Article

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
PLoS Genetics (Impact Factor: 7.53). 08/2011; 7(8):e1002218. DOI: 10.1371/journal.pgen.1002218
Source: PubMed

ABSTRACT

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.

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    • "4; Yang and Weinberg, 2008). ESRPs and RBF OX2 control the alternative splicing of several transcripts encoding cell adhesion proteins involved in the epithelial or mesenchymal phenotypes (Shapiro et al., 2011; Venables et al., 2013; Braeutigam et al., 2014). A splice variant of the tyrosine kinase receptor RON that promotes cell migration and activates EMT is controlled by antagonistic interactions involving SRSF1 and hnRNP A1, A2, and H proteins (LeFave et al., 2011; Biamonti et al., 2014). "

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    • "They found a global reduction in the heterochromatin mark H3K9Me2 and an increase in the euchromatin mark H3K4Me3 and in the transcriptional mark H3K36Me3 (McDonald et al. 2011). Given the large number of alternative splicing events that occur during EMT (Shapiro et al. 2011), it would be interesting to determine whether the global increase seen in H3K36me3 in the AML12 cell line occurs over exonic regions that are alternatively spliced during EMT. Would these marks be retained and transmitted to daughter cells as the tumors divide? "
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    ABSTRACT: Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.
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    • "In this context, it must be underlined that exons regulated during EMT are flanked by hnRNP H/F binding motifs (Shapiro et al., 2011) and that a function of hnRNP H/F in muscle cells has previously been reported (Chen et al., 1999; Paul et al., 2011). Since DDX5 and DDX17 contribute to maintain epithelial-and myoblast-specific splicing subprograms, their downregulation during EMT and myogenesis may favor the switch toward the previously reported fibroblast-and myotube-specific splicing programs (Bland et al., 2010; Shapiro et al., 2011). Even though there is a strong overlap between DDX5/DDX17-and hnRNP H/F-regulated exons, as we observed that at least 159 of the 372 DDX5/DDX17-regulated exons in MCF7 cells are percentage of input RNA, are represented as the mean values of at least three independent experiments (n R 3) and normalized to the control sample (IP in the presence of control siRNA), which was arbitrarily set to 1 ± SD (paired Student's t test: *p < 0.05). "
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