Role Of Autotaxin Research Articles

Effect of an Autotaxin Inhibitor, 2-(4-Chlorophenyl)-7-methyl-8-pentylimidazo[1,2-a] Pyrimidin-5(8H)-one (CBT-295), on Bile Duct Ligation-Induced Chronic Liver Disease and Associated Hepatic Encephalopathy in Rats.

The role of autotaxin (ATX)-lysophosphatidic acid (LPA) is yet to be explored in the context of liver cirrhosis and associated encephalopathy. Our objective of this study was to evaluate the role of an ATX inhibitor in biliary cirrhosis and associated hepatic encephalopathy in rats. The preliminary investigation revealed significant impairment in liver function, which eventually led to the development of hepatic encephalopathy. Interestingly, LPA levels were significantly increased in the plasma, liver, and brain of rats following bile duct ligation. Subsequently, we tested the efficacy of an ATX inhibitor, CBT-295, in bile duct-induced biliary cirrhosis and neuropsychiatric symptoms associated with hepatic encephalopathy. CBT-295 showed good oral bioavailability and favorable pharmacokinetic properties. CBT-295 exhibited a significant reduction in inflammatory cytokines like TGF-β, TNF-α, and IL-6 levels, also reduced bile duct proliferation marker CK-19, and lowered liver fibrosis, as evident from reduced collagen deposition. The reversal of liver fibrosis with CBT-295 led to a reduction in blood and brain ammonia levels. Furthermore, CBT-295 also reduced neuroinflammation induced by ammonia, which is characterized by a significant reduction in brain cytokine levels. It improved neuropsychiatric symptoms such as locomotor activities, cognitive impairment, and clinical grading scores associated with hepatic encephalopathy. The improvement in hepatic encephalopathy observed with the ATX inhibitor could be the result of its hepatoprotective action and its ability to attenuate neuroinflammation. Therefore, inhibition of ATX-LPA signaling can be a multifactorial approach for the treatment of chronic liver diseases.

The Role of Autotaxin and LPA Signaling in Embryonic Development, Pathophysiology and Cancer

Autotaxin (ATX) or Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) is a secreted enzyme with lysophospholipase D activity, with its primary function being the extracellular hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid [...].

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.

Role of Autotaxin in High Glucose-Induced Human ARPE-19 Cells.

Autotaxin (ATX) is an enzymatic with lysophospholipase D (lysoPLD) activity. We investigated the role of ATX in high glucose (HG)-induced human retinal pigment epithelial (ARPE-19) cells to explore the pathogenesis of diabetic retinopathy (DR). We performed a quantitative real-time polymerase chain reaction, Western blotting, immunocytochemistry, enzyme-linked immunosorbent assay, cell permeability assay, and transepithelial electrical resistance measurement in HG-induced ARPE-19 cells and compared their results with those of normal glucose and osmotic pressure controls. ATX expression and its lysoPLD activity, barrier function, and expression of vascular endothelial growth factor receptors VEGFR-1 and VEGFR-2 were downregulated, while fibrotic responses, cytoskeletal reorganization, and transforming growth factor-β expression were upregulated, in the HG group. Our results suggest that HG induces intracellular ATX downregulation, barrier dysfunction, and fibrosis, which are involved in early DR and can be targeted for DR treatment.

Immunohistochemical evaluation of autotaxin and lubricin in mild osteoarthritic rat model performing moderate physical activity

Levels of the enzyme autotaxin (ATX) are elevated in synovial fluid and plasma of osteoarthritic patients, correlating positively with radiographic and symptomatic severity of the disease. Therefore, ATX is studied as potential marker for the progression of osteoarthritis (OA), whereas the chondrocyte-secreted glycoprotein Lubricin has chondroprotective properties. The aim of this study was to evaluate the expression of ATX and Lubricin in healthy and mild OA rat articular cartilage of femur, tibia and patella, and to analyse the effect of a protocol of moderate physical activity on their expressions. Mild OA resulted from anterior cruciate ligament transection and rats exercised on a treadmill for 12 weeks. Computerized staining intensity of immunostaining was used to evaluate ATX and Lubricin expressions. Higher expressions of ATX were found in femur and tibia of OA rats, suggesting that this molecule could participate in the progression of the disease, although not involved in the patella. In the femur, physical activity performed by OA rats was able to lower ATX expression, encouraging the evidence that joint movement is beneficial for the cartilage, although no significant differences in Lubricin expression were detected in femur, tibia and patella. This evidence might shade some light about the role of ATX, Lubricin and physical exercise in OA progression.

