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

Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178, USA.
Cancer Research (Impact Factor: 9.33). 07/2009; 69(14):5743-51. DOI: 10.1158/0008-5472.CAN-08-3564
Source: PubMed


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.

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Available from: Taotao Wei, Jul 11, 2014
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    • "Breast cancer is the leading cause of cancer deaths of women , and metastatic breast cancer is invariably incurable . Metastasis begins with the migration and invasion of cancer cells into surrounding tissues and lymphatics , and then to target organs . We previously described some of the intracellular regulators of breast cancer cell metastasis ( Xie et al . , 2009 ; Zhao et al . , 2013a ) , particularly as they relate to the formation of the lamellipodia . The driving force for lamellipodia formation is localized polymerization of F - actin filaments beneath the plasma membrane , which requires an abundance of ATP ; accordingly , inhibition of energy metabolism by ESeroS - GS abolishes cancer cel"
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    ABSTRACT: Cancer cells are known to exhibit different hallmarks compared with normal cells. Therefore, targeting these features may improve the response to cancer therapy. In this study, we provided direct evidence that the α-tocopherol derivative ESeroS-GS inhibited the viability, migration, and invasion of breast cancer cells. ESeroS-GS induced cell death in different cancer cells in a dose-dependent manner but showed no significant effects on MCF-10A mammary epithelial cells. Although the ESeroS-GS-induced cell death in MDA-MB-231 breast cancer cells was accompanied with the generation of reactive oxygen species and the down regulation of mitochondrial membrane potential (MMP), no such effect on reactive oxygen species and MMP was seen in MCF-10A cells. Further studies indicated that ESeroS-GS down-regulated the expression of hexokinase II, SDH B, UQCRC2 and COX II in MDA-MB-231 cells but not in MCF-10A cells. The down-regulation of these enzymes accounts for the decreased oxidative phosphor-ylation (OXPHOS) and glycolysis in MDA-MB-231cells upon ESeroS-GS treatment. We also found that sub-toxic concentration of ESeroS-GS treatment resulted in the impairment of F-actin cytoskeleton assembly and the consequently decreased migratory and invasive ability of MDA-MB-231 cells, which might be due to the inhibition of cellular energy metabolism. These results indicate that ESeroS-GS shows potential to become a novel anti-cancer agent by targeting the energy metabolism of cancer cells. & 2014 Elsevier B.V. All rights reserved.
    European journal of pharmacology 10/2014; 745:98-107. DOI:10.1016/j.ejphar.2014.09.050 · 2.53 Impact Factor
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    • "Matrigel invasion assays were performed at 37 C using 24-well Transwell inserts coated with 30 mg of Matrigel [26] [27]. After incubation with 40 or 80 mg/ml PEG-GO for 24 h, cells (50,000) were suspended in 200 ml of serum-free medium and added to the upper chamber, and the migration inducer (NIH-3T3 cell-conditioned medium) was added to the lower chamber. "
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    ABSTRACT: Recent advances in nanomedicine provide promising alternatives for cancer treatment that may improve the survival of patients with metastatic disease. The goal of the present study was to evaluate graphene oxide (GO) as a potential anti-metastatic agent. For this purpose, GO was modified with polyethylene glycol (PEG) to form PEG-modified GO (PEG-GO), which improves its aqueous stability and biocompatibility. We show here that PEG-GO exhibited no apparent effects on the viability of breast cancer cells (MDA-MB-231, MDA-MB-436, and SK-BR-3) or non-cancerous cells (MCF-10A), but inhibited cancer cell migration in vitro and in vivo. Analysis of cellular energy metabolism revealed that PEG-GO significantly impaired mitochondrial oxidative phosphorylation (OXPHOS) in breast cancer cells; however, PEG-GO showed no effect on OXPHOS in non-cancerous cells. To explore the underlying mechanisms, a SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in PEG-GO-exposed breast cancer versus non-cancerous cells. The results indicated that PEG-GO selectively down-regulated PGC-1α in breast cancer cells and thus modified the expression of diverse energy generation-related proteins, which accounts for the inhibition of OXPHOS. The inhibition of OXPHOS by PEG-GO significantly reduced ATP production and impaired assembly of the F-actin cytoskeleton in breast cancer cells, which is required for the migratory and invasive phenotype of cancer cells. Taken together, these effects of PEG-GO on cancer cell metastasis may allow the development of a new approach to treat metastatic breast cancer.
    Biomaterials 09/2014; 35(37). DOI:10.1016/j.biomaterials.2014.08.033 · 8.56 Impact Factor
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    • "een those results and our current data could be due to cell type - specific effects . Also , RGS2 mRNA and protein levels are likely to be differentially regulated as we previously showed that RGS2 protein is rapidly degraded by the ubiquitin - proteasomal pathway . This was certainly true in the case of RGS4 in a study by the group of Yaping Tu ( Xie et al . , 2009 ) . They found that RGS4 mRNA levels in metastatic breast cancer cells were . 20 , 000 higher than in control cells . At the protein level , however , RGS4 was virtually undetectable , whereas the nonmetastatic cells demonstrated robust RGS4 protein expression ."
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    ABSTRACT: Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biological mechanisms. In the current study we utilized a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase Regulator of G protein Signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate (PMA) as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the non-selective PKC inhibitor Go6983, the PKCβ-selective inhibitor Ruboxastaurin, as well as siRNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/PLC signaling through RGS2 protein up-regulation.
    Molecular pharmacology 08/2014; 86(4). DOI:10.1124/mol.114.092403 · 4.13 Impact Factor
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