Defining the transition from deep quiescence to senescence

PROJECT SUMMARY Cell-cycle exit can be either transient (quiescence) or permanent (senescence), and these different states are thought to have distinct molecular characteristics. Further, quiescence itself is not a single homogeneous state as cells that remain quiescent for longer durations of time move progressively “deeper” into quiescence, taking longer to return to the cell cycle upon stimulation. While senescent cancer cells are tumor suppressive as they limit the proliferation of damaged cells and recruit the immune system, quiescent cancer cells are a source of drug resistance as they evade chemotherapeutic treatments targeted at cycling cells and retain their ability to proliferate in the future. The mechanisms controlling quiescence depth and the relationship between deep quiescence and senescence are still poorly understood due to the lack of robust biomarkers and necessity for single-cell assays to study questions of reversibility. However, there is a critical need for such studies as chemotherapeutic treatment results in a mix of these cell fates. By using a functional readout that gets at the core of what it means to be senescent: the long-term total lack of cell-cycle entry or progression, we found that a subset of cells pushed into deep quiescence fail to return to the cell cycle after release from treatment and that cells induced to deep quiescence by four different treatments for 2-12 days showed increasing levels of SA-βgal, the gold standard marker of senescence. Thus, we hypothesize that 1) senescence is a deep quiescence from which cells will never reawaken and 2) the transition between deep quiescence and senescence is marked by a decrease in the ratio between pro-proliferative signaling (Cyclin D) and anti-proliferative signaling (p21/p27), mediating the progressive decrease in probability of cell-cycle re-entry. Experiments proposed in Aim 1 will use time-lapse microscopy paired with post-hoc immunofluorescence imaging to measure canonical markers of senescence in cells that remain permanently arrested after release from deep quiescence. In Aim 2 we will perform RNA-sequencing on the subset of permanently arrested cells from Aim 1 and compare the transcriptomes to existing gene signatures for senescent cells. Finally, we will use time-lapse microscopy of biosensors for Cyclin D and p21/p27 to determine if the ratio of these signals predicts future cell-cycle re-entry. Collectively, these aims will help define the relationship between quiescence and senescence and contribute to a much needed quantitative and mechanistic understanding of how cells transition between transient, prolonged, and permanent cell-cycle withdrawal. If successful, the proposed work will aid in the development of more targeted and effective chemotherapy treatments and provide more accurate biomarkers to determine which cells will and will not proliferate in the future.