Gremlin Overexpression in Lung Adenocarcinoma Figure 1. Fold-change in Gremlin expression between tumor and normal sample by microarray dataset. Datasets containing tumor and normal sample and probes for GREM1 were selected and a two-tailed, unpaired t-test was conducted between log-transformed and median-centered expression values for GREM1 probes in tumor and normal samples. Datasets representing 17 tumor-normal comparisons contained significant (p,.001) differences in GREM1 expression between tumor and normal samples. doi:10.1371/journal.pone.0042264.g001 5061.56104 cells while wells containing cells receiving the control vector contained an average of 17.6763.186104 cells after 5 days (two-tailed, unpaired t-test p = 9.0461025) (Fig. 4b). A colony formation assay was also performed in the MRC-5 and BEAS-2B cell lines. Like the cell growth curve assay, a dramatic increase in colony number was identified in Gremlin-transfected cells (Fig. 4c). To confirm the findings of the colony proliferation assay, the study was again performed using new independent human lung fibroblast (HFL-1) and lung epithelial (NL-20) cell lines. These assays and immunoblot confirmation of transfection are included as figure S1. Grem1 Increases Cell Growth via a BMP-2-independent Manner To assess whether GREM1 overexpression induces proliferation by way of inhibition of BMP-2, we added recombinant BMP-2 to GREM1-transfected MRC-5 cell lines. High concentrations of BMP-2 did not attenuate GREM1 overexpression-induced cell proliferation, indicating that GREM1 increases normal lung cell growth via a BMP-2 independent pathway (Fig. S2). Discussion Lung cancer is the leading cause of cancer death in men and women, both in the U.S. and worldwide, causing deaths – more than breast, prostate, and colorectal cancers combined [28–29]. Five-year overall survival remains still less than 20% underscoring the need for a revolution in the management of patients with lung cancer [30]. Although a series of key genes have been reported as playing a role in the tumorigenesis of lung cancer, over 40% of NSCLC patients carry no known mutation or clearly targeted therapeutic indication [5]. And while surgical resection remains a mainstay of therapy, recurrence after surgery remains a serious problem even in stage I patients [31]. We hypothesized that characterization of a new molecular marker with significant overexpression in NSCLC compared to matched normal tissues could identify a novel therapeutic or diagnostic marker. The work reported here describes a potential oncogenic role for Gremlin. To begin, we conducted a systematic review of published Oncomine data to identify tumor types in which Gremlin may be upregulated in comparison to normal tissue. We identified 24 datasets published to Oncomine that contained both tumor and normal samples suitable for analysis. Comparison of fold-change (t-test, p,.001) between tumor and normal yielded 10 tumor types in which Gremlin was upregulated and 5 in which it was down regulated. Of the datasets in which Gremlin was upregulated, four datasets were of lung tumors (two adenocarcinoma datasets and two squamous cell carcinoma datasets) [9,10]. Significant upregulation in four lung tumor datasets demonstrated the potential for a significant role of Gremlin in lung cancer. Following analysis of published microarray data to identify that Gremlin is significantly overexpressed in NSCLC microarray data, we performed quantitative RT-PCR analysis in our 96 AD with matched normal and 65 SCC with matched normal samples. Gremlin expression is significantly increased in lung AD samples compared to matched normal tissues (Fig. 2, p = 1.81E-22). This is consistent with publicly available microarray data that Gremlin is highly overexpressed in tumor [11,20,32]. Unlike two published SCC microarray datasets, we did not see a significant up- regulation of SCC compared to matched normal tissue (Fig. 2, PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42264