Sue Chin Lee

Interaction of platelet-derived autotaxin with tumor integrin αVβ3 controls metastasis of breast cancer cells to bone.

Autotaxin (ATX), through its lysophospholipase D activity controls physiological levels of lysophosphatidic acid (LPA) in blood. ATX is overexpressed in multiple types of cancers, and together with LPA generated during platelet activation promotes skeletal metastasis of breast cancer. However, the pathophysiological sequelae of regulated interactions between circulating LPA, ATX, and platelets remain undefined in cancer. In this study, we show that ATX is stored in α-granules of resting human platelets and released upon tumor cell-induced platelet aggregation, leading to the production of LPA. Our in vitro and in vivo experiments using human breast cancer cells that do not express ATX (MDA-MB-231 and MDA-B02) demonstrate that nontumoral ATX controls the early stage of bone colonization by tumor cells. Moreover, expression of a dominant negative integrin αvβ3-Δ744 or treatment with the anti-human αvβ3 monoclonal antibody LM609, completely abolished binding of ATX to tumor cells, demonstrating the requirement of a fully active integrin αvβ3 in this process. The present results establish a new mechanism for platelet contribution to LPA-dependent metastasis of breast cancer cells, and demonstrate the therapeutic potential of disrupting the binding of nontumor-derived ATX with the tumor cells for the prevention of metastasis.

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.

Hits of a High-Throughput Screen Identify the Hydrophobic Pocket of Autotaxin/Lysophospholipase D As an Inhibitory Surface

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5′-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1–3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.

Mitigation of radiation injury by selective stimulation of the LPA 2 receptor

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA(2) receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA(2) receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24h after radiation exposure. Our findings suggest that by specifically activating LPA(2) receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

The autotaxin–LPA2 GPCR axis is modulated by γ-irradiation and facilitates DNA damage repair

In this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)-LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin(-)Sca-1(+)c-Kit(+) enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair.

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.

Targeting the hydrophobic pocket of autotaxin with virtual screening of inhibitors identifies a common aromatic sulfonamide structural motif.

Modulation of autotaxin (ATX), the lysophospholipase D enzyme that produces lysophosphatidic acid, with small‐molecule inhibitors is a promising strategy for blocking the ATX–lysophosphatidic acid signaling axis. Although discovery campaigns have been successful in identifying ATX inhibitors, many of the reported inhibitors target the catalytic cleft of ATX. A recent study provided evidence for an additional inhibitory surface in the hydrophobic binding pocket of ATX, confirming prior studies that relied on enzyme kinetics and differential inhibition of substrates varying in size. Multiple hits from previous high‐throughput screening for ATX inhibitors were obtained with aromatic sulfonamide derivatives interacting with the hydrophobic pocket. Here, we describe the development of a ligand‐based strategy and its application in virtual screening, which yielded novel high‐potency inhibitors that target the hydrophobic pocket of ATX. Characterization of the structure–activity relationship of these new inhibitors forms the foundation of a new pharmacophore model of the hydrophobic pocket of ATX.

Highly Potent Non-Carboxylic Acid Autotaxin Inhibitors Reduce Melanoma Metastasis and Chemotherapeutic Resistance of Breast Cancer Stem Cells

Autotaxin (ATX, aka. ENPP2) is the main source of the lipid mediator lysophosphatidic acid (LPA) in biological fluids. This study reports on inhibitors of ATX derived by lead optimization of the benzene-sulfonamide in silico hit compound 3. The new analogues provide a comprehensive structure-activity relationship of the benzene-sulfonamide scaffold that yielded a series of highly potent ATX inhibitors. The three most potent analogues (3a, IC50 ∼ 32 nM; 3b, IC50 ∼ 9 nM; and 14, IC50 ∼ 35 nM) inhibit ATX-dependent invasion of A2058 human melanoma cells in vitro. Two of the most potent compounds, 3b and 3f (IC50 ∼ 84 nM), lack inhibitory action on ENPP6 and ENPP7 but possess weak antagonist action specific to the LPA1 G protein-coupled receptor. In particular, compound 3b potently reduced in vitro chemotherapeutic resistance of 4T1 breast cancer stem-like cells to paclitaxel and significantly reduced B16 melanoma metastasis in vivo.

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.

Role of autotaxin in cancer stem cells

Stem cells are a rare subpopulation defined by the potential to self-renew and differentiate into specific cell types. A population of stem-like cells has been reported to possess the ability of self-renewal, invasion, metastasis, and engraftment of distant tissues. This unique cell subpopulation has been designated as cancer stem cells (CSC). CSC were first identified in leukemia, and the contributions of CSC to cancer progression have been reported in many different types of cancers. The cancer stem cell hypothesis attempts to explain tumor cell heterogeneity based on the existence of stem cell-like cells within solid tumors. The elimination of CSC is challenging for most human cancer types due to their heightened genetic instability and increased drug resistance. To combat these inherent abilities of CSC, multi-pronged strategies aimed at multiple aspects of CSC biology are increasingly being recognized as essential for a cure. One of the most challenging aspects of cancer biology is overcoming the chemotherapeutic resistance in CSC. Here, we provide an overview of autotaxin (ATX), lysophosphatidic acid (LPA), and their signaling pathways in CSC. Increasing evidence supports the role of ATX and LPA in cancer progression, metastasis, and therapeutic resistance. Several studies have demonstrated the ATX-LPA axis signaling in different cancers. This lipid mediator regulatory system is a novel potential therapeutic target in CSC. In this review, we summarize the evidence linking ATX-LPA signaling to CSC and its impact on cancer progression and metastasis. We also provide evidence for the efficacy of cancer therapy involving the pharmacological inhibition of this signaling pathway.