Statistical comparisons to the appropriate baseline motility (no attractant) are shown: *** for P < 0.001; ** for P < 0.01; * for P < 0.05. VEGF stimulates ATX expression and secretion in ovarian cancer cells VEGFR2 By generating exogenous LPA, ATX could contribute to the induction of VEGF in ovarian cancer cells (4). responses to VEGF, ATX, LPA, and LPC. These effects are accompanied by decreased LPA4 and VEGFR2 expression as well as by increased release of soluble VEGFR1. Since LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells. Introduction Vascular endothelial growth Rabbit polyclonal to ACD factor-A (VEGF) is a potent stimulator of angiogenesis associated with physiological processes such as wound healing and the female reproductive cycle. In addition, its expression can be increased under pathological circumstances including ischemic diseases and tumor growth. Originally identified as an inducer of vascular permeability in endothelial cells, VEGF now has established roles in endothelial cell migration, proliferation, and survival. In cancer, VEGF exhibits autocrine activities that serve to protect tumor cells from stressors such as hypoxia, chemotherapy, or radiotherapy. Anti-VEGF treatments to limit these effects are in clinical use as well as in ongoing clinical trials (1, 2). VEGF signaling is mediated by two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1), both of which are crucial for VEGF-stimulated angiogenesis and are implicated in tumor progression. VEGF gene expression can be stimulated by low glucose levels, by growth factors such as fibroblast growth factor-2 (FGF2) or platelet-derived growth factor (PDGF), and in tumor cells, by alterations in oncogenic or tumor suppressor genes. However, the strongest inducer of VEGF expression is hypoxia, a common feature of the tumor microenvironment. In ovarian cancer, VEGF contributes to malignant ascites formation by increasing peritoneal micro-vessel permeability (3) and, interestingly, lysophosphatidic acid (LPA) in concentrations reported in malignant ascites has been found to induce VEGF expression (4, Salvianolic acid A 5). The bioactive lysophospholipid LPA stimulates cell proliferation, migration, and survival and has been implicated in tumor progression (6). Signaling of LPA is mediated through classic G protein-coupled receptors belonging to the endothelial differentiation gene (EDG) family (LPA1/EDG-2, LPA2/EDG-4, and LPA3/EDG-7), and is linked to G proteins: Gq/11, Gi/o and G12/13. LPA4 (GPR23/p2y9) is more closely related to purinergic P2Y than to EDG receptors and has distinct G protein-linked signaling: Gs, Gq, Gi, and G12/13 (7, 8). Under physiological circumstances, LPA concentrations in serum are approximately 1C10 M. Although plasma concentrations are much lower (9), they can become elevated in cancer patients, particularly in ovarian cancer (10, 11). The major source of serum and plasma LPA appears to be the lysophospholipase D activity of autotaxin (ATX, NPP2) (12, 13). ATX is a member of the ecto-nucleotide pyrophosphatase and phosphodiesterase family of enzymes and is synthesized as a secreted protein (14). In contrast to other members of this group, ATX possesses lysophospholipase D activity and can catalyze hydrolysis of lysophosphatidylcholine (LPC) into LPA and sphingosylphosphorylcholine into sphingosine-1-phosphate (15C17). ATX was initially purified as a potent chemotactic factor (18) and has been found to augment invasiveness and metastatic potential in transformed cells (19). These motogenic and invasive properties require its lysophospholipase D activity (19, 20). In addition, ATX stimulates angiogenesis (21) although the mechanisms for this action have not yet been elucidated. In the present study we have focused on regulation of ATX expression in ovarian cancer cells and on the mechanisms by which it can contribute to ovarian cancer progression. Ovarian cancer ascites contains a number of bioactive molecules including VEGF, LPA, LPC, and sphingosylphosphorylcholine (22) and has elevated lysophospholipase D activity (23). We now present evidence that VEGF VEGFR2 stimulates ATX expression in the ovarian cancer cell lines CaOV3 and SKOV3. In metastatic SKOV3 cells, treatment with a VEGF blocking antibody significantly decreases ATX mRNA levels, implying that the high level of ATX detected in this cell line is due to an autocrine action of VEGF. ATX knockdown antisense morpholino oligomers (MO) results in reduced migration to VEGF, ATX, LPA and LPC. In addition, reduced ATX expression results in decreased LPA4 and VEGFR2 signaling and release of the inhibitory soluble form of VEGFR1 (sVEGFR1), Salvianolic acid A all of which may contribute to the decreased chemotactic response. These data indicate Salvianolic acid A a regulatory role for VEGF in ATX synthesis as well as a role for ATX in controlling VEGF responsiveness. Since the ATX product LPA has been previously shown to increase VEGF expression (4, 5), our data suggest a pathological positive feedback loop between ATX, LPA, and VEGF in ovarian carcinoma. Results Differential expression of VEGF and ATX in ovarian cancer cells VEGF and LPA are major components of malignant ascites.