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

Clinical Lipidology ISSN: 1758-4299 (Print) 1758-4302 (nline) Journal homepage: http://www.tandfonline.com/loi/tlip20 Role of autotaxin and lysophosphatidate in cancer progression and resistance to chemotherapy and radiotherapy Raie Bekele & Sanjay David To cite this article: Raie Bekele & Sanjay David (2012) Role of autotaxin and lysophosphatidate in cancer progression and resistance to chemotherapy and radiotherapy, Clinical Lipidology, 7:3, 313-328 To link to this article: https://doi.org/10.2217/clp.12.30 Copyright 2006 Future Medicine Ltd Published online: 18 Jan 2017. Submit your article to this journal Article views: 68 View related articles Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=tlip20

Review 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. Keywords: ceramides chemoresistance lysophosphatidic acid receptors lysophospholipase D metastasis tumor progression A growing body of evidence implicates the enzyme autotaxin (ATX) and its product lysophosphatidate (LPA) in promoting tumor progression and in counteracting the action of various chemotherapies and radiotherapy. Furthermore, increased ATX expression in tumors is associated with increased tumor aggressiveness and metastasis [1 6]. Most of this relationship depends on the production of LPA. Therefore, it is important to delineate and understand the roles of ATX and LPA in the etiology of cancer and the development of resistance to therapies. Future strategies in treating cancers could be based on blocking ATX activity, activation of LPA receptors or signaling targets that are downstream of these receptors. Many excellent reviews have been written on the functions of ATX and the role of LPA in regulating cell signaling in relation to normal physiology and cancer. This present review will include some of the latest references to advances in this field. Moreover, this review will also emphasize the role of the lipid phosphate phosphatases (LPPs) in controlling the metabolism of LPA and signaling downstream of its receptors, especially in terms of the changes that occur in cancer cells. We will also compare the signaling by LPA, which stimulates cell survival pathways, with the role of ceramides in causing cell death. Role of ATX ATX was originally isolated in 1992 from media derived from A2058 human melanoma cells. While studying the motility of tumor cells, Stracke et al. discovered that these melanoma cells secreted an autocrine motility factor, which they purified, sequenced and named ATX [7]. ATX is a member of a pyrophosphatase/ phospho diesterase family and it is also known as ectonucleotide pyrophosphatase/phosphodiesterase family member 2 (ENPP2). This family of enzymes consists of cell surface or secreted proteins that were initially described as degrading nucleotide phosphates. It is now clear that the family members can have more diverse actions. For example, later work demonstrated that ATX accounts for the lysophospholipase D activity in serum. Furthermore, the finding of a much lower K m value of ATX for lysophosphatidylcholine (LPC) compared with that of nucleotide phosphates indicated that LPC is a preferred physiological substrate and that the major function of ATX is to produce LPA [6,8]. LPA is a simple phospholipid with a phosphate head group and a single fatty acid (either saturated or unsaturated) attached at the sn-1 or sn-2 position of the glycerol backbone. Thus, there are different LPAs depending on the fatty acid and the position of the glycerol backbone to which it is attached. LPA mediates most of Raie T Bekele & David N Brindley* Signal Transduction Research Group, Department of Biochemistry, School of Translational Medicine, University of Alberta, Edmonton, T6G 2S2, Alberta, Canada *Author for correspondence: Tel.: +1 780 492 2078 Fax: +1 780 492 3378 david.brindley@ualberta.ca part of 10.2217/CLP.12.30 2012 Future Medicine Ltd Clin. Lipidol. (2012) 7(3), 313 328 ISSN 1758-4299 313

Review Bekele & Brindley the biological effects of ATX through its ability to activate at least eight G-protein-coupled receptors (GPCRs) [9 13]. LPA 1, LPA 2 and LPA 3 were first described as EDG receptor family members. The non-edg receptors include LPA 4 (GPR23/p2y9), LPA 5 (GPR92) and LPA 6 (p2y5). The other receptors include: GPR87, which has been implicated as an LPA receptor, but its physiological ligand is somewhat controversial [14]; p2y10, which appears to bind both LPA and sphingosine-1-phosphate (S1P) [15]. Activation of these receptors significantly increases tumor growth, angiogenesis, cell migration (which is a component of metastasis) and also contributes to chemoresistance [1 6]. Physiologically, ATX and LPA play major roles in forming the embryonic vasculature and stabilizing blood vessels during embryonic development. This is illustrated by the observation that ATX-deficient mice die at embryonic day 9.5 with profound vascular defects [16]. ATX is also important in tissue repair and wound healing since LPA promotes platelet aggregation, migration of fibroblasts and other cells into a wounded area and stimulates angiogenesis [17]. These same basic processes become dysfunctional and contribute to the development of tumors and their metastasis to other organs. In addition to its catalytic activity, ATX decreases adhesions of oligodendrocytes to the extracellular matrix through its C-terminal region. This action facilitates morphological remodeling [18]. Therefore, it was suggested that ATX is a matricellular protein that participates in the regulation of myelination by a novel signaling pathway, leading to changes in integrindependent focal adhesion assembly and consequently oligodendrocyte interactions with the extracellular matrix [19]. Enzymatic function of ATX & LPA formation LPC is the most abundant phospholipid in blood