bioRxiv preprint doi: https://doi.org/10.1101/062836; this version posted July 8, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Foxc1 controls cell fate decisions during transforming growth factor β induced epithelial to mesenchymal transition through the regulation of fibroblast growth factor receptor 1 expression. Alex Hopkins1, Mackenzie L. Coatham2,3, Fred B. Berry1,2* From the Department of Surgery1, Medical Genetics2 and Obstetrics and Gynecology3, University of Alberta, Edmonton AB Canada T6G 2E1 Running Title: Foxc1 regulates Fgfr1 expression in EMT To whom correspondence should be addressed: Fred Berry, Department of Surgery, 3002D Li Ka Shing Centre, University of Alberta, Edmonton, AB, Canada. T6G 2E1, Telephone: (780)492-2517; E-mail: [email protected] Key words: Transcription factor, epithelial-mesenchymal transition, metastasis, gene regulation, fibroblast growth factor receptor 1 bioRxiv preprint doi: https://doi.org/10.1101/062836; this version posted July 8, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT adopt properties of mesenchymal cells (1-4). EMT was originally observed and described as a Epithelial to mesenchymal transition transient trans-differentiation where cells from an (EMT) is an important physiological process that epithelium lose characteristic marks such as E- drives tissue formation during development but Cadherin as well as their epithelial sheet also contributes to disease pathogenesis including morphology and gain mesenchymal properties fibrosis and cancer metastasis. The forkhead box such as N-Cadherin expression and increased transcription factor gene FOXC1 is an important migratory properties (1). During development developmental regulator in the generation of EMT, is a key process in generating tissues during mesenchymal cells necessary in the formation of gastrulation, somitogenesis and neural crest cell the anterior segment of the eye, the craniofacial formation (5). EMT has also been identified as a skeleton and the meninges. Recently elevated biological step in the progression of organ fibrosis expression of FOXC1 has been detected in several and wound healing in adult tissues (6,7). Finally metastatic cancers that have undergone EMT EMT events are thought to drive metastasis in a events. We sought to determine the role of number of cancers including breast basal cell FOXC1 in the initiation of EMT events using carcinoma, hepatocellular carcinoma and NMuMG cells treated with TFGβ1. We found that pancreatic tumours (8-10). although Foxc1 expression was increased following TFGβ1 induced EMT, Foxc1 was not A key component to EMT is the required for this induction. Instead we propose characteristic decrease in E-Cadherin levels that Foxc1 is required for the specification of the resulting in the loss of epithelial adherens mesenchyme cell type, promoting an activated junctions. In concert with the loss of E-Cadherin, fibroblast phenotype over a myofibroblast progression of EMT and up regulation of the phenotype following the initiation of EMT. This mesenchymal marker N-Cadherin results in what cells type specification was achieved through the is called the "cadherin switch"(11). Epithelial cells regulation of Fibroblast growth factor (Fgfr) 1 alter cadherins to lose the resultant junctions, expression. Using an RNA sequencing approach, reorganize their cytoskeleton, and lose apical-basal we determined that levels of Fgfr1 normally polarity in favour of the front-rear polarity of activated upon TFGβ1 treatment were reduced in mesenchymal cells (2,12). Cells which have up- Foxc1-knockdown cells. Through chromatin regulated levels of N-Cadherin also up-regulate immunoprecipitation experiments we determined the intermediate filament Vimentin that is required that FOXC1 could bind to an Fgfr1 upstream for cytoskeletal re-arrangement and adoption of regulatory region. Furthermore, expression of the the spindle-like cell morphology. Multiple myofibroblast marker α-smooth muscle actin transcription factors including Snail1, Twist1 and (αSMA) was elevated in Foxc1 knockdown cells. ZEB1 and 2 have been demonstrated to directly Finally we determined that FGF2 mediated three down-regulate E-Cadherin expression and induce dimensional migratory ability was greatly EMT events (8,13-15). impaired in Foxc1-knockdown cells. Together these results define a role for Foxc1 in specifying Another key factor in the initiation of a mesenchymal cell type following TFGβ1 EMT is transforming growth factor beta 1 (TGF- mediated EMT events. β1) (16,17) (2). These secreted factors are members of a larger growth factor