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Molecular mechanism impacted by circadian disruption underscores the importance of timed anti-cancer treatment

ABSTRACT: Circadian disruption has multiple pathological consequences, but the underlying mechanisms are largely unclear. To address such mechanisms, we subjected cultured cells to chronic circadian desynchrony (CCD), mimicking a chronic jet-lag paradigm, and assayed a range of cellular functions. The results indicated a specific circadian clock-dependent increase in cell proliferation. Transcriptome analysis revealed upregulation of G1-S-phase transition genes (cMyc, CyclinD1/3, Cdt1), concomitant with increased phosphorylation of the Retinoblastoma protein (Rb) by Cyclin D kinase 4/6 (CDK4/6) and increased G1-S progression. Phospho-Rb (Ser807/811) was found to oscillate in a circadian fashion and exhibit phase-shifted rhythms in circadian desynchronized cells. A CDK4/6 inhibitor approved for cancer treatment reduced growth of cultured cells and mouse tumors in a time-of-day specific manner, but the time dependence was lost with CCD. Our study identifies a mechanism that underlies effects of circadian disruption on tumor growth and underscores the importance of treatment timed to endogenous circadian rhythms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE119668 | GEO | 2019/05/14

REPOSITORIES: GEO

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Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

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Molecular mechanism impacted by circadian disruption underscores the importance of timed anti-cancer treatment

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