MDA-9/syntenin is essential for factor VIIa-induced signaling, migration, and metastasis in melanoma cells.

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, is a novel gene that positively regulates cancer cell motility, invasion, and metastasis through distinct biochemical and signaling pathways, but how MDA-9/syntenin is regulated in response to signals with the extracellular environment and promotes tumor progression is unclear. We now demonstrate that MDA-9/syntenin is dramatically up-regulated by a combination of rFVIIa and factor F(X) in malignant melanoma. Induction of MDA-9/syntenin in melanoma was found to occur in a thrombin-independent signaling pathway and involves the PAR-1/c-Src/Rho GTPases Rac1 and Cdc42/c-Jun N-terminal kinase axis resulting in the activation of paxillin, NF-κB, and matrix metalloproteinase-2 (MMP-2). MDA-9/syntenin physically interacts with c-Src through its PDZ binding motif following stimulation of melanoma cells with rFVIIa and FX. We also document that induction of this signaling pathway is required for TF·FVIIa·Xa-induced cell migration, invasion, and metastasis by melanoma cells. The present finding uncovers a novel role of MDA-9/syntenin as an important TF·FVIIa·Xa/PAR-1-regulated gene that initiates a signaling circuit essential for cell motility and invasion of metastatic melanoma. In these contexts, targeting TF·FVIIa·Xa and its relevant downstream targets such as MDA-9/syntenin, may represent a novel therapeutic strategy to control the evolution of neoplastic cells.

Phosphorylation of synucleins by members of the Polo-like kinase family.

Phosphorylation of alpha-synuclein (alpha-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating alpha-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate alpha-syn and beta-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate alpha-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate beta-syn at Ser-118, whereas no phosphorylation of gamma-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103-140) of alpha-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of alpha-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) alpha-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in alpha-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of alpha-syn and beta-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.

Bioactive lipids lysophosphatidic acid and sphingosine 1-phosphate mediate breast cancer cell biological functions through distinct mechanisms.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are structurally related bioactive lipids with growth factor-like activities. LPA and S1P are naturally produced in vivo by blood platelets upon platelet aggregation and at least in vitro by fibroblasts, adipocytes, and multiple types of tumor cells. Breast cancer cells respond to LPA and S1P. However, their specific actions on breast cancer cell biological functions remain unclear. We therefore conducted an in vitro side-by-side study of these two lipids on breast cancer cells. LPA mediates human breast cancer MDA-BO2 cell proliferation, migration, and invasion through activation of a G(alpha i)/ERK1/2-dependent signaling pathway, whereas activation of G(alpha i)/PI3K predominates upon S1P stimulation. In MDA-BO2 cells, LPA but not S1P activities were dependent on active type 1 insulin-like growth factor and epithelial growth factor receptors. LPA and S1P act directly on endothelial cells to induce angiogenesis. We demonstrate that LPA and S1P have indirect angiogenic properties as judged by induced secretion of angiogenic factors by breast cancer cells primed with these lysophospholipids. S1P, but not LPA, controlled the expression of VEGF-A by breast cancer cells, while LPA, but not S1P, controlled the expression of GM-CSF, Gro-alpha, MCP-1, and IL-6. According to the secretion of these paracrine osteoclastic factors, LPA, but not S1P, stimulates breast cancer cell-induced osteoclastogenesis. These findings suggest that, in vivo, LPA and S1P can coordinate their action on tumor and surrounding cells to induce breast cancer progression both at primary and bone metastatic sites.

Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer.

The role of lysophosphatidic acid (LPA) in cancer is poorly understood. Here we provide evidence for a role of LPA in the progression of breast cancer bone metastases. LPA receptors LPA(1), LPA(2), and LPA(3) were expressed in human primary breast tumors and a series of human breast cancer cell lines. The inducible overexpression of LPA(1) in MDA-BO2 breast cancer cells specifically sensitized these cells to the mitogenic action of LPA in vitro. In vivo, LPA(1) overexpression in MDA-BO2 cells enhanced the growth of subcutaneous tumor xenografts and promoted bone metastasis formation in mice by increasing both skeletal tumor growth and bone destruction. This suggested that endogenous LPA was produced in the tumor microenvironment. However, MDA-BO2 cells or transfectants did not produce LPA. Instead, they induced the release of LPA from activated platelets which, in turn, promoted tumor cell proliferation and the LPA(1)-dependent secretion of IL-6 and IL-8, 2 potent bone resorption stimulators. Moreover, platelet-derived LPA deprivation in mice, achieved by treatment with the platelet antagonist Integrilin, inhibited the progression of bone metastases caused by parental and LPA(1)-overexpressing MDA-BO2 cells and reduced the progression of osteolytic lesions in mice bearing CHO-beta3wt ovarian cancer cells. Overall, our data suggest that, at the bone metastatic site, tumor cells stimulate the production of LPA from activated platelets, which enhances both tumor growth and cytokine-mediated bone destruction.

The type 1 lysophosphatidic acid receptor is a target for therapy in bone metastases.

Platelet-derived lysophosphatidic acid (LPA) supports the progression of breast and ovarian cancer metastasis to bone. The mechanisms through which LPA promotes bone metastasis formation are, however, unknown. Here we report that silencing of the type 1 LPA receptor (LPA(1)) in cancer cells blocks the production of tumor-derived cytokines that are potent activators of osteoclast-mediated bone destruction and significantly reduces the progression of osteolytic bone metastases. Moreover, functional blockade of LPA action on its cognate receptor LPA(1) using a pharmacological antagonist mimics the effects of silencing LPA(1) in tumor cells in vitro and substantially reduces bone metastasis progression in animals. Overall, these results suggest that inhibition of platelet-derived LPA action on LPA(1) expressed by tumor cells may be a promising therapeutic target for patients with bone metastases.