plasma, where it reaches concentrations of approximately 200 µm in humans [20]. The liver and probably other organs secrete unsaturated LPC [21]. Saturated LPC is mainly produced by lecithin cholesterol acyltransferase acting on the phosphatidylcholine that is present in HDLs by transferring the unsaturated fatty acid (mainly linoleate) to cholesterol [22]. These reactions provide a continuous supply of LPC that is readily accessible to most tissues. The action of ATX on extracellular LPC provides the major route for the production of circulating LPA (Figure 1). This is illustrated by experiments where ATX activity is inhibited, leading to a dramatic fall in circulating LPA [23 25]. It is proposed that the activity of ATX is partially regulated by feedback inhibition from LPA or S1P, which is the sphingolipid analog of LPA [26]. Recent work shows that the catalytic and nuclease-like domains of ATX are covalently linked by an essential disulfide bridge between Cys413 and Cys805 [27]. Residues 829 850 of ATX, which are within the nuclease-like domain, are involved in the secretion of ATX, whereas Lys852 is required for the expression of catalytic activity. Jansen et al. proposed that the nucleaselike domain of ATX is crucial for catalysis and it is a possible target to generate ATX inhibitors [27]. In addition to ATX, there are other pathways for producing extracellular LPA (Figure 1). The first involves the action of secreted phospholipase A2 (PLA2) that converts phosphatidate (PA), which is present in microvesicles released during inflammatory reactions, to LPA [28]. There is also evidence that group VIA PLA2 (Ca 2+ independent PLA2b [ipla2b]) produces extracellular LPA by human epithelial ovarian cancer cells [29,30]. Tumorigenesis and ascites formation were decreased in ipla2b (-/-) mice compared with wild-type mice [30]. LPA and LPC concentrations were decreased in the tumor microenvironment of ipla2b (-/-) mice to approximately 80% of that in wild-type mice. LPA, but not LPC, stimulated cell migration and invasion when ipla2b expression was knocked down in vitro. LPA, but not LPC, also enhanced ascites formation in vivo by approximately fivefold and tumorigenesis in ipla2b (-/-) mice [30]. LPA can also be produced inside cells from monoacylglycerol through a kinase reaction (Figure 1). Alternatively, it can be made by the first reaction in the well-known Kennedy pathway in which glycerolphosphate acyltransferases convert sn-glycerol-3-phosphate to LPA. At present, it is unclear how LPA moves out of the cell. The roles of LPPs & their importance in cancer Metabolism of extracellular LPA Plasma LPA concentrations are normally <1 µm, but they can reach >10 µm in ovarian cancer, as reviewed in [1], depending partly on LPA production by ATX. The other major component in regulating the concentration of extracellular LPA is its degradation by the LPPs (Figures 2 & 3). These 314 Clin. Lipidol. (2012) 7(3)

Autotaxin & lysophosphatidate in cancer progression & resistance to therapy Review PI4,5P PLC DG kinase PLD Phosphatidylcholine P DG PLA1 Lipins spla2 or LCAT or LPP PA AGPAT or PLA2 ipla2β LPC H P MG kinase MG LPA ATX LPP GPAT Glycerol phosphate Figure 1. Synthesis of lysophosphatidate. LPA is composed of a glycerol phosphate bound to a fatty acid chain (either saturated or unsaturated) at the sn-1 or sn-2 positions. It is synthesized outside the cell mainly by the action of ATX on LPC as described in the text. LPA can also be synthesized from PA by the actions of spla2 or ipla2b or it can be formed de novo from glycerol-3-phosphate. The metabolic interactions of LPA with PA, MG and DG are also shown. AGPAT: Acylglycerolphosphate acyltransferase; DG: Diacylglycerol; GPAT: Glycerolphosphate acyltransferase; ipla2b: Ca2+ independent phospholipase A2b; LCAT: Lecithin cholesterol acyltransferase; LPA: Lysophosphatidate; LPC: Lysophosphatidylcholine; LPP: Lipid phosphate phosphatase; MG: Monoacylglycerol; PA: Phosphatidate; PI4,5P: Phosphatidylinositol 4,5-bisphosphate; PLA1: Phospholipase A1; PLA2: Phospholipase A2; PLC: Phospholipase C; PLD: Phospholipase D; spla2: Secreted phospholipase A2. comprise a family of three enzymes that are able to dephosphorylate a large variety of bioactive lipid phosphates and pyrophosphates, including LPA [31]. The LPPs are expressed on the surface of cells, with the active phosphatase site being exposed to the outer leaflet of the plasma membrane [32]. Increasing the expression of LPP1 on the surface of fibroblasts increases their ability to degrade various extracellular lipid phosphates including LPA, PA (Figure 1) and ceramide-1-phosphate (C1P) [33]. The effect of the ecto-lpp activity on LPA is to convert it to monoacylglycerols, which, with the exception of 2-monoarachidonoylglyc erol (an endo cannabinoid), are not active signal ing molecules. This dephosphorylation of LPA is very rapid, such that the half-life of circulating LPA is approximately 3 min in mice [24,34]. The LPP isoform, LPP1, plays a major role in circu lating LPA degradation based on experiments in LPP1 hypomorph mice (Ppap2atr/tr), which have 35 95% decreases in LPP activity in most tissues except the brain [34]. Plasma LPA concen trations in these LPP1 hypomorph mice are sig nificantly increased compared with control mice, and the half-life of intravenously injected LPA is approximately fourfold higher in the Ppap2atr/tr mice compared with controls (t1/2 : 12 vs 3 min). Therefore, the balance of LPA formation by ATX versus hydrolysis by the LPPs determines the con centration of LPA in circulation as well as in the tumor microenvironment. Also the ecto-activity of LPP1 and LPP3 can contribute to the degradation of S1P [35], which is a sphingolipid analog of LPA that activates five GPCRs [36]. Increased S1P production and secre tion is associated with increased chemo resistance and stimulation of angiogenesis for the growing tumor [37]. Therefore, LPP1 and LPP3 could also function to regulate the effects of S1P on these processes. It is significant in this respect that the expres sion of LPP1 and LPP3 is very low in several www.futuremedicine.com 315

