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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Available from: Michele Balsamo, Aug 11, 2015
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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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    ABSTRACT: The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.
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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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    Genome Research 11/2013; 24(2). DOI:10.1101/gr.152132.112 · 13.85 Impact Factor
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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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    ABSTRACT: The microenvironment acts as a conduit for cellular communication, delivering signals that direct development and sustain tissue homeostasis. In pathologies such as cancer, this integral function of the microenvironment is hijacked to support tumor growth and progression. Cells sense the microenvironment via signal transduction pathways culminating in altered gene expression. In addition to induced transcriptional changes, the microenvironment exerts its effect on the cell through regulation of post-transcriptional processes including alternative splicing and translational control. Here we describe how alternative splicing and protein translation are controlled by microenvironmental parameters such as oxygen availability. We also emphasize how these pathways can be utilized to support processes that are hallmarks of cancer such as angiogenesis, proliferation, and cell migration. We stress that cancer cells respond to their microenvironment through an integrated regulation of gene expression at multiple levels that collectively contribute to disease progression.
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