MicroRNA Regulation of the Autotaxin-Lysophosphatidic Acid Signaling Axis.

The revelation that microRNAs (miRNAs) exist within the human genome uncovered an underappreciated mechanism of gene expression. For cells to regulate expression of their genes, miRNA molecules and argonaute proteins bind to mRNAs and interfere with efficient translation of the RNA transcript. Although miRNAs have important roles in normal tissues, miRNAs may adopt aberrant functions in malignant cells depending on their classification as either a tumor suppressor or oncogenic miRNA. Within this review, the current status of miRNA regulation is described in the context of signaling through the lysophosphatidic acid receptors, including the lysophosphatidic acid-producing enzyme, autotaxin. Thus far, research has revealed miRNAs that increase in response to lysophosphatidic acid stimulation, such as miR-21, miR-30c-2-3p, and miR-122. Other miRNAs inhibit the translation of lysophosphatidic acid receptors, such as miR-15b, miR-23a, and miR200c, or proteins that are downstream of lysophosphatidic acid signaling, such as miR-146 and miR-21. With thousands of miRNAs still uncharacterized, it is anticipated that the complex regulation of lysophosphatidic acid signaling by miRNAs will continue to be elucidated. RNA-based therapeutics have entered the clinic with enormous potential in precision medicine. This exciting field is rapidly emerging and it will be fascinating to witness its expansion in scope.

miR-122-5p Expression and Secretion in Melanoma Cells Is Amplified by the LPAR3 SH3-Binding Domain to Regulate Wnt1.

The lysophosphatidic acid receptor-3 (LPAR3) is a G protein-coupled receptor that mediates viability among malignant cells and aggressiveness among certain tumors. The study's objective was to determine the interplay between LPAR3 and miRNAs to impact key cellular signaling pathways. Using SK-Mel-2 and SK-Mel-5 melanoma cells, wild-type and mutated receptors were stably expressed to explore molecular mechanisms. LPAR3 signaling induced miR-122-5p intracellularly and subsequently its inclusion into exosomes. This amplification resulted in less abundant Wnt1, maintenance of GSK3 inactivation and to a lesser extent, partial degradation of ß-catenin. The surge in miR-122-5p and reduction in Wnt1 originated from signaling at the Src homology 3 (SH3) ligand-binding motif within the third intracellular loop of LPAR3, because mutant receptors did not increase miR-122-5p and had a weakened capacity to reduce Wnt1. In addition, a key mediator of melanoma survival signaling, the peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A/PGC1), was involved in miR-122-5p transcription. In conclusion, this study highlights the powerful role miRNAs have in fine-tuning specific G protein-coupled receptor-mediated signaling events by altering the transcription of signaling transduction pathway components. This study also identifies that LPAR3 increases miR-122-5p expression, which occurs mechanistically through the SH3 domain and helps explain why miR-122-5p increases are detected in cancer patient serum. IMPLICATIONS: LPAR3 is partially responsible for the production and secretion of miR-122-5p, found in the serum of a wide variety of patients with cancer.

Direct identification of microribonucleic acid miR-451 from plasma using liquid chromatography mass spectrometry.

In recent years, small endogenous RNAs have come to the forefront of both basic and translational research. For example, many studies have pointed to the potential role of microRNAs (miRNAs) as disease biomarkers. However, precise quantitative methods for the analysis of miRNAs are still lacking. In this study, we report the first mass spectrometry-based quantitation of miR-451, a circulatory microRNA. Using a highly selective sample preparation method with an average recovery of 83.6% and a novel mobile phase chemistry, we were able to reach an LOQ of 0.5 ng/mL. Because of such high sensitivity, we could detect and quantify the endogenous miR-451 from both human and rat plasma. Considering the increased precision of LC-MS compared to other methods, these results usher in a new era of miRNA biomarker discovery and validation.

Autotaxin exacerbates tumor progression by enhancing MEK1 and overriding the function of miR-489-3p.