Review Bekele & Brindley Choline Albumin LPA LPC ATX MG Integrin G α G β G γ LPA 1/2 LPA 3 LPP1/3 G α G β G γ G i/o G q G 12/13 G s PI3K Ras PLC Rho AC Akt ERK IP3 PLD camp Proliferation Ca 2+ PKA Migration Figure 2. Autotaxin lysophosphatidate signaling axis. ATX locally converts the abundant LPC to LPA, which binds to its cognate LPA 1 8 receptors (LPA 1 3 are shown) to activate multiple signaling pathways, leading to proliferation and migration of target cells. LPA 3 can couple to G s; it can also couple to G i/o and G q but not G 12/13. Moreover, LPA can directly or indirectly activate other signaling pathways not shown in the figure. LPPs on the other hand, regulate the ATX LPA signaling axis by dephosphorylating LPA to the biologically inactive MG. AC: Adenylate cyclase; ATX: Autotaxin; IP3: Inositol 3-phosphate; LPA: Lysophosphatidate; LPC: Lysophosphatidylcholine; LPP: Lipid phosphate phosphatase; MG: Monoacylglycerol; PKA: Protein kinase A; PLC: Phospholipase C; PLD: Phospholipase D. cancer cells [38 41], and this is a contributing factor in the increased plasma concentrations of LPA that are associated with ovarian cancers [36,40]. Furthermore, gonadotropin-releasing hormone has antiproliferative effects in ovarian cancer by increasing ecto-lpp expression [39]. LPP3 overexpression decreases growth and colony formation by ovarian cancer cells by degrading extracellular LPA. This action of the LPPs was proposed as a target for cancer therapy [41,42]. Consequently, the combination of high ATX and low LPPs exposes cancer cells to a microenvironment with elevated extracellular LPA concentrations. This promotes tumor growth, angiogenesis, metastasis and chemoresistance. The intracellular roles of LPPs on LPA signaling In addition to the ecto-activities of the LPPs, which help to regulate extracellular LPA concentrations and thus the activation of LPA receptors, 316 Clin. Lipidol. (2012) 7(3)

Autotaxin & lysophosphatidate in cancer progression & resistance to therapy Review the LPPs control LPA signaling downstream of LPA receptor activation. The LPPs are also located in the endoplasmic reticulum [33,43] and the Golgi network [44], with the catalytic sites presumably facing the lumenal side of these membranes. As such, intracellular LPPs could potentially regulate the levels of intracellular lipid phosphates formed after receptor activation. This controls the concentrations of the lipid phosphates relative to their dephosphorylated products, which are often also bioactive [17]. ne example of this is the formation of PA following the activation of phospholipase D (PLD) and the conversion of PA to diacylglycerol (DG) by the LPPs (Figure 1). The first convincing evidence that LPPs control intracellular rather than extracellular signaling came from studies showing that they regulate ERK activation downstream of thrombin signaling [45]. Thrombin is not a substrate of LPPs and, therefore, the ecto-lpp activity cannot affect extracellular signaling by degrading this ligand. verexpression of LPP1 and LPP2 decreases the accumulation of PA relative to DG and this is compatible with the abilities of the LPPs to convert PA to DG and to control ERK activation [46]. Subsequent work also demonstrated that mouse embryonic fibroblasts that overexpressed LPP1 showed decreased stimulation of ERK-dependent cell migration by PDGF [47]. The authors attributed this result to increases in intracellular DG concentrations in the LPP1 overexpressing cells. Sustained intracellular DG levels decreased the expression of classical PKC isoforms, which are required for PDGF-induced migration [47]. Furthermore, Long et al. showed that LPP2 and LPP3 can affect cell survival by regulating the intracellular levels of PA [48]. Increased expression of LPP1 also attenuated cell migration, a component of metastasis, which was stimulated by LPA and a phosphonate LPA analog [49]. This LPA analog can activate LPA 1/3 receptors, but it cannot be hydrolyzed by LPP1. Therefore, the attenuation of its signaling by LPP1 cannot be attributed to its ecto-activity. Instead, the authors demonstrated that the catalytic action of LPP1 decreased intra cellular signaling by blocking the activation of PLD (Figure 3), and thus PA accumulation, downstream of LPA receptor activation. This attenuated the effects of LPA and the LPA analog on cell migration, since PLD2 activity is required for LPA to stimulate this process. In addition, LPP1 overexpression also decreased the activation of ERK and Rho, two other important proteins involved in LPA-induced fibroblast migration. n the other hand, PDGF-induced migration in LPP1-overexpressing fibroblasts was not affected by LPP1 and there was no effect on PDGF-stimulated ERK activation. Conversely, PLD activation by PDGF was decreased by LPP1 overexpression; however, PLD activation is not required for PDGF to induce cell migration. Several other studies have also shown the effect of LPPs in regulating intracellular PA levels [46,48,50 52]. In the case of LPP3, there was increased conversion of PA, formed by PLD activation, to DG when LPP3 was overexpressed in either Swiss 3T3 or HEK 293 cells [53]. LPP2 MAG LPP1 SK-1 S1P LPA 1/2 PLD PA LPA Sos Ras Survival pathways LPP2 LPP1/3 ERK LPA 1/2 Cyclin A accumulation Cyclin A Entry to S-phase Activate Block LPP2 Upregulate Downregulate Figure 3. Intracellular and extracellular roles of lipid phosphate phosphatases. LPPs such as LPP1 and LPP3 regulate the levels of extracellular LPA and other lipid phosphates by removing the phosphate group, thus attenuating extracellular LPA-mediated signaling. Furthermore, LPPs also have intracellular actions in which they inactivate downstream targets of the LPA receptor such as PLD and thus block the signaling cascade elicited by LPA. Surprisingly however, LPP2 stimulates cyclin A accumulation and entry in to S-phase allowing it promote cancer progression. Cancer cells are hypersensitive to LPA signaling since they upregulate the levels of LPP2 and LPA receptors while downregulating LPP1 and 3. LPA: Lysophosphatidate; LPP: Lipid phosphate phosphatase; PA: Phosphatidate; PLD: Phospholipase D; S1P: Sphingosine-1-phosphate; SK-1: Sphingosine kinase 1. www.futuremedicine.com 317

