Xianjun Fang

Expression of Autotaxin and Lysophosphatidic Acid Receptors Increases Mammary Tumorigenesis, Invasion, and Metastases

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Lysophosphatidic Acid Receptors Determine Tumorigenicity and Aggressiveness of Ovarian Cancer Cells

BACKGROUND Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown. METHODS Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided. RESULTS SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing beta-galactosidase. CONCLUSIONS Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.

Mechanisms for Lysophosphatidic Acid-induced Cytokine Production in Ovarian Cancer Cells

A potential role for lysophosphatidic acid (LPA) in human oncogenesis was first suggested by the observation that LPA is present at elevated levels in ascites of ovarian cancer patients. In the current study, we demonstrated that LPA is a potent inducer of interleukin-6 (IL-6) and interleukin-8 (IL-8) production in ovarian cancer cells. Both IL-6 and IL-8 have been implicated in ovarian cancer progression. We characterized the IL-8 gene promoter to ascertain the transcriptional mechanism underlying LPA -induced expression of these cytokines. LPA stimulated the transcriptional activity of the IL-8 gene with little effect on IL-8 mRNA stability. The optimal response of the IL-8 gene promoter to LPA relied on binding sites for NF-κB and AP-1, two transcription factors that were strongly activated by LPA in ovarian cancer cell lines. Positive regulators of the NF-κB and AP-1 pathways synergistically activated the IL-8 gene promoter. Further, the effect of LPA on IL-6 and IL-8 generation is mediated by the Edg LPA receptors as enforced expression of LPA receptors restored LPA-induced IL-6 and IL-8 production in non-responsive cells and enhanced the sensitivity to LPA in responsive cell lines. The LPA2 receptor was identified to be the most efficient in linking LPA to IL-6 and IL-8 production although LPA1 and LPA3 were also capable of increasing the response to a certain degree. These studies elucidate the transcriptional mechanism and the Edg LPA receptors involved in LPA-induced IL-6 and IL-8 production and suggest potential strategies to restrain the expression of these cytokines in ovarian cancer.

Role of LPA4/p2y9/GPR23 in negative regulation of cell motility.

Lysophosphatidic acid (LPA) is a ligand of multiple G protein-coupled receptors. The LPA(1-3) receptors are members of the endothelial cell differentiation gene (Edg) family. LPA(4)/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA(5)/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa(4) in mice. Although LPA(4)-deficient mice displayed no apparent abnormalities, LPA(4)-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA(4), LPA(4) deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA(4) converted LPA(4)-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA(4) strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA(1) in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA(4) attenuated LPA(1)-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA(4) is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.

Autotaxin/Lysopholipase D and lysophosphatidic acid regulate murine hemostasis and thrombosis

The lipid mediator lysophosphatidic acid (LPA) is a potent regulator of vascular cell function in vitro, but its physiologic role in the cardiovasculature is largely unexplored. To address the role of LPA in regulating platelet function and thrombosis, we investigated the effects of LPA on isolated murine platelets. Although LPA activates platelets from the majority of human donors, we found that treatment of isolated murine platelets with physiologic concentrations of LPA attenuated agonist-induced aggregation. Transgenic overexpression of autotaxin/lysophospholipase D (Enpp2), the enzyme necessary for production of the bulk of biologically active LPA in plasma, elevated circulating LPA levels and induced a bleeding diathesis and attenuation of thrombosis in mice. Intravascular administration of exogenous LPA recapitulated the prolonged bleeding time observed in Enpp2-Tg mice. Enpp2+/- mice, which have ∼50% normal plasma LPA levels, were more prone to thrombosis. Plasma autotaxin associated with platelets during aggregation and concentrated in arterial thrombus, and activated but not resting platelets bound recombinant autotaxin/lysoPLD in an integrin-dependent manner. These results identify a novel pathway in which LPA production by autotaxin/lysoPLD regulates murine hemostasis and thrombosis and suggest that binding of autotaxin/lysoPLD to activated platelets may provide a mechanism to localize LPA production.

Cross-talk between LPA1 and Epidermal Growth Factor Receptors Mediates Up-regulation of Sphingosine Kinase 1 to Promote Gastric Cancer Cell Motility and Invasion

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.

Inhibition of calcium-independent phospholipase A2 suppresses proliferation and tumorigenicity of ovarian carcinoma cells.

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.

