Abstract
IntroductionmiR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.MethodsPutative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively.ResultsWe found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity.ConclusionsmiR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.
Highlights
MiR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity
We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19)
These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the epithelial to mesenchymal transition (EMT) phenotype and aggressive behavior
Summary
MiR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Epithelial to mesenchymal transition (EMT) occurs during development as it is required for formation of the neural crest and palate, among other processes [1,2] In cancer it is a pathological event associated with tumor progression and is thought to influence certain steps in the metastatic cascade, thereby contributing to the metastatic potential of carcinomas. Since ZEB1 represses E-cadherin [14] and other genes involved in polarity [15], the reduction in migratory and invasive capacity observed when miR-200c is restored to cancer cells is widely thought to be due to the ability of miR200c to target and repress ZEB1/2 which, in most cases, allows E-cadherin to be re-expressed. Even in cell lines in which E-cadherin is not restored, miR-200c still dramatically reduces migration and invasion [11], implying that additional miR-200c targets can facilitate its ability to suppress cell motility
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