Review Bekele & Brindley has been postulated to hydrolyze PA formed from PLD1 stimulation, which would reduce PA-induced recruitment of sphingosine kinase 1 (SK-1) to perinuclear membranes [54]. Also, Rastransformed fibroblasts have low LPP activities and the PA:DG ratio in these cells was increased compared with control fibroblasts after stimulation of PLD activity [38]. Basal levels of PA concentrations also increased with time in culture in these fibroblasts [55]. It was concluded from these experiments that the decrease in PA concentrations depended on the direct action of the LPPs in converting PA to DG. However, the attenuation of PLD activation downstream of the activation of a GPCR or a receptor tyrosine kinase could also contribute to the observed decrease in PA accumulation following increased LPP expression [49]. The actions of the LPPs in controlling the net accumulation of PA could have a profound affect on signaling in cancer cells. PA activates numerous downstream targets including PKC z, mtrc1, Sos, Raf, phospholipase C-g, SK-1 and ERK [1,56]. The LPPs could also control DG formation, which could alter the activation of various PKCs. LPP2, in contrast to LPP1 and LPP3, is important in regulating the cell cycle [57]. More specifically, knocking down LPP2 in fibroblasts delayed cyclin A accumulation and entry of the fibroblasts into S-phase. Conversely, overexpressing LPP2 resulted in premature entry into S-phase owing to premature cyclin A accumulation. This effect depended on the catalytic activity of LPP2, since the inactive mutant form did not affect S-phase entry. The lipid phosphate substrate of LPP2 that is involved in this phenotype is still unknown. LPP2 overexpressing fibroblasts are subsequently arrested in G2/M-phase of the cell cycle after 15 35 passages, when the cells exhibit a senescent phenotype [57]. This is reminiscent of oncogenes such as Ras and BRAF that stimulate cell proliferation followed by premature senescence, which probably serves to prevent malignancy [58,59]. Interestingly, LPP2 knockout mice are fertile and exhibit no obvious phenotype [60]. However, LPP2 regulates the timing of entry into S-phase and it is not essential for cell cycle progression. ther proteins that regulate entry into S-phase or late G1 phase, for example, cyclins D1, D2, E1 and E2 in addition to CDK2, 4 and 6, have been knocked out in mice. These mice, like the LPP2 knockout mice, also show no obvious phenotype [57]. Subsequent work identified LPP2 (PPAP2C) by gene microarray analysis as one of three potentially novel targets that have increased expression in transformed compared with nontransformed human adult mesenchymal stem cells [61]. The authors also showed increased expression of LPP2 in transformed fibroblasts and the cancer cell lines MCF7, SK-LMS1, MG63 and U2S. Knockdown of LPP2 impaired anchoragedependent growth of the cancer cell lines and also decreased the growth of primary mesenchymal stem cells, but not of differentiated human fibroblasts. Flanagan et al. [61] confirmed previous work showing that knockdown of LPP2 delayed entry into S-phase of the cell cycle [57]. They also demonstrated that the regulation of the transcription of PPAP2C is partly controlled by p53 [61]. This work established LPP2 as a putative therapeutic target for treating cancer. LPPs potentially control the accumulation of several bioactive lipid phosphates, such as LPA, C1P and S1P, in addition to PA. In the case of S1P there are also two specific S1P phosphatases [62]. Formation of intracellular LPA could occur through the action of phospholipase A1 or A2 on PA. Intracellular LPA can bind to nuclear LPA 1 receptors to modulate proinflammatory signaling [63,64] and can also induce PPARg signaling [65,66]. C1P and S1P are both involved in inflammatory signaling. C1P promotes the activation of PLA 2 to produce arachidonate while S1P induces CX2, which converts arachidonate to prostaglandin E 2 [67]. The LPPs could determine the balance of intracellular signals that dictate various cellular processes, such as inflammation, by modulating the levels of C1P and S1P compared with their products, ceramide and sphingosine. Another example is the counter-balance of signaling between ceramide and S1P in the regulation of apoptotic signaling versus cell proliferation [68]. Ceramide promotes cell death by apoptosis, whereas S1P promotes cell survival. Hence, the LPPs regulate important cell processes, such as cell survival, proliferation and migration. As mentioned above, LPP1 and LPP3 activities are decreased in several cancer cell lines. This could make cancer cells hypersensitive to survival and migratory signals in two ways [1]. First, the ability of the cancer cell to degrade extracellular LPA and S1P would be attenuated, thus enabling these extracellular messengers to have a greater effect in stimulating cell division, survival, migration and angiogenesis. Second, low LPP activity would increase the responses to agonists that activate 318 Clin. Lipidol. (2012) 7(3)

