Mechanisms Involved in Mammary Morphogenesis

在完整的腺体结构内填充中空腔是早期肿瘤发生的标志。然而，在原位癌的发展过程中，腺腔的形成及其填充的机制还知之甚少。我们建立了一个体外三维（3D）培养模型，以研究乳腺癌早期腺上皮结构（腺泡）的结构和生长特性的变化。 致癌作用我们发现，腔的形成涉及腺泡中心细胞的选择性死亡。由癌基因引起的管腔空间的填充不仅需要诱导组成性增殖，还需要允许细胞在管腔空间中存活的抗凋亡信号。我们已经鉴定了促凋亡Bcl-2蛋白Bim作为管腔形成过程中细胞凋亡的关键介质，并发现其表达受癌基因调控 在管腔空间中逃脱死亡。还发现Bim被诱导并严重参与由单层细胞从基质中脱离诱导的细胞凋亡（称为失巢凋亡的过程），并且几条证据表明，基质剥夺可能参与形态发生期间的管腔细胞死亡。该提案描述了阐明Bim在管腔形成和失巢凋亡期间诱导的细胞死亡的调节机制的计划， 与Bim合作介导细胞死亡的其他蛋白质，并确定致癌基因允许细胞逃避这些凋亡介导物诱导的细胞死亡的机制。本研究应提供与人类原位癌发生相关事件的重要信息。这些研究应提供与人类原位癌发展相关事件的重要信息。 研究Bim在失巢凋亡、管腔形成和肿瘤发生过程中的调节将涉及两种模型中Bim诱导和激活机制的研究，Bim如何被癌基因下调或失活的分析，以及阐明保护乳腺上皮细胞免受Bim诱导的死亡的基础，这些死亡在附着于基质的腺泡细胞中起作用。其他促凋亡蛋白的鉴定将涉及亲和分离技术以及诱导其他促凋亡蛋白功能丧失的策略。我们将使用逆转录病毒基因筛选，以确定额外的蛋白质，保护细胞脱离诱导死亡或腔清除腺泡。最后，我们将检查我们在永生化MCF-10A细胞中定义的过程是否在原代人类细胞中起作用。

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.