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

Irina M. Shapiro1, Albert W. Cheng1, Nicholas C. Flytzanis1, Michele Balsamo1, John S. Condeelis2, Maja H. Oktay3, Christopher B. Burge1, Frank B. Gertler1 •
Massachusetts Institute of Technology1, Albert Einstein College of Medicine2, Montefiore Medical Center3
01 Aug 2011-
TL;DR: The 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.
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.
Citations
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Journal Article•DOI•
Molecular mechanisms of epithelial–mesenchymal transition

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Samy Lamouille1, Jian Xu2, Rik Derynck1•
University of California, San Francisco1, University of Southern California2
01 Mar 2014-Nature Reviews Molecular Cell Biology
TL;DR: The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues, and the convergence of signalling pathways is essential for EMT.
Abstract: The transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT), is integral in development, wound healing and stem cell behaviour, and contributes pathologically to fibrosis and cancer progression. This switch in cell differentiation and behaviour is mediated by key transcription factors, including SNAIL, zinc-finger E-box-binding (ZEB) and basic helix-loop-helix transcription factors, the functions of which are finely regulated at the transcriptional, translational and post-translational levels. The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues. Among these, transforming growth factor-β (TGFβ) family signalling has a predominant role; however, the convergence of signalling pathways is essential for EMT.

6,036 citations

Journal Article•DOI•
Regulatory networks defining EMT during cancer initiation and progression

[...]

Bram De Craene1, Geert Berx1•
Ghent University1
01 Feb 2013-Nature Reviews Cancer
TL;DR: The EMT-associated reprogramming of cells not only suggests that fundamental changes may occur to several regulatory networks but also that an intimate interplay exists between them.
Abstract: Epithelial to mesenchymal transition (EMT) is essential for driving plasticity during development, but is an unintentional behaviour of cells during cancer progression. The EMT-associated reprogramming of cells not only suggests that fundamental changes may occur to several regulatory networks but also that an intimate interplay exists between them. Disturbance of a controlled epithelial balance is triggered by altering several layers of regulation, including the transcriptional and translational machinery, expression of non-coding RNAs, alternative splicing and protein stability.

2,136 citations

Journal Article•DOI•
Cancer Invasion and the Microenvironment: Plasticity and Reciprocity

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Peter Friedl1, Peter Friedl2, Peter Friedl3, Stephanie Alexander2, Stephanie Alexander3 •
Radboud University Nijmegen Medical Centre1, University of Texas MD Anderson Cancer Center2, University of Würzburg3
23 Nov 2011-Cell
TL;DR: The cell-matrix and cell-cell adhesion, protease, and cytokine systems that underlie tissue invasion by cancer cells are described and explained to explain how the reciprocal reprogramming of both the tumor cells and the surrounding tissue structures not only guides invasion, but also generates diverse modes of dissemination.

1,693 citations

Cites background from "An EMT-Driven Alternative Splicing ..."

  • ...…between primary and metastatic lesions within the same patient (intrapatient heterogeneity), within the same lesion (intratumoral heterogeneity), and across time (Choi et al., 2011; Honeth et al., 2008; Lopes et al., 2009; Shapiro et al., 2011; Stoecklein et al., 2008; Wang et al., 2009)....

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  • ...Once ‘‘activated,’’ these cells reorganize the structure and composition of the connective tissue by depositing extracellular matrix components (ECM), cytokines, and growth factors (Egeblad et al., 2010; He et al., 2011; Picchio et al., 2008; Shapiro et al., 2011)....

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  • ...…control on the cell cycle and cell survival, many growth factors, includingHGF, EGF, FGF, and TGFb, share signaling through ERK, JNK, Src, mTOR, and PI3K pathways toward Rac and Cdc42 activation and enhanced cytoskeletal dynamics (Massagué, 2008; Shapiro et al., 2011; Trusolino et al., 2010)....

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  • ...Leading Edge Review Cancer Invasion and the Microenvironment:...

