Cell Death Regulation in Lumen Formation and Oncogenesis

描述（由申请人提供）：完整腺体结构内的中空管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还知之甚少。我们已经开发了一种体外三维（3D）培养模型，以研究乳腺癌发生早期腺上皮结构（腺泡）的结构和生长特性的改变。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。我们已经确定了两个促凋亡蛋白，参与介导细胞死亡，在这个模型中：Bim，促凋亡BH 3的Bcl-2家族蛋白，和TRAIL，死亡配体。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。该提案描述了计划，以阐明机制，调节细胞死亡诱导的Bim和TRAIL在管腔形成和失巢凋亡，确定其他蛋白质，配合Bim介导的细胞死亡，并确定致癌基因的机制，使细胞逃避这些凋亡介质诱导的细胞死亡。本研究应提供与人类原位癌发生相关事件的重要信息。此外，了解癌基因利用的分子事件和细胞通路，使生存在管腔空间可能最终是有价值的治疗策略的设计，因为针对这些途径可能会促进肿瘤细胞的选择性死亡。促凋亡Bcl-2家族蛋白由两个亚家族组成：仅BH 3和多结构域Bcl-2样蛋白。第一个目标中的研究将从这两类参与管腔凋亡和失巢凋亡的促凋亡Bcl-2家族蛋白中鉴定蛋白，定义它们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，我们还将研究癌基因如何从这些蛋白质调控的死亡过程中逃脱。TRAIL是死亡配体家族成员，其在3D培养物中的腺泡形态发生期间被诱导，并且其诱导在时间上与未来管腔空间中凋亡细胞的出现相关。TRAIL信号传导的消除对初始管腔形成没有影响，但抑制在管腔形成后增殖到管腔空间中的细胞的凋亡。在目的II中，我们将检查如果通过表达截短的显性抑制性TRAIL受体阻断TRAIL途径，表达增殖性癌基因如细胞周期蛋白D、HPVE 7和Wntl的细胞是否能够免于管腔细胞死亡，所述细胞不能在管腔中存活。此外，我们还将研究基质附着细胞对TRAIL的不同敏感性的基础，以及允许细胞填充管腔的癌基因是否逃避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.