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A novel laminin-induced LPA autocrine loop in the migration of ovarian cancer cells ¹

SAUBHIK SENGUPTA^*, YI-JIN XIAO^* and YAN XU^*,,2

^* Department of Cancer Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, and
Department of Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio, USA

²Correspondence: Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: xuy{at}ccf.org

SPECIFIC AIMS

We examined the effects of different extracellular matrix (ECM) proteins and lysophosphatidic acid (LPA) on the migration of ovarian cancer cells. We observed that laminin, but not other ECMs, stimulated the production of LPA from ovarian cancer cells; the role of LPA receptors, G_i protein, PI3K, ß₁ integrin, iPLA₂, and cPLA₂ in LPA production and cell migration was investigated.

PRINCIPAL FINDINGS

1. LPA induced differential migration of HEY ovarian cancer cells toward different ECMs
We observed that laminin and, to a lesser extent, collagen I, collagen IV, and vitronectin (but not fibronectin) stimulated haptotaxis (directed migration toward an insoluble substrate-bound protein) of HEY ovarian cancer cells. 18:1-acyl LPA stimulated HEY cell migration to five major ECM proteins, with the order of potency (number of cells migrated) laminin > collagen I > collagen IV > vitronectin = fibronectin. The LPA-stimulated HEY cell migration through laminin was mainly via chemotaxis, since maximal cell migration was observed when LPA was present as a chemoattractant only in the lower chamber of the Transwell cells.

2. ECM induced haptotaxis and LPA-induced cell migration were pertussis toxin (PTX) sensitive
LPA-induced cell migration to ECMs was PTX-sensitive. Surprisingly, the haptotactic activities of all four ECMs (fibronectin, vitronectin, collagen IV, and laminin) tested in the absence of LPA were also sensitive to PTX. In contrast, cell adhesion to vitronectin and collagen I was not affected by PTX. Adhesion of HEY cells to other ECMs, including laminin, was very weak and was not studied further.

3. Laminin, but not other ECMs, stimulated LPA production from HEY ovarian cancer cells
We hypothesized that the PTX sensitivity of laminin-induced haptotaxis is conferred through a cross-talk between a laminin-activated integrin receptor with a G_i protein-coupled G-protein-coupled receptor (GPCR), such as an LPA receptor. This cross-talk could be mediated via the production of the ligand (such as LPA) for the GPCR. We indeed found that laminin, but no other ECMs, stimulated an average 17- and 4-fold increase of 18:1 and total LPA, respectively. We found that in HEY cells, laminin preferentially stimulated LPA with unsaturated fatty acids (such as 18:1 and 18:2 LPAs).

4. ß₁ integrin and iPLA₂ were required for laminin-induced LPA production and haptotaxis of HEY ovarian cells
A ß₁ neutralizing antibody significantly blocked the laminin-induced production of 18:1-, 18:2-, and total LPA (Fig. 1 A). HELSS [(E)-6-(bromomethylene)-3-(1-napthalenyl(-2H-tetrahydropyran-2-one)); or haloenol lactone suicide substrate], a highly specific inhibitor for iPLA₂, but not n-butanol (an inhibitor of phospholipase D) or GF109203X (a protein kinase C inhibitor), significantly inhibited the LPA production (Fig. 1A ). AACOCF₃ (an inhibitor of both iPLA₂ and cPLA₂) also inhibited the production of LPA induced by the laminin–integrin interaction in HEY cells. The inhibitors (the neutralizing antibody against ß₁ integrin and HELSS) that blocked LPA production also blocked laminin-induced cell migration (Fig. 1B, C ). Neutralizing antibodies against ß₃ or ß₄ integrins did not block cell migration (Fig. 1B ). This blockage could be reversed by the addition of exogenous LPA. AACOCF₃, on the other hand, inhibited laminin-induced cell migration in a manner similar to that of HELSS, but this blockage cannot be reversed by the addition of exogenous LPA (Fig. 1D ).

View larger version (34K): [in this window] [in a new window]   Figure 1. ß1 integrin and iPLA2 are required for laminin-induced LPA production and haptotaxis. A) Cells were pretreated with different reagents, added to laminin-coated plates, and LPA analyses were conducted. A ß1 functional blocking antibody (ß1 integrin-Ab) was added to HEY cells at a concentration of 1 µg/500,000 cells/0.5 mL. Other reagents used were n-butanol (1%), iso-butanol (1%), GF109203X (GFX, 5 µM), HELSS (1 µM), and AACOCF3 (100 µM). Cells were pretreated with these reagents/inhibitors for 30 min. B) HEY cells were pretreated with neutralizing antibodies against ß1, ß3, and ß4 integrins and cell migration assays were conducted. C) HEY cells were pretreated with different concentrations of HELSS as indicated. Cell migration to laminin in the presence or absence of LPA (20 µM) was analyzed. D) HEY cells were pretreated with different concentrations of AACOCF3. Data were summarized from 3 independent experiments. ***P <0.001; **P <0.01 (Student’s ttest).

5. PI3K is required for LPA- and laminin-induced cell migration, and G_i and PI3K are required for activation of cPLA₂
Using pharmacological inhibitors (LY294002) and genetic tools (the constitutive activated and dominant negative forms of PI3K), we found that PI3K activation was necessary for laminin- and LPA-induced cell migration. Assessed by phosphorylation, we found that cPLA₂ was activated when cells were in contact with laminin and that this activation was blocked by pretreatment with PTX and LY294002.

