Ras-Induced Autophagic Growth-Arrest in Immortalized Human Epithelial Cells

Ras 诱导永生化人上皮细胞自噬生长停滞

• 批准号: 8330969
• 负责人: Jose Gomez-Garcia
• 金额: $ 2.72万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-28 至 2012-06-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8330969
• 关键词: Apoptosis; Autophagocytosis; BRAF gene; Biological Models; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Death; Cell Line; Cell Lineage; Cells; Complex; Data; Defense Mechanisms; Epithelial Cells; Frequencies; Growth; Health; Human; Intervention; Knowledge; Lesion; MAP Kinase Gene; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mammalian Cell; Mammary gland; Mediating; Mutation; Oncogenic; Outcome; Pathway interactions; Phenotype; Preventive; RAS genes; Reaction; Signal Transduction; Staging; Telomerase; Testing; Tissues; Translation Initiation; Work; base; c-myc Genes; cell type; gain of function mutation; human FRAP1 protein; improved; malignant breast neoplasm; new therapeutic target; novel; promoter; public health relevance; response; senescence; small hairpin RNA; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Mammalian cells possess two major intrinsic mechanisms, apoptosis and senescence, to protect against uncontrolled proliferation when oncogenic mutations occur. To define whether telomerase-induced breaching of the senescence barrier would enable oncogenic Ras to promote uncontrolled cell cycle progression in cells that commonly (bronchial epithelial cells, BECs) or rarely (mammary epithelial cells, MECs) harbor oncogenic ras, we introduced oncogenic H-rasV12 into hTERT immortalized BECs and MECs. To our surprise, rather than promoting cell cycle transit, increased expression of Ras led to G1 cell cycle arrest and an autophagy-like phenotype in both cell types - a response that was independent of the canonical autophagy promoter beclin 1. During extended observation in culture (56 days), a subpopulation of H-RasV12 expressing cells reversed the autophagic phenotype, escaped from cell cycle blockade and formed multilayered foci. The data are in accord with a scenario in which activated Ras triggers at least two contradictory signals: one leading to oncogenesis and the second promoting non- canonical autophagy, cell cycle arrest and cell death. Here we propose 3 specific aims to test the hypothesis that that Ras-induced autophagic arrest and cell degradation represents a previously cryptic third line of defense against Ras oncogenesis that became apparent in cells that had breached the senescence barrier. We further hypothesize that the cell-lineage-dependent status of key cancer-related pathways determines whether Ras-arrested autophagic cells die - or overcome proliferative blockade and undergo malignant transformation. Aim1. Determine whether Ras-induced autophagic reactions are required for Ras- dependent cell cycle arrest by modulating key regulators of autophagy. Aim 2. Identify pathways downstream of activated Ras leading to autophagic cell cycle arrest by systematically inhibiting steps in the PI3K/Akt/mTOR and BRAF/MAPK/ERK pathways. Aim 3. Determine whether activation of c-Myc or eIF4F, suppression of p53 or p16Ink4 - or combinations of these oncogenic interventions - contribute to the cell-context dependent mechanism of escape from oncogenic Ras-mediated autophagy and cell cycle arrest. PUBLIC HEALTH RELEVANCE: Our work tests the idea that cells can activate a second line of defense, autopaghy, - besides senescence and apoptosis- against Ras oncogenesis, and pursues identifying the mechanism by which cancer escapes this checkpoint. This work provides the opportunity to study negative regulators of autophagy as a novel avenue of new targets for therapeutics or chemo-preventive agents to improve human health.

描述(申请人提供)：哺乳动物细胞有两个主要的内在机制，即凋亡和衰老，以防止在发生致癌突变时不受控制的增殖。为了确定端粒酶诱导的衰老屏障的破坏是否能够使致癌RAS在通常(支气管上皮细胞)或很少(乳腺上皮细胞)含有致癌RAS的细胞中促进不受控制的细胞周期进展，我们将致癌H-rasV12引入hTERT永生化的BECs和MECs中。令我们惊讶的是，RAS的表达增加非但没有促进细胞周期的传递，反而导致两种细胞的G1期细胞周期停滞和自噬样表型--这一反应不依赖于典型的自噬启动子Beclin 1。在培养的长期观察中(56天)，表达H-RasV12的细胞亚群逆转了自噬表型，逃脱了细胞周期阻断并形成了多层焦点。这些数据与激活的RAS触发至少两个相互矛盾的信号的情景是一致的：一个导致肿瘤发生，另一个促进非典型自噬、细胞周期停滞和细胞死亡。在这里，我们提出了3个特定的目标来检验这一假设，即RAS诱导的自噬停滞和细胞降解代表了先前神秘的对抗RAS癌发生的第三道防线，这种防线在已经突破衰老屏障的细胞中变得明显。我们进一步假设，关键的癌症相关通路的细胞谱系依赖状态决定了RAS抑制的自噬细胞是死亡还是克服了增殖障碍并经历了恶性转化。目的：1.通过调节自噬的关键调节因子，确定RAS诱导的自噬反应是否需要RAS依赖的细胞周期停滞。目的2.通过系统抑制PI3K/Akt/mTOR和BRAF/MAPK/ERK途径中的步骤，确定激活的RAS下游导致自噬细胞周期停滞的途径。目的3.确定c-Myc或eIF4F的激活、p53或p16Ink4的抑制或这些致癌干预的组合是否有助于逃避RAS介导的细胞自噬和细胞周期停滞的细胞上下文依赖机制。 与公共卫生相关：我们的工作测试了细胞可以激活第二道防线--除了衰老和凋亡--对抗RAS癌发生的第二道防线的想法，并致力于确定癌症逃脱这一检查点的机制。这项工作为研究自噬的负调控因素提供了机会，作为治疗或化学预防药物改善人类健康的新靶点的新途径。