Connexins are tumor suppressors, and human breast connexin 26 (Cx26) and connexin 43 (Cx43) gap junctions are often down-regulated in breast cancer. We previously showed that Cx26 and Cx43 overexpressed in MDA-MB-231 breast cancer cells inhibited tumor growth in vivo but not in twodimensional cultures. In the current study, we show that overexpression of Cx26 or Cx43 has tumor-suppressive properties in a three-dimensional environment such that they reduced anchorage-independent cell growth and induced partial redifferentiation of three-dimensional organoids of MDA-MB-231 cells. Importantly, the majority of exogenous connexins did not localize to the cell-cell interface or rescue gap junctional intercellular communication (GJIC) as assessed by dye transfer, providing evidence of a GJIC-independent mechanism of mammary tumor suppression. To further elucidate the mechanisms involved in connexin-induced three-dimensional redifferentiation of tumor cells, we examined whether connexin expression has a role in epithelial to mesenchymal transition (EMT). Cx26 and Cx43 reduced cell migration, increased cytokeratin 18 expression, and decreased vimentin levels, indicating a shift from a mesenchymal towards an epithelial phenotype. In addition, we examined the role of connexins in angiogenesis by probing an angiogenesis antibody array with conditioned media from three-dimensional MDA-MB-231 cultures. This revealed that connexin overexpression regulated various angiogenesislinked proteins. Furthermore, secreted factors from connexin overexpressing cells inhibited endothelial cell tubulogenesis and migration, and xenografts of Cx43 overexpressing MDA-MB-231 cells showed reduced tumor angiogenesis. In summary, Cx26 and Cx43 inhibit the malignant properties of MDA-MB-231 cells via GJIC-independent mechanisms, including regulation of EMT and angiogenesis. (Cancer Res 2006; 66(20): 9886-94)

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Membrane type 1-matrix metalloproteinase (MT1-MMP) has been suggested to play an important role in angiogenesis, but the mechanisms involved remain incompletely understood. Using an in vitro model of angiogenesis in which cell migration of bovine aortic endothelial cells (BAECs) and their morphogenic differentiation into capillary-like structures on Matrigel are induced by overexpression of MT1-MMP, we show that the plateletderived bioactive lipid sphingosine 1-phosphate (S1P) is the predominant serum factor essential for MT1-MMPdependent migration and morphogenic differentiation activities. In the presence of S1P, MT1-MMP-dependent cell migration and morphogenic differentiation were inhibited by pertussis toxin, suggesting the involvement of G i -protein-coupled receptor-mediated signaling. Accordingly, cotransfection of BAECs with MT1-MMP and a constitutively active G␣ i2 (Q205L) mutant increased cell migration and morphogenic differentiation, whereas treatment of BAECs overexpressing MT1-MMP with antisense oligonucleotides directed against S1P 1 and S1P 3 , the predominant S1P receptors, significantly inhibited both processes. These results demonstrate that MT1-MMP-induced migration and morphogenic differentiation involve the cooperation of the enzyme with platelet-derived bioactive lipids through S1P-mediated activation of G␣ icoupled S1P 1 and S1P 3 receptors. Given the important contribution of platelets to tumor angiogenesis, the stimulation of endothelial MT1-MMP function by S1P may thus constitute an important molecular event linking hemostasis to angiogenesis. IntroductionAngiogenesis, the formation of new vessels from pre-existing endothelium, is an essential component of pathologic conditions such as rheumatoid arthritis, diabetic retinopathies, inflammation, arteriosclerosis and tumor growth, and metastasis. 1 Tumor angiogenesis is an extremely complex process in which tumor cells secrete a number of stimulatory cytokines, such as vascular endothelial growth factor (VEGF), 2 that induce proliferation, migration, and survival of the endothelial cells (ECs). 3 VEGF is also a potent inducer of vascular permeability, resulting in the extravasation of plasma fibrinogen in the extravascular space and in the formation of a cross-linked fibrin provisional matrix that is essential for neovascularization. 4 Since new vessels are highly permeable, fibrin is continuously present into the tumor stroma and reflects the activation of the coagulation system, 5 suggesting an important regulatory function of the hemostatic system in angiogenesis and tumor growth. Such a close association between human cancer and hemostasis is illustrated by the observation that cancer coagulopathies occur in more than 80% of patients with disseminated cancer and account for a significant percentage of the morbidity and mortality of this disease. 6,7 The mechanisms involved in the pathogenesis of hemostatic disorders in cancer remains, however, incompletely understood but likely involve alterations in the function of some components ...

Background: Pannexins functions in skeletal myogenesis are unknown.Results: Panx1 and Panx3 species are co-expressed in skeletal muscle. Their levels are modulated during myoblast differentiation regulating either myoblast proliferation and/or differentiation status.Conclusion: Panx1 and Panx3 channels are novel regulators of skeletal muscle myoblast differentiation and proliferation.Significance: Skeletal muscle development and health depend on functional pannexin channels.