Cell Death Regulation in Lumen Formation and Oncogenesis

描述（由申请人提供）：完整腺体结构内的空腔填充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尚不清楚。我们开发了一个体外三维（3D）培养模型来研究乳腺癌变早期腺上皮结构（acini）的结构和生长特性的改变。我们发现管腔的形成与腺泡中心细胞的选择性死亡有关。由癌基因引起的腔隙填充不仅需要诱导组成性增殖，还需要抗凋亡信号，使细胞能够在腔隙中存活。我们在该模型中发现了两种参与介导细胞死亡的促凋亡蛋白：Bim，一种促凋亡的仅bh3 - Bcl-2家族蛋白，和TRAIL，一种死亡配体。研究还发现，Bim被诱导并关键参与了由单层细胞从基质中脱离引起的细胞凋亡（这一过程被称为anoikis），有几条证据表明，基质剥夺可能参与了形态发生期间的腔细胞死亡。该提案描述了阐明在管腔形成和凋亡过程中由Bim和TRAIL诱导的细胞死亡调节机制的计划，确定与Bim合作介导细胞死亡的其他蛋白质，并确定癌基因允许细胞逃避由这些凋亡介质诱导的细胞死亡的机制。这项研究应该提供与人类原位癌肿瘤发展相关事件的重要信息。此外，了解致癌基因利用的分子事件和细胞途径，使其在管腔空间中存活，最终可能对设计治疗策略有价值，因为靶向这些途径可能促进肿瘤细胞的选择性死亡。促凋亡bcl -2家族蛋白包括两个亚家族：仅bh3和多结构域bax样蛋白。第一个Aim的研究将从这两类促凋亡Bcl-2家族蛋白中识别出参与细胞凋亡和细胞凋亡的蛋白，确定其在这些死亡过程中的作用，并追踪导致其激活或抑制的途径。此外，我们将研究癌基因如何从这些蛋白调控的死亡过程中逃脱。TRAIL是死亡配体家族成员，在3D培养的腺泡形态发生过程中被诱导，其诱导与未来管腔内凋亡细胞的出现具有时间相关性。取消TRAIL信号对初始管腔形成没有影响，但会抑制管腔形成后增殖到管腔空间的细胞凋亡。在Aim II中，我们将研究表达增殖癌基因如细胞周期蛋白D、HPVE7和Wntl等不能在管腔中存活的细胞，如果TRAIL通路被截断的显性抑制性TRAIL受体的表达阻断，是否能保护细胞免于管腔细胞死亡。此外，我们将研究基质附着细胞对TRAIL差异敏感性的基础，以及允许细胞填充管腔的癌基因是否能逃避TRAIL诱导的细胞死亡。

DESCRIPTION (provided by applicant): 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 two proapoptotic proteins that are involved in mediating cell death in this model: Bim, a proapoptotic BH3-only Bcl-2 family protein, and TRAIL, a death ligand. 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 and TRAIL 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. In addition, an understanding of the molecular events and cellular pathways utilized by oncogenes to allow survival in the luminal space may ultimately be valuable in the design of therapeutic strategies because targeting such pathways may promote selective death of tumor cells. Proapoptotic Bcl-2-family proteins are comprised of two subfamilies: BH3-only and multidomain Bax-like proteins. The studies in the first Aim will identify proteins from these two classes of proapoptotic Bcl-2 family proteins that are involved in luminal apoptosis and anoikis, define their role of in these death processes, and trace the pathways leading to their activation or inhibition. In addition, we will examine how oncogenes also escape from death processes regulated by these proteins. TRAIL is death-ligand family member that is induced during acinar morphogenesis in 3D cultures and its induction correlates temporally with the appearance of apoptotic cells in the future luminal space. Abrogation of TRAIL signaling has no effect on initial lumen formation but inhibits apoptosis of cells that proliferate into the luminal space after the lumen has formed. In Aim II, we will examine whether cells expressing proliferative oncogenes like cyclin D, HPVE7, and Wntl, that are unable to survive in the lumen, are protected from luminal cell death if TRAIL pathways are blocked by expression of truncated dominant inhibitory TRAIL receptors. In addition we will investigate the basis for the differential sensitivity of matrix-attached cells to TRAIL, and whether oncogenes that allow cells to fill the lumen escape TRAIL-induced cell death.