Lysophosphatidic Acid Is a Major Regulator of Growth-Regulated Oncogene α in Ovarian Cancer

Growth-regulated oncogene α (GROα), a member of the chemokine superfamily, is commonly expressed in transformed cells and contributes to angiogenesis and tumorigenesis. Here, we report that increased GROα levels are detected in the plasma and ascites of ovarian cancer patients. Ovarian cancer cell lines in culture express and secrete GROα. However, when they are starved in serum-free medium, ovarian cancer cells ceased producing GROα, suggesting that GROα is not constitutively expressed but rather is produced in response to exogenous growth factors in ovarian cancer cells. The prototype peptide growth factors present in serum such as platelet-derived growth factor, insulin-like growth factor I, and insulin do not stimulate GROα production by ovarian cancer cells. In contrast, lysophosphatidic acid (LPA), a glycerol backbone phospholipid mediator present in serum and ascites of ovarian cancer patients, is a potent inducer of GROα expression in ovarian cancer cell lines. Treatment of ovarian cancer cells with LPA leads to transcriptional activation of the GROα gene promoter and robust accumulation of GROα protein in culture supernatants. The action of LPA on GROα expression is mediated by LPA receptors, particularly the LPA2 receptor in that ectopic expression of these receptors restores the LPA-dependent GROα production in nonresponsive cells. Down-regulation of LPA2 expression by small interfering RNA (siRNA) in ovarian cancer cells desensitizes GROα production in response to LPA. The effect of serum on GROα production is also significantly decreased by siRNA inhibition of LPA2 expression. These studies identify LPA as a primary regulator of GROα expression in ovarian cancer. (Cancer Res 2006; 66(5): 2740-8)

Sp-1 and c-Myc Mediate Lysophosphatidic Acid–Induced Expression of Vascular Endothelial Growth Factor in Ovarian Cancer Cells via a Hypoxia-Inducible Factor-1–Independent Mechanism

Purpose: Lysophosphatidic acid (LPA), which is present in ascites of ovarian cancer patients, stimulates expression of vascular endothelial growth factor (VEGF). VEGF is essential for the development and abdominal dissemination of ovarian cancer. We examined how LPA drives VEGF expression to gain a better understanding of tumor angiogenesis under normoxic conditions. Experimental Design: ELISA, Northern blotting, immunoblotting, quantitative PCR, and promoter reporter analysis in combination with small interfering RNA and pharmacologic inhibitors were used to examine LPA-induced VEGF expression and the underlying mechanisms. Results: LPA stimulated expression of multiple VEGF variants. A 123-bp fragment proximal to the transcriptional initiation site was identified to be functional promoter region responsible for the response to LPA. The fragment harbors consensus sites for several transcription factors including c-Myc and Sp-1 but not hypoxia-inducible factor-1. Blockade of Rho, ROCK, or c-Myc reduced LPA-dependent VEGF production and promoter activation, suggesting that the G12/13-Rho-ROCK-c-Myc cascade partially contributes to VEGF induction by LPA. More significantly, the multiple Sp-1 sites within the responsive region of the VEGF promoter were essential for LPA-mediated transcription. LPA induced Sp-1 phosphorylation and DNA-binding and transcriptional activities. The silencing of Sp-1 expression with small interfering RNA or inhibition of Sp-1 with pharmacologic inhibitors blocked VEGF production induced by LPA. Conclusions: LPA stimulates hypoxia-inducible factor-1-independent VEGF expression to promote tumor angiogenesis through activation of the c-Myc and Sp-1 transcription factors.

Glycogen Synthase Kinase 3β Is a Negative Regulator of Growth Factor-induced Activation of the c-Jun N-terminal Kinase

The c-Jun N-terminal kinase (JNK)/stress activated protein kinase is preferentially activated by stress stimuli. Growth factors, particularly ligands for G protein-coupled receptors, usually induce only modest JNK activation, although they may trigger marked activation of the related extracellular signal-regulated kinase. In the present study, we demonstrated that homozygous disruption of glycogen synthase kinase 3β (GSK-3β) dramatically sensitized mouse embryonic fibroblasts (MEFs) to JNK activation induced by lysophosphatidic acid (LPA) and sphingosine-1-phosphate, two prototype ligands for G protein-coupled receptors. To a lesser degree, a lack of GSK-3β also potentiated JNK activation in response to epidermal growth factor. In contrast, the absence of GSK-3β decreased UV light-induced JNK activation. The increased JNK activation induced by LPA in GSK-3β null MEFs was insufficient to trigger apoptotic cell death or growth inhibition. Instead, the increased JNK activation observed in GSK-3β–/– MEFs was associated with an increased proliferative response to LPA, which was reduced by the inhibition of JNK. Ectopic expression of GSK-3β in GSK-3β-negative MEFs restrained LPA-triggered JNK phosphorylation and induced a concomitant decrease in the mitogenic response to LPA compatible with GSK-3β through the inhibition of JNK activation, thus limiting LPA-induced cell proliferation. Mutation analysis indicated that GSK-3β kinase activity was required for GSK-3β to optimally inhibit LPA-stimulated JNK activation. Thus GSK-3β serves as a physiological switch to specifically repress JNK activation in response to LPA, sphingosine-1-phosphate, or the epidermal growth factor. These results reveal a novel role for GSK-3β in signal transduction and cellular responses to growth factors.