Abstract TP245: Unfavorable Role Of Autotaxin Lysophosphatidic Acid Axis During Ischemic Reperfusion In Stroke.

Introduction: Stroke is a leading cause of death, also serious cause of long-term disability in the US. r-TPA and mechanical thrombectomy apply to 5-10% of patients, while others have the risk of intracerebral hemorrhage. Ischemic reperfusion following stroke exacerbates the condition and initiates various molecular signals. However, with a better therapeutic regimen, the detrimental effects can be limited. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is formed by the enzyme Autotaxin (ATX). ATX-LPA axis affects endothelial permeability in ischemic stroke. Hypothesis: ATX-LPA axis favors endothelial permeability in ischemic stroke. Methods: Transient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. LPA was analyzed in plasma with mass spectrometry and in tissue with immunohistochemistry. AR-2 probe fluorescence assay was used to measure ATX activity. Evans Blue Permeability in vivo was measured with infrared imager. Mitochondrial bioenergetics was measured with an XF24 analyzer. Permeability was studied using Electric Cell Substrate Impedance System (ECIS). Results: ATX enzymatic activity is prominently raised in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated (P&lt;0.01) in I/R mice. LPA was increased in brain tissue along the vasculature, indicating an unfavorable role of ATX in maintaining endothelial integrity. LPA receptors were regulated differentially following stroke. Permeability measured in MBMEC showed that LPA treatment increased the permeability in a time and concentration-dependent manner. LPA treatment also increased superoxide in MBMEC suggesting redox imbalance with LPA exposure. Mitochondrial superoxide elevated following I/R was limited with ATX inhibitors which also restricted the permeability compared to that of the I/R mice Conclusion: LPA produced by ATX plays a significant role in ischemic stroke, increasing BBB permeability and elevating mitochondrial dysfunction. Targeting the ATX-LPA axis could be a better therapeutic avenue for stroke management.

Disrupted Blood-Brain Barrier and Mitochondrial Impairment by Autotaxin-Lysophosphatidic Acid Axis in Postischemic Stroke.

BackgroundThe loss of endothelial integrity increases the risk of intracerebral hemorrhage during ischemic stroke. Adjunct therapeutic targets for reperfusion in ischemic stroke are in need to prevent blood‐brain barrier disruption. Recently, we have shown that endothelial permeability is mediated by lysophosphatidic acid (LPA), but the role of autotaxin, which produces LPA, remains unclear in stroke. We investigate whether autotaxin/LPA axis regulates blood‐brain barrier integrity after cerebral ischemia.Methods and ResultsIschemic stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by 24‐hour reperfusion. The therapeutic efficacy of autotaxin/LPA receptor blockade was evaluated using triphenyl tetrazolium chloride staining, Evans blue permeability, infrared imaging, mass spectrometry, and XF24 analyzer to evaluate blood‐brain barrier integrity, autotaxin activity, and mitochondrial bioenergetics. In our mouse model of ischemic stroke, the mRNA levels of autotaxin were elevated 1.7‐fold following the cerebral ischemia and reperfusion (I/R) group compared with the sham. The enzymatic activity of autotaxin was augmented by 4‐fold in the I/R group compared with the sham. Plasma and brain tissues in I/R group showed elevated LPA levels. The I/R group also demonstrated mitochondrial dysfunction, as evidenced by decreased (P<0.01) basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity. Treatment with autotaxin inhibitors (HA130 or PF8380) or autotaxin/LPA receptor inhibitor (BrP‐LPA) rescued endothelial permeability and mitochondrial dysfunction in I/R group.ConclusionsAutotaxin‐LPA signaling blockade attenuates blood‐brain barrier disruption and mitochondrial function following I/R, suggesting targeting this axis could be a new therapeutic approach toward treating ischemic stroke.