Autotaxin & lysophosphatidate in cancer progression & resistance to therapy Review GPCRs (e.g., LPA and S1P) and receptor tyrosine kinases (e.g., EGF and PDGF) by favoring intracellular signaling by lipid phosphates formed downstream of the activation of these receptors (Figure 3). The role of ATX in cancer progression Tumor progression is a multistep process that arises from accumulating mutations leading to oncogene activation and tumor suppressor inactivation, ultimately resulting in the neoplastic transformation of cells. This is followed by a transition to an invasive (metastatic) tumor with an ability to migrate and penetrate through basement membranes and invade surrounding tissues. Neoplastic transformation, in addition to oncogene activation, often requires other collaborative pathways such as the ATX LPA signaling axis to promote proliferation and support metastasis [69]. The role of ATX in cancer progression has been documented since its discovery as autocrine motility factor secreted by melanoma cells [7]. ur understanding of its role was enhanced significantly with the discovery that its true physiological role is to convert the abundantly circulating LPC into bioactive LPA. riginally, LPC was thought to be a bioactive signaling molecule. However, inhibiting ATX activity abrogates most of its effects and, therefore, many biological effects of LPC are now attributed to LPA, especially in the context of cell migration and protection from apoptosis. The crystal structure of ATX indicates the presence of somatomedin B like (SMB) domains [70]. The SMB2 domain of ATX was shown to bind to b1- and b3-integrins, which increased the concentration of ATX on the surface of platelets [71]. The latter authors proposed that this interaction localizes ATX on the surface of cells, thus delivering LPA in the vicinity of its receptors. In the case of aggressive tumors, this interaction could localize ATX and produce a microenvironment for increased LPA production. It is also suggested that this interaction will restructure the SMB domains, which regulate the LPA exit site and expedite product release [72]. Hence, the ATX b3-integrin interaction could be especially important, considering that most aggressive tumors such as melanomas and carcinomas of the prostate, breast, cervix and pancreas express the integrin a v b 3 subtype, which promotes proliferation, survival, migration, invasion and chemoresistance [73 75]. In short, ATX s major role in cancer progression is closely tied to LPA production and also to its interaction with target cells that facilitates delivery of LPA locally to its receptors. In addition, other functions of ATX have been proposed that are not directly related to LPA production and are mediated by ATX effects on cell adhesion [76]. The role of LPA in cancer progression LPA binds and signals through at least eight GPCRs on the cell surface: LPA 1 (EDG2), LPA 2 (EDG4), LPA 3 (EDG7), LPA 4 (GPR23/p2y9), LPA 5 (GRP92), LPA 6 (p2y5), LPA 7 and LPA 8. The expression of these receptors is cell type specific, allowing different cells to respond differently to LPA [9 13]. In addition, each of these receptors could couple to at least four distinct G proteins, G i/o, G s, G q and G 12/13, thus allowing LPA to have a variety of biological actions. Coupling of the GPCR with G i can induce activation of either Ras or PI3K and decreased adenylate cyclase activity. G q activation will increase the activity of phospholipase Cb, which is implicated in Ca 2+ mobilization. G 12/13 activates Rho signaling with effects on migration and G s stimulates adenylate cyclase, resulting in an increase in camp (Figure 2) [77,78]. Thus, LPA, through activating different G proteins, can mediate cell division and migration on target cells. The GPCRs LPA 1, LPA 2 and LPA 3 belong to the EDG family and are the most widely expressed and well-characterized LPA receptors. Thus, we will focus mainly on them and, to a lesser extent, on LPA 4. LPA receptors LPA 1 LPA 1 is a 41 kda, high-affinity LPA receptor consisting of 364 amino acids [79]. It has a very high sequence similarity with LPA 2 and LPA 3 and can couple with G i/o, G q and G 12/13 [77,80]. LPA 1 is the most widely expressed receptor with functions in normal development that became clearly evident in LPA 1 knockout mice, which showed partial lethality and weight loss attributed to defective suckling [81]. LPA modulates the colony scattering of epithelial cancers, which is a prerequisite for cell invasion. Recent work demonstrates that LPA 1 mediates LPA-stimulated cell scattering of gastrointestinal carcinomas [82]. These effects depend on the activation of Src kinase and MEK. LPA 2 LPA 2 is a 39-kDa protein consisting of 348 amino acids in mice. Similar to the LPA 1 receptor, it can www.futuremedicine.com 319

Review Bekele & Brindley couple with G i/o, G q and G 12/13 [78,83]. LPA 2 null mice show no phenotypic aberration compared with wild-type mice. Moreover, the LPA 1 and LPA 2 double-knockout mice are phenotypically identical to the LPA 1 knockout mice [84], indicating that either LPA 3 or LPA 4 have redundant functions or the role of the LPA 2 receptor is primarily in pathophysiological conditions such as tumorigenesis [77]. The C-terminal tail of the LPA 2 but not the LPA 1/3 receptors contains a specific PDZ domain that allows it to interact with NHERF2 [85]. In addition, this C-terminal tail also specifically interacts with the carboxyl cysteine-rich domain of the proapoptotic protein Siva-1 [86], allowing it to uniquely modulate signaling from LPA. LPA 3 LPA 3 is a 40-kDa protein with 353 amino acids. It is unique from LPA 1 or LPA 2 in its ability to couple with G s. This coupling allows LPA 3 to stimulate adenylate cyclase and increase the levels of camp, as opposed to LPA 1 and LPA 2, which decrease camp concentrations. In addition, the LPA 3 receptor lacks the ability to couple with G 12/13 and so it does not activate the Rho signaling pathway. Similar to LPA 1 and LPA 2, LPA 3 couples to G i/o and G q [77,78,87]. Targeted