Disruption of circadian rhythm in hypoxia

 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，以符合昼夜循环所决定的模式。在哺乳动物中， 异二聚体Clock-Bmal 1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适应性优势是生物钟几乎无处不在的基础。然而，这种广泛的日常转录-翻译动员和时间限制可能会造成脆弱性。细胞的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，昼夜节律的破坏（例如轮班工作）与癌症发病率的增加有关，并且时钟网络组件的失调有助于小鼠的肿瘤发生，并且经常在人类恶性肿瘤中观察到。在代谢应激的低氧和酸性肿瘤环境中禁用生物钟可能会提高癌细胞存活率，并允许暂时不受约束的肿瘤进展。低氧条件稳定缺氧诱导因子（HIF-1 α），其执行增加糖酵解至乳酸的通量的转录程序。初步数据表明，稳定的HIF可逆地暂停核心时钟振荡，通过产酸代谢变化驱动的HIF β。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，显示这种相同的HIF β-和酸依赖性抑制昼夜节律振荡，这表明这种反应可能是很好的保守。实现这一建议的目的将进一步表征这种酸和缺氧介导的生物钟节律性崩溃，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过程将决定在HIF-1 α稳定和酸暴露期间时钟网络振荡和正常昼夜代谢物受到干扰的程度。这些扰动可能揭示了生物钟驱动活动的变化，这些变化增强了压力下的生存能力。目标2将使用细胞工程和实时昼夜节律报告器来测试低细胞外pH阻碍有效乳酸挤出并通过抑制NAD+水平来禁用时钟振荡的机制假设。在目标3中，器官型人类皮肤的创造将允许测试生物钟决定的假设， 晚期肿瘤中的时间限制（如在实体瘤的酸性和缺氧核心中会发生的）增强了肿瘤进展。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，失能的异相节律可以为选择性靶向这些众所周知的治疗抗性低氧和酸性癌细胞提供新的时间窗。

 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.