Osteoclast-Derived Autotaxin, a Distinguishing Factor for Inflammatory Bone Loss.

The severity of rheumatoid arthritis (RA) correlates directly with bone erosions arising from osteoclast (OC) hyperactivity. Despite the fact that inflammation may be controlled in patients with RA, those in a state of sustained clinical remission or low disease activity may continue to accrue erosions, which supports the need for treatments that would be suitable for long-lasting inhibition of OC activity without altering the physiologic function of OCs in bone remodeling. Autotaxin (ATX) contributes to inflammation, but its role in bone erosion is unknown. ATX was targeted by inhibitory treatment with pharmacologic drugs and also by conditional inactivation of the ATX gene Ennp2 in murine OCs (ΔATXC tsk ). Arthritic and erosive diseases were studied in human tumor necrosis factor-transgenic (hTNF+/- ) mice and mice with K/BxN serum transfer-induced arthritis. Systemic bone loss was also analyzed in mice with lipopolysaccharide (LPS)-induced inflammation and estrogen deprivation. Joint inflammation and bone erosion were assessed by histology and micro-computed tomography. The role of ATX in RA was also examined in OC differentiation and activity assays. OCs present at sites of inflammation overexpressed ATX. Pharmacologic inhibition of ATX in hTNF+/- mice, as compared to vehicle-treated controls, significantly mitigated focal bone erosion (36% decrease; P < 0.05) and systemic bone loss (43% decrease; P < 0.05), without affecting synovial inflammation. OC-derived ATX was revealed to be instrumental in OC bone resorptive activity and was up-regulated by the inflammation elicited in the presence of TNF or LPS. Specific loss of ATX in OCs from mice subjected to ovariectomy significantly protected against the systemic bone loss and erosion that had been induced with LPS and K/BxN serum treatments (30% reversal of systemic bone loss [P < 0.01]; 55% reversal of erosion [P < 0.001]), without conferring bone-protective properties. Our results identify ATX as a novel OC factor that specifically controls inflammation-induced bone erosions and systemic bone loss. Therefore, ATX inhibition offers a novel therapeutic approach for potentially preventing bone erosion in patients with RA.

Serum Autotaxin in Non-Alcoholic Fatty Liver Disease in Chinese Population: A Case-Control Study

Background: Non-alcoholic fatty liver disease (NAFLD) has been emerging as popular chronic liver disease with no curative agent and represent serum biomarkers. Methods: 76 NAFLD patients and 76 health controls visited the First Affiliated Hospital of Zhejiang University in 2016 were included in this study. Demographic information, medical history, clinical evaluations, blood routine, serum biochemical analysis and serum Autotaxin level were obtained. Findings: Serum Autotaxin level in NAFLD patients was significantly lower than that in controls (174.3±59.1 vs. 197.7±58.9 ng/mL, P < 0.05). Prevalence NAFLD significantly decreased (62.9%, 47.1%, 39.6%, Ptrend < 0.05), as Autotaxin level increased. ROC curve of Autotaxin was developed to predict the presence of NAFLD. Area under ROC was 0.606. Logistic analysis demonstrated an independent role of Autotaxin in predicting NAFLD. Interpretation: This case control study revealed a negative relationship between serum Autotaxin level and NAFLD, independent of age, sex, body mass index and other metabolic factors. As serum Autotaxin level increased, NAFLD prevalence decreased, indicating a potential biomarker as well as therapeutic role of Autotaxin in NAFLD. Funding Statement: This study was supported by funding from Science Foundation of Health Bureau of Zhejiang Province (No. 2017183691 to Yi Chen) and National Key Basic Research Development Program (No. 81700504 to Yi Chen). The funders did not play any role in the study design, data collection and analysis, decisions regarding data release or manuscript preparation. Declaration of Interests: None of the authors has any conflict of interest to claim. Ethics Approval Statement: Study protocol was approved by Hospital Ethics Committee and performed in accordance with Declaration of Helsinki. All subjects gave informed consent before enrollment.

