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Title: Predictive reporter system for investigating dose dependency in oncogene-induced senescence
Author: Chan, Sue Li Adelyne
ISNI:       0000 0004 9354 0283
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Oncogene-induced senescence (OIS) is a well-described autonomous tumour suppressor mechanism which removes cells harbouring oncogenic mutations from the proliferation pool. However, senescent cells remain metabolically active and express factors of the senescence-associated secretory phenotype (SASP), which can conversely have a pro-tumorigenic impact on the cellular microenvironment. Yet, aberrant oncogene activation alone is insufficient for triggering OIS, as studies both in vitro and in vivo have demonstrated that low-dose oncogene cells continue proliferating, in some cases proceeding to full malignancy. The molecular mechanisms governing this dose-dependent switch remain unclear. To address this question, we developed a cell system where a constitutively active fluorescence marker (mVenus) stoichiometrically reflects the level of inducible oncogenic HRASG12V. This allows pre-induction sorting into subpopulations expressing differing levels of mVenus, which is reflected in differences in HRAS dose following induction. Using this system, we first validated that subpopulations expressing different levels of RAS are distinct in their proliferative / senescence phenotypes, implying a theoretical, cell-autonomous ‘oncogenic tipping point’ above which cells enter into OIS. We then demonstrate the potential utility of our proposed ‘predictive reporter’ system in addressing gene dose-related biological questions. RNA sequencing of sorted subpopulations demonstrates the effect of RAS dose on gene expression, where between-subpopulation differences suggests that this sorting strategy may serve as a viable intermediate between bulk and single-cell sequencing technologies. These global dose-dependent differences were validated using single-cell RNA sequencing, which also demonstrated transcriptomic heterogeneity that exists independently of RAS dose. Finally, we demonstrate that the reconstruction of subpopulations into a mixed population with cells of varying RAS doses can have implications on phenotype, and that the current system is a flexible tool that facilitates these sorts of ‘cellular ecosystem’ type experiments in vitro.
Supervisor: Narita, Masashi Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: senescence ; cancer ; oncogene