Haihong Jiang

Breast Cancer Migration and Invasion Depend on Proteasome Degradation of Regulator of G-Protein Signaling 4

Aberrant signaling through G-protein coupled receptors promotes metastasis, the major cause of breast cancer death. We identified regulator of G-protein signaling 4 (RGS4) as a novel suppressor of breast cancer migration and invasion, important steps of metastatic cascades. By blocking signals initiated through G(i)-coupled receptors, such as protease-activated receptor 1 and CXC chemokine receptor 4, RGS4 disrupted Rac1-dependent lamellipodia formation, a key step involved in cancer migration and invasion. RGS4 has GTPase-activating protein (GAP) activity, which inhibits G-protein coupled receptor signaling by deactivating G-proteins. An RGS4 GAP-deficient mutant failed to inhibit migration and invasion of breast cancer cells in both in vitro assays and a mouse xenograft model. Interestingly, both established breast cancer cell lines and human breast cancer specimens showed that the highest levels of RGS4 protein were expressed in normal breast epithelia and that RGS4 down-regulation by proteasome degradation is an index of breast cancer invasiveness. Proteasome blockade increased endogenous RGS4 protein to levels that markedly inhibit breast cancer cell migration and invasion, which was reversed by an RGS4-targeted short hairpin RNA. Our findings point to the existence of a mechanism for posttranslational regulation of RGS4 function, which may have important implications for the acquisition of a metastatic phenotype by breast cancer cells. Preventing degradation of RGS4 protein should attenuate aberrant signal inputs from multiple G(i)-coupled receptors, thereby retarding the spread of breast cancer cells and making them targets for surgery, radiation, and immune treatment.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G protein-coupled receptors (GPCRs) promotes breast cancer metastasis. G proteins convey GPCR signals by dissociating into Gα and Gβγ subunits. The aim of the present study was to determine whether blockade of Gβγ signaling suppresses breast cancer cell migration and invasion, which are critical components of metastasis. Conditioned media (CM) of NIH-3T3 fibroblasts are widely used as chemoattractants in in vitro cancer metastasis studies. Expression of a Gβγ scavenger peptide attenuated NIH-3T3 CM-induced migration and invasion of both metastatic breast cancer MDA-MB-231 and MDA-MB-436 cells by 40 to 50% without effects on cell viability. Migration and invasion of cells in response to NIH-3T3 CM were also blocked by 8-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-1-naph-thalene-carboxylic acid) (M119K), a Gβγ inhibitor, with maximum inhibition exceeding 80% and half-maximal inhibitory concentration (IC50) values of 1 to 2 μM. M119K also attenuated Rac-dependent formation of lamellipodia, a key structure required for metastasis. Constitutively active Rac1 rescued Gβγ blockade-mediated inhibition of breast cancer cell migration, whereas dominant negative Rac1 inhibited cell migration similar to Gβγ blockade. Furthermore, M119K suppressed Gi protein-coupled CXC chemokine receptor 4 (CXCR4)-dependent MDA-MB-231 cell migration by 80% with an IC50 value of 1 μM, whereas tyrosine kinase receptor-dependent cell migration was significantly less inhibited. However, CXCR4-dependent inhibition of adenylyl cyclase, a Giα-mediated response in MDA-MB-231 cells, was not blocked by M119K but was blocked by pertussis toxin, which selectively inactivates Giα. This report is the first to directly demonstrate the role of Gβγ in cancer cell migration and invasion and suggests that targeting Gβγ signaling pathways may provide a novel strategy for suppressing breast cancer metastasis.

Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness

BACKGROUND Drugs targeting individual G protein-coupled receptors are used as asthma therapies, but this strategy is limited because of G protein-coupled receptor signal redundancy. Regulator of G protein signaling 2 (RGS2), an intracellular selective inhibitor of multiple bronchoconstrictor receptors, may play a central role in the pathophysiology and treatment of asthma. OBJECTIVE We defined functions and mechanisms of RGS2 in regulating airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. METHODS Real-time PCR and Western blot were used to determine changes in RGS2 expression in ovalbumin-sensitized/-challenged mice. We also used immunohistochemistry and real-time PCR to compare RGS2 expression between human asthmatic and control subjects. The AHR of RGS2 knockout mice was assessed by using invasive tracheostomy and unrestrained plethysmography. Effects of loss of RGS2 on mouse airway smooth muscle (ASM) remodeling, contraction, intracellular Ca(2+), and mitogenic signaling were determined in vivo and in vitro. RESULTS RGS2 was highly expressed in human and murine bronchial epithelium and ASM and was markedly downregulated in lungs of ovalbumin-sensitized/-challenged mice. Lung tissues and blood monocytes from asthma patients expressed significantly lower RGS2 protein (lung) and mRNA (monocytes) than from nonasthma subjects. The extent of reduction of RGS2 on human monocytes correlated with increased AHR. RGS2 knockout caused spontaneous AHR in mice. Loss of RGS2 augmented Ca(2+) mobilization and contraction of ASM cells. Loss of RGS2 also increased ASM mass and stimulated ASM cell growth via extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. CONCLUSION We identified RGS2 as a potent modulator of AHR and a potential novel therapeutic target for asthma.

Targeting phosphoinositide 3-kinase γ in airway smooth muscle cells to suppress interleukin-13-induced mouse airway hyperresponsiveness

We recently reported that phosphoinositide 3-kinase γ (PI3Kγ) directly regulates airway smooth muscle (ASM) contraction by modulating Ca2+ oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 μg per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3Kγ-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca2+ transient and increased Ca2+ oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3Kγ inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3Kγ by 70% only reduced the initial Ca2+ transient by 20 to 30% but markedly attenuated Ca2+ oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3Kγ in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3Kγ inhibitor can ameliorate AHR in a murine model of asthma.

Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kγ can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kγ protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kγ inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 μM. In contrast, inhibitors of PI3Kα, PI3Kβ, or PI3Kδ, at concentrations 40-fold higher than their reported IC50 values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kγ-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca2+ transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kγ directly controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kγ inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

Regulator of G-Protein Signaling 2 Repression Exacerbates Airway Hyper-Responsiveness and Remodeling in Asthma

G protein-coupled receptors (GPCRs) are important regulators of cell functions in asthma. We recently reported that regulator of G-protein signaling (RGS) 2, a selective modulator of Gq-coupled GPCRs, is a key regulator of airway hyper-responsiveness (AHR), the pathophysiologic hallmark of asthma. Because RGS2 protein levels in airway cells were significantly lower in patients with asthma compared with patients without asthma, we further investigated the potential pathological importance of RGS2 repression in asthma. The human RGS2 gene maps to chromosome 1q31. We first screened patients with asthma for RGS2 gene promoter single-nucleotide polymorphisms (SNPs) and found significant differences in the distribution of two RGS2 SNPs (A638G, rs2746071 and C395G, rs2746072) between patients with asthma and nonasthmatic subjects. These two SNPs are always associated with each other and have the same higher prevalence in patients with asthma (65%) as compared with nonasthmatic subjects (35%). Point mutations corresponding to these SNPs decrease RGS2 promoter activity by 44%. The importance of RGS2 down-regulation was then determined in an acute IL-13 mouse model of asthma. Intranasal administration of IL-13 in mice also decreased RGS2 expression in lungs by ∼50% and caused AHR. Although naive RGS2 knockout (KO) mice exhibit spontaneous AHR, acute IL-13 exposure further increased AHR in RGS2 KO mice. Loss of RGS2 also significantly enhanced IL-13-induced mouse airway remodeling, including peribronchial smooth muscle thickening and fibrosis, without effects on goblet cell hyperplasia or airway inflammation in mice. Thus, genetic variations and increased inflammatory cytokines can lead to RGS2 repression, which exacerbates AHR and airway remodeling in asthma.

Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis

BackgroundPirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti-fibrotic effects of pirfenidone.MethodsWe used the GeneChip microarray to screen for genes that were rapidly up-regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real-time PCR and western blots. Biochemical and functional analyses were used to establish their anti-fibrotic effects in cellular and animal models of pulmonary fibrosis.ResultsWe identified Regulator of G-protein Signaling 2 (RGS2) as an early pirfenidone-induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin-induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin-induced pulmonary fibrosis in wild-type but not RGS2 knockout mice.ConclusionsEndogenous RGS2 exhibits anti-fibrotic functions. Upregulated RGS2 contributes significantly to the anti-fibrotic effects of pirfenidone.

Upregulated P-Rex1 exacerbates human airway smooth muscle hyperplasia in asthma

P-Rex1, a Rac-selective activator is aberrantly upregulated in airway smooth muscle of subjects with asthma compared to non-asthmatics, and augments cell proliferation, thus contributing to airway smooth muscle hyperplasia and remodeling in severe asthma.

Phorbol myristate acetate suppresses breast cancer cell growth via down-regulation of P-Rex1 expression

Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) is highly overexpressed in estrogen receptor-positive and ErbB2-overexpressing human luminal breast tumors. The pre...

Abstract 5188: Phosphoinositide 3-kinase γ: Implications in breast cancer metastasis

Metastasis is the major cause of breast cancer mortality. Type I phosphatidylinositol 3-kinases (PI3Ks), including α, β, γ and δ, play a pivotal role in diverse cell processes such as proliferation and motility. Of these PI3Ks, PI3Kγ is especially intriguing because it is: 1) normally expressed primarily in white blood cells which have a physiological need to migrate and 2) is specifically activated by G-protein coupled receptors (GPCRs). We recently reported that aberrant G i -coupled receptor signaling promotes breast cancer metastasis (Xie et al., Cancer Res. 69: 5743-5751). The goal of the present study was to determine roles of PI3Kγ in breast cancer metastasis. We first examined the levels of PI3Kγ in different breast cancer cell lines by RT-PCR and Western blot analysis. We then immunohistochemically analyzed PI3Kγ protein expression in histologically benign and neoplastic cells of archival human breast tissues. We also investigated effects of PI3Ks inhibitors on migration and invasion abilities of breast cancer cells in Transwell chamber assays. Finally, we determined effects of PI3Ks inhibitors on lamellipodia formation, a key step involved in cancer metastasis. Our data indicated that PI3Kγ mRNA and protein were not detected in a normal human breast epithelial cell line (MCF-10A) or non-metastatic breast cancer cells (MCF-7 and T47D), but was found in metastatic breast cancer MDA-MB-231 cells and MDA-MB-436 cells. In contrast, PI3Kα, β and δ are ubiquitously expressed in these cell lines. In addition, immunohistochemical staining using a PI3Kγ-specific antibody showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma and in breast cancer metastases to a regional lymph node as compared to the adjacent benign breast tissues or the noninvasive ductal carcinoma in situ. Thus, PI3Kγ expression level correlates with the metastatic potential of breast cancer cells. Interestingly, migration and invasion of metastatic breast cancer cells were inhibited by a selective inhibitor of PI3Kγ but not α or β isoforms. Furthermore, blocking PI3Kγ but not α and β disrupted the Rac-dependent lamellipodia formation induced by the activation of G i -coupled receptor CXC chemokine receptor 4, but not epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that up-regulated PI3Kγ conveys the metastatic signal initiated from GPCRs in breast cancer cells and suggest that PI3Kγ may be a novel therapeutic target for the treatment of breast cancers. Supported by Nebraska State LB692 (YX). 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 5188.