BackgroundA 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.ResultsIn 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.ConclusionsTaken 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.
Background: Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development.
In vivo and in vitro studies suggest a crucial role for Sphingosine 1-phosphate (S1P) and its receptors in the development of the nervous system. Dihydrosphingosine 1-phosphate (dhS1P), a reduced form of S1P, is an agonist at S1P receptors, but the pharmacology and physiology of dhS1P has not been widely studied. The mycotoxin fumonisin B1 (FB1) is a potent inhibitor of ceramide synthases and causes selective accumulation of dihydrosphingosine and dhS1P. Recent studies suggest that maternal exposure to FB1 correlates with the development of neural tube defects (NTDs) in which the neural epithelial progenitor cell layers of the developing brain fail to fuse. We hypothesize that the altered balance of S1P and dhS1P in neural epithelial cells contributes to the developmental effects of FB1. The goal of this work was first to define the effect of FB1 exposure on levels of sphingosine and dh-sphingosine and their receptor active 1-phosphate metabolites in human embryonic stem cell-derived neural epithelial progenitor (hES-NEP) cells; and second, to define the relative activity of dhS1P and S1P in hES-NEP cells. We found that dhS1P is a more potent stimulator of inhibition of cAMP and Smad phosphorylation than is S1P in neural progenitors, and this difference in apparent potency may be due, in part, to more persistent presence of extracellular dhS1P applied to human neural progenitors rather than a higher activity at S1P receptors. This study establishes hES-NEP cells as a useful human in vitro model system to study the mechanism of FB1 toxicity and the molecular pharmacology of sphingolipid signaling.
The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.
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