Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

<p>Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance. </p><p> Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.</p> / Dissertation

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Durant l’exécution de l’apoptose, plus de 2 000 protéines sont protéolysées par les caspases, une famille de protéases à cystéine. Le clivage de plusieurs d’entre elles a pour effet d’interrompre les processus régulant le trafic intracellulaire. Durant mes études, je me suis intéressée à deux substrats potentiels des caspases, soit les sorting nexins SNX1 et SNX2. Leur clivage en N-terminal avait auparavant été identifié par protéomie dans des extraits cellulaires apoptotiques, respectivement aux sites LFAD91[flèche vers le bas]A et VSLD84[flèche vers le bas]S. Conjointement avec le complexe rétromère, SNX1 et SNX2 jouent un rôle essentiel dans le transport rétrograde de cargos, tel le récepteur lysosomal CI-MPR, des endosomes vers le TGN, évitant ainsi leur dégradation aux lysosomes. Notamment, l’association entre SNX1 et SNX2 et le complexe rétromère, via la sous-unité Vps35, requerrait leur domaine N-terminal, or, celui-ci est clivé durant l’apoptose. Dans le but de déterminer l’impact de la protéolyse de SNX1 et SNX2 sur la fonction du complexe rétromère et le transport rétrograde, nous avons étudié leur clivage par les caspases. Nos résultats indiquent qu’in vitro, les caspases initiatrices 8, 9 et 10 protéolysent SNX1 et SNX2 tandis que seule la caspase-6 exécutrice clive SNX2. Plusieurs fragments de SNX1 sont générés par le clivage à 16 sites, dont le site LFAD91[flèche vers le bas]A en N-terminal ainsi que plusieurs suivant un résidu glutamate. Durant l’apoptose, SNX2 est entre autres clivée par la caspase-6, et ce au site VSLD84[flèche vers le bas]S en N-terminal. Nous avons par la suite étudié l’effet de la protéolyse de SNX1 et SNX2 sur la fonction du complexe rétromère. Nos résultats démontrent que SNX2 tronquée, imitant le clivage au site VSLD84[flèche vers le bas]S, n’interagit plus avec Vps35, la sous-unité centrale du complexe rétromère. De plus, la déplétion de SNX1 et SNX2, récapitulant potentiellement leur protéolyse, a pour effet de délocaliser Vps26, une autre sous-unité du complexe rétromère. Par ailleurs, nous avons évalué l’effet de la protéolyse de SNX2 sur la régulation du récepteur Met, lequel serait régulé entre autres par SNX1 et SNX2. La déplétion de SNX2 induit une augmentation de la phosphorylation du récepteur Met et de ERK1/2 suivant sa stimulation. De plus, l’ARNm de SNX1 et SNX2 sont tous deux réduits dans les tissus de tumeurs de patients atteints du cancer colorectal (CCR) et une diminution des niveaux de SNX2 corrèle avec une hausse de mortalité chez ces patients. Pour conclure, notre étude démontre un effet direct de la protéolyse de SNX2 sur le complexe rétromère durant l’apoptose et suggère un lien entre SNX2 et la pathogenèse du CCR. / Abstract: During the execution of apoptosis, more than 2,000 proteins are proteolysed by caspases, a family of cysteine proteases. The cleavage of several of them results in the interruption of intracellular trafficking processes. During my studies, I investigated two potential caspases substrates, namely the sorting nexin SNX1 and SNX2. Their cleavage at their N-terminus has previously been identified in apoptotic cell lysates by proteomics, respectively at LFAD91[down arrow]A and VSLD84[down arrow]S sites. Together with the retromer complex, SNX1 and SNX2 play an essential role in the retrograde transport of cargos, such as the lysosomal receptor CI-MPR, from endosomes to TGN, thus avoiding their degradation by lysosomes. In particular, the association between SNX1 and SNX2 and the retromer complex, via the Vps35 subunit, seems to require their N-terminal domain, which is thought to be cleaved during apoptosis. To determine the impact of SNX1 and SNX2 proteolysis on the function of the retromer complex and retrograde transport, we have first studied their cleavage by caspases. Our results indicate that in vitro, initiator caspases 8, 9, and 10 proteolyze SNX1 and SNX2 while only executioner caspase-6 cleaves SNX2. Several fragments of SNX1 are generated by the cleavage of up to 16 sites, including at the N-terminus LFAD91[down arrow]A site and following glutamate residues. During apoptosis, SNX2 is directly cleaved by caspase-6 at the site VSLD84[down arrow]S in its N-terminus. We next investigated the effect of SNX1 and SNX2 proteolysis on the function of retromer complex. Our results demonstrate that truncated SNX2, mimicking cleavage at the VSLD84[down arrow]S site, no longer interacts with Vps35, the central subunit of retromer complex. Furthermore, depletion of SNX1 and SNX2, potentially recapitulating their proteolysis, redistributes Vps26, another retromer subunit. In addition, we evaluated the effect of SNX2 proteolysis on the regulation of Met receptor, which has been shown to be regulated by SNX1 and SNX2. SNX2 depletion induces an increase in Met and ERK1/2 phosphorylation after stimulation. In addition, both SNX1 and SNX2 mRNAs are reduced in tumor tissues of colorectal cancer patients and decreased expression levels of SNX2 correlates with increased mortality. In conclusion, our study demonstrates a direct effect of SNX2 proteolysis on retromer complex association during apoptosis and suggests a link between SNX2 and the pathogenesis of colorectal cancer.