deletion of LPA 3 in mice showed no phenotypic abnormalities compared with wild-type or hetero zygous mice but had effects on embryo implantation, resulting in reduced litter size, which was attributed to a reduction in prostaglandin synthesis [88]. LPA 4 LPA 4 does not belong to the EDG family of receptors and it lies in a different evolutionary branch of the lysophospholipid receptor family, with close similarity to the platelet-activating factor receptor [78]. LPA 4 null mice, like the LPA 2 and LPA 3 null mice, displayed no obvious abnormalities. LPA 4 is expressed at very low levels in most human tissues with the exception of the ovary [89]. Most mammalian cells and tissues coexpress different LPA receptors and also have varying preferences for the different species of LPA; consequently, the signaling pathway activated under a specific context, such as tumorigenesis, could depend on the species of LPA in the microenvironment and the cooperative or competitive interactions of the different receptors for effectors proteins. Such an interaction is observed in LPA 4 null mouse embryonic fibroblasts, which become hypersensitive to LPA-induced migration as compared with the wild-type [89]. This suggests a competitive interaction of LPA receptors for effector proteins in the mouse embryonic fibroblasts. Hence, the LPA receptors, with their ability to control cell division and migration, can influence tumorigenesis. In fact, LPA 1 and LPA 2 receptors are often overexpressed in cancers of the thyroid [90], breast and ovary [91 94]. LPA 1, LPA 2 and LPA 4 have also been shown to co operate with the wellknown oncogene, cmyc, and transform mouse embryonic fibroblast to become anchorage-independent for growth on soft agar [69], which is a hallmark of neoplastic transformation. Moreover, studies in mice have revealed that mammary-specific expression of LPA 1, LPA 2 and LPA 3 receptors and ATX leads to the production of late-onset mammary tumors [95]. The altered expression of these receptors coupled to increased expression of ATX and localized production of LPA allows the growing tumor to thrive through increased and sustained activation of the ERK and PI3K pathways, leading to an overall increase in proliferation [69,94,95]. In addition, these signaling pathways will also lead to a more robust activation of the Rho signaling pathway, as well as increased PLD activation, which is now emerging as a key regulator of metastasis and anchorage-independent growth [96,97]. LPA also stimulates increased production of upa and activation of MMP-9 through stimulation of the Rho RCK pathway in ovarian cancer [98], which promotes invasion [99,100]. Harma et al. demonstrated a metastasis suppressor function for LPA 1 and Ga 12/13 signaling in regulating cell motility and invasion versus epithelial maturation. Ga 12/13 and Ga i were identified as key mediators of LPA signaling via stimulation of RhoA and Rho kinases, RCK1 and 2, activating Rac1, whereas inhibition of adenylate cyclase and accumulation of camp may be secondary events [101]. The authors concluded that LPA and LPA 1 effectively promote epithelial maturation and block invasion of some malignant prostate cancer cells in 3D culture [101]. LPA has a potent action in inducting the formation of actin stress fibers and focal adhesions. LPA failed to promote focal contacts or focal adhesions in cancer cells after adhesion to fibronectin when the actin-binding and crosslinking protein, AFAP-110, was knocked down [102]. The authors concluded that AFAP-110 plays an important role in MDA-MB-231 breast cancer cell adhesion, possibly by regulating stress filament crosslinking, which would promote focal adhesion formation. 320 Clin. Lipidol. (2012) 7(3)

Autotaxin & lysophosphatidate in cancer progression & resistance to therapy Review LPA affects tumor progression by enabling cells to grow and migrate, and also by allowing nascent tumor cells to survive through stimulating angiogenesis by the secretion of VEGF [94] and activation of SK 1, which forms S1P [49,103,104]. Finally, LPA stimulates the increased secretion of proinflammatory cytokines, such as IL 8 and IL 6 [105]. This is especially important as inflammation is now being recognized as a key mediator of tumor progression and metastasis. For instance, IL 8 levels are significantly higher in 67% of patients with breast cancer and this elevated level is correlated with accelerated disease progression and a higher tumor load [106]. Putting all the evidence together, it is clear that LPA signaling plays a key role in tumor progression. The effect of ATX LPA signaling & ceramides in chemotherapy & radiotherapy resistance Depending on the type and stage of the cancer, patients are prescribed different regimens of chemotherapy or radiotherapy, with an overall goal of killing rapidly growing cancer cells. Thus, we will focus on the common effects of the various chemotherapeutics and radiotherapy, and how signaling from the ATX LPA axis could perturb their effect. Many chemotherapeutic agents, including Taxol (Bristol-Myers Squibb, NJ, USA), doxorubicin and tamoxifen, as well as radiation therapy share the effects of increasing ceramide formation as part of their therapeutic action in stimulating apoptosis and/or necrosis (Figure 4) [107 114]. Ceramides are sphingolipids that release cytochrome C from mitochondria and activate caspases to initiate apoptosis [111,115]. Growth factors, including LPA, counteract these effects by increasing PI3K activity, which provides an antiapoptotic signal and decreases ceramide production in response to the chemotherapeutic agents [116]. We also showed that LPA and S1P antagonize the inhibitory actions of ceramides on cell division and that ceramides block the activation of PLD by LPA and S1P [117,118]. The latter action of ceramides depends on blocking the activation