ATX-LPA Signaling Research Articles

Autotaxin, Pruritus and Primary Biliary Cholangitis (PBC).

Autotaxin (ATX) is a 125-kD type II ectonucleotide pyrophosphatase/phosphodiesterase (ENPP2 or NPP2) originally discovered as an unknown "autocrine motility factor" in human melanoma cells. In addition to its pyrophosphatase/phosphodiesterase activities ATX has lysophospholipase D (lysoPLD) activity, catalyzing the conversion of lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). ATX is the only ENPP family member with lysoPLD activity and it produces most of the LPA in circulation. In support of this, ATX heterozygous mice have 50% of normal LPA plasma levels. The ATX-LPA signaling axis plays an important role in both normal physiology and disease pathogenesis and recently has been linked to pruritus in chronic cholestatic liver diseases, including primary biliary cholangitis (PBC). Several lines of evidence have suggested that a circulating puritogen is responsible, but the identification of the molecule has yet to be definitively identified. In contrast, plasma ATX activity is strongly associated with pruritus in PBC, suggesting a targetable molecule for treatment. We review herein the biochemistry of ATX and the rationale for its role in pruritus.

Abstract 5170: The autotaxin-lysophosphatidic acid signaling pathways in ovarian carcinoma

Abstract Ovarian carcinoma ranks as the fifth most common cancer among women. This is attributed to late presentation of the patients. The disease is characterized by widespread intraperitoneal metastases and accumulation of effusion fluids mainly in the peritoneal cavity Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression and resistance that act via specific cell-surface receptors present in many transformed cell types. LPA is produced by two main pathways, by PLA-2 and by Autotaxin (LysoPLD). Once its produced, it acts on six specific G protein coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. Previous studies have shown high levels of LPA in the effusions of ovarian carcinoma patients. The aim of our study was to examine the role of ATX-LPA signaling in the progression of serous ovarian carcinoma. A total of 230 samples of serous ovarian carcinoma from the three different anatomic sites of the disease, were used .Effusion fluids were frozen and stored separately. All specimens were submitted for routine diagnostic purpose to the Division of Pathology at the Norwegian Radium Hospital during the period 1998-2008. mRNA levels of the six different LPA receptors were measured by RT-PCR, protein levels of Autotaxin were measured by Western blotting. Results show that LPA2R is higher in effusion-derived tumor cells compared to solid lesions (p<0.001), while the opposite was found for LPA3R (p<0.001) and LPA6R (p<0.001). Clinico-pathological analysis showed that LPA3R levels are significantly higher in pre-chemo patients (p = 0.038). LPA2R (p = 0.021) and LPA5R (p = 0.035) were found higher in cases with larger residual disease volume. LPA5R (p = 0.032) was higher in cases with primary chemo-resistance and LPA1R (p = 0.05) was marginally associated with better overall survival. Autotaxin protein levels were lower in effusion-derived tumor cells compared to solid lesions (p<0.001), yet higher levels of Autotaxin were found in peritoneal lesions compared to pleural ones (p = 0.033). In this study we showed significant changes in mRNA LPAR levels and Autotaxin protein levels with the progression of the disease that correlates to clinic-pathological parameters. To our best knowledge, this is the first description of the ATX-LPA signaling pathway in ovarian carcinoma with clinical relevance. Citation Format: Hadil Onalla, Claes Tropé, Reuven Reich, Ben Davidson. The autotaxin-lysophosphatidic acid signaling pathways in ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2015-5170

Autotaxin: structure-function and signaling

Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase-2, is a secreted lysophospholipase D (lysoPLD) that hydrolyzes extracellular lysophospholipids into the lipid mediator lysophosphatidic acid (LPA), a ligand for specific G protein-coupled receptors. ATX-LPA signaling is essential for development and has been implicated in a great diversity of (patho)physiological processes, ranging from lymphocyte homing to tumor progression. Structural and functional studies have revealed what makes ATX a unique lysoPLD, and how secreted ATX binds to its target cells. The ATX catalytic domain shows a characteristic bimetallic active site followed by a shallow binding groove that can accommodate nucleotides as well as the glycerol moiety of lysophospholipids, and by a deep lipid-binding pocket. In addition, the catalytic domain has an open tunnel of unknown function adjacent to the active site. Here, we discuss our current understanding of ATX structure-function relationships and signaling mechanisms, and how ATX isoforms use distinct mechanisms to target LPA production to the plasma membrane, notably binding to integrins and heparan sulfate proteoglycans. We also briefly discuss the development of drug-like inhibitors of ATX.

Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis.

Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX-LPA signaling was mediated by PI3K/Akt-Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient-derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation.