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.

Selective Inhibition of Autotaxin Is Efficacious in Mouse Models of Liver Fibrosis.

Autotaxin (ATX) is a secreted glycoprotein that converts lysophosphatidylcholine (LPC) to the bioactive phospholipid lysophosphatidic acid (LPA) and is the major enzyme generating circulating LPA. Inhibition of LPA signaling has profound antifibrotic effects in multiple organ systems, including lung, kidney, skin, and peritoneum. However, other LPA-generating pathways exist, and the role of ATX in localized tissue LPA production and fibrosis remains unclear and controversial. In this study, we describe the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel small-molecule ATX inhibitor, PAT-505 [3-((6-chloro-2-cyclopropyl-1-(1-ethyl-1H-pyrazol-4-yl)-7-fluoro-1H-indol-3-yl) thio)-2-fluorobenzoic acid sodium salt]. PAT-505 is a potent, selective, noncompetitive inhibitor that displays significant inhibition of ATX activity in plasma and liver tissue after oral administration. When dosed therapeutically in a Stelic Mouse Animal Model of nonalcoholic steatohepatitis (NASH), PAT-505 treatment resulted in a small but significant improvement in fibrosis with only minor improvements in hepatocellular ballooning and hepatic inflammation. In a choline-deficient, high-fat diet model of NASH, therapeutic treatment with PAT-505 robustly reduced liver fibrosis with no significant effect on steatosis, hepatocellular ballooning, or inflammation. These data demonstrate that inhibiting autotaxin is antifibrotic and may represent a novel therapeutic approach for the treatment of multiple fibrotic liver diseases, including NASH.

Inhibition of autotaxin delays breast tumor growth and lung metastasis in mice

Autotaxin is a secreted enzyme that produces most extracellular lysophosphatidate, which stimulates 6 G-protein-coupled receptors. Lysophosphatidate promotes cancer cell survival, growth, migration, invasion, metastasis, and resistance to chemotherapy and radiotherapy. The present work investigated whether inhibiting autotaxin could decrease breast tumor growth and metastasis. We used a new autotaxin inhibitor (ONO-8430506; IC90=100 nM), which decreased plasma autotaxin activity by >60% and concentrations of unsaturated lysophosphatidates by >75% for 24 h compared with vehicle-treated mice. The effects of ONO-8430506 on tumor growth were determined in a syngeneic orthotopic mouse model of breast cancer following injection of 20,000 BALB/c mouse 4T1 or 4T1-12B cancer cells. We show for the first time that inhibiting autotaxin decreases initial tumor growth and subsequent lung metastatic nodules both by 60% compared with vehicle-treated mice. Significantly, 4T1 cells express negligible autotaxin compared with the mammary fat pad. Autotaxin activity in the fat pad of nontreated mice was increased 2-fold by tumor growth. Our results emphasize the importance of tumor interaction with its environment and the role of autotaxin in promoting breast cancer growth and metastasis. We also established that autotaxin inhibition could provide a novel therapeutic approach to blocking the adverse effects of lysophosphatidate in cancer.

Significance of Autotaxin Activity and Overexpression in Comparison to Soluble Intercellular Adhesion Molecule in Thyroid Cancer