Upregulated expression of autotaxin, a secreted phospholipase and phosphodiesterase enzyme, appears in malignant disease. The identification of a circulating miRNA signature should distinguish autotaxin-mediated disease and also elucidate unknown molecular mechanisms that rationalize its malignant potential. Using female transgenic 'AT-ATX' mice, whereby human wild-type autotaxin is expressed in liver under the control of the alpha-1 antitrypsin promoter, transgenic animals express augmented autotaxin in circulation and a percentage develop tumors. Serum collected at necropsy had circulating miRNAs analyzed for statistical significance. The ensuing autotaxin-mediated miRNome differentiated between groups: healthy FVB/N mice versus AT-ATX mice with and without tumors. Intriguingly, miR-489-3p was sharply increased in AT-ATX tumor-bearing mice. Tissue analysis showed a correlation between miR-489-3p expression in tumors and surrounding milieu with autotaxin concentration in circulation. Sequence alignment suggested miR-489-3p targets MEK1, which was confirmed through in vitro studies. Exogenously added miR-489-3p, which decreases MEK1 in normal cells, dramatically increased MEK1 expression in cells stably expressing autotaxin. Taken together, this suggests that autotaxin overrides the normal regulatory function of miR-489-3p to inhibit MEK1 via coordinately increased miR-489-3p appearing in serum.

Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p.

Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.

Suppression of the GTPase-activating protein RGS10 increases Rheb-GTP and mTOR signaling in ovarian cancer cells.

The regulator of G protein signaling 10 (RGS10) protein is a GTPase activating protein that accelerates the hydrolysis of GTP and therefore canonically inactivates G proteins, ultimately terminating signaling. Rheb is a small GTPase protein that shuttles between its GDP- and GTP-bound forms to activate mTOR. Since RGS10 suppression augments ovarian cancer cell viability, we sought to elucidate the molecular mechanism. Following RGS10 suppression in serum-free conditions, phosphorylation of mTOR, the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), p70S6K and S6 Ribosomal Protein appear. Furthermore, suppressing RGS10 increases activated Rheb, suggesting RGS10 antagonizes mTOR signaling via the small G-protein. The effects of RGS10 suppression are enhanced after stimulating cells with the growth factor, lysophosphatidic acid, and reduced with mTOR inhibitors, temsirolimus and INK-128. Suppression of RGS10 leads to an increase in cell proliferation, even in the presence of etoposide. In summary, the RGS10 suppression increases Rheb-GTP and mTOR signaling in ovarian cancer cells. Our results suggest that RGS10 could serve in a novel, and previously unknown, role by accelerating the hydrolysis of GTP from Rheb in ovarian cancer cells.

Cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer.

More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.

Vinyl sulfone analogs of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma.

Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 to 0.39 µM, consistent with a Ki of 3.50 µM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.

PKA-type I selective constrained peptide disruptors of AKAP complexes.

A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.

The Src homology 3 binding domain is required for lysophosphatidic acid 3 receptor-mediated cellular viability in melanoma cells.

The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells.

Molecular epigenetics in the management of ovarian cancer: are we investigating a rational clinical promise?

Epigenetics is essentially a phenotypical change in gene expression without any alteration of the DNA sequence; the emergence of epigenetics in cancer research and mainstream oncology is fueling new hope. However, it is not yet known whether this knowledge will translate to improved clinical management of ovarian cancer. In this malignancy, women are still undergoing chemotherapy similar to what was approved in 1978, which to this day represents one of the biggest breakthroughs for treating ovarian cancer. Although liquid tumors are benefiting from epigenetically related therapies, solid tumors like ovarian cancer are not (yet?). Herein, we will review the science of molecular epigenetics, especially DNA methylation, histone modifications and microRNA, but also include transcription factors since they, too, are important in ovarian cancer. Pre-clinical and clinical research on the role of epigenetic modifications is also summarized. Unfortunately, ovarian cancer remains an idiopathic disease, for the most part, and there are many areas of patient management, which could benefit from improved technology. This review will also highlight the evidence suggesting that epigenetics may have pre-clinical utility in pharmacology and clinical applications for prognosis and diagnosis. Finally, drugs currently in clinical trials (i.e., histone deacetylase inhibitors) are discussed along with the promise for epigenetics in the exploitation of chemoresistance. Whether epigenetics will ultimately be the answer to better management in ovarian cancer is currently unknown; but we hope so in the future.

Transcriptional suppression, DNA methylation, and histone deacetylation of the regulator of G-protein signaling 10 (RGS10) gene in ovarian cancer cells.

RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.

Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model.

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression-whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reduces in vitro BrdU-positive HeyA8-MDR cells, although this did not correlate with a reduction in tumor volume observed using an in vivo mouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multicomponent approach for modulating tumor progression.

MicroRNA-30c-2* expressed in ovarian cancer cells suppresses growth factor-induced cellular proliferation and downregulates the oncogene BCL9.