family, which includes the well-identified morphogens TGF-β, INTRODUCTION bone morphogenetic proteins and the activin/inhibin subfamily. These factors act Epithelial to mesenchymal transition physiologically as paracrine or autocrine signals (EMT) is a biologically important process and are known to contribute to embryonic whereby epithelial cells alter their morphology and development, tissue homeostasis, as well as tumor 2 bioRxiv preprint doi: https://doi.org/10.1101/062836; this version posted July 8, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. suppression and metastasis (2). TGF-β1 acts RESULTS primarily through the binding to type II TGF-β To determine whether Foxc1 expression receptors, which in turn phosphorylate and was elevated during EMT, we utilized the well- activate type I receptors. These activated type I established NMuMG cell model. These epithelial receptors then initiate a signal transduction cells will undergo a transition to a mesenchymal cascade resulting in the phosphorylation of phenotype in response to the EMT inducer, TGF- SMAD2 or SMAD3 proteins. Once β1. We detected changes in cytoskeletal phosphorylated, SMAD2/3 protein can associate architecture characteristic of EMT in NMuMG with SMAD4 and translocate to the nucleus and cells after treatment with TGF-β1 (5 ng/ml) for 24 regulate gene expression. In response to TGF-β1- hours (Fig 1A). Foxc1 mRNA was elevated after SMAD2/3 signalling, expression of many 24 and 48 hours of induction (Fig 1B). This transcription factor genes that initiate EMT such change expression was also accompanied by an as Snail1 and Zeb1/2 are rapidly induced (2,16). induction of the mesenchymal cells markers Snail1, Vimentin and Cdh2 (N-cadherin) The forkhead box transcription factor expression as well as a reduction in the epithelial FOXC1 is required for the development and cells marker Cdh1 (E-cadherin) mRNA levels. formation of tissues derived from neural crest and Expression of Foxc1 mRNA was unchanged at 4 h paraxial mesenchyme including the anterior and 12 h of TGF-β1 treatment. In contrast, Snail1 segment of the eye, the meninges, the axial mRNA levels were rapidly elevated 4 h post TGF- skeleton and craniofacial skeleton (18-21). As β1 treatment. Over time, levels of Foxc1 mRNA much of the formation of these tissues is driven by decreased in untreated cells, whereas E-cadherin EMT events, it stands to reason that FOXC1 may levels increased. Given the increase in Foxc1 function in EMT. More recently, elevated mRNA levels following TGF-β1-induced EMT we expression of FOXC1 was detected in basal cell- asked whether decreasing Foxc1 levels would like breast carcinomas and other metastatic prevent EMT induction. To test this idea, we cancers that have underwent EMT (22-28). transiently transfected NMuMG cells with Foxc1 Furthermore reduction of FOXC1 expression in siRNA or stably transduced cells with Foxc1 some cancer cell lines can lead to a reversal of a shRNAs resulting in a 50% and 80% reduction of mesenchymal phenotype (23). However in many Foxc1 mRNA levels, respectively (Supplemental of these experiments, the cells had underwent Fig 1A, Figure 2A). Very little FOXC1 protein EMT events prior alterations in FOXC1 levels. was detected in the stable expressing Foxc1 Thus little information is known regarding the role shRNA cells (Fig 2B). As a control, we for FOXC1 in response to physiological induction transduced cells with a vector expressing shRNA of EMT events. To assess this question, we targeting EGFP. When Foxc1 knock down cells utilized the TGF-β1 induction of EMT in the were treated with TGF-β1 for 24 hours, we mouse mammary epithelial cell line NMuMG to detected no differences in expression profiles investigate the role of Foxc1 in EMT events. We characteristic of EMT. Levels of Snail1, Vimentin found that expression of Foxc1 was indeed and N-Cadherin mRNA were increased in both induced by TGF-β1 treatment. However, loss of control (shEGFP) and Foxc1 (shFOXC1) Foxc1 function through RNA interference did not knockdown cells following TGF-β1 treatment prevent the induction of EMT events in response (Figs 2C and Supplemental S1). E-cadherin to TGF-β1. Instead we found that Foxc1 regulated mRNA levels decreased following TGF-β1 in both the specificity of the mesenchymal cell phenotype. the shEGFP and shFOXC1 NMuMG knockdown Loss of Foxc1 expression led to a reduction of cells. We examined whether the morphological Fibroblast Growth Factor receptor (FGFR) 1 phenotype characteristic of EMT was affected in expression and promoted a less invasive response to reduced Foxc1