Cellular Mechanisms in Epithelial Glandular Morphogenesis and Preinvasive Cancer

Epithelial cancer progression is a consequence of deregulation of a number of cellular processes, and is accompanied by the perturbation of epithelial glandular architecture. The breakdown of adhesive contacts, internalization of cell-surface proteins, recycling of cargo back to the plasma membrane, and intracellular signaling are all events that are derailed during epithelial glandular disruption. Multiple platforms have emerged that enable the study of cellular events preceding and contributing to glandular disruption, as it pertains to early epithelial tumor progression. While investigations of monolayers provide insight into interactions between cells, the 3D morphogenesis culture system allows for further analysis of hyperactivated cellular signaling and its effects on acinar structure development, reminiscent of glandular remodeling during epithelial cancer progression. Ultimately, the studies made in vitro are validated with the use of mouse models to demonstrate the role of key signaling cascades in tumor development.

This dissertation contributes to the understanding of epithelial glandular morphogenesis by utilizing monolayers, 3D culture systems, and a mouse model of mammary ductal carcinoma in situ. In response to chronic Wnt stimulation, an observed characteristic of some tumor microenvironments, the small GTP-binding protein ARF6, promotes the internalization of cell surface proteins by employing distinct endocytic mechanisms in polarized epithelial cells. MCF10DCIS.com cells were used to study the role of these pathways in a model of Ductal Carcinoma in situ (DCIS). ARF6 knockdown or dominant inhibition of the protein results in striking alterations of the DCIS acinar phenotype and decreased cell proliferation in vitro. These defects are coupled with a marked perturbation of cargo recycling along the endocytic pathways. Finally, in a mouse mammary model of DCIS, ARF6 depletion or its inhibition results is profound changes to the development and progression of DCIS with pronounced reduction in tumor cell invasion. Taken together our studies suggest a vital role for ARF6 signaling and endocytic recycling in the onset and progression of mammary epithelial glandular cancers.