GPRC5A Research Articles

21P Urethane-induced lung carcinogenesis in genetically edited C57Bl/6 mice with CHEK2 and GPRC5A heterozygous inactivating mutations

Some lung cancer (LC) patients carry germline truncating mutations in DNA repair or other cancer-related genes, however it is unclear whether these allelic variants play a causative role in LC predisposition, or, alternatively, are occasionally present in these subjects due to a chance. Case-control comparison is often not efficient due to rarity of gene-inactivating variants in population, therefore alternative approaches are required. We evaluated whether germ-line inactivation of GPRC5A and CHEK2 genes facilitates LC in mice. C57BL/6 mice with heterozygous inactivating mutations in the GPRC5A or CHEK2 genes were created using the CRISPR/Cas9 system. Donor eggs were obtained from the first generation of crossing mice of C57BL/6 (males) and CBA (females) inbred strains. Offspring mice F1 bearing inactivating mutations in GPRC5A and CHEK2 were used in further breeding with C57BL/6 wild-type strain for the study purposes. Altogether, 33 females and 21 males without mutations, 11 females and 19 males with mutations in CHEK2, and 13 females and 14 males with mutations in GPRC5A were analyzed. At the age of 3 months, mice were intraperitoneally injected with urethane (1 g/kg). The experiment was stopped after 45 weeks, all animals were autopsied and the lung tumor nodes were counted. In the wild-type (control) mice, the number of affected animals was 16/33 (48%) in females and 12/21 (57%) in males (pooled males/females: 28/54 (52%)). The rate of tumor development was not increased in CHEK2-heterozygous mice (females: 6/11 (55%); males: 7/19 (37%); pooled males/females: 13/30 (43%)). However, animals carrying GPRC5A heterozygous inactivating mutations showed a trend towards elevated occurrence of carcinogen-induced lung neoplasms (females: 9/13 (69%); males: 11/14 (79%); pooled males/females: 20/27 (74%); р = 0.06 when compared to controls). These data support evidences for the involvement of GPRC5A gene in pathogenesis of lung cancer.

MicroRNA-342-5p protects against myocardial ischemia-reperfusion injury by targeting the GPRC5A pathway.

Myocardial ischemia/reperfusion (MI/R) injury usually occurs in patients with cardiovascular disease. However, myocardial reperfusion insult often induces apoptosis. It is assumed that that microRNAs (miRNAs) are involved in the pathological and physiological processes associated with myocardial ischemia/reperfusion (MI/R). In the present study, we found decreased expression of miR-342-5p in hypoxia/reoxygenation (H/R) cardiomyocyte model (H9C2 cells) and MI/R mouse model. Alternatively, overexpression of miR-342-5p was found to ameliorate myocardial cell damage in both in vivo and in vitro. In addition, G protein-coupled receptor, family C, group 5, member A (GPRC5A) was identified as a direct target of miR-342-5p. The up-regulation of GPRC5A functioned to inhibit the previously observed protective effect of miR-342-5p in the H9C2 H/R model. Our results revealed that miR-342-5p may be a potential target for MI/R injury prevention and therapy of MI/R injury.

GPRC5A deficiency leads to dysregulated MDM2 via activated EGFR signaling for lung tumor development.

GPRC5A, a retinoic acid induced gene, is preferentially expressed in lung tissue. Gprc5a gene deletion leads to spontaneous lung tumor development. However, the mechanism of Gprc5a-mediated lung tumor suppression is not fully understood. Here we showed that MDM2, a p53-negative regulator, was dysregulated in Gprc5a-knockout (ko) mouse tracheal epithelial cells (KO-MTEC) compared to wild type ones. Targeting MDM2 in 1601-a Gprc5a-ko mouse derived lung tumor cell line-and A549-human lung cancer cells, by MDM2 inhibitor Nutlin-3a or small hairpin RNA (sh-RNA)-restored p53 signaling pathway, reduced cancer stem cell markers, and inhibited tumorigenicity. This suggests that dysregulated MDM2 pathway is essential for the oncogenic activities of these cells. MDM2 was found to be stabilized mainly by activated EGFR signaling as targeting EGFR by Erlotinib or sh-RNA repressed MDM2 in a transcription-independent manner. Importantly, overexpression of MDM2 and reduced GPRC5A expression at both protein and mRNA levels were frequently found in clinical human lung cancer tissues. Taken together, GPRC5A deficiency contributes to dysregulated MDM2 via activated EGFR signaling, which promotes lung tumor development.

Genome-Wide Gene Expression Changes in the Normal-Appearing Airway during the Evolution of Smoking-Associated Lung Adenocarcinoma.

