Gprc5a functions as a tumor suppressor in mouse lung, and human GPRC5A may share this property. The Gprc5a-deficient mouse is a novel model to study lung carcinogenesis and chemoprevention.

Mouse models can be useful for increasing the understanding of lung tumorigenesis and assessing the potential of chemopreventive agents. We explored the role of inflammation in lung tumor development in mice with knockout of the tumor suppressor Gprc5a. Examination of normal lung tissue and tumors from 51 Gprc5a+/+ (adenoma incidence 9.8%; adenocarcinoma 0%) and 38 Gprc5a−/− mice (adenoma 63%, adenocarcinoma 21%) revealed macrophages infiltration into lungs of 45% of the Gprc5a−/− mice and 8% of Gprc5a+/+ mice and the direct association of macrophages with 42% of adenomas and 88% of adenocarcinomas in the knockout mice. Gprc5a−/− mouse lungs contained higher constitutive levels of proinflammatory cytokines and chemokines and were more sensitive than lungs of Gprc5a+/+ mice to stimulation of NF-κB activation by lipopolysaccharide (LPS) in vivo. Studies with epithelial cells cultured from tracheas of Gprc5a−/− and Gprc5a+/+ mice revealed that Gprc5a loss is associated with increased cell proliferation, resistance to cell death in suspension, and increased basal, TNFα-induced, and LPS-induced NF-κB activation, which were reversed partially in Gprc5a−/− adenocarcinoma cells by re-expression of Gprc5a. Compared to Gprc5a+/+ cells, the Gprc5a−/− cells produced higher levels of chemokines and cytokines and their conditioned medium induced more extensive macrophage migration. Silencing Gprc5a and the p65 subunit of NF-κB in Gprc5a+/+ and Gprc5a−/− cells, respectively reversed these effects. Thus, Gprc5a−/− loss enhances NF-κB activation in lung epithelial cells leading to increased autocrine and paracrine interactions, cell autonomy and enhanced inflammation, which may synergize in the creation of a tumor promoting microenvironment.

Retinoic acid regulates the expression of genes involved in cell proliferation, differentiation and survival by direct control of gene transcription via activation of nuclear retinoid receptors bound to response elements in the promoters of target genes or by indirect mechanisms. Herein, we investigated the mechanism by which retinoic acid induces the expression of the human tumor suppressor GPRC5A. The proximal 5' upstream region of the GPRC5A gene was found to contain two potential RAR/RXR binding sites (RAREs) and one VDR/RXR binding site with direct repeat 5 (DR5) motifs designated DR5I (-489 to -473), DR5II (-136 to -120) and DR5III (-81 to -65). DR5II and DR5III but not DR5I were conserved among vertebrates. However, only DR5III (5'-TGT CCC TCT GCT CAC CC-3') was found to be the functional RARE for mediating induction of GPRC5A as indicated by electrophoretic mobility shift assay using wild type and mutated synthetic oligonucleotides representing different fragments of the promoter for competition with retinoic acid receptor beta RARE. Chromatin immunoprecipitation assay confirmed the binding of retinoic acid receptors alpha and gamma and retinoid X receptors alpha and beta to DR5III in intact cells. These results demonstrate the importance of functional analysis for validating the activity of predicted response elements.

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Lung carcinogenesis occurs through a multi-step process, which involves the activation or over-expression of oncogenes and the inactivation or silencing of tumor-suppressor genes. Mouse models for human lung cancer have proven to be valuable tools for understanding the basic tumor biology as well as for the development and validation of new approaches to cancer prevention and therapy. Previously, we found that mice with a knockout (KO) of G-protein coupled receptor 5A (Gprc5a) develop lung tumors after a long latent period (12 to 24 months). To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n=5) of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A signature of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The signature also separated both mouse and human adenocarcinomas from normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of UBE2C, MCM2, and FEN-1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively. Our findings support the following conclusions: 1) lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK, 2) gene expression changes induced by tobacco carcinogen(s) are conserved between mouse and human lung epithelial cells, 3) the Gprc5a-KO mouse model recapitulates human lung adenocarcinoma. Thus, the Gprc5a-KO mouse model could be useful for cancer chemoprevention studies. Supported by the Samueal Waxman Cancer Research Foundation and the NCI Cancer Center Core Grant P30-CA16672. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2466.