MicroRNAs (miRNAs) are small noncoding RNAs that function as master regulators of posttranscriptional gene expression with each miRNA negatively regulating hundreds of genes. Lysophosphatidic acid (LPA) is a mitogenic lipid present within the ovarian tumor microenvironment and induces LPA receptor activation and intracellular signaling cascades like ERK/MAPK, leading to enhanced cellular proliferation. Here, we show that in SKOV-3 and OVCAR-3 cells, LPA stimulation at concentrations ranging from 1 nmol/L to 20 µmol/L for 30 to 60 minutes increases miR-30c-2*, and this effect is mediated through a combination of receptors because knock down of multiple LPA receptors is required for inhibition. The epidermal growth factor and platelet-derived growth factor also increase miR-30c-2* transcript expression, suggesting a broader responsive role for miR-30c-2*. Thus, we investigated the functional role of miR-30c-2* through ectopic expression of synthetic miRNA precursors of mature miRNA or antagomir transfection and observed that microRNA-30c-2* reduces, and the antagomir enhances, cell proliferation and viability in OVCAR-3, cisplatin-insensitive SKOV-3 and chemoresistant HeyA8-MDR cells. Ectopic expression of miR-30c-2* reduces BCL9 mRNA transcript abundance and BCL9 protein. Consistent with this observation, miR-30c-2* ectopic expression also reduced BCL9 luciferase reporter gene expression. In comparison with IOSE cells, all cancer cells examined showed increased BCL9 expression, which is consistent with its role in tumor progression. Taken together, this suggest that growth factor induced proliferation mediates a neutralizing response by significantly increasing miR-30c-2* which reduces BCL9 expression and cell proliferation in SKOV-3 and OVCAR-3 cells, likely as a mechanism to regulate signal transduction downstream.

The Role of Proteomics in the Diagnosis and Treatment of Women's Cancers: Current Trends in Technology and Future Opportunities.

Technological and scientific innovations over the last decade have greatly contributed to improved diagnostics, predictive models, and prognosis among cancers affecting women. In fact, an explosion of information in these areas has almost assured future generations that outcomes in cancer will continue to improve. Herein we discuss the current status of breast, cervical, and ovarian cancers as it relates to screening, disease diagnosis, and treatment options. Among the differences in these cancers, it is striking that breast cancer has multiple predictive tests based upon tumor biomarkers and sophisticated, individualized options for prescription therapeutics while ovarian cancer lacks these tools. In addition, cervical cancer leads the way in innovative, cancer-preventative vaccines and multiple screening options to prevent disease progression. For each of these malignancies, emerging proteomic technologies based upon mass spectrometry, stable isotope labeling with amino acids, high-throughput ELISA, tissue or protein microarray techniques, and click chemistry in the pursuit of activity-based profiling can pioneer the next generation of discovery. We will discuss six of the latest techniques to understand proteomics in cancer and highlight research utilizing these techniques with the goal of improvement in the management of women's cancers.

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.

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

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.

Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid.

BACKGROUND: Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective. RESULTS: Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor. CONCLUSIONS: Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.

Lysophosphatidic acid-induced transcriptional profile represents serous epithelial ovarian carcinoma and worsened prognosis.

BACKGROUND: Lysophosphatidic acid (LPA) governs a number of physiologic and pathophysiological processes. Malignant ascites fluid is rich in LPA, and LPA receptors are aberrantly expressed by ovarian cancer cells, implicating LPA in the initiation and progression of ovarian cancer. However, there is an absence of systematic data critically analyzing the transcriptional changes induced by LPA in ovarian cancer. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, gene expression profiling was used to examine LPA-mediated transcription by exogenously adding LPA to human epithelial ovarian cancer cells for 24 h to mimic long-term stimulation in the tumor microenvironment. The resultant transcriptional profile comprised a 39-gene signature that closely correlated to serous epithelial ovarian carcinoma. Hierarchical clustering of ovarian cancer patient specimens demonstrated that the signature is associated with worsened prognosis. Patients with LPA-signature-positive ovarian tumors have reduced disease-specific and progression-free survival times. They have a higher frequency of stage IIIc serous carcinoma and a greater proportion is deceased. Among the 39-gene signature, a group of seven genes associated with cell adhesion recapitulated the results. Out of those seven, claudin-1, an adhesion molecule and phenotypic epithelial marker, is the only independent biomarker of serous epithelial ovarian carcinoma. Knockdown of claudin-1 expression in ovarian cancer cells reduces LPA-mediated cellular adhesion, enhances suspended cells and reduces LPA-mediated migration. CONCLUSIONS: The data suggest that transcriptional events mediated by LPA in the tumor microenvironment influence tumor progression through modulation of cell adhesion molecules like claudin-1 and, for the first time, report an LPA-mediated expression signature in ovarian cancer that predicts a worse prognosis.