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


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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    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.
    Cell Reports 06/2014; 7(6). DOI:10.1016/j.celrep.2014.05.010 · 8.36 Impact Factor
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    • "Saha et al. compiled the asthma biomarkers from genomics, proteomics, epigenetics and few experimentally validated datasets, and derived significant enrichment of pathways, including adipokine and ROS pathways [107]. Similarly, gene profiling studies on cancer-related type III EMT have shown that TGFβ induces global changes in mRNA expression, mRNA splicing [108] and microRNA expression [109]. Parallel proteomics studies have demonstrated that type III EMT induces growth factor independence pathways downstream of IL-6 signaling [110], which has led to a better understanding of cellular and signaling processes involved in cancer metastases [111,112]. "
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    ABSTRACT: A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma.
    World Allergy Organization Journal 06/2014; 7(1):13. DOI:10.1186/1939-4551-7-13
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    • "It has been observed that cancer cells tend to shift the splice isoform repertoire toward those normally expressed in early developmental stages.20 EMT has been associated with global changes in splicing programs involving genes important for cellular morphology and movement.27 This review will summarize the current state of knowledge of aberrant splicing regulation of genes and pathways in prostate cancer (PCa) and will discuss emerging evidence for its role in the development of castration resistance. "
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    ABSTRACT: Alternative splicing (AS) is a crucial step in gene expression. It is subject to intricate regulation, and its deregulation in cancer can lead to a wide array of neoplastic phenotypes. A large body of evidence implicates splice isoforms in most if not all hallmarks of cancer, including growth, apoptosis, invasion and metastasis, angiogenesis, and metabolism. AS has important clinical implications since it can be manipulated therapeutically to treat cancer and represents a mechanism of resistance to therapy. In prostate cancer (PCa) AS also plays a prominent role and this review will summarize the current knowledge of alternatively spliced genes with important functional consequences. We will highlight accumulating evidence on AS of the components of the two critical pathways in PCa: androgen receptor (AR) and phosphoinositide 3-kinase (PI3K). These observations together with data on dysregulation of splice factors in PCa suggest that AR and PI3K pathways may be interconnected with previously unappreciated splicing regulatory networks. In addition, we will discuss several lines of evidence implicating splicing regulation in the development of the castration resistance.
    Asian Journal of Andrology 05/2014; 16(4). DOI:10.4103/1008-682X.127825 · 2.60 Impact Factor
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