Autotaxin–Lysophosphatidic Acid Signaling Axis Mediates Tumorigenesis and Development of Acquired Resistance to Sunitinib in Renal Cell Carcinoma

Sunitinib is currently considered as the standard treatment for advanced renal cell carcinoma (RCC). We aimed to better understand the mechanisms of sunitinib action in kidney cancer treatment and in the development of acquired resistance. Gene expression profiles of RCC tumor endothelium in sunitinib-treated and -untreated patients were analyzed and verified by quantitative PCR and immunohistochemistry. The functional role of the target gene identified was investigated in RCC cell lines and primary cultures in vitro and in preclinical animal models in vivo. Altered expression of autotaxin, an extracellular lysophospholipase D, was detected in sunitinib-treated tumor vasculature of human RCC and in the tumor endothelial cells of RCC xenograft models when adapting to sunitinib. ATX and its catalytic product, lysophosphatidic acid (LPA), regulated the signaling pathways and cell motility of RCC in vitro. However, no marked in vitro effect of ATX-LPA signaling on endothelial cells was observed. Functional blockage of LPA receptor 1 (LPA1) using an LPA1 antagonist, Ki16425, or gene silencing of LPA1 in RCC cells attenuated LPA-mediated intracellular signaling and invasion responses in vitro. Ki16425 treatment also dampened RCC tumorigenesis in vivo. In addition, coadministration of Ki16425 with sunitinib prolonged the sensitivity of RCC to sunitinib in xenograft models, suggesting that ATX-LPA signaling in part mediates the acquired resistance against sunitinib in RCC. Our results reveal that endothelial ATX acts through LPA signaling to promote renal tumorigenesis and is functionally involved in the acquired resistance of RCC to sunitinib.

Abstract 3884: Targeting ATX and LPA receptors melanoma invasion and metastasis.

Abstract ATX, a lysophospholipase D that generates LPA, is highly up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancers secrete ATX, and it contributes to their invasive properties. LPA produced by ATX activates LPA1-8 G protein-coupled receptors on the cell surface. Many cancers over-express multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. Therefore, ATX-LPA signaling axis is an emerging target for cancer therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and metastasis in vivo. In this study, we sought to understand the roles of the LPA GPCRs both in the tumor cells and tissues targeted by metastatic cancer cells. Using the in vivo B16 melanoma metastasis model syngeneic to the C57BL/6 mouse strain, we found that the incidence of lung metastasis was greatly reduced in the LPA5KO mice compared to WT, LPA1KO, and LPA2KO mice, respectively. In addition, we found that the selective inhibitor of LPA1&3, Ki16425 also reduced lung metastasis, suggesting that inhibition of LPA5 and LPA1 receptors in the target tissues may prevent cancer metastasis. B16 melanoma cells predominantly express LPA5 receptor. These cells do not invade across a matrigel layer in response to LPA. We found that siRNA knockdown of LPA5 receptors in B16 melanoma cells relieves this inhibition and resulted in an increase in invasion. These results suggest that the LPAR, in particular LPA5 receptors in tumor and stromal cells may play different roles in invasion and metastasis. Citation Format: Gabor Tigyi, Yuko Fujiwara, SueChin Lee, Jianxiong Liu, Renukadevi Patil, Renuka Gupte, Duane D. Miller, Tamas Oravecz, Louisa Balazs. Targeting ATX and LPA receptors melanoma invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3884. doi:10.1158/1538-7445.AM2013-3884

Abstract 5680: Identification and characterization of a novel class of ATX inhibitors.

Abstract The lysophospholipase D (autotaxin, ATX) plays an important role in cancer invasion and metastasis. A High-throughput screen of over 10,000 compounds from the University of Cincinnati Drug Discovery Center, identified several small-molecule inhibitors with IC50 values ranging from nanomolar to low micromolar. The pharmacology of the two most potent compounds, 966791(1) and 918103(2), were further characterized in enzyme, cellular, and whole animal systems. Both of these compounds, significantly reduced cell invasion in melanoma cancer cell lines without any apparent cytotoxicity. Compound (1) was shown to be a competitive inhibitor of ATX for both FS-3 and Pnp-TMP substrates, while compound (2) was competitive only for FS-3. This suggest that compound (2) targets the hydrophobic pocket not the active site of ATX, which was supported by docking of compounds 1 and 2, along with mutagenesis data. The potency of the compounds was comparable to each other in each of the assays. Current ATX inhibitors reported in literature target the active site in the catalytic domain. In our search for ATX inhibitors we identified compounds able to inhibit both the active site and hydrophobic pocket. We conclude that these data suggest compounds solely inhibiting the hydrophobic pocket are sufficient to disrupt the ATX-LPA signaling axis can serve as specific small-molecule modulators for mechanistic studies of ATX as well as a potential leads for the development of anti-cancer drugs. Citation Format: James Fells, Sue Chin Lee, Derek Norman, Yuko Fujiwara, Ryoko Tsukahara, Jianxiong Liu, Jason Kirby, Sandra Nelson, Gabor Tigyi. Identification and characterization of a novel class of ATX inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5680. doi:10.1158/1538-7445.AM2013-5680

Structure–function relationships of autotaxin, a secreted lysophospholipase D

Autotaxin (ATX or ENPP2) is an ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) that functions as a secreted lysophospholipase D to produce the multifunctional lipid mediator lysophosphatidic acid (LPA) from more complex lysophospholipids. LPA acts on distinct G protein-coupled receptors thereby activating multiple signaling cascades and cellular responses. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes, ranging from vascular and neural development to lymphocyte homing, fibrosis and cancer. Despite much progress in understanding LPA receptor signaling, the precise mode of action of ATX has long remained elusive due to the lack of structural data. In particular, it has been unclear what makes ATX a unique lysophospholipase D and how the enzyme is targeted to LPA-responsive cells. Recent structural studies have begun to clarify these issues. Here we discuss new insights and inferences from the ATX structure.

