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BACKGROUND - A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance.
RESULTS - In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways.
CONCLUSIONS - Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
OBJECTIVE - The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening.
METHODS - The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting.
RESULTS - Pretreatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%.
CONCLUSION - Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure.
Published by Mosby, Inc.
BACKGROUND - Lung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA(2)) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA(2) as a novel molecular target for antiangiogenesis therapy.
METHODS - Glioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA(2) expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA(2)α(+/+)) or deficient in (cPLA(2)α(-/-)) cPLA(2)α, the predominant isoform in endothelium (n = 6-7 mice per group). The effect of inhibiting cPLA(2) activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA(2) inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA(2)α(+/+) and cPLA(2)α(-/-) mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA(2)α(-/-) MPMEC. All statistical tests were two-sided.
RESULTS - GL261 tumor progression proceeded normally in cPLA(2)α(+/+) mice, whereas no GL261 tumors formed in cPLA(2)α(-/-) mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA(2)α(-/-) mice. Immunohistochemical examination of the remaining tumors from cPLA(2)α(-/-) mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA(2)α(+/+) mice. Inhibition of cPLA(2) activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average number of days to reach tumor volume of 700 mm(3), CDIBA vs vehicle: 16.8 vs 11.8, difference = 5, 95% confidence interval = 3.6 to 6.4, P = .04) and a decrease in tumor size (eg, GL261 model: mean volume on day 21, CDIBA vs vehicle: 40.1 vs 247.4 mm(3), difference = 207.3 mm(3), 95% confidence interval = 20.9 to 293.7 mm(3), P = .021). cPLA(2) deficiency statistically significantly reduced MPMEC proliferation and invasive migration (P = .002 and P = .004, respectively). Compared with untreated cells, cPLA(2)α(-/-) MPMEC treated with lysophosphatidylcholine and lysophosphatidic acid displayed increased cell proliferation (P = .011) and invasive migration (P < .001).
CONCLUSIONS - In these mouse models of brain and lung cancer, cPLA(2) and lysophospholipids have key regulatory roles in tumor angiogenesis. cPLA(2) inhibition may be a novel effective antiangiogenic therapy.
There has been a renewal of interest in interactions of membrane proteins with detergents and lipids, sparked both by recent results that illuminate the structural details of these interactions and also by the realization that some experimental membrane protein structures are distorted by detergent-protein interactions. The integral membrane enzyme diacylglycerol kinase (DAGK) has long been thought to require the presence of lipid as an obligate "cofactor" in order to be catalytically viable in micelles. Here, we report that near-optimal catalytic properties are observed for DAGK in micelles composed of lysomyristoylphosphatidylcholine (LMPC), with significant activity also being observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. All three of these detergents were also sustained high stability of the enzyme. NMR measurements revealed significant differences in DAGK-detergent interactions involving LMPC micelles versus micelles composed of dodecylphosphocholine. These results highlight the fact that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and also suggest that C(14)-based detergents may be worthy of more widespread use in studies of membrane proteins.
