Phillip Callihan

Regulators of G-Protein signaling RGS10 and RGS17 regulate chemoresistance in ovarian cancer cells

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.

Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology.

BackgroundLysophospholipids 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.ResultsOur results demonstrate that Lysophosphatidic Acid (LPA) and Sphingosine-1-phosphate (S1P) receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR)- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK.ConclusionThus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

Regulation of stem cell pluripotency and differentiation by G protein coupled receptors

Stem cell-based therapeutics have the potential to effectively treat many terminal and debilitating human diseases, but the mechanisms by which their growth and differentiation are regulated are incompletely defined. Recent data from multiple systems suggest major roles for G protein coupled receptor (GPCR) pathways in regulating stem cell function in vivo and in vitro. The goal of this review is to illustrate common ground between the growing field of stem cell therapeutics and the long-established field of G protein coupled receptor signaling. Herein, we briefly introduce basic stem cell biology and discuss how several conserved pathways regulate pluripotency and differentiation in mouse and human stem cells. We further discuss general mechanisms by which GPCR signaling may impact these pluripotency and differentiation pathways, and summarize specific examples of receptors from each of the major GPCR subfamilies that have been shown to regulate stem cell function. Finally, we discuss possible therapeutic implications of GPCR regulation of stem cell function.

Convergent Regulation of Neuronal Differentiation and Erk and Akt Kinases in Human Neural Progenitor Cells by Lysophosphatidic Acid, Sphingosine 1-Phosphate, and LIF: Specific Roles for the LPA1 Receptor

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.

Sphingosine-1-Phosphate Signaling in Neural Progenitors

Sphingosine-1-phosphate (S1P) and its receptors are important in nervous system development. Reliable in vitro human model systems are needed to further define specific roles for S1P signaling in neural development. We have recently reported that human embryonic stem cell-derived neuroepithelial progenitor cells (hES-NEP) express functional S1P receptors. These cells can be further differentiated to a neuronal cell type, and therefore represent a good model system to study the role of S1P signaling in human neural development. The following sections describe in detail the culture of hES-NEP cells and two assays to measure S1P signaling in these cells.

Abstract 2140: Regulator of G-protein signaling 2 blunts cellular viability mediated by a stabilized lysophosphatidic acid analogue (2S-OMPT) in the presence of chemotherapy in an ovarian cancer cell model of chemoresistance

Lysophosphatidic acid (LPA) enhances growth, survival, viability and proliferation in numerous models. Thus, the receptors for LPA are also implicated in oncogenic signaling and represent a group of “druggable” therapeutic targets in cancer. Regulator of G protein Signaling 2 (RGS2) is a GTPase activating protein (GAP) that deactivates the G alpha subunits of heterotrimeric G proteins, turning off G protein-coupled receptor (GPCR) signaling. In our previous studies examining computational bioinformatics, we demonstrated that reduced RGS gene expression develops with the occurrence of drug resistance in ovarian cancer cell lines. The purpose of our study was to investigate whether the LPA receptors are also involved in mediating viability as a result of enhanced GPCR signaling due to reduced RGS proteins in this chemoresistance model. This is important since RGS proteins can deactivate G alpha subunits of many different GPCRs, and are not specific to merely the LPA receptors. If a “druggable” GPCR is mediating viability in chemoresistance, it is critical to identify which ones are responsible. To test this idea we used the metabolically stabilized LPA analogue L-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothionate (2S-OMPT) as a potent non-hydrolyzable agonist for LPA receptors at a concentration (10 µM) sufficient to activate multiple LPA receptors. We hypothesized that if LPA signaling was not involved, then OMPT would override any contribution of RGS2 on modulating the viability response in the presence of chemotherapy. Using a tet-off promoter system, we generated an inducible stable model of RGS2-expressing HeyA8-MDR cells, which we have previously confirmed are resistant to taxanes. After 48 h OMPT pre-treatment, HeyA8-MDR-RGS2-inducible cells were exposed to cisplatin for 24 h and evaluated for viability. IC 50 values for cisplatin were lower in the RGS2-induced HeyA8-MDR cells that were not pretreated with OMPT, a demonstration of enhanced sensitivity to cisplatin in the presence of RGS2. All conditions pre-treated with OMPT demonstrated enhanced viability even in the presence of chemotherapy; however, ovarian cancer cells that were pretreated with OMPT and cultured in tet-free medium to induce RGS2 expression showed a significant blunting of viability. This supports the interaction between LPA receptors and RGS2 in the control of drug sensitivity in ovarian cancer chemoresistance and provides a mechanism for our current and previous observations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2140. doi:1538-7445.AM2012-2140