title: Connexins act as tumor suppressors in three-dimensional mammary cell organoids by regulating differentiation and angiogenesis.
abstract: 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 two-dimensional 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 angiogenesis-linked 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.
authors.affiliation: Departments of Anatomy and Cell Biology and Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McLachlan
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.forename: Hong-Ling
authors.initials: HL
authors.lastname: Wang
authors.forename: Stephanie
authors.initials: S
authors.lastname: Langlois
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D000072259:Connexin 26; D018031:Connexin 43 / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D017630:Connexins / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D004847:Epithelial Cells / Q000473:pathology; D017629:Gap Junctions / Q000378:metabolism; D006801:Humans; D008648:Mesoderm / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology; D014183:Transplantation, Heterologous
chemical_list: D018031:Connexin 43; D017630:Connexins; C500216:GJA1 protein, human; C000607042:GJB2 protein, human; C000607043:Gjb2 protein, mouse; D000072259:Connexin 26