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Journal Article•DOI•
The human transcriptome across tissues and individuals

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Marta Melé1, Pedro G. Ferreira, Ferran Reverter2, Ferran Reverter3, David S. DeLuca4, Jean Monlong2, Jean Monlong5, Michael Sammeth2, Taylor Young4, Jakob M. Goldmann6, Jakob M. Goldmann2, Dmitri D. Pervouchine7, Dmitri D. Pervouchine2, Timothy J. Sullivan4, Rory Johnson2, Ayellet V. Segrè4, Sarah Djebali2, Anastasia Niarchou8, Anastasia Niarchou9, Fred A. Wright10, Tuuli Lappalainen, Miquel Calvo3, Gad Getz4, Gad Getz1, Emmanouil T. Dermitzakis9, Emmanouil T. Dermitzakis8, Kristin G. Ardlie4, Roderic Guigó •
Harvard University1, Pompeu Fabra University2, University of Barcelona3, Broad Institute4, McGill University5, Radboud University Nijmegen6, Moscow State University7, University of Geneva8, Swiss Institute of Bioinformatics9, North Carolina State University10
08 May 2015-Science
TL;DR: Tissues exhibit characteristic transcriptional signatures that show stability in postmortem samples that are dominated by a relatively small number of genes, though few are exclusive to a particular tissue and vary more across tissues than individuals.
Abstract: Transcriptional regulation and posttranscriptional processing underlie many cellular and organismal phenotypes. We used RNA sequence data generated by Genotype-Tissue Expression (GTEx) project to investigate the patterns of transcriptome variation across individuals and tissues. Tissues exhibit characteristic transcriptional signatures that show stability in postmortem samples. These signatures are dominated by a relatively small number of genes—which is most clearly seen in blood—though few are exclusive to a particular tissue and vary more across tissues than individuals. Genes exhibiting high interindividual expression variation include disease candidates associated with sex, ethnicity, and age. Primary transcription is the major driver of cellular specificity, with splicing playing mostly a complementary role; except for the brain, which exhibits a more divergent splicing program. Variation in splicing, despite its stochasticity, may play in contrast a comparatively greater role in defining individual phenotypes.

1,131 citations

Journal Article•DOI•
Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression.

[...]

Xin Ye1, Robert A. Weinberg1•
Massachusetts Institute of Technology1
01 Nov 2015-Trends in Cell Biology
TL;DR: It is proposed that epithelial-mesenchymal plasticity is likely controlled by multiple varients of the core EMT program, and foresee the need to resolve the various programs and the molecular mechanisms that underlie them.

811 citations

Cites background from "An EMT-Driven Alternative Splicing ..."

  • ...…that of the ESRP (epithelial splicing regulatory proteins), RBFOX, MBNL (Muscleblind-like), CELF (CUG-BP- and ETR-3-like factors), and hnRNP (heterogeneous nuclear ribonucleoproteins) families, thereby promoting production of mRNA isoforms specific to the mesenchymal state (Shapiro et al., 2011)....

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  • ...The EMT program also elicits profound changes in the transcriptome within a cell by altering the expression of splicing factors, including that of the ESRP (epithelial splicing regulatory proteins), RBFOX, MBNL (Muscleblind-like), CELF (CUG-BP- and ETR-3-like factors), and hnRNP (heterogeneous nuclear ribonucleoproteins) families, thereby promoting production of mRNA isoforms specific to the mesenchymal state (Shapiro et al., 2011)....

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References
More filters
Journal Article•DOI•
Mapping and quantifying mammalian transcriptomes by RNA-Seq.

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Ali Mortazavi1, Brian A. Williams1, Kenneth McCue1, Lorian Schaeffer1, Barbara J. Wold1 •
California Institute of Technology1
29 Jun 2008-Nature Methods
TL;DR: Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors.
Abstract: We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other

12,293 citations

"An EMT-Driven Alternative Splicing ..." refers methods in this paper

  • ...We first estimated gene expression changes during EMT using ‘Reads Per Kilobase of Exon Model per Million Mapped Reads’ (RPKM), a measure of expression that reflects the molar concentration of a transcript in the sample by normalizing read counts for mRNA length and for the total read number in the sample [32]....

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Journal Article•DOI•
Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications

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Therese Sørlie, Charles M. Perou1, Robert Tibshirani2, Turid Aas3, Stephanie Geisler3, Hilde Johnsen4, Trevor Hastie2, Michael B. Eisen5, Matt van de Rijn2, Stefanie S. Jeffrey2, T. Thorsen3, Hanne Quist4, John C. Matese2, Patrick O. Brown2, David Botstein2, Per Eystein Lønning3, Anne Lise Børresen-Dale •
University of North Carolina at Chapel Hill1, Stanford University2, University of Bergen3, University of Oslo4, University of California, Berkeley5
11 Sep 2001-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.
Abstract: The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.

10,791 citations

Journal Article•DOI•
The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells

[...]

