Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffinembedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma. [Mol Cancer Ther 2008;7(10):3352 -62]

Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin, and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa like Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor 11 (TLR11) generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long β-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the IL-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the β-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and β-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite C. parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from P. falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin, and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the β-hairpin, and diverges widely from the actin-binding surfaces of vertebrate profilins.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k 1 >500 M ؊1 s ؊1) and is hydrolyzed slowly (k 2 ‫؍‬ 0.024 s ؊1). Release of the first hydrolysis product is random and rapid (>1 s Autotaxin (ATX),4 also known as nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), was identified as a secreted autocrine motility-stimulating factor in melanoma cell cultures (1) and has subsequently been shown to play critical roles in angiogenesis, apoptosis, cancer metastasis, development, neuropathic pain, and wound healing (reviewed in Refs. 2-5). ATX displays both nucleotide phosphodiesterase activity (6) and a robust lysophospholipase D activity (lyso-PLD (7)). ATX phosphodiesterase activity is weak and is not considered relevant for in vivo function (7-9). Rather, the physiological activities of ATX have been attributed to synthesis of lysophosphatidic acid (LPA), a growth factor/chemokine that binds several endothelial differential gene family receptors (LPA1-5) (reviewed in Ref. 4), and initiates a variety of signaling cascades (4, 5) from lysophosphatidylcholine. ATX is the primary source of plasma LPA synthesis (10, 11).The plasma LPC concentration (50 -200 M) is comparable with the K M value for steady-state LPC hydrolysis by ATX (7,(12)(13)(14)(15). ATX binds LPA product more strongly than LPC substrate (12, 16), which has led to the hypothesis that product feedback inhibition regulates ATX activity and LPA production in vivo (16). However, rapid degeneration of serum LPA by lipid phosphate phosphohydrolase 1 (LPP1) (17, 18) would diminish LPA product inhibition of ATX.Rapid LPA degradation upon release from ATX also limits the effective target area of newly synthesized LPA, such that LPA signaling is restricted to within the diffusional area of the ATX⅐lipid complex from substrate binding locations. If LPC binding, hydrolysis, and LPA product release are rapid, LPA release and downstream signaling would be local (i.e. limited to sites of LPC binding). If, however, LPC substrate binding were more rapid than LPA release and bound LPA/LPC were inaccessible to degradation by LPP1, ATX with bound LPC/LPA could diffuse, thereby spreading LPA signaling to distal sites and cells. Recent in vivo studies show that competitive inhibition of ATX accelerates LPA degradation (19), consistent with the possibility of global ATX/LPA signaling via exclusion from LPP1.In this study, we measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates and LPC...