Gremlin is Overexpressed in Lung Adenocarcinoma and Increases Cell Growth and Proliferation in Normal Lung Cells Michael S. Mulvihill1., Yong-Won Kwon3., Sharon Lee1, Li Tai Fang 1 , Helen Choi1,2, Roshni Ray1, Hio Chung Kang2, Jian-Hua Mao3, David Jablons1,2*, Il-Jin Kim1,2* 1 Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, California, United States of America, 2 Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States of America, 3 Life Sciences Division, Lawrence Berkeley National Laboratory (LBNL), Berkeley, California, United States of America Abstract Background: Gremlin, a member of the Dan family of BMP antagonists, is a glycosylated extracellular protein. Previously Gremlin has been shown to play a role in dorsal-ventral patterning, in tissue remodeling, and recently in angiogenesis. Evidence has previously been presented showing both over- and under-expression of Gremlin in different tumor tissues. Here, we sought to quantify expression of Gremlin in cancers of the lung and performed in vitro experiments to check whether Gremlin promotes cell growth and proliferation. Methodology/Principal Findings: Expression of Gremlin in 161 matched tumor and normal lung cancer specimens is quantified by quantitative real-time PCR and protein level is measured by immunohistochemistry. GREM1 was transfected into lung fibroblast and epithelial cell lines to assess the impact of overexpression of Gremlin in vitro. Results: Lung adenocarcinoma but not squamous cell carcinoma shows a significant increase in Gremlin expression by mRNA and protein level. Lung fibroblast and epithelial cell lines transfected with GREM1 show significantly increased cell proliferation. Conclusions/Significance: Our data suggest that Gremlin acts in an oncogenic manner in lung adenocarcinoma and could hold promise as a new diagnostic marker or potential therapeutic target in lung AD or general thoracic malignancies. Citation: Mulvihill MS, Kwon Y-W, Lee S, Fang LT, Choi H, et al. (2012) Gremlin is Overexpressed in Lung Adenocarcinoma and Increases Cell Growth and Proliferation in Normal Lung Cells. PLoS ONE 7(8): e42264. doi:10.1371/journal.pone.0042264 Editor: Kamyar Afarinkia, Univ of Bradford, United Kingdom Received February 17, 2012; Accepted July 4, 2012; Published August 1, 2012 Copyright: 2012 Mulvihill 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. Funding: This work was supported by the Barbara Isackson Lung Cancer Research Fund, and The Eileen D. Ludwig Endowed Fund for Thoracic Oncology Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: David.Jablons@ucsfmedctr.org (DJ); kimij@cc.ucsf.edu (IJK) . These authors contributed equally to this work. Introduction The discovery of molecular alterations specific to cancerous and pre-cancerous cells has yielded insight into the role played by oncogenes and tumor suppressor genes in the initiation and progression of human cancers [1,2]. Frequently, oncogenes are derived from proto-oncogenes in processes such as point mutations, gene amplifications, or gene rearrangements [3,4]. These structural changes leading to the development of an oncogene then result in quantitative and qualitative changes in the expression of the related protein product. In lung cancer, important oncogenes have previously been identified and used for targeted therapy. EGFR is mutated in around 20% of lung adenocarcinoma (AD) patients [5]. Patients with EGFR mutations have shown a positive response to therapy with erlotinib, although many of these patients relapse later, frequently due to a secondary EGFR mutation, T790M [6]. An oncogenic fusion gene, EML4-ALK, was recently identified [7]. Crizotinib can now be used for the treatment of patients with the EML4-ALK fusion [8]. However, the frequency of EML4-ALK in the Western population is only around 1–7% [7], which means that more than 40% of non-small cell lung cancer (NSCLC) patients without EGFR or EML4-ALK mutations are left without any available targeted therapy [7]. As such, there is an urgent need for the development of new diagnostic markers and potential therapeutic targets to reduce the mortality of lung cancer. To identify novel genes that may potentially play a role in carcinogenesis, we sought to identify genes that were highly upregulated in comparison to matched normal tissue. Gremlin (GREM1) was one of the best candidates with significant over- expression in lung cancer compared to matched normal tissues in several published adenocarcinoma microarray datasets [9,10] Gremlin was initially identified in a Xenopus expression cloning screen and referred to as drm (down-regulated in mos-transformed cells) [11,12]. A 25 kDa protein, it carries a carboxy-terminal cysteine-rich motif that is homologous with a protein domain PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42264