Epithelial–mesenchymal transition (EMT) and its reversal, mesenchymal–epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial–mesenchymal plasticity and gene expression.

The epithelial–mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapies, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover prolonged, sub-cytotoxic exposure to OpA is sufficient to suppress EMT-imparted CSC features including sphere formation and resistance to doxorubicin. In vivo growth of CSC-rich mammary cell tumors, is suppressed by OpA treatment. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.

Background: Epithelial-mesenchymal transition (EMT) facilitates cellular movements critical for proper development; however, in a carcinoma, EMT promotes metastatic dissemination. Stable intermediate states (partial-EMT) are increasingly implicated in metastatic dissemination while reversal of EMT, termed mesenchymal-epithelial transition (MET), is increasingly implicated in metastatic colonization. To understand the partial and reversible nature of EMT, we characterized chromatin accessibility dynamics, transcriptome changes, protein expression patterns, as well as E-cadherin expression, localization, and gene-level dynamics in mammary epithelial cells undergoing stepwise reversible EMT. Results: While shorter EMT induction induced internalization of E-cadherin protein, surface expression was recovered upon MET without loss of transcript or bulk protein. Conversely, a longer EMT induced stable repression of E-cadherin indicated by loss of chromatin accessibility and induced global expansion of accessible sites across the genome, facilitated by increased engagement of multiple transcription factor families, including AP-1 and SMAD. We observe enrichment for binding sites for the insulator proteins CTCF and BORIS was significantly diminished in both stemness-enriched partial-EMT and partial-MET states and determined that CTCF repression imparts alterations in some histone covalent modifications concomitant with those observed during TGFβ-induced EMT.Conclusions: These findings are indicative of a major role for chromatin looping and reorganization in plasticity, stemness, and partial EMT phenotypes. BackgroundEpithelial-mesenchymal transition (EMT) is a conserved cellular process that drives programs such as gastrulation and wound healing. During EMT, epithelial cells alter their gene expression and morphology, lose cell-cell contacts, and adopt a mesenchymal-like state (1).Because this process promotes invasion, intravasation, and resistance to anoikis in tumor cells, the EMT program is also implicated in metastatic dissemination (2)(3)(4). Recent work has contributed to a revised model of metastasis in which reversal of EMT, mesenchymal-epithelial transition (MET), is necessary for colonization of cells which arrive at the metastatic site by means of an EMT (5-8). Thus, understanding the factors that impact the reversibility of EMT is critical to developing better anti-metastasis treatments.EMT propels cells through a progressive adoption of gene expression changes leading to phenotypic alterations. While a hallmark of EMT is the suppression of genes such as Ecadherin (CDH1) and epithelial cell adhesion molecule (EPCAM), there are also profound changes throughout the epigenome, transcriptome, spliceosome, and protein translation machinery (9). EMT can be initiated by microenvironmental signals such as TGFβ, EGF, hypoxia, and tissue stiffness (10-13), and is effected through networks of EMT-transcription factor proteins (EMT-TFs) such as SNAIL, SLUG, TWIST1, ZEB1, SIX1, SOX10, and FOXC2 (14-18). These transcripti...

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapy, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover, prolonged, sub-cytotoxic exposure to OpA is sufficient to reduce migration, sphere formation, and resistance to doxorubicin. OpA is well-tolerated in mice and treatment with OpA alone reduces tumor burden. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.in survival 14-17 . To improve TNBC patient outcomes, novel and specific approaches targeted at CSCs are needed.One proposed mechanism driving the emergence of CSC-like cells is the epithelialmesenchymal transition, EMT 18,19 . EMT is a trans-differentiation process characterized by spindle-like morphology, loss of apical-basal polarity, increased motility, and a tolerance to anoikis. These phenotypic shifts are driven by gene expression changes mediated by transcription factors Snail (SNAI1), Twist (TWIST1), and ZEB1, effects of which include upregulation of vimentin and N-cadherin, and downregulation of epithelial markers E-cadherin and miR-200c 20-26 . Cells that have undergone an EMT typically acquire CSC properties including decreased sensitivity to conventional chemotherapies used to treat TNBC. This chemoresistance is driven by drug efflux pumps, enhanced DNA repair capacity, mesenchymal-like properties, and epigenetic changes 16,27-32 . There are currently no approved therapies that specifically target CSCs. A leading pre-clinical compound is salinomycin, reported to decrease the subpopulation of CSCs, tumor initiating capability, and chemoresistance, with negligible side effects 33 . Other naturally occurring compounds such as curcumin, and quercetin have been reported to reduce the effects of EMT by inhibiting key proteins associated with migration (Snail, MMP-2/9), anoikis tolerance (Bcl-2), cell-to-cell adhesion (N-cadherin), and signaling cascades (JAK/STAT, ERK) 34-37 .Ophiobolin A (OpA) is a natural product produced from fungi in the genera Aspergillus, Bipolaris, Cephalosporium, Cochliobolus, is a secondary metabolite that has long been studied for its phytotoxic effects in a variety of plants and has begun to be evaluated as a cytotoxic compound 38 . Recently published cell Western blotting and antibodiesCells were lysed in the presence of 100 microliters radio-immunoprecipitation (RIPA) buffer containing protease inhibitors (Alfa Aesar, Stoughton, MA, USA) on ice. Protein was quantified using the Bradford Assay (BioRad,...

Epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We found that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET and is lower in triple-negative breast cancer cell lines with EMT features. Through analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs but also by diminished enrichment for CTCF binding motifs. Through loss-of-function analysis we demonstrate that suppression of CTCF alters cellular plasticity, facilitating entrance into a partial EMT state. These findings are indicative of a role of CTCF and chromatin reorganization for epithelial-mesenchymal plasticity.