Smoking perpetuates in cytologically normal airways a molecular "field of injury" that is pertinent to lung cancer and early detection. The evolution of airway field changes prior to lung oncogenesis is poorly understood largely due to the long latency of lung cancer in smokers. Here, we studied airway expression changes prior to lung cancer onset in mice with knockout of the Gprc5a gene (Gprc5a-/-) and tobacco carcinogen (NNK) exposure and that develop the most common type of lung cancer, lung adenocarcinoma, within 6 months following exposure. Airway epithelial brushings were collected from Gprc5a-/- mice before exposure and at multiple times post-NNK until time of lung adenocarcinoma development and then analyzed by RNA sequencing. Temporal airway profiles were identified by linear models and analyzed by comparative genomics in normal airways of human smokers with and without lung cancer. We identified significantly altered profiles (n = 926) in the NNK-exposed mouse normal airways relative to baseline epithelia, a subset of which were concordantly modulated with smoking status in the human airway. Among airway profiles that were significantly modulated following NNK, we found that expression changes (n = 22) occurring as early as 2 months following exposure were significantly associated with lung cancer status when examined in airways of human smokers. Furthermore, a subset of a recently reported human bronchial gene classifier (Percepta; n = 56) was enriched in the temporal mouse airway profiles. We underscore evolutionarily conserved profiles in the normal-appearing airway that develop prior to lung oncogenesis and that comprise viable markers for early lung cancer detection in suspect smokers. Cancer Prev Res; 11(4); 237-48. ©2018 AACR.

Repression of GPRC5A is associated with activated STAT3, which contributes to tumor progression of head and neck squamous cell carcinoma

BackgroundG protein–coupled receptor family C group 5 member A (GPRC5A), a retinoic acid-inducible gene, is a lung tumor suppressor. Previously, we showed that repression of GPRC5A expression was associated with pathologic differentiation grade of oral squamous cell carcinomas (OSCC) and overexpression of GPRC5A gene inhibited the malignant phenotype in OSCC cells, suggesting that GPRC5A also functions as a tumor suppressor in oral cancer. However, the molecular mechanisms underlying GPRC5A deficiency in head and neck squamous cell carcinoma (HNSCC) are still unclear.MethodsIn this study, we used Western blot analysis and immunohistochemical (IHC) staining to investigate the expression of GPRC5A in both HNSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental HNSCC cells were characterized for their biological activities in anchorage-independent growth.ResultsIHC analysis showed that, GPRC5A expression was high in normal tissue, but gradually decreased in oral leukoplakia, a precancerous stage, and greatly suppressed in primary cancer. Repression of GPRC5A was correlated with activated STAT3, which associates with aggressive clinicopathological features in HNSCC patients. Moreover, overexpression of GPRC5A suppressed IL-6-induced-STAT3 activation and inhibited anchorage-independent growth in HNSCC cells.ConclusionsRepressed GPRC5A associates with increased tumor grade and activated STAT3, which may be used as a prognostic marker for tumor progression of HNSCC.

GPRC5A and lung cancer

The G protein-coupled receptor family C,member 5,group A (GPRC5A) gene is known as retinoic acid-induced gene,which is mainly distributed in lung tissue.The expression of GPRC5A in lung cancer is significantly decreased compared with normal lung.GPRC5A leads to lung cancer through knockout mice,which is proven to be a suppressor gene of lung cancer.GPRC5A may be a novel biomarker for the diagnosis of lung cancer and a new target for the treatment of lung cancer. Key words: Lung neoplasms; Receptors, G-protein-coupled

Abstract 4995: Loss of the lung-specific tumor suppressor Gprc5a causes persistent activation of Stat3 leading to increased survival signaling in normal and malignant lung epithelial cells

