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: 8.17). 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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    • "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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    • "Differential exon inclusion was obtained using the Percentage Splicing Index (PSI) as previously defined (Wang et al. 2008; Shapiro et al. 2011). This index reflects the inclusion level of the exon and is defined as PSI = #inclusion_reads/(#inclusion_reads + #exclusion_reads); Inclusion reads correspond to the reads that fall in the exon region (a), plus the reads that support the exon junctions (b). "
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    • "Most recently, transcriptome-wide analysis of splicing changes in a Twist-induced model of EMT revealed hundreds of genes with altered splicing patterns, many of which are regulated by three splicing factors ESRP1, RBFOX2, and PTB. Furthermore, a set of nine of these splicing events with validated and robust changes during EMT had predictive value in classifying breast cancer cell lines as either luminal (generally poorly metastatic) or basal (generally more aggressive and metastatic) (Shapiro et al. 2011 "
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