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Integrated Cistromic and Expression Analysis of Amplified NKX2-1 in Lung Adenocarcinoma Identifies LMO3 As a Functional Transcriptional Target

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Integrated Cistromic and Expression Analysis of Amplified NKX2-1 in Lung Adenocarcinoma Identifies LMO3 As a Functional Transcriptional Target Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Watanabe, Hideo, Joshua M. Francis, Michele S. Woo, Banafsheh Etemad, Wenchu Lin, Daniel F. Fries, Shouyong Peng, et al. “Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target.” Genes & Development 27, no. 2 (January 24, 2013): 197-210. As Published http://dx.doi.org/10.1101/gad.203208.112 Publisher Cold Spring Harbor Laboratory Press Version Final published version Citable link http://hdl.handle.net/1721.1/85103 Terms of Use Article is available under a Creative Commons license; see publisher's site for details. Detailed Terms http://creativecommons.org/ Downloaded from genesdev.cshlp.org on January 28, 2014 - Published by Cold Spring Harbor Laboratory Press Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target Hideo Watanabe, Joshua M. Francis, Michele S. Woo, et al. Genes Dev. 2013 27: 197-210 originally published online January 15, 2013 Access the most recent version at doi:10.1101/gad.203208.112 Supplemental http://genesdev.cshlp.org/content/suppl/2013/01/09/gad.203208.112.DC1.html Material References This article cites 71 articles, 32 of which can be accessed free at: http://genesdev.cshlp.org/content/27/2/197.full.html#ref-list-1 Open Access Freely available online through the Genes & Development Open Access option. Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the top Service right corner of the article or click here. To subscribe to Genes & Development go to: http://genesdev.cshlp.org/subscriptions Copyright © 2013 by Cold Spring Harbor Laboratory Press Downloaded from genesdev.cshlp.org on January 28, 2014 - Published by Cold Spring Harbor Laboratory Press Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target Hideo Watanabe,1,2,3 Joshua M. Francis,1,2,3 Michele S. Woo,1,2 Banafsheh Etemad,1 Wenchu Lin,1,2 Daniel F. Fries,1,3 Shouyong Peng,1,3 Eric L. Snyder,4 Purushothama Rao Tata,5 Francesca Izzo,1,6 Anna C. Schinzel,1,6 Jeonghee Cho,1,2 Peter S. Hammerman,1,2,3 Roel G. Verhaak,1,2,3 William C. Hahn,1,2,3,6 Jayaraj Rajagopal,5 Tyler Jacks,4,7 and Matthew Meyerson1,2,3,8,9 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; 2Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; 3Cancer Program, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA; 4David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; 5Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, Boston, Massachusetts 02114, USA; 6RNAi Facility, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; 7Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; 8Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA- binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas. [Keywords: lung cancer; lineage-specific oncogene; transcription factor; cistromic analysis; carcinogenesis] Supplemental material is available for this article. Received August 20, 2012; revised version accepted December 20, 2012. Genomic alterations of transcription factors are known scription factors, genomic alterations are only associated to play key roles in the development of many human with particular types of cancers: For example, AR ampli- cancers. For example, TP53 (the gene encoding p53) is the fication is linked to mechanisms of resistance in recurrent most frequently mutated gene in all cancers (Levine and prostate cancers (Visakorpi et al. 1995), PAX5 deletion is Oren 2009), MYC is frequently amplified in many types of linked to acute lymphocytic leukemia (Mullighan et al. cancers (including breast carcinomas and small-cell lung 2007), and RUNX1 translocation is linked to acute mye- carcinomas) and is translocated in Burkitt’s lymphomas logenous leukemia (Miyoshi et al. 1991). In addition, there (Dalla-Favera et al. 1982; Taub et al. 1982), and ETS family has been emerging evidence that a lineage-restricted members are frequently translocated in prostate cancer genomic amplification of developmental transcription (Tomlins et al. 2005), Ewing’s sarcoma (Delattre et al. factors occurs frequently in solid tumors, as exemplified 1992), and leukemias (Peeters et al. 1997). For some tran- by MITF in melanomas and SOX2 in lung and esophageal squamous cell carcinomas (Garraway et al. 2005; Bass et al. 2009). 9Corresponding author NKX2-1 is the most significantly focally amplified gene E-mail [email protected] in lung adenocarcinomas, with amplification detected in Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.203208.112. Freely ;12% of cases (Kendall et al. 2007; Tanaka et al. 2007; available online through the Genes & Development Open Access option. Weir et al. 2007; Kwei et al. 2008). NKX2-1, also referred GENES & DEVELOPMENT 27:197–210 Ó 2013 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/13; www.genesdev.org 197 Downloaded from genesdev.cshlp.org on January 28, 2014 - Published by Cold Spring Harbor Laboratory Press Watanabe et al. to as TTF-1 (for thyroid transcription factor 1), is well adenocarcinoma (Yamaguchi et al. 2012); however, the known as a molecular marker for lung adenocarcinoma transcriptional consequences of NKX2-1 amplification and is particularly useful in clinical diagnosis of meta- in lung adenocarcinoma and the mechanism underlying static carcinomas, where its identification supports the its oncogenic activity in this disease have not been tumor originating in the lung (Bejarano et al. 1996; established. Holzinger et al. 1996). Nkx2-1 is required for the de- In the normal lung, NKX2-1 induces a subset of gene velopment of the trachea, brain, and thyroid in early expression changes involved in the differentiation of murine embryonic development and for peripheral lung- alveolar type II cells. Among the directly induced genes branching morphogenesis later in development (Costa reported are LAMP3, CEACAM6, and CIT (Kolla et al. et al. 2001; Maeda et al. 2007). Mice lacking Nkx2-1 die 2007), and an NKX2-1 overexpression signature in BEAS-2B at birth of respiratory failure with hypoplastic lungs bronchoepithelial cells includes focal adhesion and oxi- that stem from an undivided foregut (Yuan et al. 2000). dative phosphorylation pathways (Hsu et al. 2009). Pro- NKX2-1 may belong to the class of lineage survival moter regions directly bound by Nkx2-1 in developing oncogenes, which are ordinarily required for the differen- lungs have been also reported, which include the pro- tiation and survival of particular cell lineages and later moters of E2f3, Ccnb1, and Ccnb2 genes (Tagne et al. become subject to focal amplification in cancers within 2012). Mechanistically, transcriptional activity of Nkx2-1 their own lineage (Garraway and Sellers 2006). While the has been shown to be facilitated by interaction with specific cell of origin that gives rise to lung adenocarci- several cellular proteins, including nuclear hormone re- nomas has yet to be precisely characterized, NKX2-1 is ceptors such as the retinoic acid receptor (RAR), zinc required for the survival of lung adenocarcinoma cells finger transcription factors such as Gata-6, and coactiva- with amplification of NKX2-1 (Kendall et al. 2007; Tanaka tors such as Src (Maeda et al. 2007). et al. 2007; Weir et al. 2007; Kwei et al. 2008). Here, using integrated cistromic and gene expression The role of NKX2-1 in cancer pathogenesis is complex analysis, we show that NKX2-1 amplification is associ- and remains poorly understood. Activating translocations ated with overexpression of the LMO3 gene, a member of NKX2-1 have been reported in ;3% of acute pre-T-cell of the LMO family of oncogenes that are translocated in lymphoblastic leukemias (T-ALL) (Homminga et al. 2011), T-ALL (Boehm et al. 1988b; McGuire
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