Jiong Deng

β-catenin interacts with and inhibits NF-κB in human colon and breast cancer

Abstract β-catenin plays an important role in development and homeostasis. Deregulated β-catenin is involved in oncogenesis. In this study, we found that β-catenin can physically complex with NF-κB, resulting in a reduction of NF-κB DNA binding, transactivation activity, and target gene expression. Repressed NF-κB activity is found in human colon cancer cells in which β-catenin is activated. Importantly, activated β-catenin was found to inhibit the expression of NF-κB target genes, including Fas and TRAF1. Furthermore, a strong inverse correlation was identified between the expression levels of β-catenin and Fas in colon and breast tumor tissues, suggesting that β-catenin regulates NF-κB and its targets in vivo. Thus, β-catenin may play an important role in oncogenesis through the crossregulation of NF-κB.

Inhibition of Nuclear Factor-κB Activity Is Involved in E1A-mediated Sensitization of Radiation-induced Apoptosis

The adenoviral E1A protein has been implicated in the potentiation of apoptosis induced by various external stimuli, but the exact mechanism of that potentiation is not clear. In this study, we compared the sensitivity to ionizing γ-irradiation of E1A transfectants with that of parental cells in a human ovarian cancer cell line (SKOV3.ip1); we found that the E1A transfectants became sensitive to radiation-induced apoptosis. Recently, activation of the transcription factor nuclear factor-κB (NF-κB) has been shown to play a key role in the anti-apoptotic pathway of radiation-induced apoptosis. In an attempt to determine whether NF-κB was involved in the E1A-mediated sensitization of radiation-induced apoptosis, we found that radiation-induced activation of NF-κB occurred in the parental cells but was blocked in the E1A transfectants. Furthermore, parental cells cotransfected with NF-κB and E1A were better protected from undergoing apoptosis upon irradiation than those transfected with E1A alone. Thus, our results suggest that inhibition of NF-κB activation by E1A is a plausible mechanism for E1A-mediated sensitization of radiation-induced apoptosis.

A Hypoxia-Independent Hypoxia-Inducible Factor-1 Activation Pathway Induced by Phosphatidylinositol-3 Kinase/Akt in HER2 Overexpressing Cells

HER2 overexpression, a known prognostic factor in many human cancers, can activate phosphatidylinositol-3 kinase (PI-3K)/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia-inducible factors (HIFs) promote angiogenesis and energy metabolism and thereby enhance tumor growth and metastasis. HIFs, composed of alpha and beta subunits, are activated in most human cancers, including those that overexpress HER2. Previous reports have suggested that increased PI-3K/Akt or decreased PTEN activity may activate the HIF pathway in various tumors, but the detailed mechanism is still not completely understood. Here we reported an interaction between the HIF and PI-3K/Akt pathways in HER2-overexpressing cancer cells. Our results indicate that HER2 overexpression, which results in constitutively active Akt, turns on HIF-1alpha independently of hypoxia, and this activation is weaker than that under hypoxic condition. Further investigation showed that Akt is required for the hypoxia-independent HIF activity. The PI-3K/Akt pathway did not affect the HIF-1alpha binding with its E3 ligase von Hippel-Lindau but enhanced the binding affinity between the HIF-1 alpha and beta subunits. Furthermore, we found that Akt interacts with HIF-1beta and regulates HIF activity. Our results indicated that HER2 can induce HIF activation via the activation of Akt suggesting that activation of HER2/Akt pathway may promote angiogenesis independent of hypoxia, which may have important implications for the oncogenic activity of HER2 and Akt.

Crossregulation of NF-κB by the APC/GSK-3β/β-catenin pathway

Glycogen synthase kinase‐3β (GSK‐3β) and adenomatous polyposis coli (APC) play an important role in the regulation of β‐catenin. Inhibition of or defects in their functions can lead to activation of β‐catenin. β‐catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF‐κB). However, the regulatory roles of GSK‐3β/APC on the NF‐κB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK‐3β/APC might regulate NF‐κB activity through β‐catenin. We found that inhibition of GSK‐3β suppressed NF‐κB activity, whereas reexpression of APC restored NF‐κB activity in APC mutated cells. The regulatory effects were through β‐catenin because depletion of β‐catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF‐κB target TRAF1 was inversely correlated with activated β‐catenin. Thus, APC/GSK‐3β, through β‐catenin, may crossregulate NF‐κB signaling pathway.

Knockout of the Tumor Suppressor Gene Gprc5a in Mice Leads to NF-κB Activation in Airway Epithelium and Promotes Lung Inflammation and Tumorigenesis

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 macrophage 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 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, tumor necrosis factor α–induced, and lipopolysaccharide-induced NF-κB activation, which were reversed partially in Gprc5a−/− adenocarcinoma cells by reexpression of Gprc5a. Compared with 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. Cancer Prev Res; 3(4); 424–37. ©2010 AACR.

Crossregulation of NF-kappaB by the APC/GSK-3beta/beta-catenin pathway.

Glycogen synthase kinase‐3β (GSK‐3β) and adenomatous polyposis coli (APC) play an important role in the regulation of β‐catenin. Inhibition of or defects in their functions can lead to activation of β‐catenin. β‐catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF‐κB). However, the regulatory roles of GSK‐3β/APC on the NF‐κB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK‐3β/APC might regulate NF‐κB activity through β‐catenin. We found that inhibition of GSK‐3β suppressed NF‐κB activity, whereas reexpression of APC restored NF‐κB activity in APC mutated cells. The regulatory effects were through β‐catenin because depletion of β‐catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF‐κB target TRAF1 was inversely correlated with activated β‐catenin. Thus, APC/GSK‐3β, through β‐catenin, may crossregulate NF‐κB signaling pathway.

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.

Lung tumor suppressor GPRC5A binds EGFR and restrains its effector signaling

GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a(-/-) mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a(-/-) mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a(-/-) MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a(-/-) mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non-small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801-14. ©2015 AACR.

Crossregulation of NF-?B by the APC/GSK-3?/?-catenin pathway

Glycogen synthase kinase‐3β (GSK‐3β) and adenomatous polyposis coli (APC) play an important role in the regulation of β‐catenin. Inhibition of or defects in their functions can lead to activation of β‐catenin. β‐catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF‐κB). However, the regulatory roles of GSK‐3β/APC on the NF‐κB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK‐3β/APC might regulate NF‐κB activity through β‐catenin. We found that inhibition of GSK‐3β suppressed NF‐κB activity, whereas reexpression of APC restored NF‐κB activity in APC mutated cells. The regulatory effects were through β‐catenin because depletion of β‐catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF‐κB target TRAF1 was inversely correlated with activated β‐catenin. Thus, APC/GSK‐3β, through β‐catenin, may crossregulate NF‐κB signaling pathway.

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.