shared by members of secreted proteins such as DAN and Cerberus [13]. Drm was identified as a novel gene that is suppressed in cells transformed by v-ras, v-src, v-raf, and v-fos. It was shown that DRM can inhibit the growth of normal but not transformed cells in culture [11]. A possible tumor-suppressor role was proposed for drm on the basis of its down-regulation in these transformed cell lines and it was hypothesized that high levels of drm inhibit the growth or viability of normal cells, but that transformed cells are resistant to this inhibitory effect [11]. Studies of normal development of the limb have implicated a role for Gremlin in proper establishment of limb bud morphology. Gremlin indirectly enhances FGF-mediated limb outgrowth while simultaneously inhibiting chondrogenesis and cell death [14,15]. Sonic Hedgehog (SHH) has also been shown to upregulate and maintain Gremlin expression, enabling Gremlin to relieve the repressive effects of BMP-4 on FGF-4 expression, yielding a net positive feedback to increase SHH [14,16]. The importance of the interaction between Gremlin and the BMP signaling pathway in the normal development of the proximal-distal patterning of the lung has previously been investigated. Gremlin acts as a functional physiological antagonist that restricts BMP-4 activity to the distal bud, thereby regulating the number of branching epithelial sacs [17]. Similarly, antago- nism of BMP-4 signal using the BMP antagonist Xnoggin results in a severe reduction in distal epithelial cell types and an increase in proximal cell types [18]. Overexpression of Gremlin in the distal lung epithelium using an SP-C promoter in mice results in transgenic lungs that phenotypically resemble proximal airways epithelium with decreased squamous epithelium [19]. Deletion of GREM1 in mouse embryonic stem cells results in a neonatal lethal phenotype characterized by a reduction in differentiated alveoli and multi-layered epithelium in comparison to wild-type embryos [2]. RNA and protein analysis indicates that Gremlin is frequently undetectable in multiple malignant cell lines, including neuroblas- toma, glioblastoma, medulloblastoma, and colon adenocarcinoma [20]. The pattern of expression suggests a possible role as an inhibitor of tumor progression in these lineages. Overexpression of Gremlin was sufficient to inhibit the neoplastic phenotype of both Daoy and Soas-2 cell line [21]. Analysis of publicly-available microarray data also suggests a significant downregulation of Gremlin in tumors of the CNS [22]. While these studies have suggested a possible tumor-suppressive role for Gremlin, recent work has shown an oncogenic role for Gremlin in other tumor types. Gremlin is over-expressed in stromal cells associated with basal cell carcinomas of the skin, and can promote cell proliferation in this model [23]. Gremlin is also upregulated in the lung cancer cell line A549 per a semi- quantitative northern blot analysis [24]. It was recently reported that Gremlin is overexpressed in many malignant mesothelioma (MM) tissue specimens [25]. Inhibition of Gremlin via shRNA significantly inhibited proliferation of MM cell lines. The function of Gremlin in cancer cells is controversial and appears to work in a tissue-specific manner. In lung cancer, nothing to date is known about the role of Gremlin. Thus, we performed genetic and histological analysis of Gremlin in a large number of lung AD and squamous cell carcinoma (SCC) samples. mRNA and protein expression analysis of Gremlin were done in 161 matched tumor-normal pairs. Of these, 96 pairs were of AD, while 65 of the pairs were from SCC. The potential oncogenic function of Gremlin was analyzed by GREM1 transfection in multiple normal lung fibroblast and epithelial cells. Gremlin Overexpression in Lung Adenocarcinoma Results Expression Analysis of Gremlin in Published Microarray Data A systematic review of publicly available microarray data within the Oncomine database (www.oncomine.org) was conducted. Appropriate datasets were identified using a search for tumor – normal comparisons of datasets containing probes targeting GREM1 (Gremlin). Expression values were log-transformed and median-centered per array. Our search identified 24 datasets appropriate for analysis. Comparison of fold-change (t-test, p,.001) between tumor and normal yielded 4 lung cancer datasets in which Gremlin expression was significantly changed. Two datasets involving lung adenocarcinoma specimens with matched normal tissue showed significant upregulation [9,19], as did two squamous cell carcinoma datasets that did not utilize paired tumor – normal specimens, instead providing normal specimens matched to adenocarcinoma samples [26,27]. A total of 17 datasets with significant fold-change in Gremlin expression between tumor and normal were identified. Cited literature was then reviewed to confirm that the analysis was as documented in the Oncomine annotation. Fold-change representation of change in Gremlin expression across datasets available in Oncomine is included in figure 1. Gremlin is Overexpressed in Lung AD, not SCC To establish the change in expression of Gremlin between tumor and matched normal tissue, a quantitative real-time PCR experiment (Taqman) was performed. Ninety-six adenocarcinoma samples were determined to have significantly higher (p = 1.81E- 22) expression of Gremlin when compared to matched normal samples. Sixty-five samples of squamous cell carcinoma were determined to not have any significant difference in expression between tumor and matched normal sample. Change in GAPDH- normalized expression by quantitative real-time PCR is repre- sented in figure 2. High Gremlin Protein Expression in lung AD, not SCC Immunohistochemistry (IHC) was selected to assess if up- regulation of the Gremlin mRNA resulted in a commensurate increase in protein expression. Immunohistochemical staining permitted visualization and localization of Gremlin expression in tumor samples and in matched normals. A total of 24 pairs of adenocarcinoma with matched normal and 8 pairs of squamous cell carcinoma with matched normal tissue were stained for Gremlin protein expression. Gremlin immunoreactivity was observed to be stronger in sections from adenocarcinoma than in matched normal sections. Approximately 67% (16 of 24 samples) of adenocarcinoma samples demonstrated increased Gremlin expression, while 25% (2 of 8 samples) of squamous cell carcinoma samples demonstrated increased expression. Represen- tative immunohistochemical data of adenocarcinoma, squamous cell carcinoma, and matched normal tissue are shown in figure 3. Cell Growth and Colony Formation Assay The effects of Gremlin overexpression on cell proliferation were studied using a growth curve assay. The efficiency of Gremlin transfection was checked by immunoblot (Fig. 4a). In the MRC5 cell line, wells containing cells transfected with GREM1 vector contained an average of 61.266.256104 cells after 5 days while wells containing cells receiving vector alone contained an average of 4.8360.286104 cells (two-tailed, unpaired t-test p = 9.8861025). In the BEAS-2B cell line, wells containing cells transfected with the GREM1 vector contained an average of PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42264