Uncovering the Mechanisms of Science Activation in p-16 Positive Cancer Cells.

PhD Theses MedicalPro-senescence therapy, the process of activating a stable cell cycle arrest in cancer cells, increasingly draws interest as a novel mechanism for cancer treatment, especially for cancers with unmet clinical needs, such as p16-positive basal-like breast cancer (BLBC). Characteristically, senescent cells feature a secretome profile termed the senescence-associated secretory phenotype (SASP). The composition of this secretome differs across cell types and senescence induction mechanism. However, in senescent cancer cells this profile remains largely unexplored. Previous work in the Bishop laboratory identified that knockdown of the small ribosomal subunit proteins, RPS3A or RPS7, activated senescence in the p16-positive BLBC cell model, MDA-MB-468. The aim of this project was to uncover molecular events that drive and contribute to establishing RSIS in MDA-MB-468 cells, and to profile the secretome of these cells to further our understanding of the SASP of senescent cancer cells. Using siRNA to investigate the kinetics of RSIS induction demonstrated that senescence initiation is associated with a translocation of p16 to the nucleus and increased cellular expression of a well-known SASP molecule, IL-6. To unbiasedly explore the secretome profile of RSIS MDA-MB-468 cells, three independent inducible shRNA cell lines targeting RPS3A or RPS7, and non-targeting control shRNA cell lines were generated, and mass spectrometry performed. This investigation revealed a distinct secretome profile which was intriguingly, functionally enriched for immune 5 activation. Within this thesis, genetic targets which induce a senescence-associated phenotypic change in p16-positive HeLa cells was also explored in a phenocopying analysis performed on the genome-wide siRNA screen dataset. This analysis corroborated ribosomal targets and strikingly, identified spliceosome machinery targets as modulators of senescence. Finally, the consequential changes to the proteome following RSIS initiation were examined using the inducible depletion of RPS3A or RPS7. This analysis revealed that knockdown of both RP targets independently led to upregulation of mitochondrial biogenesis and calcium signalling. I propose a model in which these molecular events contribute to facilitating the nuclear translocation of p16 which in turn drives senescence induction. Overall, in this thesis I have generated and interrogated datasets to progress our knowledge of the mechanisms and networks governing senescence activation in p16- positive cancer cells. In the future, this body of work will hopefully contribute to advancing pro-senescence therapy as a novel therapeutic strategy for cancers with unmet clinical burdens