Chemotherapeutics Apoptotic pathway Survival Radiation LPA LPA 2 DNA damage PI3K LPA 2 Siva-1 Degradation PLD Proteasome p53 Ceramide Sphingosine PA SK-1 S1P Siva-1 Apoptosis Survival Figure 4. The effects of lysophosphatidate signaling on chemotherapy and radiotherapy. Both chemotherapy and radiotherapy induce apoptosis of target cells through activating the p53 pathway or increasing ceramide accumulation, in addition to several other mechanisms not shown in the figure. LPA causes resistance to these therapies through activating pathways, such as that initiated by PI3K, which blocks ceramide formation, or activating the PLD pathway, which stimulates the synthesis of the antiapoptotic lipid, S1P. LPA also stimulates the degradation of proapoptotic proteins, such as p53 and Siva-1, which also contributes to its resistance to chemotherapy and radiotherapy. LPA: Lysophosphatidate; PA: Phosphatidate; PLD: Phospholipase D; S1P: Sphingosine-1-phosphate; SK-1: Sphingosine kinase 1. www.futuremedicine.com 321

Review Bekele & Brindley of PLD1 and PLD2 by phosphatidylinositol 4,5-bisphosphate [119]. Ceramides also decrease the association of PLD1 with Rac, Rho, Cdc42, PKCa and PKCb [120], which could contribute to an inhibition of cell migration. These studies establish that the mutual antagonism between ceramides versus S1P and LPA is mediated, at least in part, at the level of PLD (Figure 4). The balance between ceramide and S1P concentrations is considered to be an important rheostat that controls the death versus the survival of cancer cells [121,122]. Ceramides are readily converted to sphingosine and then to S1P when ceramidases and SK-1, respectively, are activated (Figure 4). Breast cancer cells increase S1P production when activated by survival signals from LPA, estrogens or EGF [37,104,123 127]. LPA should have a dual action in adjusting this rheostat towards cell survival. First, LPA blocks ceramide formation [116], which should decrease the ceramide-induced inhibition of PLD activation. Second, LPA-induced PLD activation should activate SK-1 and S1P formation, since PA is an activator of SK-1 (Figures 2 & 3) [128]. This should increase the ratio of S1P:ceramide and thus favor cell survival. Chemotherapy and radiotherapy also lead to increased accumulation and activation of the p53 tumor suppressor, especially with respect to radiotherapy, which classically activates the p53 pathway by inducing DNA damage. LPA signaling should counteract this effect, since LPA increases the proteasomal degradation of p53 leading to decreased p53-mediated transcription [129]. In this latter study, LPA protected A549 lung carcinoma cells from the effects of actinomycin D in inducing the expression of p53 and causing apoptosis. In addition, LPA produces resistance to the effects of Taxol in killing breast cancer cells through apoptosis and necrosis [116,130]. LPA also protects against the effects of doxorubicin (also known as Adriamycin ) [131] and carboplatin in killing ovarian cancer cells [132]. Furthermore, activation of LPA 2 has been implicated in protection against radiationinduced intestinal injury. In fact, the LPA mimic, octadecenyl thiophosphate, which activates LPA 2, reduces death caused by lethal dose (LD 100/30 ) radiation by 50% [133]. This result indicates that increased LPA signaling should also decrease the efficacy of killing tumors by radiotherapy. This could be attributed to the unique ability of LPA 2 to bind to the proapoptotic protein Siva-1, given that Siva-1 will be degraded along with the receptor after internalization, contributing to the chemoresistance associated with LPA signaling [78]. Conclusion The accumulating body of evidence clearly implicates the ATX LPA signaling axis in tumor progression and resistance to various therapeutic agents and to radiotherapy. Cancer cells are able to utilize this signaling pathway through their expression of LPA receptors such as LPA 1 and LPA 2. In addition, cancer cells often increase their expression of ATX while downregulating the expression of LPP1, which normally degrades LPA and attenuates its signaling. These adaptations allow the growing tumor to exist in a microenvironment that favors locally high LPA concentrations and results in increased LPA signaling. High concentrations of LPA promote aberrant signaling in cancer cells, allowing it to interact with other oncogenes and promote uncontrolled proliferation of the tumor through activation of signaling pathways such as PI3K, PLD and ERK pathways. LPA signaling also stimulates cancer cells to migrate and invade neighboring tissues through activation of Rho and PLD signaling pathways, and this contributes to the increased metastatic potential of the tumor. Furthermore, ATX and LPA induce the cancer cells to express VEGF [134,135] and S1P [104], which stimulate endothelial cells to form new blood vessels that feed the growing tumor in a process called angiogenesis. VEGF also stimulates ATX production, leading to increased LPA in a feed-forward system [136]. LPA is able to achieve such a multitude of biological activity through binding to its different GPCRs that, in turn, couple to different G-proteins (G i/o, G s, G q and G 12/13 ). After the tumor has progressed, depending on its grade, stage and localization, patients are prescribed various treatments programs, which include surgery, chemotherapy and/or radiotherapy. Nevertheless, highly increased signaling by LPA and other growth factors counteracts the efficacy of chemotherapy and radiotherapy. This is clearly illustrated by studies showing that LPA reverses Taxol-, cisplatin- and Adriamycin-induced apoptosis and radiation-induced injury. Future perspective Although we are still developing our understanding of signaling pathways that are activated in cancer cells by LPA, there is now sufficient evidence to establish that LPA is a significant growth 322 Clin. Lipidol. (2012) 7(3)

