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Global Regulation of the Histone Mark H3k36me2 Underlies Epithelial Plasticity and Metastatic Progression

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Global Regulation of the Histone Mark H3k36me2 Underlies Epithelial Plasticity and Metastatic Progression Published OnlineFirst March 18, 2020; DOI: 10.1158/2159-8290.CD-19-1299 RESEARCH ARTICLE Global Regulation of the Histone Mark H3K36me2 Underlies Epithelial Plasticity and Metastatic Progression Salina Yuan1,2,3, Ramakrishnan Natesan3,4,5, Francisco J. Sanchez-Rivera6, Jinyang Li1,2,3, Natarajan V. Bhanu5,7, Taiji Yamazoe1,2,3, Jeffrey H. Lin1,3, Allyson J. Merrell1,2,3, Yogev Sela1,2,3, Stacy K. Thomas1,3, Yanqing Jiang3,4, Jacqueline B. Plesset3,4, Emma M. Miller8, Junwei Shi3,4,5, Benjamin A. Garcia5,7, Scott W. Lowe6,9, Irfan A. Asangani3,4,5, and Ben Z. Stanger1,2,3 Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 18, 2020; DOI: 10.1158/2159-8290.CD-19-1299 ABSTRACT Epithelial plasticity, reversible modulation of a cell’s epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and eras- ing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differen- tiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with mas- ter regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. SIGNIFIcance: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epi- thelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer. INTRODUCTION proteins such as N-cadherin (Ncad), Vimentin, and Fibronec- tin, are classically induced by a small cohort of master EMT- Cancer cells are capable of dramatic changes in phenotype related transcription factors (“EMT-TF”) including SNAI1/2, and function, a phenomenon known as cellular plasticity (1). TWIST1/2, and ZEB1/2 (2). However, the relative importance Epithelial-to-mesenchymal transition (EMT; i.e., epithelial of each of these transcription factors varies depending on plasticity) and its reverse process (MET) are among the most experimental and clinical contexts, leading to seemingly con- widely studied examples of cellular plasticity; these pro- flicting reports on the roles they play in EMT and cancer pro- grams are utilized during embryonic development for tissue gression (4–6). Such findings raise the possibility that a more morphogenesis and are frequently reactivated during tumor generalized mechanism or common molecular feature might progression (2). In particular, histologic and transcriptomic exist that could unify the activity of these EMT-TFs. signatures relevant to EMT are frequently associated with Epigenetic modifiers and complexes can enact broad, metastasis, therapeutic resistance, and other factors portend- genome-wide changes in transcription; such global epige- ing poor prognosis and patient mortality (3). netic alterations have been reported to underlie other cell The cellular hallmarks of EMT, which include repression fate changes, particularly during normal embryonic devel- of epithelial proteins such as E-cadherin (Ecad), Occludin, opment (7, 8). In tumor cells, several EMT-TFs have previ- and tight junction proteins, and induction of mesenchymal ously been reported to functionally cooperate with various epigenetic modifiers (9); however, such studies have focused on interactions at the promoters of preselected epithelial 1Department of Medicine, University of Pennsylvania, Philadelphia, Penn- and mesenchymal genes (10–16). And although previous sylvania. 2Department of Cell and Developmental Biology, University of profiling efforts have identified genome-scale changes in Pennsylvania, Philadelphia, Pennsylvania. 3Abramson Family Cancer DNA methylation (17) and certain histone marks (18) during Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania. experimentally induced EMT, it remains uncertain whether 4Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania. 5Penn Epigenetics Institute, University of Pennsylvania, these epigenetic changes are necessary and/or sufficient to Philadelphia, Pennsylvania. 