Autotaxin: Its Role in Biology of Melanoma Cells and as a Pharmacological Target

Autotaxin (ATX) is an extracellular lysophospholipase D (lysoPLD) released from normal cells and cancer cells. Activity of ATX is detected in various biological fluids. The lysophosphatidic acid (LPA) is the main product of ATX. LPA acting through specific G protein-coupled receptors (LPA1-LPA6) affects immunological response, normal development, and malignant tumors' formation and progression. In this review, the impact of autotoxin on biology of melanoma cells and potential treatment is discussed.

The structure of autotoxin is presented in Figure 1. At the N-terminus, ATX possesses hydrophobic signal sequence (SS) followed by two somatomedin B-like domains (SBLD) with RGD tripeptide motif suggesting that this domain may play a role in cell-extracellular matrix interactions. At catalytic domain (CD), Thr 210 and N542-linked glycan are suggested to be responsible for hydrolytic activity of ATX, but motif G/FXGXXG is responsible for metal binding. ATX activity is stimulated by divalent cations for example, Ca 2+ , Mg 2+ , and Co 2+ . C-terminally to CD is the nucleaselike domain (NLD). It contains EF-hand-like motif and is structurally similar to DNA and RNA-nonspecific endonucleases; however, it lacks the catalytic activity. In humans, NLD is covalently linked with catalytic domain via disulfide  bridge (C413-C805). The ATX gene is located on chromosome 8 at position 8q24.1 and ATX has three alternative splicing isoforms in humans: ATX teratocarcinoma-derived ATX-t (925 a.a); melanoma-derived ATX-m (863 a.a), and brain specific, ATX-γ (888 a.a) [22][23][24][25].

Role of Autotoxin during Normal Development
ATX has a critical role in formation of vasculature by vasculogenesis and angiogenesis. ATX knockout mice (atx −/− ) are lethal around embryonic day 10.5. Admittedly, ATX is a major producing enzyme for LPA, nevertheless the phenotypes of LPA receptors knockout mice is less severe, suggesting that ATX-induced cellular signal may involve others pathways. This speculation is supported by results of experiments where modification of LPA level in blood (2fold increase) by driving ATX expression is not sufficient to induce tumorigenesis [13,[26][27][28].

Role of Autotoxin in Biology of Melanoma Cells
ATX was identified in the cultured cell supernatant of human melanoma cells (A2058) as a cell motility-stimulating factor acting at pM-nM concentrations in pertussis toxin-sensitive manner [29]. Further studies have provided evidence that LPA, product of ATX, mediates chemotaxis and proliferation of melanoma cells [30]. The recent experiments suggest that ATX expression is one of the factors involved in metastasis of melanoma cells (Figure 2). Inhibition of ATX production blocks LPA-induced migration of melanoma cells [31]. It has been detected that melanoma metastatic specimens have increased ATX level, and ATX expression in primary melanoma is higher than in melanoma in situ [32]. Moreover, reduced expression of ATX predicts survival in uveal melanoma [33]. It has been shown that ATX-stimulated motility is suppressed by an LPA 1 -selective antagonist, Ki16425, in melanoma cells [34]. Accumulating evidence suggest the various intracellular signaling pathways may be involved in ATX-induced motility of melanoma cell. It has been shown that this action is mediated through G-protein coupled isoform of phosphatidylinositol 3-kinase γ (PI3Kγ) suggesting involvement of proteins located downstream of PI3Kγ, for example, small G proteins [35]. Accordingly, there is evidence that ATX induces Cdc42/Rac1/p21-activated kinase (PAK1) complex formation [36]. The experimental data suggest that this complex is required for LPA-induced activation of focal adhesion kinase (FAK) [37]. The changes of PAK1 and FAK activity affect cytoskeleton proteins and structural integrity of melanoma cells. Moreover, ATX in melanoma cells induces the expression and activity of urokinase-type plasminogen activator (UPA) in a dosedependent manner. This action is mediated by G i proteins and PI3K/Akt signaling involving translocation of p65 into the nucleus and DNA binding of necrosis factor kappa B [38]. In general, action of LPA on melanoma cells enhances their metastaic potential (Figure 3). Recent experiments have provided evidence about the role of LPA receptors in biology of melanoma cells [32]. Downregulation of LPA 3 and using sequence-specific small interfering RNA (siRNA) reduces melanoma cells viability and proliferation. The effects of LPA receptors activation in melanoma cells are presented in

