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Modeling mammary epithelial cell polarization and the role of podocalyxin in breast tumor progression Graves, Marcia Lynn

Abstract

The mammary gland consists of an organized network of epithelial ducts and lobules. This histoarchitecture can be recapitulated in vitro by culturing mammary epithelial cells as 3D spheroids embedded in a reconstituted basement membrane. I first used this assay to characterize the role of cell-cell and cell-ECM adhesion in the formation and polarization of the apical junction complexes in normal mammary epithelial cells. Cell-cell adhesion alone was sufficient to initiate polarized junction assembly. However, the addition of exogenous ECM generated a spatial polarity signal dependent on laminin-1 and α6 and β1 integrins. This caused clusters of mammary epithelial cells to re-localize the junctional complexes to the center of the spheroid prior to lumen formation. In ductal breast carcinoma, a critical hallmark is the loss of normal polarized tissue architecture without the induction of an epithelial-to-mesenchymal transformation (EMT). Thus, misregulation of molecules that function as polarity determinants may contribute to ductal tumor progression. Podocalyxin is an anti-adhesive glycoprotein that may be involved, as it is important in epithelial morphogenesis, and its overexpression in clinical breast tumors is associated with poor outcome. Despite this, overexpression of podocalyxin in normal mammary epithelial cells did not disrupt 3D morphogenesis or apicobasal polarity. However, its overexpression in non-metastatic breast tumor cells did perturb the architecture and growth of tumor spheroids in vitro and it facilitated subcutaneous tumor growth in vivo without causing an EMT. Mechanistically, podocalyxin localized to and expanded non-adhesive membrane domains and induced microvillus formation that was dependent on its extracellular domain and Rho GTPase-regulated actin polymerization. Podocalyxin also recruited its intracellular binding partners NHERF-1 and ezrin via its cytoplasmic tail. Strikingly, the formation of this protein complex was not required for microvillus formation. Additionally, podocalyxin delayed cell-cell aggregation and decreased the initial adhesion, spreading and strength of attachment of tumor cells to fibronectin where it restricted β1 integrin localization to the basal/attached domain. These alterations in adhesion possibly contributed to podocalyxin's ability to increase growth factor-dependent tumor cell migration. Altogether, these data indicate that podocalyxin overexpression may facilitate a ductal tumor-like progression that involves EMT-independent alterations in tissue architecture.

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