Autophagy and Epithelial Cell Fate During Anoikis and 3D Morphogenesis

描述(申请人提供)：自噬是一种严格控制的溶酶体降解过程，细胞在各种形式的胁迫下消化自己的内容。在真核细胞中，自噬主要起到营养耗竭或应激时的保护性反应的作用。总体而言，关于这一基本过程如何影响癌症的启动、进展和对治疗的反应，仍有许多有待了解。在这个提案中，我们将验证自噬通过两个机制影响上皮癌发展的假说-1)增强上皮细胞在ECM脱离诱导的应激和凋亡(ANOIKI)中生存的能力和2)限制增殖。这一假说是基于使用独特的3D上皮培养系统进行的初步研究，以检验致癌侮辱如何影响腺体结构中正常中空管腔的形成。在这个模型中，管腔的形成涉及中央细胞的选择性死亡，而不是与细胞外基质(ECM)直接接触。最近，我们发现在管腔形成过程中有高水平的自噬，这表明这一关键过程也与上皮细胞的命运有关。在随后的工作中，我们首次证实了自噬在细胞外基质剥夺(失巢)过程中被强烈诱导。基于这些初步数据，在目标1中，我们将评估自噬是否通过减轻失巢和3D管腔形成过程中细胞外基质脱离的压力来促进细胞存活。此外，我们还获得了自噬调节分子Beclin/ATG6(已知的肿瘤抑制因子)在3D培养中抑制细胞增殖的初步证据。在第二个目标中，我们将询问其他自噬调节因子(称为ATGs)是否在生长因子撤除和3D形态形成过程中抑制增殖，以明确地确定自噬可以抑制上皮细胞的增殖，并寻找能够抑制肿瘤细胞扩张的新的自噬调节因子。在目标3中，我们将描述调节自噬如何影响癌基因表达细胞的存活和增殖，以确定如何利用自噬来治疗在已知癌症途径中存在扰动的肿瘤细胞。总体而言，拟议的实验将提供关于自噬在人类癌症发生和发展过程中的确切作用的独特信息。最终，这些研究可能会导致新的治疗策略，通过操纵自噬来防止癌细胞的不受控制的扩张。

DESCRIPTION (provided by applicant): Autophagy is a tightly regulated lysosomal degradation process in which a cell digests its own contents under various forms of duress. In eukaryotic cells, autophagy primarily functions as a protective response during nutrient depletion or stress. Overall, much remains to be learned about how this fundamental process affects cancer initiation, progression and response to therapy. In this proposal, we will test the hypothesis that autophagy influences epithelial cancer development through two mechanisms-1) enhancing the ability of epithelial cells to survive ECM detachment-induced stress and apoptosis (anoikis) and 2) limiting proliferation. This hypothesis is based on initial studies using a unique 3D epithelial culture system to examine how oncogenic insults affect the formation of the normally hollow lumen in glandular structures. In this model, lumen formation involves the selective death of central cells lacking direct contact with extracellular matrix (ECM). Recently, we have discovered high levels of autophagy during lumen formation, indicating that this critical process also contributes to epithelial cell fate. In subsequent work, we are the first to establish that autophagy is strongly induced during ECM deprivation (anoikis). Based on this preliminary data, in Aim 1, we will evaluate whether autophagy promotes cell survival via mitigating the stresses of ECM detachment during anoikis and 3D lumen formation. In addition, we have obtained preliminary evidence that the autophagy regulatory molecule Beclin/ATG6 (a known tumor suppressor) inhibits cell proliferation in 3D culture. In Aim 2. we will interrogate whether other autophagy regulators (called ATGs) inhibit proliferation during growth factor withdrawal and 3D morphogenesis in order to definitively establish that autophagy can restrain epithelial proliferation and to identify new autophagy regulators that are able to inhibit neoplastic cell expansion. In Aim 3. we will delineate how modulating autophagy influences survival and proliferation in oncogene-expressing cells in order to determine how autophagy can be exploited to therapeutically target tumor cells harboring perturbations in known cancer pathways. Overall, the proposed experiments will provide unique information on the precise roles of autophagy during the development and progression of human carcinomas. Ultimately, these studies may lead to new treatment strategies to prevent the uncontrolled expansion of cancer cells via manipulating autophagy.