The objective of this study was to evaluate the role of autotaxin (ATX) activity and gene expression compared to soluble intercellular adhesion molecule-1 (sICAM-1) in thyroid carcinoma. Sixty-five patients with thyroid swelling were included. There were 20 cases of simple multinodular goiter (group I), 15 cases of follicular adenoma (group II) and 30 cases of thyroid cancer (group III). Group III was further subdivided into negative and positive lymph nodes (group IIIa and IIIb; 22 and 8 cases, respectively). sICAM-1 concentration and ATX activity were measured using colorimetric enzyme-linked immunosorbent assay (ELISA), while ATX gene expression was detected by real-time polymerase chain reaction (PCR). sICAM-1 level, ATX activity and gene expression were significantly elevated in patients with thyroid carcinoma compared to other groups. The ATX activity showed significantly higher sensitivity and specificity than sICAM-1 (100% and 97.1% vs 93.3% and 88.6%, respectively). Both sICAM-1 and ATX values were significantly higher in patients with positive lymph nodes compared to those without lymph node involvement (p<0.001). Higher levels of ATX activity and gene expression were significantly correlated with larger tumor size and undifferentiated pathological subtype in thyroid carcinoma. In this respect, ATX was superior to sICAM-1. Our data suggest that ATX activity and gene expression are reliable diagnostic and prognostic tools in thyroid carcinoma compared to sICAM-1.

Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.

Role of autotaxin and lysophosphatidate in cancer progression and resistance to chemotherapy and radiotherapy

There is an accumulating body of evidence linking the secreted enzyme autotaxin (ATX) and its product lysophosphatidate (LPA) to tumor progression, metastasis and resistance to chemotherapy or radiotherapy. ATX achieves this mainly by converting the abundant lysophosphatidylcholine in the circulation to the potent bioactive signaling molecule, LPA. ATX is also bound to integrins on cell surfaces, which enables it to deliver LPA locally to at least eight G-protein-coupled receptors. These receptors activate a variety of signaling cascades, which stimulate cell division, survival and migration. Cancer cells also often show decreased expression of LPP-1 and -3, which both dephosphorylate extracellular LPA and also block its signaling downstream of receptor activation. This contributes to the hypersensitivity of cancer cells to the effects of LPA signaling, which coupled with increased ATX expression, promotes their metastasis and survival.

Autotaxin controls caudal diencephalon‐mesencephalon development in the chick

The diencephalon is the embryonic anlagen of the higher integration centers of the brain. Recent studies have elucidated how the cells in the rostral diencephalon acquire their regional identities. However, the understanding of the mechanisms under which the caudal diencephalon is formed is still limited. Here we focus on the role of Autotaxin (ATX), a lysophospholipid-generating exoenzyme, whose mRNA is detected in the caudal diencephalon. RNA interference against ATX altered the expression pattern of Pax6-regualted genes, Tcf4, Lim1, and En1, implying that ATX is required for the maintenance of the regional identity of the caudal diencephalon and the diencephalon-mesencephalon boundary (DMB). Furthermore, ATX-RNAi inhibited neuroepithelial cell proliferation on both sides of the DMB. We propose a dual role of ATX in chick brain development, in which ATX not only contributes to the formation of caudal diencephalon as a short-range signal, but also regulates the growth of mesencephalon as a long-range signal.

Abstract 1304: Induction of pericyte migration by autotaxin in angiogenesis

Abstract Angiogenesis is essential for tumor cell growth and progression. Recent studies have shown that autotaxin (ATX) is a critical angiogenic factor since ATX knock-out mice exhibit severe vascular defects. ATX also has the ability to stimulate tumor cell motility by enzymatically generating lysophosphatidic acid (LPA). To form a new and stable blood vessel, interactions between pericytes and endothelial cells play important roles. A previous study has shown that endothelial cells can be induced to generate ATX; however little is known about how ATX and LPA are involved in these interactions. The focus of our study is to define the role of ATX in angiogenesis by examining pericyte and endothelial cell response to ATX. To test the effects on cell migration, we treated pericytes and endothelial cells with ATX and other angiogenic factors (including Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF)). Pericytes responded strongly to ATX and LPA stimulation, but human umbilical vein endothelial cells (HUVEC) and skin microvescular cells (HDMEC) showed little or no effect. PDGF elicits a qualitatively similar response. In contrast, endothelial cells migrated when treated with VEGF, but pericytes showed no such stimulation. Pericytes treated with ATX and PDGF together show an additive response. Moreover, in a checkerboard assay the migratory response to ATX or LPA is chemokinetic, whereas PDGF is chemotactic. We then utilized RT-PCR to examine the mRNA levels of four major LPA receptors in pericytes when treated with angiogenic factors. Treatment with PDGF increases LPAR1 mRNA levels and decreases LPAR4 levels. Treatment with ATX also increases LPAR1, but shows no change in LPAR4 levels. By contrast, VEGF treatment has no effect on the LPAR1 mRNA level, but increases the LPAR4 mRNA level. The amount of migration appears to correlate with an increase of LPAR1 and perhaps a decrease of LPAR4. As such, certain angiogenic factors might affect cell motility, in part, through the differential expression of various LPA receptors. These studies suggest that the ATX/LPA signaling pathway effects the formation of mature blood vessels by promoting the pericyte coverage of immature blood vessels. The role of the ATX/LPA pathway in the formation of mature blood vessels provides the scientific rationale for targeting the pathway in the development of novel anti-angiogenic compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1304.