Abstract The G protein-coupled receptor, family C, group 5, member A (GPRC5A), a retinoic acid induced gene expressed primarily in human and mouse lung tissue. Our previous studies demonstrated that mice with a deletion of the Gprc5a gene develop spontaneous lung adenomas and adenocarcinomas. Since Gprc5a is expressed preferentially in epithelial cells in lung tissues, we isolated epithelial cells from tracheas of Gprc5a+/+ and Gprc5a−/− mice and used them for an analysis of phenotypic and genotypic differences that might explain the mechanisms of Gprc5a actions. We found that normal Gprc5a−/− cells can survive better than Gprc5a+/+ cells in basal medium deprived of supplemented growth factors. Furthermore, the Gprc5a−/− cells but not the Gprc5a+/+ cells were able to form colonies in semi-solid medium. To explore possible mechanisms that underlay the distinct phenotypes of the above cells, we compared and contrasted the survival signaling pathway mediated by the Signal Transducer and Activator of Transcription 3 (STAT3), which is triggered by certain cytokines and growth factors to regulate the expression of genes implicated in cell growth differentiation, survival, inflammation and transformation. Increased Stat3 activity by phosphorylation has been identified in a variety of tumors including non-small cell lung cancers (NSCLCs) and it has been linked to inflammation and tumor progression. The constitutive activity (phosphorylation of tyrosine 705) of Stat3 and the expression of several Stat3 regulated genes including survivin, Bcl-XL, and Mcl1 at both the mRNA and protein levels was higher in the Gprc5a−/− cells compared to normal Gprc5a+/+ cells. Stat3 activation was triggered by autocrine Leukemia inhibitory factor (Lif), a member of the interleukine-6 (IL-6) family of cytokines, as revealed after adding neutralizing antibodies against different Stat3 activating cytokines to the growth medium. Although Gprc5a+/+ cells produced and secreted sufficient amounts of Lif into their conditioned medium, their response to Lif was transient, whereas the Gprc5a−/− cells showed a prolonged response evidenced by a persistent Stat3 activation. Lung cancer cells isolated from an adenocarcinorma of Gprc5a−/− mice exhibited Lif-mediated Stat3 activation similar to the normal Gprc5a−/− cells. Blocking Stat3 activation using AG490, an inhibitor of Stat3 signaling, or using a dominant negative Stat3 (mutant Y705F) in the Gprc5a−/− normal and cancer cells increased apoptosis and inhibited anchorage-independent growth. These results demonstrate that Stat3 activation may be important for the early stages of lung tumorigenesis in Gprc5a−/− mice and by implication that the tumor suppressive effects of Gprc5a are mediated, at least in part, by suppression of Stat3 signaling. Supported in part by the Samuel Waxman Cancer Research Foundation. 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 4995.

Abstract PL02-03: Insights from a new mouse model into the role of inflammation at early stages of lung carcinogenesis

Abstract PL02-03: Insights from a new mouse model into the role of inflammation at early stages of lung carcinogenesis

Mechanisms underlying the induction of the putative human tumor suppressor GPRC5A by Retinoic acid

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’- TGTCCCTCTGCTCACCC-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 β RARE. Chromatin immunoprecipitation assay confirmed the binding of retinoic acid receptors α and γ and retinoid X receptors α and β to DR5III in intact cells. These results demonstrate the importance of functional analysis for validating the activity of predicted response elements.

A New Tumor Suppressor Gene, Selective for Lung Cancer

sion of this gene occurred in human lung (among more than a dozen human tissues examined), and, moreover, the important observation was made that GPRC5A mRNA was expressed at only very low levels in human lung cancer cells (three of five head and neck squamous cell carcinoma and four of six non – small cell lung cancer cell lines). The authors hypothesized that this decrease in mRNA expression might be associated with the malignant transformation of airway epithelium. The article by Tao et al. ( 2 ) in the current issue of the Journal now provides excellent, detailed experimental evidence for this hypothesis. The key experimental tool in this article has been the use of mice (B6/129sv) in which the Gprc5a gene has been knocked out by homologous recombination. The Lotan group is not the fi rst to have used knockout technology to study the biology of Gprc5a in the mouse lung. Thus, in an elegant study, Xu et al. ( 3 ) used classical recombinant technology to show that Gprc5a is expressed abundantly and specifi cally in the lung during embryonic development and in adult mice. These authors found that the onset of Gprc5a expression in the embryonic mouse lung coincided with the onset of differentiation in the lung, and they suggested that this gene plays an important role in lung maturation and differentiation. The loss of such a differentiating function during carcinogenesis is in accord with classical concepts of cancer as a disease of differentiation ( 4 , 5 ). However, most notably, the lungs of 3-month-old Gprc5a − / − mice appeared histologically normal in spite of the fact that this gene is expressed at high levels in Gprc5a +/+ mice of this age, in both type I and type II pneumocytes. The reason for this lack of phenotype at 3 months of age is unclear, but the authors suggest that the expression of a related gene that regulates G protein signaling (Raig3, also known as Gprc5c) may

Novel reciprocal regulation of cAMP signaling and apoptosis by orphan G-protein-coupled receptor GPRC5A gene expression

GPRC5A is a member of G-protein-coupled receptors, which was originally identified as an all- trans-retinoic acid-induced gene. Although recent studies reported that this gene was highly expressed in the cancer cell lines and that GPRC5A might positively regulate cell proliferation, its mechanism remains unknown. We investigated the upstream and downstream signaling of GPRC5A and its biological function, and found that cAMP signaling is the novel GPRC5A induction pathway. When GPRC5A gene was overexpressed, intracellular cAMP concentration was decreased, and Gsα gene expression was downregulated. On the other hand, RNA interference of GPRC5A increased mRNA levels of Gsα and intracellular cAMP, reduced cell number, and induced apoptosis. Conversely, cell number was increased by GPRC5A overexpression. We first report the novel negative feedback model of cAMP signaling through GPRC5A gene expression. This evidence explains one of the mechanisms of the GPRC5A-regulated cell growth in some cancer cell lines.