<p>The DNA of a cell operates as its blueprint, providing coded information for the production of the RNA and proteins that allow the cell to function. Cells can face a myriad of insults to their genomic integrity during their lifetimes, from simple errors during growth and division to reactive oxygen species to chemotherapeutic reagents. To deal with these mutagenic insults and avoid passing them on to progeny, cells are equipped with multiple defenses. Checkpoints can sense problems and halt a cell’s progression through the cell cycle in order to allow repairs. More drastically, cells can also prevent passing on mutations to progeny by triggering apoptosis, or programmed cell death. This work will present two separate discoveries regarding the regulation of DNA damage-induced apoptosis and the regulation of the spindle checkpoint.</p><p> The protease caspase-2 has previously been shown to be an important regulator of DNA damage-induced apoptosis. In unstressed cells caspase-2 is present as an inactive monomer, but upon sensing a stress caspase-2 dimerizes and becomes catalytically active. The mechanisms that regulate this dimerization are poorly understood. The first research chapter details our development of a novel method to study dimerized caspase-2, which in turn identified TRAF2 as a direct activator of caspase-2. Specifically, we utilized the Bimolecular Fluorescence Complementation technique, wherein complementary halves of the Venus fluorophore are fused to caspase-2: when caspase-2 dimerizes, the non-fluorescent halves fold into a functional Venus fluorophore. We combined this technique with a Venus-specific immunoprecipitation that allowed the purification of caspase-2 dimers. Characterization of the caspase-2 dimer interactome by MS/MS identified several members of the TNF Receptor Associated Factor (TRAF) family, specifically TRAF1, 2, and 3. Knockdown studies revealed that TRAF2 plays a primary role in promoting caspase-2 dimerization and downstream apoptosis in response to DNA damage. Identification of a TRAF Interacting Motif (TIM) on caspase-2 indicates that TRAF2 directly acts on caspase-2 to induce its activation. TRAF2 is known to act as an E3 ubiquitin ligase as well as a scaffold for other E3 ubiquitin ligases. Indeed, we identified three lysine residues in the caspase-2 prodomain (K15, K152, and K153) important for its ubiquitination and complex formation. Together these results revealed a novel role for TRAF2 as a direct activator of caspase-2 apoptosis triggered by DNA damage.</p><p> During mitosis, when the cell prepares to divide, great care is taken to ensure that the chromosomes are properly segregated between the two daughter cells by the mitotic spindle. This is primarily accomplished through the spindle checkpoint, which becomes activated when the mitotic spindle is not properly attached to each chromosome’s kinetochore. When activated, the primary effector of the spindle checkpoint, the mitotic checkpoint complex (MCC), inhibits the anaphase-promoting complex (APC/C) by binding to the APC/C co-activator, CDC20. This prevents the APC/C from targeting critical pro-mitotic proteins, like cyclin B and securin, to promote mitotic exit. Although the function of the MCC is well understood, its regulation is not, especially in regard to protein phosphatases To investigate this, we activated the spindle checkpoint with microtubule inhibitors and then treated with a variety of phosphatase inhibitors, examining the effect on the MCC and APC/C. We found that two separate inhibitors, calyculin A and okadaic acid (1uM), were able to promote the dissociation of the MCC. This led to the activation of the APC/C, but the cells remained in mitosis as evidenced by high levels of Cdk1 activity and chromosome condensation. This is the first time that phosphatases have been shown to be essential to maintaining the MCC and an active spindle checkpoint.</p> / Dissertation