The epithelial brush border (BB) Na(+)/H(+) exchanger NHE3 is associated with the actin cytoskeleton by binding both directly and indirectly to ezrin; indirect binding is via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BBs is restricted by the actin cytoskeleton and NHERF binding such that only approximately 30% of NHE3 in the apical domain of an OK cell line stably expressing NHERF2 is mobile, as judged by FRAP analysis. Given that levels of NHE3 are partially regulated by changes in trafficking, we investigated whether the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation to allow NHE3 trafficking. The agonist studied was lysophosphatidic acid (LPA), an inflammatory mediator, which acutely stimulates NHE3 activity by increasing the amount of NHE3 on the BBs by stimulated exocytosis. LPA acutely stimulated NHE3 activity in OK cells stably expressing NHERF2. Two conditions that totally prevented LPA stimulation of NHE3 activity only partially prevented stimulation of NHE3 mobility: the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the NHE3F1 double mutant which has minimal direct binding of NHE3 to ezrin. These results show that LPA stimulation of NHE3 mobility occurs in two parts: (1) PI3K-dependent exocytic trafficking to the BB and (2) an increase in surface mobility of NHE3 in BBs under basal conditions. Moreover, the LPA stimulatory effect on NHE3 mobility required NHERF2. Although NHE3 and NHERF2 co-precipitated under basal conditions, they failed to co-precipitate 30 minutes after addition of LPA, whereas the physical association was re-established by 50-60 minutes. This dynamic interaction between NHERF2 and NHE3 was confirmed by acceptor photobleaching Förster Resonance energy Transfer (FRET). The restricted mobility of NHE3 in BBs under basal conditions as a result of cytoskeleton association is therefore dynamic and is reversed as part of acute LPA stimulation of NHE3. We suggest that this acute but transient increase in NHE3 mobility induced by LPA occurs via two processes: addition of NHE3 to the BB by exocytosis, a process which precedes binding of NHE3 to the actin cytoskeleton via NHERF2-ezrin, and by release of NHERF2 from the NHE3 already localized in the apical membrane, enabling NHE3 to distribute throughout the microvilli. These fractions of NHE3 make up a newly identified pool of NHE3 called the 'transit pool'. Moreover, our results show that there are two aspects of LPA signaling involved in stimulation of NHE3 activity: PI3K-dependent stimulated NHE3 exocytosis and the newly described, PI3K-independent dissociation of microvillar NHE3 from NHERF2.
A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.
Aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses have yet to be fully identified. We show that lysophosphatidic acid levels increase in bronchoalveolar lavage fluid following lung injury in the bleomycin model of pulmonary fibrosis, and that mice lacking one of its receptors, LPA1, are markedly protected from fibrosis and mortality in this model. The absence of LPA1 led to reduced fibroblast recruitment and vascular leak, two responses that may be excessive when injury leads to fibrosis rather than to repair, whereas leukocyte recruitment was preserved during the first week after injury. In persons with idiopathic pulmonary fibrosis, lysophosphatidic acid levels in bronchoalveolar lavage fluid were also increased, and inhibition of LPA1 markedly reduced fibroblast responses to the chemotactic activity of this fluid. LPA1 therefore represents a new therapeutic target for diseases in which aberrant responses to injury contribute to fibrosis, such as idiopathic pulmonary fibrosis.
We describe a model system in which cancer cell colonies disperse into single, highly migratory cells in response to lysophosphatidic acid (LPA). Though LPA is known to stimulate chemotaxis and chemokinesis, a colony dispersal effect has not been reported, to our knowledge. Cancer colony dispersal by LPA is comprised of an ordered sequence of events: (1) stimulation of membrane ruffling and formation of lamellipodia, (2) dissolution of adherens junctions, (3) single cell migration in a mesenchymal-like morphology we term "ginkgo-leaf." The net result is dispersal of carcinoma cells from a compact colony. We analyzed these three steps using live-cell imaging and computer-assisted quantification and measured the following parameters: onset of lamellipodia formation, lamellipodia velocity, colony dispersal, trans-epithelial resistance, migrating cell number and speed. Because hepatocyte growth factor (HGF) was described as an epithelial scatter factor, we compared it to LPA in our system and found that HGF has no epithelial colony dispersal properties and that this effect is strictly related to LPA. Given its striking similarity to tumor cell budding observed in patients, we propose that LPA-colony dispersal may provide a cellular mechanism underlying cancer invasion and as such deserves further studies.
Copyright (c) 2005 Wiley-Liss, Inc.
RhoA organizes actin stress fibres and is necessary for cell transformation by oncogenes such as src and ras. Moreover, RhoA is implicated in cadherin clustering during the formation of adherens junctions. The catenin p120 has also been implicated in cadherin clustering through an unknown mechanism. Here we show that p120 selectively inhibits RhoA activity in vitro and in vivo. RhoA inhibition and the interaction of p120 with cadherins are mutually exclusive, suggesting a mechanism for regulating the recruitment and exchange of RhoA at nascent cell-cell contacts. By affecting RhoA activation, p120 could modulate cadherin functions, including suppression of invasion, neurite extension and junction formation.