Mechanisms Involved in Mammary Morphogenesis

在完整的腺体结构内填充空腔是早期肿瘤发生的标志。然而，在原位癌的发展过程中，腺腔的形成及其充盈的机制还知之甚少。我们已经建立了一种体外三维(3D)培养模型来研究腺上皮结构(Acini)在乳房早期的构筑和生长特性的变化。 致癌。我们发现，腔的形成涉及腺泡中心细胞的选择性死亡。由癌基因引发的腔间隙的填充，不仅需要诱导结构性增殖，还需要抗凋亡信号，使细胞能够在腔间隙存活。我们已经确定促凋亡的Bcl-2蛋白Bim是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的调节 逃脱了流明空间中的死亡。BIM也被发现通过单层细胞从基质中分离(称为失巢凋亡)诱导并关键地参与了诱导的细胞凋亡，一些证据表明基质剥夺可能参与了形态发生过程中腔细胞的死亡。这项提案描述了计划阐明在管腔形成和失巢凋亡过程中BIM诱导细胞死亡的机制，识别 与Bim协作以介导细胞死亡的其他蛋白质，并确定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。这项研究应该提供与人类原位癌发展相关的事件的重要信息。这些研究应该提供与人类原位癌发展相关的事件的重要信息。 为了研究Bim在失巢失巢、管腔形成和肿瘤形成过程中的调节作用，将包括在两种模型中研究Bim诱导和激活的机制，分析Bim是如何被癌基因下调或失活的，以及阐明在附着于基质的腺泡细胞中保护乳腺上皮细胞免受Bim诱导死亡的基础。鉴定其他促凋亡蛋白将涉及亲和分离技术以及导致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查来识别额外的蛋白质，这些蛋白质可以保护细胞免受脱离引起的死亡或腺泡腔的清除。最后，我们将检查我们在永生化的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.