Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.
Epithelial-to-mesenchymal transition (EMT) is the process by which cancer cells lose their epithelial characteristics and obtain a mesenchymal phenotype that is thought to allow them to migrate away from the primary tumor. A better understanding of how EMT is controlled would be valuable in predicting the likelihood of metastasis and in designing targeted therapies to block metastatic progression. While there have been many studies on the contribution of changes in gene expression to EMT, much less is known regarding the role of alternative splicing of mRNA during EMT. Alternative splicing can produce different protein isoforms from the same gene that often have distinct activities and functions. Here, we used a recently developed method to characterize changes in alternative splicing during EMT and found that thousands of multi-exon genes underwent alternative splicing. Alternative isoform expression was confirmed in human breast cancer cell lines and in primary human breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. Since EMT is considered an early step in metastatic progression, novel markers of EMT that we identified in human breast cancer samples might become valuable prognostic and diagnostic tools if confirmed in a larger cohort of patients.