Gremlin Overexpression in Lung Adenocarcinoma Figure 4. Effect of Grem1 overexpression on MRC5 and BEAS-2B cell proliferation. MRC5 and BEAS-2B pBabepur-flag-Grem1 cells overexpressing flag-tagged Grem1. (A) Grem1 protein expression by immunoblot analysis. B-actin protein expression was the loading control. (B) Growth curve of MRC5 and BEAS-2B cells infected with control vectors or flag-tagged Grem1. Cells (56103 cells/well) were seeded onto 6-well plates. For each day, three plates were counted and averaged. Error bars show standard deviation of triplicate samples. (two-tailed, unpaired t-test between GREM1 and vector MRC5 p = 9.8861025 BEAS-2B p = 9.0461025) (C) Colony formation assays documenting the effect of Grem1 overexpression on the growth of MRC5 and BEAS-2B cells. doi:10.1371/journal.pone.0042264.g004 p = 0.705) [26,27]. It is possible that either sample size or a lack of paired samples in published microarray data may contribute to these findings. While our study utilized 65 pairs of SCC and matched normal tissues, samples from Garber et al and Bhatta- charjee et al appear to have used 13 and 21 tumor specimens, respectively, but expression levels are compared with normal specimen expression levels pooled from all lung tumor subtypes included in the respective studies [26,27]. The other possibility is a difference of platform. We conducted a quantitative RT-PCR analysis using a Taqman system while comparative studies utilized a microarray platform. Next, we checked the protein expression of Gremlin by IHC. We analyzed 24 pairs of AD and 8 pairs of SCC. As shown in Figure 3, Gremlin expression was observed in 67% of the AD samples to be greater than in matched normal slides. In SCC, only 2 of 8 samples demonstrated increased immunoreactivity in tumor specimens (Fig. 3). We conducted a series of in vitro experiments with Gremlin transfection to assess the effect of Gremlin overexpression in lung fibroblast and epithelial cells. Transfection of GREM1 significant- ly increased cell growth (Fig. 4b) in both normal lung fibroblast (MRC-5) and epithelial (BEAS-2B) cells. Moreover, many colonies were found in GREM1-transfected cells while almost no (MRC-5) or low (BEAS-2B) numbers of colonies were found in vector- transfected cells (Fig. 4c). The use of two additional independent cell lines – HLF-1 and NL-20– reiterated these findings (Fig. S1). These in vitro data support our hypothesis that overexpression of Gremlin in lung cancer is involved in lung tumorigenesis and promotes cell growth and proliferation. While previous work has shown an anti-proliferative role in malignant non-thoracic cell lines, this current study and the recent work of Wang et al suggests an oncogenic role for Gremlin in thoracic malignancies [25]. This difference in tumor-suppressive or tumor-proliferative role for Gremlin appears to be a cell-type dependent effect. Topol et al and Chen et al have shown previously that overexpression of Gremlin can inhibit proliferation in tumor-derived cell lines Daoy (primitive neuroectodermal) and Saos-2 (osteoblastic) [11,21]. This finding is in concordance with published microarray data showing that Gremlin is consistently downregulated in tumors of the CNS in comparison to matched normals [28]. Thus, overexpression of Gremlin in certain organs such as lung may promote tumor growth, while down-regulation of Gremlin in other organs such as brain may inhibit tumor growth. The mechanism by which Gremlin enhances proliferation in lung AD remains unclear. Recent developments in the role of BMP2/4 in tumorigenesis and Gremlin’s role in angiogenesis may provide novel directions to elucidate the mechanism by which Gremlin can induce proliferation in lung AD. The ability of Gremlin to antagonize members of the BMP family has previously been well-documented [12]. However, the role of the BMP2/4 proteins in tumorigenesis is controversial [33]. As recently reviewed by Singh and Morris [33], the BMP2/4 proteins are commonly shown to have anti-tumorigenic roles, such as the negative regulation of A549 cell line growth by BMP4 in Buckley, et al [34]. However, BMP2 has been shown in some settings to enhance tumor growth in vivo due to activation of Smad-1/5 PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42264