Mandi M. Murph

Expression of Autotaxin and Lysophosphatidic Acid Receptors Increases Mammary Tumorigenesis, Invasion, and Metastases

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Lysophosphatidic Acid Receptors Determine Tumorigenicity and Aggressiveness of Ovarian Cancer Cells

BACKGROUND Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown. METHODS Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided. RESULTS SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing beta-galactosidase. CONCLUSIONS Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.

Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

ATX-LPA receptor axis in inflammation and cancer

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) mediates a plethora of physiological and pathological activities via interactions with a series of high affinity G protein-coupled receptors (GPCR). Both LPA receptor family members and autotaxin (ATX/LysoPLD), the primary LPA-producing enzyme, are aberrantly expressed in many human breast cancers and several other cancer lineages. Using transgenic mice expressing either an LPA receptor or ATX, we recently demonstrated that the ATX-LPA receptor axis plays a causal role in breast tumorigenesis and cancerrelated inflammation, further validating the ATX-LPA receptor axis as a rich therapeutic target in cancer.

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.

Targeting the lipids LPA and S1P and their signalling pathways to inhibit tumour progression.

The bioactive lipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), the enzymes that generate and degrade them, and the receptors that receive their signals are all potential therapeutic targets in cancer. LPA and S1P signalling pathways can modulate a range of cellular processes that contribute to tumourigenesis, such as proliferation and motility, and components of the signalling pathways often show aberrant expression and altered activity upon malignant transformation. This article reviews LPA- and S1P-mediated activities that might contribute to the aetiology of cancer, and examines the potential of the many antagonists that have been developed to inhibit LPA and S1P signalling pathways. In addition, the outcomes of various clinical trials using LPA- and S1P-associated targets in cancer and other diseases are described, and future directions are discussed.

Lysophosphatidic Acid Decreases the Nuclear Localization and Cellular Abundance of the p53 Tumor Suppressor in A549 Lung Carcinoma Cells

Lysophosphatidic acid (LPA) is a bioactive lipid that promotes cancer cell proliferation and motility through activation of cell surface G protein–coupled receptors. Here, we provide the first evidence that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells, which express endogenous LPA receptors. The LPA effect depends on increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. Inhibition of phosphatidylinositol 3-kinase protected cells from the LPA-induced reduction of p53, which implicates this signaling pathway in the mechanism of LPA-induced loss of p53. LPA partially protected A549 cells from actinomycin D induction of both apoptosis and increased p53 abundance. Expression of LPA1, LPA2, and LPA3 receptors in HepG2 hepatoma cells, which normally do not respond to LPA, also decreased p53 expression and p53-dependent transcription. In contrast, neither inactive LPA1 (R124A) nor another Gi-coupled receptor, the M2 muscarinic acetylcholine receptor, reduced p53-dependent transcription in HepG2 cells. These results identify p53 as a target of LPA action and provide a new dimension for understanding how LPA stimulates cancer cell division, protects against apoptosis, and thereby promotes tumor progression. (Mol Cancer Res 2007;5(11):1201–11)

Cancer Stem Cell Radioresistance and Enrichment: Where Frontline Radiation Therapy May Fail in Lung and Esophageal Cancers

Many studies have highlighted the role cancer stem cells (CSC) play in the development and progression of various types of cancer including lung and esophageal cancer. More recently, it has been proposed that the presence of CSCs affects treatment efficacy and patient prognosis. In reviewing this new area of cancer biology, we will give an overview of the current literature regarding lung and esophageal CSCs and radioresistance of CSC, and discuss the potential therapeutic applications of these findings.

Sharpening the edges of understanding the structure/function of the LPA1 receptor: Expression in cancer and mechanisms of regulation

Since the molecular cloning of the vzg-1/Edg-2/LPA1 gene, studies have attempted to characterize LPA1 receptor functionality into a single categorical role, different from the other Edg-family LPA receptors. The desire to categorize LPA1 function has highlighted its complexity and demonstrated that the LPA1 receptor does not have one absolute function throughout every system. The central nervous system is highly enriched in the LPA1 receptor, suggesting an integral role in neuronal processes. Metastatic and invasive breast cancer also appears to have LPA-mediated LPA1 receptor functions that enhance phenotypes associated with tumorigenesis. LPA1 possesses a number of motifs conserved among G protein-coupled receptors (GPCRs): a DRY-like motif, a PDZ domain, Ser/Thr predicted sites of phosphorylation, a di-leucine motif, double cysteines in the tail and conserved residues that stabilize structure and determine ligand binding. The third intracellular loop of the LPA1 receptor may be the crux of receptor signaling and attenuation with phosphorylation of Thr-236 potentially a key determinant of basal LPA1 signaling. Mutagenesis data supports the notion that Thr-236 regulates this process since mutating Thr-236 to Ala-236 increased basal and LPA-mediated serum response factor (SRF) signaling activity and Lys-236 further increased this basal signaling. Here we describe progress on defining the major functions of the LPA1 receptor, discuss a context dependent dualistic role as both a negative regulator in cancer and a proto-oncogene, outline its structural components at the molecular amino acid level and present mutagenesis data on the third intracellular loop of the receptor.

Of spiders and crabs: the emergence of lysophospholipids and their metabolic pathways as targets for therapy in cancer.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), two small lysophospholipids, are potent inducers of many of the hallmarks of cancer including cell proliferation, survival, migration, invasion, and neovascularization in in vitro and in vivo tumor models. Furthermore, the enzymes metabolizing LPA and S1P and their receptors are aberrant in multiple cancer lineages and exhibit transforming activity altering patterns and targets for metastasis. Several recent studies show the remarkable activity of new chemical genomics and/or potential novel drugs in preclinical models. Combined with the physiologic and pathophysiologic activities of LPA and S1P, these studies suggest the implementation of preclinical and clinical evaluation of LPA and S1P as therapeutic targets.