Abstract 336: CD73 induces actin polymerization to protect epithelial cell-cell adhesions: Loss of this physiological reflex in endometrial carcinoma

Abstract

Abstract We previously reported that ecto-5′nucleotidase (CD73)-generated adenosine prevents hypoxia-mediated breakdown of the epithelial barrier in normal endometrium in vivo and that CD73 is down-regulated in advanced stage endometrial carcinomas (EC). Loss of CD73 occurred specifically in the carcinoma cells. CD73-generated adenosine is heightened in tissues overwhelmed by inflammation, ischemia, or hypoxia. We hypothesized that the loss of CD73 in EC supports tumor progression by negating the reflex of extracellular adenosine to protect epithelial cell integrity. CD73 was associated with epithelial differentiation in vitro in mesenchymal-to-epithelial transitioning cells. Well-differentiated EC cells showed localization of CD73 to cell-cell contacts. Transmission electron microscopy (TEM) together with enzyme histochemistry revealed CD73 was localized to filopodia at cell-cell contacts. CD73 activity also associated with membrane zippers, a filopodia-based mechanism for forming cell-cell adhesions. CD73 was strongly associated with membrane F-actin. CD73 inhibitor, α, β-methylenediphosphate (AoPCP), reduced total F-actin in hypoxic (1% O2, 5% CO2) EC cells. Phalloidin staining showed AoPCP was specifically reducing membrane F-actin. EC cells expressed two adenosine receptors, A1R and A2BR. Inhibition of A1R by the receptor antagonist, DPCPX, decreased total F-actin. A1R antagonism reduced membrane F-actin. TEM showed A1R antagonism shortened the length of cell-cell filopodia. N(6)-cyclopentyladenosine (CPA), an A1R agonist, increased total F-actin and was membrane specific. This led to the assembly of the actin-related protein 2/3 complex, an actin polymerization complex of filopodia. Rho GTPase, cell division control protein 42 (Cdc42) co-immunoprecipitated with neuronal Wiskott-Aldrich syndrome protein (N-WASP). CPA increased E-cadherin, β-catenin and Na+K+ ATPase in membrane fractions. EC cell migration and invasion was increased by CD73 siRNA and AoPCP and was reversed by 5′-N-ethylcarboxamidoadenosine, an adenosine receptor analog. In summary, we show that CD73-generated adenosine induces an important physiological reflex, inherent to non-neoplastic epithelial cells, to re-form cell-cell adhesions in response to hypoxic stress. Epithelial cells are inherently programmed to maintain cell-cell adhesions, and like other epithelial characteristics this reflex must be down-regulated for tumor progression. Understanding the biology of extracellular adenosine in tumors has the potential to contribute to the discovery of new therapeutic targets, as agonists and antagonists of adenosine receptors are currently being investigated in clinical trials of non-cancer diseases. (NIH 1P50CA098258-01) Citation Format: Jessica L. Bowser, Michael R. Blackburn, Kenneth Dunner, Russell R. Broaddus. CD73 induces actin polymerization to protect epithelial cell-cell adhesions: Loss of this physiological reflex in endometrial carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2015-336