6. PTX-sensitive cells in laminin-induced haptotaxis produced LPA
Laminin induced haptotaxis in seven additional cell lines tested (three ovarian cancer cell lines: OCC1, Ovca432, and Ovca420; four breast cancer cell lines: GI-101A, T47D, MDA-MB-231, and MCF7). Laminin-induced haptotaxis in OCC1 and ovca432 was PTX sensitive, similar to that observed in HEY cells. In contrast, laminin-induced haptotaxis in ovca420, T47D, MDA-MB231, and MCF7 cells was PTX insensitive. Whereas OCC1 and ovca432 cells respond to LPA in cell migration, the PTX-insensitive cells (ovca420, T47D, MDA-MB231, and MCF7) did not. GI101A cells were partially sensitive to PTX in laminin- and LPA-induced cell migration and responded weakly to LPA in cell migration assays. All PTX-sensitive cell lines (HEY, OCC1, ovca432, and GI101A) produced increased amounts of 18:1-,18:2-, and total LPAs when they came in contact with laminin.

7. LPA₃, but not LPA₁ or LPA₂, was necessary for LPA-induced ovarian cancer cell migration
All four LPA nonresponsive cell lines (ovca420, T47D, MDA-MB-231, and MCF7) express LPA₁ and LPA₂, but not LPA₃. All LPA-responsive cell lines, including HEY, OCC1, ovca432, and GI101A, express LPA₃, although GI-101A expresses very low levels or no LPA₁ and OCC1 expresses low levels of LPA₂. These data imply that LPA₃, but not LPA₁ or LPA₂, plays an important role in LPA-induced cell migration. Transfecting LPA₃ into ovca420 cells enabled them to respond to LPA in cell migration assays and this LPA-induced cell migration was inhibited by PTX.

CONCLUSION AND SIGNIFICANCE

LPA is an important bioactive lipid molecule. We have shown that LPA is elevated in ascites and blood from patients with ovarian cancer. LPA is potentially involved in virtually all important aspects of tumor development, including cell proliferation, survival, angiogenesis, and migration. However, the production and signaling pathways of LPA under pathophysiological conditions are still poorly understood. We describe here for the first time that laminin, an important ECM molecule, appears to be a physiological stimulus that induces LPA production in ovarian cancer cells. More important, we provide evidence that LPA produced in such a way is a mediator of cell migration induced by laminin in the same cells via activating its own receptor. We have demonstrated an autocrine loop of LPA in HEY ovarian cancer cells that is potentially involved in tumor metastasis.

Integrins and G-protein-coupled receptors (GPCRs) play pivotal roles in mediating cell migration stimulated by extracellular signaling molecules. We present data here to show a novel mechanism of cross-talk between a ß₁ integrin and a GPCR (via production of a GPCR ligand). Although similar cross-talk mechanisms have been shown between receptor tyrosine kinases and GPCRs, to our knowledge this is the first report of such a mechanism for the cross-talk between an integrin receptor and a GPCR. The specific integrin involved in LPA production needs to be identified and the mechanisms of other ECMs-induced PTX-sensitive haptotaxis remain to be determined.

The role of individual LPA receptors in cell migration had not been clearly elucidated before. We demonstrate here that LPA₃ plays a unique role in LPA-induced cell migration. This notion is supported by our transfection studies and observation that laminin selectively stimulates the production of LPA with unsaturated fatty acid chains (18:1 and 18:2 LPA) from ovarian cancer cells. LPA₃, but not LPA₁ or LPA₂, has a preference for LPAs with unsaturated fatty acids. We have shown that increased LPA species with unsaturated fatty acid chains may be associated with late-stage or recurrent ovarian cancer and are more potent in stimulating cell proliferation and migration. Furthermore, malignant ovarian tissues express markedly elevated levels of LPA₃ (but not LPA₁). The importance of LPA and LPA receptors in ovarian cancer development warrants further study. The molecular mechanisms underlying the receptor subtype specificity remain to be investigated. We have shown here that an iPLA₂ is likely to be involved in LPA production. The mechanisms by which an integrin activates iPLA₂ remain to be determined. G_i and PI3K are both required for cell migration in ovarian cancer cells. cPLA₂ appears to be a downstream effector of the G_i-PI3K pathway. Taken together, we reveal a novel autocrine loop in the migration of ovarian cancer cells and identify pathways leading to LPA production and cell migration (Fig. 2 ). The pathophysiological importance of these findings remains to be determined by in vivo studies.

View larger version (19K): [in this window] [in a new window]   Figure 2. A schematic of the laminin-induced LPA autocrine loop in migration of HEY cells. When ovarian cancer cells come in contact with laminin, a ß1 integrin is activated. This activation leads to LPA production via iPLA2 activity. LPA produced and secreted extracellularly stimulates its receptor, LPA3, which is coupled to a Gi protein. This leads to the activation of PI3K, which is upstream of cPLA2. Gi protein, PI3K, and cPLA2, but not Akt, are required for LPA-induced cell migration.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1145fje; doi: 10.1096/fj.02-1145fje

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