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

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          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.

          Author Summary

          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.

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          Most cited references43

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          Alternative splicing: new insights from global analyses.

          Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions. Recent progress in these areas is summarized in this review.
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            New signals from the invasive front.

            Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. Revealing their mechanisms is one of the main challenges for exploratory and applied cancer research. Recent experimental progress has identified a number of molecular pathways and cellular mechanisms that underlie the multistage process of metastasis formation: these include tumour invasion, tumour-cell dissemination through the bloodstream or the lymphatic system, colonization of distant organs and, finally, fatal outgrowth of metastases.
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              Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription.

              Many cofactors bind the hormone-activated estrogen receptor (ER), yet the specific regulators of endogenous ER-mediated gene transcription are unknown. Using chromatin immunoprecipitation (ChIP), we find that ER and a number of coactivators rapidly associate with estrogen responsive promoters following estrogen treatment in a cyclic fashion that is not predicted by current models of hormone activation. Cycles of ER complex assembly are followed by transcription. In contrast, the anti-estrogen tamoxifen (TAM) recruits corepressors but not coactivators. Using a genetic approach, we show that recruitment of the p160 class of coactivators is sufficient for gene activation and for the growth stimulatory actions of estrogen in breast cancer supporting a model in which ER cofactors play unique roles in estrogen signaling.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                August 2011
                August 2011
                18 August 2011
                : 7
                : 8
                : e1002218
                Affiliations
                [1 ]Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                [2 ]Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                [3 ]Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York, United States of America
                [4 ]Department of Pathology, Montefiore Medical Center, Bronx, New York, United States of America
                [5 ]Department of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                University of Pennsylvania, United States of America
                Author notes

                Conceived and designed the experiments: IMS AWC CBB FBG. Performed the experiments: IMS NCF MB. Analyzed the data: IMS AWC. Contributed reagents/materials/analysis tools: MHO JSC. Wrote the paper: IMS AWC FBG CBB.

                Article
                PGENETICS-D-10-00244
                10.1371/journal.pgen.1002218
                3158048
                21876675
                dd222abe-4a7e-45e4-86c3-6c6e2e615c21
                Shapiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 2 November 2010
                : 17 June 2011
                Page count
                Pages: 21
                Categories
                Research Article
                Biology
                Genomics
                Genome Sequencing
                Molecular Cell Biology
                Gene Expression
                RNA processing
                Medicine
                Oncology
                Cancer Detection and Diagnosis
                Cancer Screening
                Early Detection
                Basic Cancer Research

                Genetics
                Genetics

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