Mammary Tumorigenesis through LPA Receptor Signaling

Lysophosphatidic acid (LPA) is a lipid growth factor that is produced by an extracellular phospholipase, termed autotaxin (ATX), and acts via G protein-coupled receptors. In this issue of Cancer Cell, Liu et al. show that transgenic overexpression of ATX or LPA receptors leads to invasive and metastatic mammary cancer.

Different Roles of Two Autotaxin Isoforms in Proliferation, Migration and Adhesion in the Non-Mutational Tyrosine Kinase Inhibitor Resistant Acute Lymphoblastic Leukemia Cell Line SupB15.

The Bcr-Abl oncogene is present in 30–40% of adult patients with acute lymphoblastic leukemia (ALL). The Abl kinase inhibitor imatinib-based therapy has become standard for this subset ALL. Acquired resistance to imatinib occurs frequently and is associated with mutations in the tyrosine kinase domain (TKD) approximately in about 80% of patients. In contrast, TKD mutations are uncommon in primary imatinib resistance which appears to be multifactorial, although the underlying mechanisms have been incompletely elucidated.We have established a Ph+ cell line for the analysis of non-mutational resistance mechanisms of imatinib resistance: SupB15RT, a Bcr-Abl expressing lymphoblastic cell line derived from SupB15WT cell line by gradually increasing the exposure to imatinib. SupB15RT shows cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. We have shown that several commonly implicated mechanisms of imatinib resistance do not play a role in conferring the imatinib resistance in SupB15RT cells. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affymetrix- Microarrays, we identified 29 differentially regulated genes. Autotaxin (ATX) is one of the most highly up-regulated genes in imatinib resistant SupB15RT cells, and suggested a contribution to imatinib resistance.ATX is an exo-enzyme (pyrophosphophatase/phosphodiesterase). It plays a role in tumor progression and migration as a tumor cell autocrine motilty factor in various solid tumor cell types. ATX is involved in the synthesis of the signaling molecule, lysophosphatidic acid (LPA) which promotes survival and motility. It was the aim of this study to determine whereas ATX plays a functional role for imatinib resistance in Ph+ ALL.Using RT-PCR we demonstrated that 2 isoforms of ATX are expressed in SupB15RT cells: ATXshort and ATXlong. ATXlong (863 aa) contains highly basic insertion in the catalytic domain (52 residues). We retroviraly transfected BaF3 cells with p185 and/or ATXshort or ATXlong to analyze its influence on growth, adhesion and migration in mouse cell model. In comparison to wild type BaF3 cells the proliferation of BaF3 cells expressing ATXshort is enhanced (1,5-fold), whereas ATXlong expressing BaF3 cells showed no difference in proliferation in comparison to Mock infected cells. The proliferation of p185 expressing BaF3 cells co-expressing ATXshort or ATXlong is not inhibited by the treatment with 1μM imatinib after 3 days in contrast to p185 expressing BaF3 cells. In adhesion experiments, BaF3 cells expressing ATXshort showed a higher attachment independent of p185 expression. We also performed migration experiments using transwell assays. These assays showed more migration with cells co-expressing p185 and ATXlong compared to p185 alone. This is in agreement with our results for SupB15RT vs. SupB15WT with a 3-fold migration increase of SupB15RT. Application of 10% fetal calf serum (FCS) in migration experiments resulted in a 1,5-fold higher migration of the ATXlong expressing BaF3 cells compared to culture without FCS. One explanation for this finding may be that FCS contains lysophosphatidic choline (LPC) which is converted to LPA by ATX.Although expression of both 2 isoforms of ATX is important for the increased proliferation, it seems that the 2 isoforms have different cellular functions in Ph+ lymphoblastic cells. ATXshort seems to enhance adhesion whereas ATXlong plays an important role in motility. Taken together our results indicate a role for ATX in TK- inhibitor resistant SupB15RT cells through LPA signaling via LPA receptors. The ratio between ATXshort and ATXlong probably is important for the intracellular signaling and has to be explored.

