Erzsebet Szabo

Autotaxin and LPA1 and LPA5 receptors exert disparate functions in tumor cells versus the host tissue microenvironment in melanoma invasion and metastasis.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. Implications: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis. Mol Cancer Res; 13(1); 174–85. ©2014 AACR.

miR-203 Functions as a Tumor Suppressor by Inhibiting Epithelial to Mesenchymal Transition in Ovarian Cancer

OBJECTIVE Ovarian cancer is a gynecological malignancy that has a high mortality rate in women due to metastatic progression and recurrence. miRNAs are small, endogenous, noncoding RNAs that function as tumor suppressors or oncogenes in various human cancers by selectively suppressing the expression of target genes. The objective of this study is to investigate the role of miR-203 in ovarian cancer. METHODS miR-203 was expressed in ovarian cancer SKOV3 and OVCAR3 cells using lentiviral vector and cell proliferation, migration, invasion were examined using MTT, transwell and Matrigel assays, respectively. Tumor growth was examined using Xenograft mouse model. RESULTS miR-203 expression was downregulated, whereas expression of its target gene Snai2 was upregulated in human ovarian serous carcinoma tissue as compared to normal ovaries. In addition, high miR-203 expression was associated with long-term survival rate of ovarian cancer patients. miR-203 overexpression inhibited cell proliferation, migration, and invasion of SKOV3 and OVCAR3 ovarian cancer cells. Furthermore, miR-203 overexpression inhibited the epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Silencing Snai2 with lentiviral short hairpin (sh) RNA mimics miR-203-mediated inhibition of EMT and tumor cell invasion. Xenografts of miR-203-overexpressing ovarian cancer cells in immunodeficient mice exhibited a significantly reduced tumor growth. CONCLUSION miR-203 functions as a tumor suppressor by down regulating Snai2 in ovarian cancer.

Mitigation of the hematopoietic and gastrointestinal acute radiation syndrome by octadecenyl thiophosphate, a small molecule mimic of lysophosphatidic acid.

We have previously demonstrated that the small molecule octadecenyl thiophosphate (OTP), a synthetic mimic of the growth factor-like mediator lysophosphatidic acid (LPA), showed radioprotective activity in a mouse model of total-body irradiation (TBI) when given orally or intraperitoneally 30 min before exposure to 9 Gy γ radiation. In the current study, we evaluated the effects of OTP, delivered subcutaneously, for radioprotection or radiomitigation from −24 h before to up to +72 h postirradiation using a mouse TBI model with therapeutic doses at around 1 mg/kg. OTP was injected at 10 mg/kg without observable toxic side effects in mice, providing a comfortable safety margin. Treatment of C57BL/6 mice with a single dose of OTP over the time period from −12 h before to +26 h after a lethal dose of TBI reduced mortality by 50%. When administered at +48 h to +72 h postirradiation (LD50/30 to LD100/30), OTP reduced mortality by ≥34%. OTP administered at +24 h postirradiation significantly elevated peripheral white blood cell and platelet counts, increased crypt survival in the jejunum, enhanced intestinal glucose absorption and reduced endotoxin seepage into the blood. In the 6.4–8.6 Gy TBI range using LD50/10 as the end point, OTP yielded a dose modification factor of 1.2. The current data indicate that OTP is a potent radioprotector and radiomitigator ameliorating the mortality and tissue injury of acute hematopoietic as well as acute gastrointestinal radiation syndrome.

Rapid disruption of intestinal epithelial tight junction and barrier dysfunction by ionizing radiation in mouse colon in vivo: protection by N-acetyl-l-cysteine.

The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and β-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.

Design and synthesis of sulfamoyl benzoic acid analogues with subnanomolar agonist activity specific to the LPA2 receptor.

Lysophosphatidic acid (LPA) is a growth factor-like mediator and a ligand for multiple GPCR. The LPA2 GPCR mediates antiapoptotic and mucosal barrier-protective effects in the gut. We synthesized sulfamoyl benzoic acid (SBA) analogues that are the first specific agonists of LPA2, some with subnanomolar activity. We developed an experimental SAR that is supported and rationalized by computational docking analysis of the SBA compounds into the LPA2 ligand-binding pocket.

The LPA2 receptor agonist Radioprotectin-1 spares Lgr5-positive intestinal stem cells from radiation injury in murine enteroids