6Department of Cancer Biology and Genetics, induce cancer cell plasticity or whether they represent cor- Memorial Sloan Kettering Cancer Center, New York, New York. 7Department related (secondary) events. In this study, we took an unbiased of Biochemistry and Molecular Biophysics, University of Pennsylvania, genetic approach to identify epigenome-wide mechanisms 8 Philadelphia, Pennsylvania. Haverford College, Haverford, Pennsylvania. driving epithelial plasticity and metastatic progression, and 9Howard Hughes Medical Institute, New York, New York. directly interrogated the functional role of specific epigenetic Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). modifications in cancer cell identity. Corresponding Author: Ben Z. Stanger, Perelman School of Medicine at the University of Pennsylvania, 421 Curie Boulevard, BRB II/III, Philadel- phia, PA 19104. Phone: 215-746-5560; E-mail: [email protected] RESULTS Cancer Discov 2020;10:1–18 Modeling Epithelial Plasticity In Vitro doi: 10.1158/2159-8290.CD-19-1299 To better understand epigenetic mechanisms underlying ©2020 American Association for Cancer Research. epithelial plasticity, we established an experimental system JUne 2020 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 18, 2020; DOI: 10.1158/2159-8290.CD-19-1299 RESEARCH ARTICLE Yuan et al. relying on stochastic changes in cell state rather than genetic depleted in the Ecad− fraction. Conversely, sgRNAs targeting or chemical induction. We began by utilizing a panel of Ecad exhibited the opposite pattern (Fig. 1F). Furthermore, single-cell clones derived from the KPCY (LSL-KrasG12D; sgRNAs targeting ribosomal proteins were depleted in both Tp53loxP/+; Pdx1-cre; LSL-RosaYFP/YFP) mouse model of pancre- fractions, whereas the representation of control nontargeting atic ductal adenocarcinoma (PDAC; ref. 19). Clones retained sgRNAs did not change (Supplementary Fig. S3A and S3B). both epithelial and mesenchymal subpopulations, as defined Among the sgRNAs showing significant enrichment or by the presence or absence of surface Ecad (20) and other depletion in the Ecad+ or Ecad− populations (Supplementary classic epithelial or mesenchymal markers (Fig. 1A and B; Table S1), nuclear receptor binding SET domain protein Supplementary Fig. S1A). To confirm that epithelial plasticity 2 (Nsd2) emerged as the top gene for which all targeting is associated with global differences in chromatin landscape, sgRNAs were significantly enriched in the Ecad+ subpopula- we performed Assay for Transposase-Accessible Chromatin tion (Fig. 1F; Supplementary Fig. S3C), second only to Zeb1. using sequencing (ATAC-seq) on FACS-sorted Ecad+ versus NSD2 is a histone methyltransferase that dimethylates his- Ecad− cells and identified chromatin regions exhibiting dif- tone H3 at lysine-36 (H3K36me2), a mark associated with ferential accessibility. Ecad− cells exhibited greater accessibil- actively transcribed genes (21), and acts as an oncogenic ity in genes associated with a mesenchymal phenotype and/ driver in multiple myeloma (22) and a regulator of invasion or EMT (Fig. 1C; Supplementary Fig. S1B). In contrast, Ecad+ and metastasis in prostate cancer (23–25). Conversely, sgR- cells exhibited greater accessibility in genes associated with NAs targeting lysine-specific demethylase 2A Kdm2a( ) were an epithelial phenotype, including Ecad itself (Fig. 1C). Thus, significantly overrepresented in the Ecad− subpopulation FACS sorting based on surface Ecad expression isolates cell (Fig. 1F; Supplementary Fig. S3D). KDM2A is a demethylase populations with distinct chromatin profiles, reflecting tran- that preferentially targets H3K36me2 (26–28). Both Nsd2 and scriptional programs related to epithelial-to-mesenchymal Kdm2a have paralogs with similar catalytic activities, but none state. of these were enriched or depleted in our screen (Supplemen- Next, we sought to ensure that the Ecad+ and Ecad− frac- tary Fig. S3C–S3F), possibly due to low levels of expression tions do not represent fixed, nonplastic subpopulations. To (Supplementary Fig. S3G). These results suggest that NSD2 this end, we sorted pure populations of Ecad+ or Ecad− cells. and KDM2A act nonredundantly on H3K36 as determinants Over a 2-to-4-week period after initial sorting, all cultures of the
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