Taoyan Men

Gprc5a Deletion Enhances the Transformed Phenotype in Normal and Malignant Lung Epithelial Cells by Eliciting Persistent Stat3 Signaling Induced by Autocrine Leukemia Inhibitory Factor

Signal transducers and activators of transcription 3 (Stat3) is activated by cytokines and growth factors in lung cancers and regulates expression of genes implicated in cell growth, survival, and transformation. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors, indicating that Gprc5a is a tumor suppressor. Herein, we show that epithelial cells from Gprc5a knockout mouse lung (Gprc5a(-/-) cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semisolid medium than their counterparts from wild-type mice (Gprc5a(+/+) cells). Stat3 tyrosine 705 phosphorylation and expression of several Stat3-regulated antiapoptotic genes were higher in Gprc5a(-/-) than in Gprc5a(+/+) cells. Both cell types secreted leukemia inhibitory factor (Lif); however, whereas Stat3 activation was persistent in Gprc5a(-/-) cells, it was transient in Gprc5a(+/+) cells. Lung adenocarcinoma cells isolated from Gprc5a(-/-) mice also exhibited autocrine Lif-mediated Stat3 activation. The level of Socs3, the endogenous Stat3 inhibitory protein, was higher in Gprc5a(+/+) than in Gprc5a(-/-) cells, and expression of the tumor suppressor stabilized Socs3. Inhibition of Stat3 signaling in Gprc5a(-/-) normal and cancer cells by the Janus-activated kinase 2 inhibitor AG490 or by a dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited colony formation. These results show that persistent Stat3 activation is important for the survival and transformation of Gprc5a(-/-) lung cells and suggest that the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling through Socs3 stabilization.

Enhancement of lung tumorigenesis in a Gprc5a Knockout mouse by chronic extrinsic airway inflammation

BackgroundAlthough cigarette smoking is the principal cause of lung carcinogenesis, chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, has been identified as an independent risk factor for lung cancer. Bacterial colonization, particularly with non-typeable Haemophilus influenzae (NTHi), has been implicated as a cause of airway inflammation in COPD besides cigarette smoke. Accordingly, we hypothesized that lung cancer promotion may occur in a chronic inflammatory environment in the absence of concurrent carcinogen exposure.ResultsHerein, we investigated the effects of bacterial-induced COPD-like inflammation and tobacco carcinogen-enhanced tumorigenesis/inflammation in the retinoic acid inducible G protein coupled receptor knock out mouse model (Gprc5a-/- mouse) characterized by late-onset, low multiplicity tumor formation. Three-month-old Gprc5a-/- mice received 4 intraperitoneal injections of the tobacco-specific carcinogen, NNK, followed by weekly exposure to aerosolized NTHi lysate for 6 months. The numbers of inflammatory cells in the lungs and levels of several inflammatory mediators were increased in Gprc5a-/- mice treated with NTHi alone, and even more so in mice pretreated with NNK followed by NTHi. The incidence of spontaneous lung lesions in the Gprc5a-/- mice was low, but NTHi exposure led to enhanced development of hyperplastic lesions. Gprc5a-/- mice exposed to NNK alone developed multiple lung tumors, while NTHi exposure increased the number of hyperplastic foci 6-fold and the tumor multiplicity 2-fold. This was associated with increased microvessel density and HIF-1α expression.ConclusionWe conclude that chronic extrinsic lung inflammation induced by bacteria alone or in combination with NNK enhances lung tumorigenesis in Gprc5a-/- mice.

Comparative functional genomics analysis of NNK tobacco-carcinogen induced lung adenocarcinoma development in Gprc5a-knockout mice

Background Improved understanding of lung cancer development and progression, including insights from studies of animal models, are needed to combat this fatal disease. 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). Methodology/Principal Findings 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 gene expression signature, NNK-ADC, 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 NNK-ADC expression signature also separated both mouse and human adenocarcinomas from adjacent normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of Ube2c, Mcm2, and Fen1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively. Conclusions/Significance Our findings demonstrate that lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK and that gene expression changes induced by tobacco carcinogen(s) may be conserved between mouse and human lung epithelial cells. Further experimentation to prove the reliability of the Gprc5a knockout mouse model for the study of tobacco-induced lung carcinogenesis is warranted.

Antitumor Effect of Retinoic Acid Receptor-β2 Associated with Suppression of Cyclooxygenase-2

Retinoic acid receptor-β2 (RAR-β2) is a putative tumor suppressor gene in various cancers. To determine the underlying molecular mechanisms, we transfected RAR-β2 cDNA into esophageal cancer TE-1 and TE-8 cells and found that RAR-β2 suppressed tumor cell growth in vitro and tumor formation in nude mice in TE-8 cells, whereas the stable transfection of RAR-β2 did not restore retinoid sensitivity or inhibit tumor formation in nude mouse in TE-1 cells. Molecularly, we revealed that RAR-β2 antitumor activity was associated with expression and suppression of cyclooxygenase-2 (COX-2) in these tumor cell lines. Moreover, antisense RAR-β2 cDNA induced COX-2 expression in TE-3 cells. Furthermore, when COX-2 expression is first blocked by using antisense COX-2 expression vector, the effect of RAR-β2 is diminished in these tumor cells. In addition, we analyzed expression of RAR-β2 and COX-2 mRNA in tissue specimens and found that RAR-β2 expression is associated with low levels of COX-2 expression in esophageal cancer tissues. Induction of RAR-β2 expression in oral leukoplakia tissues after the patients treated with 13-cis RA correlated with a reduction in COX-2 expression and clinical response. Our findings indicate that some of RAR-β2 antitumor activities are mediated by suppression of COX-2 expression in some of these esophageal cancer cells. After correlating antitumor effect of RAR-β2 with COX-2 expression in the published studies, we also found the association. Thus, further studies will determine whether manipulation of COX-2 expression in different cancers can antagonize RAR-β2 activity.

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

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 2466: Tobacco carcinogen-enhanced lung tumorigenesis in Gprc5a knockout mice recapitulates human lung cancer development

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.