Disruption of circadian rhythm in hypoxia

缺氧时昼夜节律紊乱

• 批准号: 9197571
• 负责人: Zandra Elene Walton
• 金额: $ 3.04万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197571
• 关键词: 3-Dimensional; Acidosis; Acids; Buffers; Cancerous; Cell Culture Techniques; Cell Cycle Arrest; Cell Line; Cell Survival; Cell physiology; Cells; Circadian Rhythms; Citric Acid Cycle; Data; Diagnostic Neoplasm Staging; Disabled Persons; Down-Regulation; Drops; Dyes; Energy Metabolism; Environment; Enzymes; Epidermis; Epithelium; Exposure to; Feedback; Gene Expression; Glucose; HRAS gene; Hour; Human; Hypoxia; Hypoxia Inducible Factor; Incidence; Ion Cotransport; Knock-out; Lactate Dehydrogenase; Lactate Transporter; Lactic acid; Life; Luciferases; Malignant - descriptor; Malignant Neoplasms; Mammals; Mediating; Metabolic; Metabolic stress; Metabolism; Modeling; Modification; Molecular; Mus; Neuroblastoma; Normal tissue morphology; Nutrient; Organism; Output; Oxygen; Pattern; Periodicity; Phase; Phenocopy; Process; Production; Proteins; RNA; Reporter; Resistance; Role; Skin; Solid Neoplasm; Squamous cell carcinoma; Staging; Sterile coverings; Stress; Suspension substance; Suspensions; Testing; Therapeutic; Time; Toxic effect; Transcript; Tumor stage; Up-Regulation; Work; cancer cell; cellular engineering; circadian pacemaker; cofactor; cost; deprivation; extracellular; fitness; inhibitor/antagonist; keratinocyte; killings; knock-down; metabolomics; neoplastic cell; novel; novel therapeutics; osteosarcoma; overexpression; programs; promoter; protein metabolite; public health relevance; response; shift work; skin organogenesis; small hairpin RNA; stressor; transcription factor; tumor; tumor progression; tumorigenesis

 DESCRIPTION (provided by applicant): Circadian rhythms enable organisms to anticipatorily time cellular activities to coincide with patterns dictated by the day-night cycle. In mammals, the diurnal activity of the heterodimeric Clock-Bmal1 transcription factor drives clock function and the circadian oscillation of thousands of transcripts per cell and associated enzymatic activities, metabolite levels, and cellular functions. The fitness advantage engendered by such temporal organization underlies the near ubiquity of circadian clocks in living things. However, such extensive daily transcriptional-translational mobilizations and temporal constraints may create vulnerability. Whether the cellular molecular clock continues to cycle normally or is suspended under metabolic stresses is unknown. Intriguingly, circadian disruption (e.g. shift work) is associated with increased cancer incidence, and deregulation of clock network components contributes to tumorigenesis in mice and is frequently observed in human malignancy. Disabling the clock in the metabolically stressful hypoxic and acidic tumor environment may enhance cancer cell survival and permit temporally unbridled tumor progression. Low oxygen conditions stabilize hypoxia inducible factors (HIF) which carry out a transcriptional program that increases glycolytic flux to lactic acid. Preliminary data indicate stabilization of HIF reversily suspends core clock oscillation through acid-producing metabolic changes driven by HIF. Human neuroblastoma and osteosarcoma cell lines, as well as fresh mouse skin explants, display this same HIF- and acid-dependent dampening of circadian oscillation, suggesting this response may be well conserved. Fulfillment of the aims of this proposal will further characterize this acid- and hypoxia-mediated collapse of clock rhythmicity and define its role in cancer. In Aim 1, multiday RNA/protein and metabolomics timecourses will determine the extent to which clock network oscillations and normally circadian metabolites are perturbed during HIF stabilization and acid exposure. These perturbations could reveal modifications of clock-driven activities that enhance survival during stress. Aim 2 will use cell engineering and real-time circadian reporters to test the mechanistic hypothesis that low extracellular pH hinders efficient lactate extrusion and disables clock oscillation through dampening of NAD+ levels. In Aim 3, creation of organotypic human skin will allow testing of the hypothesis that loss of clock-dictated temporal constraints in late-stage tumors, as would occur in the acidic and hypoxic core of solid tumors, enhances tumor progression. Understanding the mechanism by which clock oscillation is lost and identifying important downstream metabolite effectors may reveal therapeutic means to disrupt this potentially survival- and progression-enhancing process. Additionally, the disabled, out- of-phase rhythm may provide novel chronotherapeutic windows for selective targeting of these notoriously treatment-resistant hypoxic and acidic cancer cells.

 描述（由申请人提供）：昼夜节律使生物体能够预期细胞活动的时间，以与昼夜周期规定的模式相一致。在哺乳动物中， 异二聚体 Clock-Bmal1 转录因子的昼夜活动驱动时钟功能和每个细胞数千个转录物的昼夜节律振荡以及相关的酶活性， 代谢水平和细胞功能。这种时间组织产生的适应性优势是生物钟几乎无处不在的基础。然而，如此广泛的日常转录翻译动员和时间限制可能会造成脆弱性。细胞分子钟是继续正常循环还是在代谢应激下暂停尚不清楚。有趣的是，昼夜节律紊乱（例如轮班工作）与癌症发病率增加有关，而时钟网络组件的失调会导致小鼠肿瘤的发生，并且在人类恶性肿瘤中经常观察到。在代谢应激的缺氧和酸性肿瘤环境中禁用生物钟可能会增强癌细胞的存活率并允许暂时不受限制的肿瘤进展。低氧条件可稳定缺氧诱导因子 (HIFα)，该因子执行转录程序，增加糖酵解生成乳酸的通量。初步数据表明，HIFα 的稳定通过 HIFα 驱动的产酸代谢变化可逆地暂停核心时钟振荡。人类神经母细胞瘤和骨肉瘤细胞系，以及新鲜的小鼠皮肤外植体，都表现出同样的 HIFα 和酸依赖性昼夜节律振荡抑制，表明这种反应可能得到很好的保存。该提案目标的实现将进一步表征这种酸和缺氧介导的时钟节律崩溃，并确定其在癌症中的作用。在目标 1 中，多日 RNA/蛋白质和代谢组学时间进程将确定时钟网络振荡和正常昼夜节律代谢物在 HIFα 稳定和酸暴露期间受到干扰的程度。这些扰动可能揭示时钟驱动活动的改变，从而提高压力下的生存能力。目标 2 将使用细胞工程和实时昼夜节律报告器来测试以下机制假设：低细胞外 pH 值会阻碍有效的乳酸排出，并通过抑制 NAD+ 水平来禁用时钟振荡。在目标 3 中，创建器官型人类皮肤将允许测试时钟丢失导致的假设 晚期肿瘤的时间限制，就像实体瘤的酸性和缺氧核心中发生的那样，会加速肿瘤的进展。了解时钟振荡丢失的机制并识别重要的下游代谢效应物可能会揭示破坏这种潜在的生存和进展增强过程的治疗方法。此外，失能的异相节律可能为选择性靶向这些众所周知的难治性缺氧和酸性癌细胞提供新的时间治疗窗口。