Autotoxin as a Pharmacological Target
The approved melanoma therapy lacks significant efficiency, hence, new therapeutic targets are under investigation. The current research focuses on the autotoxin-LPA axis [39]. Because LPA acts on multiple receptors with overlapping activities, currently the LPA receptors are not attractive as a pharmacological target, and the main stream of investigation concerns ATX activity.

L-Histidine.
L-histidine inhibits activities of ATX in a non-competitive manner with IC 50 ∼ 4 mM and ATXstimulated migration of human melanoma cells; 10 mM L-histidine induces 90%-95% reduction in stimulated motility. The proposed mechanism of L-histidine action is based on inhibition of a process that is required for the hydrolysis of both nucleotides and phospholipids [40].  of a decreased turnover number and decreased affinity of the active site for its substrates. Of note, LPA both inhibits ATX and activates LPA receptors (EC 50 for LPA 18 : 1 at LPA 1−4 ∼ 0.007-0.5 μM), therefore it may induce the opposite of the intended effect [6,41].

Nonlipid Small Molecule.
It has been recently shown that thiazolidinediones compounds with incorporated boric acid moiety into catalic T210 residue (HA 130) inhibit ATX-mediated LPA production with IC 50 ∼ 30 nM [46]. Intravenous injection of HA 130 decreases 3.8-fold plasma LPA level in mice at 10 min. Furthermore, HA 130 inhibits ATX-mediated melanoma cells migration without affecting LPA receptor signaling pathways.
A report has been recently published describing the pharmacokinetic and pharmacodynamic properties of PF-8380 [47]. It inhibits activity of isolated ATX or ATX activity in blood with IC 50 ∼ 3 and 100 nM, respectively. There are no data about influence on melanoma cells, hoverer, PF-8380 (30 mg/kg) taken orally decreases the plasma LPA level about 95%, suggesting its potential usage in melanoma treatment.
There are several small-molecule, nonlipid ATX inhibitors including hexachlorophene, merbromin, bithionol, and others under investigation [48,49]. Their mechanism of action differ (competitive, noncompetitive or mixed inhibition) and the most potent compounds inhibit ATX activity with IC 50 at micromolar range. Their biological action was confirmed in experiments in vivo measuring effects on melanoma cell motility and invasion. A recently developed new TX autotaxin inhibitor pipemidic acid inhibits ATX with IC 50 ∼ 900 nM [50]. The natural phenolic antioxidants, including flavonols, possess inhibitory properties against ATX; however, the effect on ATX activity is about 2-fold lower than LPA 16 : 1 (1-palmitoleoyl-sn-glycerol-3phosphate). Moreover, it has been estimated that it would be difficult to affect ATX activity in vivo by flavonoids supplementation in diet because plasma concentration of flavonoids in plasma may reach 10 μM [51].
The recently published crystallography results are used in ligand-based computational approaches for optimization of the current ATX inhibitors and development of new ones [52,53].
Taken together, the increasing incidence of melanoma and poor average survival of metastatic melanoma are the main reason for the development of the new chemical compounds used in melanoma treatment. Autotaxin, melanoma cell motility-stimulating factor, and their receptors seem to be promising targets for pharmacological treatment 4 Enzyme Research of melanoma. Much more research is needed for synthesis and pharmacological characterization of new specific ATX or LPA receptors inhibitors.