Sendurai A. Mani1, Wenjun Guo1, Mai Jing Liao1, Elinor Ng Eaton1, Ayyakkannu Ayyanan2, Alicia Y. Zhou1, Mary W. Brooks1, Ferenc Reinhard1, Cheng Cheng Zhang1, Michail Shipitsin3, Lauren L. Campbell3, Kornelia Polyak3, Cathrin Brisken2, Jing Yang4, Robert A. Weinberg1 •
Massachusetts Institute of Technology1, École Polytechnique Fédérale de Lausanne2, Harvard University3, University of California, San Diego4
16 May 2008-Cell
TL;DR: It is reported that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers, and it is shown that those cells have an increased ability to form mammospheres, a property associated with mammARY epithelial stem cells.

8,052 citations

"An EMT-Driven Alternative Splicing ..." refers background in this paper

  • ...model in which mammary epithelial cells (HMLE) expressing Twist fused to a modified estrogen receptor (ER) undergo EMT when the fusion protein is activated by addition of the ER ligand 4-hydroxytamoxifen (4-OHT; tamoxifen)...

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Journal Article•DOI•
Alternative Isoform Regulation in Human Tissue Transcriptomes

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Eric T. Wang1, Rickard Sandberg2, Rickard Sandberg1, Shujun Luo3, Irina Khrebtukova3, Lu Zhang3, Christine Mayr1, Stephen F. Kingsmore4, Gary P. Schroth3, Christopher B. Burge1 •
Massachusetts Institute of Technology1, Karolinska Institutet2, Illumina3, National Center for Genome Resources4
27 Nov 2008-Nature
TL;DR: An in-depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments yielding a digital inventory of gene and mRNA isoform expression suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.
Abstract: Through alternative processing of pre-messenger RNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analyses in which sequence reads are mapped to exon-exon junctions indicated that 92-94% of human genes undergo alternative splicing, 86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that most alternative splicing and alternative cleavage and polyadenylation events vary between tissues, whereas variation between individuals was approximately twofold to threefold less common. Extreme or 'switch-like' regulation of splicing between tissues was associated with increased sequence conservation in regulatory regions and with generation of full-length open reading frames. Patterns of alternative splicing and alternative cleavage and polyadenylation were strongly correlated across tissues, suggesting coordinated regulation of these processes, and sequence conservation of a subset of known regulatory motifs in both alternative introns and 3' untranslated regions suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.

4,711 citations

"An EMT-Driven Alternative Splicing ..." refers background in this paper

  • .... More than 90% of human genes are estimated to undergo alternative splicing, with a majority of alternative splicing events exhibiting tissue-specific splicing differences...

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Journal Article•DOI•
Twist, a Master Regulator of Morphogenesis, Plays an Essential Role in Tumor Metastasis

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Jing Yang1, Sendurai A. Mani1, Joana Liu Donaher1, Sridhar Ramaswamy2, Sridhar Ramaswamy3, Raphael Itzykson4, Christophe Côme5, Pierre Savagner5, Inna Gitelman6, Andrea L. Richardson3, Robert A. Weinberg1 •
Massachusetts Institute of Technology1, Broad Institute2, Harvard University3, École Normale Supérieure4, French Institute of Health and Medical Research5, Ben-Gurion University of the Negev6
25 Jun 2004-Cell
TL;DR: A mechanistic link between Twist, EMT, and tumor metastasis is established, suggesting that Twist contributes to metastasis by promoting an epithelial-mesenchymal transition (EMT).

3,670 citations

"An EMT-Driven Alternative Splicing ..." refers background or methods in this paper

  • ...Ectopic Twist expression in epithelial cells results in loss of E-cadherin-mediated cell-cell adhesion, acquisition of mesenchymal markers and increased motility of isolated cells [18], a hallmark of the mesenchymal phenotype....

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  • ...phenotype, including N-cadherin and vimentin [16,18,48,49]....

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  • ...As expected, control HMLE/pBP epithelial cells expressed high levels of E-cadherin while HMLE/pBP-Twist mesenchymal cells expressed high levels of N-cadherin (Figure 6A; [18])....

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  • ...We introduced ESRP1-EGFP into HMLE/pBP-Twist cells, immortalized human mammary epithelial cells that ectopically express Twist [18], and analyzed expression of canonical EMT markers....

    [...]

  • ...As expected, E-cadherin was downregulated, while N-cadherin was upregulated during EMT [18]; actin transcript levels remained unchanged (Figure S2A)....

    [...]

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