Understanding the molecular interplay between senescence, rejuvenation, and healthy ageing

Senescence is classically defined as an irreversible cell cycle arrest. There is now convincing evidence that senescent cells accumulate during human ageing, potentially driving age-related dysfunction through depletion of mitotically active cells and stimulation of chronic inflammation. Recently, a landmark paper demonstrated that removal of p16INK4A (p16)-positive senescent cells in mice prolonged healthy lifespan, suggesting a direct link between senescence and age-related dysfunction. As such, restraining the senescent pool or slowing their rate of accumulation presents an attractive therapeutic strategy for extending healthspan. Previously, our laboratory has demonstrated that siRNA transfection can reverse deep senescence in p16-positive primary adult human mammary epithelial cells using a panel of senescence markers. Subsequent siRNA screening revealed 28 hits which strongly induced reversal in the deeply senescent HMECs. In this project, siRNA knockdown of p16 combined with p21WAF1/CIP1 (p21) was found to reverse deep replicative senescence in primary adult human mammary fibroblasts, as defined by a panel of senescence markers. This discovery provided the opportunity to screen for novel siRNAs which induce reversal in both cell types. Screening in the deeply senescent HMFs of the 28 shortlisted candidates and 33 protein interactors identified using bioinformatics revealed 45 siRNAs which significantly increased cell number compared to the negative siRNA control. Subsequent immunofluorescence staining and high content analysis of the top 14 candidates identified 10 hit siRNAs which induced senescence reversal as defined by a panel of markers. Interestingly, these 10 hits were enriched for cytoskeletal and cell adhesion processes, suggesting an interplay between external forces and senescence induction. The top siRNA hit, early growth response 2 or EGR2, a transcription factor, was validated and selected for further exploration as a novel driver of senescence. Bioinformatics analysis revealed an enrichment for EGR2 binding sites within genes dynamically expressed in HMEC senescence, including p16, p21, and nine hits identified to reverse senescence in HMFs and HMECs. Furthermore, deeply senescent HMFs and HMECs were found to have a significantly increased nuclear EGR2 foci number compared to proliferating cells, and this was significantly decreased in reversed HMFs. In conclusion, it is proposed that EGR2 may represent a novel driver of both HMF and HMEC senescence.