Jillian H. Hurst

Regulator of G-protein signaling (RGS) proteins in cancer biology

The regulator of G-protein signaling (RGS) family is a diverse group of multifunctional proteins that regulate cellular signaling events downstream of G-protein coupled receptors (GPCRs). In recent years, GPCRs have been linked to the initiation and progression of multiple cancers; thus, regulators of GPCR signaling are also likely to be important to the pathophysiology of cancer. This review highlights recent studies detailing changes in RGS transcript expression during oncogenesis, single nucleotide polymorphisms in RGS proteins linked to lung and bladder cancers, and specific roles for RGS proteins in multiple cancer types.

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.

Regulator of G-protein signalling expression and function in ovarian cancer cell lines

Regulator of G-protein signalling (RGS)2 proteins critically regulate signalling cascades initiated by G-protein coupled receptors (GPCRs) by accelerating the deactivation of heterotrimeric G-proteins. Lysophosphatidic acid (LPA) is the predominant growth factor that drives the progression of ovarian cancer by activating specific GPCRs and G-proteins expressed in ovarian cancer cells. We have recently reported that RGS proteins endogenously expressed in SKOV-3 ovarian cancer cells dramatically attenuate LPA stimulated cell signalling. The goal of this study was twofold: first, to identify candidate RGS proteins expressed in SKOV-3 cells that may account for the reported negative regulation of G-protein signalling, and second, to determine if these RGS protein transcripts are differentially expressed among commonly utilized ovarian cancer cell lines and non-cancerous ovarian cell lines. Reverse transcriptase-PCR was performed to determine transcript expression of 22 major RGS subtypes in RNA isolated from SKOV-3, OVCAR-3 and Caov-3 ovarian cancer cell lines and non-cancerous immortalized ovarian surface epithelial (IOSE) cells. Fifteen RGS transcripts were detected in SKOV-3 cell lines. To compare the relative expression levels in these cell lines, quantitative real time RT-PCR was performed on select transcripts. RGS19/GAIP was expressed at similar levels in all four cell lines, while RGS2 transcript was detected at levels slightly lower in ovarian cancer cells as compared to IOSE cells. RGS4 and RGS6 transcripts were expressed at dramatically different levels in ovarian cancer cell lines as compared to IOSE cells. RGS4 transcript was detected in IOSE at levels several thousand fold higher than its expression level in ovarian cancer cells lines, while RGS6 transcript was expressed fivefold higher in SKOV-3 cells as compared to IOSE cells, and over a thousand fold higher in OVCAR-3 and Caov-3 cells as compared to IOSE cells. Functional studies of RGS 2, 6, and 19/GAIP were performed by measuring their effects on LPA stimulated production of inositol phosphates. In COS-7 cells expressing individual exogenous LPA receptors, RGS2 and RSG19/GAIP attenuated signalling initiated by LPA1, LPA2, or LPA3, while RGS6 only inhibited signalling initiated by LPA2 receptors. In SKOV-3 ovarian cancer cells, RGS2 but not RGS6 or RGS19/GAIP, inhibited LPA stimulated inositol phosphate production. In contrast, in CAOV-3 cells RGS19/GAIP strongly attenuated LPA signalling. Thus, multiple RGS proteins are expressed at significantly different levels in cells derived from cancerous and normal ovarian cells and at least two candidate RGS transcripts have been identified to account for the reported regulation of LPA signalling pathways in ovarian cancer cells.

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.

Lysophosphatidic Acid Stimulates Cell Growth by Different Mechanisms in SKOV-3 and Caov-3 Ovarian Cancer Cells: Distinct Roles for Gi- and Rho-Dependent Pathways

Background/Aims: Lysophosphatidic acid (LPA) is an autocrine growth signal critical to the initiation and progression of ovarian cancer. In the current study, we investigated the receptors and signaling cascades responsible for mediating LPA-stimulated cell growth in SKOV-3 and Caov-3 ovarian cancer cell lines. Methods: Pharmacological inhibitors of distinct LPA and epidermal growth factor receptors, G proteins and kinases were tested for their effect on LPA-stimulated cell growth, MAP kinase activation and Akt activation in SKOV-3 and Caov-3 cells. Results: Distinct agonist pharmacological profiles were observed. Saturated and unsaturated LPA species were equally potent in Caov-3 cells, while saturated LPA was less potent than unsaturated LPA in SKOV-3 cells. Further, the LPA1/LPA3 receptor antagonist Ki16425 was more potent in SKOV-3 cells. The effect of LPA on cell growth in both cell lines was dependent on phosphatidylinositol-3 kinases and MAP kinases. However, LPA-stimulated SKOV-3 cell growth required Gi G proteins, while Caov-3 cell growth was dependent on the Rho effector p160 Rho kinase. Finally, we demonstrated that regulator of G protein signaling proteins significantly regulated Gi-dependent LPA-stimulated cell growth in SKOV-3 cells. Conclusions: LPA-stimulated cell growth is mediated by distinct but overlapping receptors and signaling pathways in these two model ovarian cancer cell lines.

Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity

Background: Ubiquitination is a post-translational modification that regulates protein behavior. Results: Pheromone stimulation induces dynamic ubiquitination of the MAPKK Ste7; disruption of this modification leads to altered MAPK signal specificity. Conclusion: Dynamic ubiquitination is required to maintain the strength and fidelity of the pheromone response. Significance: This study identifies a novel regulatory mechanism in MAPK cascades, a signaling module that is central to human physiology and disease. Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCFCdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCFCdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCFCdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCFCdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.

Cancer immunotherapy innovator James Allison receives the 2015 Lasker~DeBakey Clinical Medical Research Award

The 2015 Lasker~DeBakey Clinical Medical Research Award honors James P. Allison, PhD, for pioneering a new approach to cancer immunotherapy (Figure 1). Allison has made many seminal contributions to immunology, including the identification of the receptor on T cells that recognizes and binds antigens; the discovery that T cells require a second molecular signal from the costimulatory molecule CD28 to launch a response to a bound antigen; elucidation of the function of cytotoxic T lymphocyte antigen-4 (CTLA-4), which acts as a built-in off-switch on T cells; and the development of a CTLA-4–blocking antibody, which unleashes T cells, allowing them to eliminate cancer cells. A therapeutic CTLA-4 antibody (ipilimumab, also known as Yervoy) is now used to treat advanced melanoma and is currently under investigation for the treatment of many other malignancies. Allison’s work has caused a paradigm shift in the treatment of cancer in which treatment is focused on helping the patient’s immune system defeat the tumor instead of targeting the tumor itself.

Pioneering geneticist Mary-Claire King receives the 2014 Lasker~Koshland Special Achievement Award in Medical Science

The 2014 Lasker~Koshland Special Achievement Award in Medical Science has been awarded to Dr. Mary-Claire King, a pioneer in human genetics (Figure ​(Figure1).1). Dr. King discovered the BRCA1 gene locus that underlies hereditary breast cancer; additionally, she has been instrumental in utilizing DNA sequencing to identify missing persons. In her address to the American Society for Human Genetics in 2012, Dr. King stated that “In genetics, there are hard problems and incredibly hard problems, but we do not acknowledge any unsolvable problems. The most daunting task for us is not tackling new discovery but rather integrating discovery into a meaningful social context” (1). Dr. King has consistently taken on incredibly hard problems in genetics, and her work has had a profound influence on our understanding of the genetics of human disease, on the use of DNA to advance human rights and prosecute abuses of those rights, and on our view of the frontiers in genomic technologies. Figure 1 Mary-Claire King is the winner of the 2014 Lasker~Koshland Special Achievement Award in Medical Science for bold and imaginative contributions to medical science and society.

Richard Scheller and Thomas Südhof receive the 2013 Albert Lasker Basic Medical Research Award

Neural communication underlies all brain activity. It governs our thoughts, feelings, sensations, and actions. But knowing the importance of neural communication does not answer a central question of neuroscience: how do individual neurons communicate? We know that communication between two neurons occurs at specialized cell junctions called synapses, at which two communicating neurons are separated by the synaptic cleft. The presynaptic neuron releases chemicals, known as neurotransmitters, into the synaptic cleft in which neurotransmitters bind to receptors on the surface of the postsynaptic neuron. Neurotransmitter release occurs in response to an action potential within the sending neuron that induces depolarization of the nerve terminal and causes an influx of calcium. Calcium influx triggers the release of neurotransmitters through a specialized form of exocytosis in which neurotransmitter-filled vesicles fuse with the plasma membrane of the presynaptic nerve terminal in a region known as the active zone, spilling neurotransmitter into the synaptic cleft. By the 1950s, it was clear that brain function depended on chemical neurotransmission; however, the molecular activities that governed neurotransmitter release were virtually unknown until the early 1990s. This year, the Lasker Foundation honors Richard Scheller (Genentech) and Thomas Sudhof (Stanford University School of Medicine) for their “discoveries concerning the molecular machinery and regulatory mechanisms that underlie the rapid release of neurotransmitters.” Over the course of two decades, Scheller and Sudhof identified and characterized a set of proteins that mediate the fusion of neurotransmitter-filled synaptic vesicles with the plasma membranes of presynaptic nerve terminals. These proteins participate in the formation and regulation of a membrane-bridging complex, known as the soluble NSF attachment protein (SNAP) receptor (SNARE) complex. It is now known that this mechanism is used to mediate various forms of exocytosis throughout the body.

A modern Cosmas and Damian: Sir Roy Calne and Thomas Starzl receive the 2012 Lasker~Debakey Clinical Medical Research Award

Cosmas and Damian, the patron saints of surgeons, were twin brothers who dedicated their lives to healing the sick (Figure ​(Figure1).1). They are most revered for performing the first transplant operation, when they replaced the gangrenous leg of the sacristan Justinian with one from a recently deceased soldier. In the 1940s and 1950s, when Thomas Starzl and Sir Roy Calne (Figure ​(Figure2)2) were training to be surgeons, successful transplantation was still in the domain of the miraculous; in fact, both Calne and Starzl had been told that the procedure was impossible. However, within a period of 30 years, they were able to overcome surgical and biological obstacles to develop liver transplantation procedures that have saved the lives of thousands of patients and made the transplantation of multiple organs a reality.