Disruption of circadian rhythm in hypoxia

 描述(申请人提供)：昼夜节律使生物体能够预测细胞活动的时间，以与昼夜循环所规定的模式相一致。在哺乳动物中， 异二聚体Clock-BMal1转录因子的昼夜活性驱动时钟功能和每个细胞数千个转录本的昼夜振荡以及相关的酶活性， 代谢物水平和细胞功能。这种时间组织所产生的健康优势，奠定了生物钟在生物中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。细胞分子时钟是继续正常循环还是在代谢压力下暂停尚不清楚。有趣的是，昼夜节律的扰乱(例如轮班工作)与癌症发病率的增加有关，而时钟网络组件的放松调控有助于小鼠的肿瘤发生，并经常在人类恶性肿瘤中观察到。在新陈代谢紧张、低氧和酸性的肿瘤环境中禁用时钟可能会提高癌细胞的存活率，并允许肿瘤暂时无节制地发展。低氧条件稳定低氧诱导因子()，低氧诱导因子执行一种转录程序，增加糖酵解到乳酸的通量。初步数据表明，低氧诱导因子的稳定通过低氧诱导因子驱动的产酸代谢变化逆转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，显示出同样的低氧诱导因子和酸依赖的昼夜振荡抑制，表明这种反应可能是很保守的。这项提议的目标的实现将进一步表征这种由酸和低氧介导的时钟节律性的崩溃，并确定其在癌症中的作用。在目标1中，多天RNA/蛋白质和代谢组学的时间进程将决定在低氧诱导稳定和酸暴露期间时钟网络振荡和正常昼夜代谢产物受到扰动的程度。这些干扰可能揭示了时钟驱动活动的改变，从而提高了应激状态下的存活率。目的2将使用细胞工程学和实时昼夜节律记者来测试低细胞外pH阻碍有效的乳酸排出并通过抑制NAD+水平而禁用时钟振荡的机制假说。在目标3中，创造器官类型的人类皮肤将允许测试丢失由时钟决定的假说 晚期肿瘤的时间限制，就像发生在实体肿瘤的酸性和缺氧核心一样，促进了肿瘤的进展。了解时钟振荡丢失的机制并确定重要的下游代谢物效应物，可能会揭示破坏这一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臭名昭著的耐药低氧和酸性癌细胞提供新的时间治疗窗口。

 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.