, significantly reduced E-cadherin expression and protein level, and increased Slug and Vimentin levels (28), thus driving EMT. NFATc transcriptional activity was shown to be capable of maintaining Ecadherin levels even in the presence of TGFβ induced EMT (28), suggesting that NFATc acted as a "molecular brake" or "guardian" of epithelial traits, preventing a complete EMT (39). Consistently, NFATc1 was identified to be a master regulator of chromatin remodeling to regulate hybrid E/M phenotypes in skin cancer in vivo; the proportion of hybrid E/M phenotypes was also shown to be increased by GRHL2, OVOL1/2, and ΔNP63α at the expense of complete EMT cells, thus lending further credence to our results indicating a functional equivalence between NFATc1 and previously identified PSFs such as GRHL2, OVOL1/2, ΔNP63α, and NRF2 (6).…”

“…On one hand, overexpression of NFATc increased the levels of TWIST, ZEB1, SNAI1; its downregulation decreased the levels of these EMT-TFs as well as mesenchymal markers such as N-cadherin and Vimentin ZEB (27). On the other hand, NFATc transcriptional activity was also shown to be crucial for maintaining E-cadherin levels (28), which can inhibit ZEB1 indirectly (29,30) and restrict EMT. Moreover, NFATc can activate SOX2 (27,31) which can upregulate levels of miR-200 (32); overexpression of miR-200 can drive MET (33).…”

Quantifying Cancer Epithelial-Mesenchymal Plasticity and Its Association with Stemness and Immune Response

“…Mathematical modeling approaches have been further used to characterize phenotypic stability factors (PSFs) that can promote and stabilize hybrid E/M states. These PSFs include the transcription factors OVOL, GRHL2, NRF2, NP63, NUMB and miR-145/OCT4 [50][51][52][53][54]. These PSFs can function in two related manners.…”