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.

Acute oncogenic stress can activate autophagy and facilitate permanent arrest of the cell cycle through a failsafe mechanism known as oncogene-induced senescence (OIS). Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS) and has been reported to encode oncoproteins within its latency program that engage host autophagy and OIS pathways; however, the mechanisms by which KSHV oncoproteins promote KS tumorigenesis remain unclear. Here, I demonstrate that ectopic expression of the latent KSHV protein viral cyclin (v-cyclin) deregulates the cell cycle, induces DNA-damage responses (DDRs) and promotes OIS through an autophagy-dependent mechanism. During latency, v-cyclin is co-expressed from a single transcript with a viral homolog of FLICE-inhibitory protein (v-FLIP) that blocks autophagy by binding and inhibiting Atg3. Co-expression of v-FLIP has no effect on DDRs, but efficiently blocks v-cyclin-induced autophagy and senescence. Remarkably, suppression of v-FLIP function during KSHV latency, through specific inhibitory peptides, rescues host cell autophagy and induces senescence of infected cells. Together, these data reveal a coordinated viral gene-expression program that subverts autophagy, impairs senescence, and facilitates the proliferation of KSHV-infected cells.

Senescence is a cell stress program characterized by a stable cell cycle arrest and thus aims to protect against replication of potentially harmful cells. In oncogene-induced senescence (OIS) the cell cycle arrest is brought about by activation of an oncogene. This in turn initiates a DNA damage response and subsequently, the DDR induces p53-p21 and RB tumour suppressor pathways. The metabolism of senescent cells is highly altered, notably there is increased secretion of proteins and increased functional activity of certain metabolic enzymes. There have been many recent studies investigating the role of specific metabolic pathways in OIS and how they may be targeted for therapeutic benefit. This thesis aims to identify novel metabolic regulators of OIS, by combining high throughput RNAi screening and LC-MS based methods. This thesis has identified and validated 17 essential OIS metabolic genes; in this list, there was enrichment for genes involved in lipid biosynthetic processes. Lipid metabolism was an attractive focus for this thesis as it has not been extensively studied in current literature. Next, ceramide synthase 4 (CERS4) was extensively validated as a key enzyme for both OIS and replicative senescence. Using LC-MS based lipidomics, CERS4-driven rewiring of lipid metabolism in OIS was revealed and this corresponded with an accumulation of ceramides due to increased de novo ceramide synthesis. It was then confirmed OIS-related ceramide is mechanistically linked to cell cycle via the PP1-RB-E2F axis. Ceramide activates PP1, which physically binds to RB in a CERS4-dependent manner. PP1 is then able to dephosphorylate and activate RB, which inhibits transcription of E2F targets (cell cycle genes). Overall, this thesis identifies a metabolic checkpoint that links altered lipid metabolism with OIS.

L'adénocarcinome canalaire pancréatique (PDAC) est une maladie agressive dans le monde. En raison du manque d'outils de détection hautement sensibles et spécifiques à un stade précoce de la maladie et de traitements peu curatifs, la plupart des patients sont diagnostiqués à un stade incurable avec une mortalité élevée. Comprendre la pathologie moléculaire, en particulier le stade précoce de cette maladie mortelle, est d’une importance capitale pour améliorer les bons pronostics de ces patients. La première et principale partie de mes travaux reposait sur une analyse de profilage génétique dans laquelle ils mettaient en évidence le rôle de la signalisation TGFβ / Activine et du récepteur ALK4 au cours du développement de la PDAC, en plus des quatre gènes oncogéniques courants (KRAS, CDKN2A, TP53 et SMAD4). ). D'autre part, INHBA (codant pour ActivinA) en tant que membre clé des ligands de la superfamille TGFβ est une cible de l'activation oncogénique de Kras dans les cellules du canal pancréatique et a été désigné comme composant potentiel de la SASP par une analyse au sécrétome de cellules subissant une sénescence induite par Kras. . Le but de mes travaux est donc d’étudier l’impact de la signalisation ActivinA lors de l’initiation de la PDAC. Dans la première partie de mon projet, j'ai démontré qu'ActivinA était un facteur SASP protecteur de la tumeur, produit par les cellules sénescentes d'ADM / PanIN, qui limitent leur prolifération et leur expansion dans les lésions hautement prolifératives via son récepteur ALK4 lors de l'initiation du PDAC. De plus, nos travaux ont révélé que les cellules sénescentes Dclk1 + limitent l'expansion des lésions prénéoplasiques et ont un impact partiel sur le composant stromal soutenu par la signalisation ActivinA. En outre, j'ai également exploré les fonctions autocrines et paracrines de la signalisation ActivinA au dernier stade de la PDAC. Nous avons constaté que le manque de signalisation ActivinA-ALK4 chez la souris entraînait une augmentation de la capacité de prolifération des cellules tumorales et une accumulation réduite de cellules stromales, ce qui pourrait aider à expliquer leurs métastases variées et un temps de survie relativement plus long. En conclusion, mon travail donne un autre aperçu de la signalisation ActivinA et Dclk1 dans l'initiation de la PDAC et leur existence dans des cellules sénescentes au sein de lésions précurseurs pancréatiques de bas grade devrait être considérée comme une étape cruciale pour développer de nouvelles stratégies diagnostiques et thérapeutiques pour les patients atteints de PDAC / Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease worldwide and remains the ranking of 7th leading cause of cancer related death in the past 5 years. Because of the lack of highly sensitive and specific detective tools in an early stage of this disease and low curative treatments, most of the patients are diagnosed in an incurable stage and end up with a high mortality. Understanding the molecular pathology especially the early stage of this lethal disease is of great importance to improve the outcome of these patients. The first and major part of my work was based on gene profiling analysis in which they emphasized the role of TGFβ/Activin signalling and ALK4-receptor during the development of PDAC in addition to the four common oncogenic genes (KRAS, CDKN2A, TP53 and SMAD4). On the other hand, INHBA(encoding for ActivinA) as a key member of TGFβ-superfamily ligands is a target of Kras oncogenic activation in pancreatic duct cell and has been pointed a potential SASP candidate component through a secretome analysis of cells undergoing Kras induced senescence. Therefore, the aim of my work is to investigate the impact of ActivinA-signalling during PDAC initiation. In the first part of my project, I demonstrated that ActivinA as a tumour-protecting SASP factor, produced by ADM/PanINs senescent cells, that limit their proliferation and expansion into highly proliferative lesions through its ALK4 receptor in the initiation of PDAC. Furthermore, our work revealed that senescent Dclk1+ cells limit the expansion of preneoplastic lesions and partly impact the stromal component which is sustained by ActivinA-signalling. Besides, I also explored the autocrine and paracrine functions of ActivinA-signalling in the late stage of PDAC. We found that lack of ActivinA-ALK4 signalling in mice leads to an increased proliferative capacity in tumor cells and a reduced accumulation of stromal cells, which might help to explain their diverse metastasis and a relative longer survival time. To conclude, my work gives another insight of ActivinA-signalling and Dclk1 in the onset of pancreatic cancer and their existence in senescent cells within low grade pancreatic precursor lesions should be considered a crucial step for developing novel diagnostic and therapeutic strategies for PDAC patients

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.