A Role of Autotaxin in Non-Mutational Resistance in Imatinib-Resistant Acute Lymphoblastic Leukemia (ALL) Cell Line SupB15RT.

The Bcr-Abl oncogene is present in 30-40% of adult patients with acute lymphoblastic leukemia (ALL). Therapy with imatinib has become standard for Ph+ ALL but resistance to the tyrosine kinase inhibitor occurs for the majority of patients. In about 80% of patients with acquired resistance mutations in the tyrosine kinase domain (TKD) have been found. In contrast, primary resistance to imatinib appears to be multifactorial and precise mechanisms have been incompletely elucidated. We have established an imatinib-resistant cell line (SupB15RT) which was derived from the previously well characterized SupB15 cell line (SupB15WT) by gradually increasing the exposure to imatinib. We found that several commonly implicated mechanisms of imatinib resistance, i.e. Bcr-Abl gene amplification, point mutations in the TKD, Bcr-Abl overexpression, up-regulation of multidrug resistance gene proteins or ineffective inhibition of Bcr-Abl tyrosine phosphorylation do not play a role in conferring the imatinib-resistant phenotype in SupB15RT cells. Thus, the SupB15RT cells represent a suitable model for the analysis of resistance mechanisms in Ph+ ALL with primary imatinib resistance. Interestingly, SupB15RT cells show cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. Analysis of signal transduction pathways downstream of Bcr-Abl revealed that imatinib exposure was not associated with down-regulation of pSTAT-5 and pErk in the imatinib-resistant SupB15RT cells, in contrast to SupB15WT. Phosphorylation of Akt was inhibited by 0.5μM imatinib in SupB15WT cells, whereas imatinib in concentrations up to 5μM failed to suppress Akt phosphorylation in SupB15RT cells, indicating constitutive activation of Akt kinase during imatinib treatment. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affimetrix-Microarrays, we identified 29 differentially regulated (at least 3-fold) genes. One of the most highly up-regulated genes in imatinib-resistant SupB15RT cells was Autotaxin (ATX), a nucleotide pyrophosphatase/ phosphodiesterase 2. This exo-enzyme was originally identified as a tumor cell autocrine motility factor, which is involved in tumor progression and migration in various tumor cell types. ATX is a lysophospholipase D which is involved in the synthesis of lysophosphatidic acid (LPA), a signaling molecule that promotes survival, growth, differentiation, and motility. We investigated if LPA imparted imatinib resistance in SupB15WT cells by modulation of growth, survival and migration. When SupB15WT cells were treated with LPA, alone or in combination with imatinib, SupB15WT cell proliferation was increased both in the absence as well as in the presence of imatinib. The dose-dependent increase of proliferation after LPA treatment was 1.9–2.6-fold (1–10μM LPA) in the presence of 1μM imatinib. In addition we performed migration experiments using Transwell assays. We detected a 3-fold increase in migration of SupB15RT vs. SupB15WT cells. We found no influence on apoptosis in imatinib treated SupB15WT cells treated with LPA compared with cells not treated with LPA. Taken together, our results indicate a role of ATX in imatinib-resistant SupB15RT cells, preferentially by stimulating proliferation and migration through LPA signaling via LPA receptors and activation of PI3K and Akt.