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Title: Identification and study of a role for toll-like receptors in oncogene-induced senescence
Author: Hari, Priya
ISNI:       0000 0004 7429 5921
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2018
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Oncogene-induced senescence (OIS) is a fail-safe mechanism activated to halt the proliferation of cells at risk of malignant transformation. It is a cell cycle arrest program of biological changes to the cell comprising of the activation of tumour suppressor pathways, altered cellular metabolism, extensive chromatin remodelling and the activation of a senescence-associated secretory phenotype (SASP). The vast array of proteins secreted by the cells not only play a cell-autonomous role in reinforcing the senescence phenotype, but also modulate the cell's micro-environment by inducing senescence in neighbouring cells, promoting angiogenesis, and initiating an immune response through the recruitment of immune cells. To this end, senescence is a complex phenotype that has countless pathophysiological implications and understanding its molecular mechanisms of activation could prove to be fruitful for understanding its diverse functions. Components of the innate immune system have been shown to play an essential role in the development of the SASP through its processing and activation of Caspase 1 that in turn leads to activation of IL-1B. A gene set enrichment analysis of OIS cells showed significant upregulation in the Pattern Recognition Receptor (PRR) family, from the innate immune response. Hence, we explored the role of innate immune receptors in OIS. Methods and Materials IMR90 human diploid fibroblast cells, stably transfected with an oncogenic ER:RAS fusion protein undergo OIS upon treatment with 4-hydroxytamoxifen. A loss of function siRNA screen was conducted targeting components of the innate immune systems, including pattern recognition receptors. This served as a proof-of-principle screen for a larger screen of proteases and ubiquitin conjugation enzymes. Potential regulators of OIS were identified through siRNA that bypassed the proliferative arrest associated with OIS. We chose to focus on studying the role of TLR2 and TLR10 in senescence. A transcriptome analysis was carried out to identify genes regulated by these TLRs and further biological manipulation was used to confirm the mechanism through which these receptors control senescence. Results Toll-like receptor 2 (TLR2) and TLR10 have been identified as regulators of OIS. Their overexpression in IMR90 cells induces a premature form of senescence where the cells have significantly reduced proliferative activity and display senescence-associated β galactosidase activity. Moreover, the knockdown of TLR2 and TLR10 results in suppression of tumour suppressor pathway genes, reduced signaling through the pathway and blunting of the SASP. High TLR2 expression in patients with lung adenocarcinoma is associated with a higher survival rate. Concomitantly, the screening also identified Caspase 4, a critical component of non-canonical inflammasome signaling, to be regulated by TLR2 and TLR10 in OIS. A full transcriptome analysis of cells with TLR2 and TLR10 knockdown revealed serum amyloid amylase 1 (SAA1) and SAA2 are upregulated in OIS and were also confirmed to be activating ligands of TLR2. The activation of TLR2 by SAA, followed by the engagement of the non-canonical inflammasome by LPS electroporation induced senescence in proliferating IMR90 cells. Conclusion Our results suggest that the TLR2 and TLR10 act as potential tumour suppressor genes, signaling upstream of the inflammasome to initiate the production of inflammatory cytokines, and thereby the SASP. The production of the SASP develops a positive feedback loop, generating the damage-associated molecular pattern (DAMP) A-SAA that initiates an immune response signal cascade and subsequently activates senescence.
Supervisor: Acosta, Juan Carlos ; Brunton, Valerie Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: oncogene-induced senescence ; SASP proteins ; senescence-associated secretory phenotype ; angiogenesis ; Caspase 1 ; immune receptors