Autotaxin and LPA receptor signaling in cancer

Lysophosphatidic acid (LPA; monoacyl-glycerol-3-phosphate) is a lipid mediator that functions as a mitogen and motility factor for many cell types. LPA signals through six specific G protein-coupled receptors, named LPA(1-6), which trigger both overlapping and distinct signaling pathways. LPA is produced from extracellular lysophosphatidylcholine by a secreted lysophospholipase D, named autotaxin (ATX), originally identified as an "autocrine motility factor" for tumor cells. ATX-LPA signaling is vital for embryonic development and promotes tumor formation, angiogenesis, and experimental metastasis in mice. Elevated expression of ATX and/or aberrant expression of LPA receptors are found in several human malignancies, while loss of LPA(6) function has been implicated in bladder cancer. In this review, we summarize our present understanding of ATX and LPA receptor signaling in cancer.

Structure-Based Design of Novel Boronic Acid-Based Inhibitors of Autotaxin

Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug target. We recently described a boronic acid-based inhibitor of ATX, named HA155 (1). Here, we report the design of new inhibitors based on the crystal structure of ATX in complex with inhibitor 1. Furthermore, we describe the syntheses and activities of these new inhibitors, whose potencies can be explained by structural data. To understand the difference in activity between two different isomers with nanomolar potencies, we performed molecular docking experiments. Intriguingly, molecular docking suggested a remarkable binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for inhibitor design.

Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

BackgroundAutotaxin (ATX) possesses lysophospholipase D (lyso PLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation and tumor progression. Osteopontin (OPN) is an important chemokine involved in the survival, proliferation, migration, invasion and metastasis of gastric cancer cells. The focus of the present study was to investigate the relationship between the ATX-LPA axis and OPN.ResultsIn comparison with non-treated cells, we found that the ATX-LPA axis up-regulated OPN expression by 1.92-fold in protein levels and 1.3-fold in mRNA levels. The ATX-LPA axis activates LPA2, Akt, ERK and ELK-1 and also protects SGC7901 cells from apoptosis induced by Taxol treatment.ConclusionsThis study provides the first evidence that expression of OPN induced by ATX-LPA axis is mediated by the activation of Akt and MAPK/ERK pathways through the LPA2 receptor. In addition, OPN is required for the protective effects of ATX-LPA against Taxol-induced apoptosis and ATX-LPA-induced migration of SGC7901 cells.

Structural basis of substrate discrimination and integrin binding by autotaxin

Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We identify a hydrophobic lipid-binding pocket and map key residues required for catalysis and selection between nucleotide and phospholipid substrates. We show that ATX interacts with cell-surface integrins via its N-terminal somatomedin-B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling, and enable new approaches to target ATX with small-molecule therapeutics.

Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC(50) approximately 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.

Autotaxin expression and its connection with the TNF-alpha-NF-κB axis in human hepatocellular carcinoma

BackgroundAutotaxin (ATX) is an extracellular lysophospholipase D that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been shown to be involved in many cancers. However, the functional role of ATX and the regulation of ATX expression in human hepatocellular carcinoma (HCC) remain elusive.ResultsIn this study, ATX expression was evaluated in tissues from 38 human HCC and 10 normal control subjects. ATX was detected mainly in tumor cells within tissue sections and its over-expression in HCC was specifically correlated with inflammation and liver cirrhosis. In addition, ATX expression was examined in normal human hepatocytes and liver cancer cell lines. Hepatoma Hep3B and Huh7 cells displayed stronger ATX expression than hepatoblastoma HepG2 cells and normal hepatocytes did. Proinflammtory cytokine tumor necrosis factor alpha (TNF-α) promoted ATX expression and secretion selectively in Hep3B and Huh7 cells, which led to a corresponding increase in lysophospholipase-D activity. Moreover, we explored the mechanism governing the expression of ATX in hepatoma cells and established a critical role of nuclear factor-kappa B (NF-κB) in basal and TNF-α induced ATX expression. Further study showed that secreted enzymatically active ATX stimulated Hep3B cell invasion.ConclusionsThis report highlights for the first time the clinical and biological evidence for the involvement of ATX in human HCC. Our observation that links the TNF-α/NF-κB axis and the ATX-LPA signaling pathway suggests that ATX is likely playing an important role in inflammation related liver tumorigenesis.

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.