The filling of a hollow lumen within an intact glandular structure is a hallmark of early tumorigenesis. However the mechanisms involved in the formation of a glandular lumen and its filling during the development of carcinoma-in-situ are poorly understood. We have developed an in vitro three-dimensional (3D) culture model to investigate alterations in the architecture and growth properties of glandular epithelial structures (acini) during early stages of mammary carcinogenesis. We have found that formation of the lumen involves selective death of cells in the center of the acini. Filling of the luminal space, as provoked by oncogenes, requires not only induction of constitutive proliferation but also anti-apoptotic signals that allow cells to survive in the luminal space. We have identified the proapoptotic Bcl-2 protein Bim as critical mediator of apoptosis during lumen formation and found that its expression is regulated by oncogenes that escape death in the luminal space. Bim was also found to be induced and critically involved in apoptosis induced by detachment of monolayer cells from matrix (a process referred to as anoikis) and several lines of evidence suggest that matrix deprivation may be involved in luminal cell death during morphogenesis. This proposal describes plans to elucidate the mechanisms that regulate cell death induced by Bim during both lumen formation and anoikis, identify other proteins that collaborate with Bim to mediate cell death, and determine the mechanisms whereby oncogenes allow cells to escape cell death induced by these apoptotic mediators. This study should provide important information relating to events associated with the development of carcinoma-in-situ tumors in humans. These investigations should provide important information relating to events associated with the development of carcinoma-in-situ tumors in humans. Studies to examine the regulation of Bim during anoikis, lumen formation and oncogenesis will involve investigations of the mechanism involved in Bim induction and activation in both models, analysis of how Bim is downregulated or inactivated by oncogenes, and elucidation of the basis for protection of mammary epithelial cells from Bim-induced death operating in acinar cells that are attached to matrix. Identification of other proapoptotic proteins will involve affinity isolation techniques as well as strategies that induce loss of function of other pro-apoptotic proteins. We will use retroviral genetic screens to identify additional proteins that protect cells from detachment induced death or luminal clearing in acini. Lastly, we will examine whether the processes that we define in the immortalized MCF-10A cells are operative in primary human cells.
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