Rapidly proliferating cells are highly sensitive to ionizing radiation and can undergo apoptosis if the oxidative and genotoxic injury exceed the defensive and regenerative capacity of the cell. Our earlier work has established the antiapoptotic action of the growth factor-like lipid mediator lysophosphatidic acid (LPA). Activation of the LPA2 GPCR has been hypothesized to elicit antiapoptotic and regenerative actions of LPA. Based on this hypothesis we developed a novel nonlipid agonist of LPA2, which we designated Radioprotectin-1 (RP-1). We tested RP-1 at the six murine LPA GPCR subtypes using the transforming growth factor alpha shedding assay and found that it had a 25 nM EC50 that is similar to that of LPA18:1 at 32 nM. RP-1 effectively reduced apoptosis induced by γ-irradiation and the radiomimetic drug Adriamycin only in cells that expressed LPA2 either endogenously or after transfection. RP-1 reduced γ-H2AX levels in irradiated mouse embryonic fibroblasts transduced with the human LPA2 GPCR but was ineffective in vector transduced MEF control cells and significantly increased clonogenic survival after γ-irradiation. γ-Irradiation induced the expression of lpar2 transcripts that was further enhanced by RP-1 exposure within 30 min after irradiation. RP-1 decreased the mortality of C57BL/6 mice in models of the hematopoietic and gastrointestinal acute radiation syndromes. Using Lgr5-EGFP-CreER;Tdtomatoflox transgenic mice, we found that RP-1 increased the survival and growth of intestinal enteroids via the enhanced survival of Lgr5+ intestinal stem cells. Taken together, our results suggest that the LPA2-specific agonist RP-1 exerts its radioprotective and radiomitigative action through specific activation of the upregulated LPA2 GPCR in Lgr5+ stem cells.

Tu1377 Rapid Disruption of Colonic Epithelial Tight Junctions by Ionizing Radiation in Mice In Vivo: Protection by N-Acetyl L-Cysteine

Protective Effects of Partially Hydrolyzed Guar Gum Against Intestinal Epithelial Barrier Dysfunction Atsushi Majima, Yuji Naito, Osamu Handa, Yuriko Onozawa, Yukiko Uehara, Hideki Horie, Mayuko Morita, Yasuki Higashimura, Katsura Mizushima, Tetsuya Okayama, Kazuhiro Katada, Kazuhiro Kamada, Kazuhiko Uchiyama, Ishikawa Takeshi, Tomohisa Takagi, Yoshito Itoh, Zenta Yasukawa, Makoto Tokunaga, Tsutomu Okubo

Regulation of tumor cell – Microenvironment interaction by the autotaxin-lysophosphatidic acid receptor axis

The lipid mediator lysophosphatidic acid (LPA) in biological fluids is primarily produced by cleavage of lysophospholipids by the lysophospholipase D enzyme Autotaxin (ATX). LPA has been identified and abundantly detected in the culture medium of various cancer cell types, tumor effusates, and ascites fluid of cancer patients. Our current understanding of the physiological role of LPA established its role in fundamental biological responses that include cell proliferation, metabolism, neuronal differentiation, angiogenesis, cell migration, hematopoiesis, inflammation, immunity, wound healing, regulation of cell excitability, and the promotion of cell survival by protecting against apoptotic death. These essential biological responses elicited by LPA are seemingly hijacked by cancer cells in many ways; transcriptional upregulation of ATX leading to increased LPA levels, enhanced expression of multiple LPA GPCR subtypes, and the downregulation of its metabolic breakdown. Recent studies have shown that overexpression of ATX and LPA GPCR can lead to malignant transformation, enhanced proliferation of cancer stem cells, increased invasion and metastasis, reprogramming of the tumor microenvironment and the metastatic niche, and development of resistance to chemo-, immuno-, and radiation-therapy of cancer. The fundamental role of LPA in cancer progression and the therapeutic inhibition of the ATX-LPA axis, although highly appealing, remains unexploited as drug development to these targets has not reached into the clinic yet. The purpose of this brief review is to highlight some unique signaling mechanisms engaged by the ATX-LPA axis and emphasize the therapeutic potential that lies in blocking the molecular targets of the LPA system.

Abstract B28: Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis

Autotaxin (ATX), a lysophospholipase D that generates the growth factor-like mediator lysophosphatidic acid LPA, is up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancer cells secrete ATX, and it contributes to their invasive properties. The biological functions of ATX are mediated by LPA acting on G protein-coupled receptors (LPAR1-8). Many cancers overexpress multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and lung metastasis in vivo. In our present study, we sought to understand the roles of LPAR both in the tumor cells and stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express the LPA5 receptor. We found that these cells do not invade across a matrigel layer in response to LPA. shRNA knockdown of LPA5 receptors in B16F10 attenuates this inhibition, suggesting that the LPA5 receptor acts as an anti-invasive receptor in these tumor cells. Interestingly, we identified a different role for host LPA5 receptor. We found that the incidence of B16F10 lung metastasis was significantly reduced in the LPA5 knockout mice compared to wild type mice. In addition, LPA1 knockout mice also showed diminished B16F10 lung metastasis suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of these knockout animals was apparent at 24 hours after injection. However, deletion of LPA1 or LPA5 did not affect the subcutaneous growth of B16F10 tumors. In summary, our results suggest that LPAR, in particular LPA5 and LPA1 receptors in tumor and stromal cells may play different roles in invasion and the seeding of metastasis. Citation Format: Sue-Chin Lee, Yuko Fujiwara, Jianxiong Liu, Junming Yue, Ryoko Tsukahara, Erzsebet Szabo, Renukadevi Patil, Duane D. Miller, Louisa Balazs, Tamas Oravecz, Gabor J. Tigyi. Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B28. doi:10.1158/1538-7445.CHTME14-B28