Autotaxin & lysophosphatidate in cancer progression & resistance to therapy Review factor that causes the proliferation of tumor cells, their metastasis and chemo resistance. The development of therapies for treating cancers by blocking LPA formation and signaling, as part of cancer treatment, is at a very early stage. Selective inhibitors can be used to block the signaling cascade elicited by LPA. This could be achieved by inhibiting either ATX or blocking the LPA receptors. It is not our objective in this review to discuss all of the inhibitors of ATX that have been developed. These inhibitors include cyclic PA, l-histidine and small-molecule inhibitors including hexachlorophene, merbromin, pipemidic acid and flavonols. There are already several reviews dealing with this topic (e.g., [137]). Instead, we will only discuss the actions of some of these inhibitors. Analogs of LPA have been developed to inhibit ATX at nanomolar concentrations [23] and some of these act by mimicking the feedback inhibition of ATX by LPA [26]. Syn- and anti-bromophosphonate-lpas inhibited tumor growth in both colon cancer cells in liver and breast cancer [138,139]. Bromophosphonate-LPA not only inhibits ATX, but it also has the advantage of blocking signaling by LPA 1 4 receptors. ther classes of ATX inhibitors have been developed that do not resemble LPA. These include the boronic acid-based inhibitors, but so far these inhibitors exhibit biological efficacy for only short periods [24]. Another small-molecule inhibitor of ATX, bithionol, inhibited cell growth and migration of melanoma, ovarian and breast cancer cells [140]. n the other hand, there are ATX inhibitors such as PF-8380 that reduce LPA levels in plasma and the site of inflammation in vivo, indicating that the local LPA environment could also be targeted by ATX inhibitors [25]. Finally, the recent development of longer-acting ATX inhibitors such as benzyl and naphthalene methylphosphonic acid inhibitors provide a promising improvement in the series of ATX inhibitors [141]. Nonetheless, it remains to be seen whether such inhibitors, or several other ATX inhibitors, are able to decrease tumor development in animal models or a clinical setting. Inhibiting ATX has the potential advantage that it would attenuate the activation of all LPA receptors; however, such inhibition could impact on other physiological activities that require LPAmediated signaling. Thus, developing inhibitors against LPA receptors could also have potential benefits as chemotherapeutic agents, especially since receptors such as LPA 2 have been shown to have unique roles under pathophysiological conditions. However, this could prove to be difficult owing to the expression of various LPA receptors on the cell surface, meaning such inhibitors should exhibit inhibition on multiple LPA receptors. Nevertheless, the selective inhibitor AM966, a potent antagonist of LPA 1 receptor, was shown to inhibit lung fibrosis in the mouse, indicating selective inhibition might have advantages under certain contexts [142]. Thus, selective inhibitors, such as AM966, could probably be employed in cancers with predominant LPA 1 expression. Executive summary Role of autotaxin Autotaxin (ATX) is a secreted enzyme that has lysophospholipase D activity and converts lysophosphatidylcholine to lysophosphatidate (LPA). Its overexpression in tumors increases tumor growth and metastasis. Function of lysophosphatidate formation Lysophosphatidate (LPA) activates at least eight receptors, which are coupled to G i/o, G s, G q or G 12/13. Activation of LPA receptors provides signals for cell division, survival, migration, metastasis, angiogenesis and chemoresistance. Roles of lipid phosphate phosphatases Lipid phosphate phosphatases (LPPs) are enzymes that dephosphorylate extracellular LPA and hence decrease the availability of LPA that can activate its receptors. The catalytic activity of the LPPs also decreases the activation of signaling pathways downstream of the stimulation of G-protein-coupled receptors and receptor tyrosine kinases. Importance of LPPs & their significance in cancer Low activity of LPP1 and LPP3 contributes to the hypersensitivity of tumor cells to the actions of several growth factors, including LPA. Conversely, increased expression of LPP2 is associated with cell transformation and accelerated entry into S-phase of the cell cycle. Effect of LPA signaling & ceramides in chemotherapy & radiotherapy resistance Ceramides are proapoptotic lipids that accumulate in cells undergoing chemotherapeutic treatments or radiotherapy. The stimulation of ceramide accumulation provides part of the signal used for killing cancer cells. Several growth factors, including LPA, counteract this proapoptotic signal. Conclusion The ATX LPA signaling axis is a key mediator of cancer progression and resistance to chemotherapy and radiotherapy. Blocking the ATX action or LPA signaling provides a promising approach for the development of new therapeutics to treat cancer. www.futuremedicine.com 323

Review Bekele & Brindley verall, ATX inhibitors and LPA receptor inhibitors could possibly be used on their own or as adjuvants to increase the efficacy of existing chemotherapeutic agents to prevent tumor growth and metastasis. Financial & competing interests disclosure DN Brindley has served as a consultant to no Pharmaceuticals. Grant support from the Canadian Institutes of Health Research, the Canadian Breast Cancer Foundation and the Alberta Cancer Foundation is gratefully acknowledged. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. References Papers of special note have been highlighted as: of interest of considerable interest 1 Samadi N, Bekele R, Capatos D, Venkatraman G, Sariahmetoglu M, Brindley DN. 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