In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies identified serine 199, serine 201, serine 227, and serine 234 as critical residues regulating the alternative splicing of caspase 9, as well as playing a role in the anchorage-independent growth of A549 cells. Since dysregulation of this splicing mechanism correlated with NSCLC tumors/cell lines and constitutively active Akt, oncogenic factors for NSCLC known to activate the PI3-Kinase/Akt pathway were examined in HBEC-3KT cells. In contrast to k-ras V12 expression, the overexpression/mutation of EGFR affected the alternative splicing of caspase 9 in a pro-oncogenic manner, dramatically lowering the caspase 9a/9b mRNA ratio. Stable downregulation of caspase 9b by shRNA blocked the ability of E746-A750 del EGFR expressing HBEC-3KTs to induce anchorage-independent growth, suggesting a role for caspase 9b as a cooperative oncogenic factor. These findings were further corroborated by the ability of caspase 9b expression to completely block the inhibition of clonogenic colony formation by erlotinib. Therefore, this study demonstrates that oncogenic factors activating the PI3-Kinase/Akt pathway regulate the alternative splicing of caspase 9, to produce caspase 9b, via a coordinated mechanism involving the phosphorylation of SRp30a. In additional studies, we demonstrate that the PI3-Kinase/PKCι pathway, a pathway important for cancer cell survival and transformation of lung epithelial cells, regulates the alternative splicing of Bcl-x pre-mRNA via modulation of SAP155 expression to produce an anti-apoptotic phenotype in NSCLC. Therefore, these studies link oncogenic mechanisms in NSCLC to the therapeutically relevant and distal target mechanisms of caspase 9 and Bcl-x pre-mRNA splicing.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Background Glioblastoma Multiforme (GBM) is the most commonly occurring tumour of the central nervous system (CNS). Currently GBM is considered an incurable malignancy with patients experiencing abysmal life expectancies. Lack of progress in the discovery of novel treatments has led to the repurposing of existing licenced medication as a possible alternative option. Metformin is from the biguanide family of drugs and is the most common medication used in the treatment of type 2 diabetes. Clinical studies have reported that, in type 2 diabetic patients, metformin might reduce cancer incidence and severity. Currently, metformin is being assessed in clinical trials as a treatment for a range of cancer types including GBM. The antineoplastic mechanisms utilized by metformin and other biguanides have not been fully elucidated. Methods The effects of metformin were evaluated, alone and in combination with other agents, on a panel of GBM cell cultures. Functional analysis of metformin mechanism of action was assessed through measurement of apoptosis, depolarisation of the mitochondria membrane, caspase pathway activation, cell cycle progression and the expression levels of micoRNAs. Results Analysis of fourteen GBM cell cultures showed a cytotoxic response to metformin that was significantly linked to the P53 status (p=0.0024). In combination drug testing, one of the four drugs showed a synergistic pairing with metformin. The kinase inhibitor sorafenib, showed synergism (CI ≤ 1) in eight GBM cell cultures. Flow cytometry of metformin treated GBM cells showed no significant increase (p > 0.005) in apoptotic cell populations. Caspase 3/7 levels showed no significant increase post metformin treatment (p > 0.005). Metformin caused depolarisation of the mitochondrial membrane in six GBM cell cultures. Four microRNAs were shown to have expression levels changes post-metformin treatment. Upregulation of expression was identified in miR-140, miR-192, let-7c. Downregulation was identified in miR-222. Conclusions Metformin was shown to have cytotoxic effect on a GBM cell cultures and has potential as GBM therapeutic agent and possible treatment synergy with sorafenib. The significance of P53 status to metformin sensitivity may suggest that its use should be directed to a sub-set of GBM patients. Mechanism for cell death by metformin was shown not to rely on apoptotic pathways but caspase 3/7 independent depolarization of mitochondrial cell membranes and cell cycle arrest. Investigations into autophagy may help to further define the pathways metformin is utilising to promote cell death. The impact of metformin on the expression profile of miR-222, miR-192 and let-7c is in line with clinical studies of other cancer types. This shows possible insight into the cancer independent actions of metformin. The interplay recorded between glucose availability and cell death indicates a possible key factor in the utilisation of metformin as a therapeutic agent. This finding may warrant the addition of dietary control regimes in clinical trials to maximise metformin efficacy. This work highlights the strong potential for biguanides in the development of new drug treatments and in expanding our knowledge of cancer metabolism.

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.