Disruption of circadian rhythm in hypoxia

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 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.