The Bcl-2 family of proteins strictly regulates the intrinsic pathway of apoptosis. Direct physical interactions between Bcl-2 proteins regulate mitochondrial outerpermeabilisation (MOMP), which occurs in response to various cell stresses andapoptotic stimuli. How changes in Bcl-2 protein activity regulate apoptosiscommitment is still unclear, especially with regard to how they interact with eachother within the context of the mitochondrial membrane. Recent studies haveshown that Bcl-2 proteins exist in a dynamic equilibrium between the mitochondriaand the cytosol. In this thesis, by using FRAP, I have measured changes in Bcl-XLand Mcl-1 dynamics in single cells. Surprisingly, individual cells within a populationshow widely differing Bcl-XL and Mcl-1 dynamics. There is a corelation betweenBcl-XL and Mcl-1 dynamics with BH3-only protein expression. Anti-apoptotic andpro-apoptotic Bcl-2 proteins stabilise each other on the OMM. Together, theseresults indicate that cells constantly fine tune mitochondrial priming and thatanalysing anti-apoptotic Bcl-2 proteins by FRAP allows this to be measured at asingle cell level in real time before MOMP.

Apoptosis is a type of programmed cell death which plays a fundamental role in maintaining homeostasis in multi-cellular organisms. The Bcl-2 family has been identified as the central players in regulation of apoptosis. It consists of anti-apoptotic proteins (e.g Bcl-XL) and pro-apoptotic proteins which are further classified as BH3 proteins (e.g Bim, Bid) and effector proteins (e.g Bax, Bak). BH3-proteins regulate apoptosis by activating the pro-apoptotic proteins Bax and Bak to permeabilize mitochondria, and/or by inhibiting anti-apoptotic proteins such as Bcl-XL and Bcl-2. In this study, we employed fluorescence spectroscopy and functional assays with full-length Bim and showed that major Bim isoforms have similar activities in vitro. Bim preferably activates Bax over Bak while Bid preferably activates Bak. Bim displayed a unique binding to Bcl-XL so that the Bim-Bcl-XL complex is resistant to BH3-mimic drug ABT-263 treatment while Bid does not. A Bcl-XL enhancer BH3 TCTP was also shown to interact with Bim-Bcl-XL and Bid-Bcl-XL complex differently, where a single mutation abolished its enhancement of Bid-Bcl-XL but not Bim-Bcl-XL. Closer investigation of the dual apoptotic functions of the BH3-protein Bim revealed that the C-terminal membrane binding domain (MBD) is unexpectedly also involved both in binding of Bim to Bax in solution and in activating Bax. Multiple mutations in this domain reduced or abolished binding to membranes but did not affect binding to Bax or correlate with Bax activation. Deletion eliminated binding to and activation of Bax, but not binding to or inhibition of Bcl-XL. Thus MBD mediates binding to both membranes and Bax separately. On the other hand, although the MBD is not the determining factor for interactions with Bcl-XL, our data demonstrates Bim MBD also plays a major role in binding to Bcl-XL. The C-terminal MBD was shown to be contributing to the ABT-263 resistance of Bim-Bcl-XL complex by directly interacting with Bcl-XL. We discovered additional interactions between the MBD of Bim and both Bcl-XL and Bcl-2. Our data suggested a novel topology and mechanism for the Bim-MBD that positions the central hydrophobic residues of the MBD appropriately for binding to Bcl-XL. / Thesis / Doctor of Philosophy (PhD) / Apoptosis is a type of programmed cell death which plays a fundamental role in maintaining homeostasis in multi-cellular organisms. The Bcl-2 family has been identified as the central players in regulation of apoptosis. It consists of anti-apoptotic proteins (e.g Bcl-XL) and pro-apoptotic proteins which are further classified as BH3 proteins (e.g Bim, Bid) and effector proteins (e.g Bax, Bak). Here we showed that Bim is constitutively active in vitro and preferably activates Bax, distinguishing itself from Bid which preferably activate Bak. We showed that the C-terminus membrane binding domain (MBD) of Bim plays a crucial role in reguating binding and activation of Bax, as well as providing extra binding support to Bcl-XL, independent of its membrane binding feature.

Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin. Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells. Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.

Dissecting the Mechanisms of Direct Activation for Proapoptotic BAK and BAX / Chemistry and Chemical Biology

Breast cancer is the second leading cause of death amongst women ages 20 to 59. Despite advancements in cancer therapies, more research is necessary to improve the diagnoses and treatment of several types of breast cancer. Paclitaxel (Taxol) is a commonly utilized anti-cancer drug for various types of solid tumors. However, the molecular mechanism utilized by paclitaxel to induce cell death is still elusive. Previous studies in our laboratory have shown that the pro-apoptotic BCL-2 family protein, BAK (BCL-2 homologous antagonist/killer) plays an important role in paclitaxel-induced cell death. In untreated breast cancer cells, BAK is associated with the anti-apoptotic BCL-2 family protein MCL-1 (myeloid leukemia cell differentiation protein). BAK is activated with paclitaxel treatment in concert with loss of MCL-1 expression. In addition, it has been shown that the pro-apoptotic BH3-only BCL-2 family protein Noxa, specifically interacts with MCL-1 to inactivate MCL-1 function. Based on these observations, we hypothesized that modulation of Noxa/MCL-1 axis could mimic paclitaxel-induced cell death. Here, we found that down-regulation of MCL-1 induced cell death in all breast cancer cell lines that we tested, but not in a non-transformed breast epithelial cell line. In contrast, Noxa overexpression induced MCL-1 degradation and cell death in some cell lines (Noxa-sensitive), while in others Noxa overexpression neither changed MCL-1 levels nor induced cell death (Noxa-resistant). Noxa strongly interacted with MCL-1 in the Noxa-sensitive cell line, but not in the Noxa-resistant cell line. Based on these findings, the overexpression of Noxa might have two different mechanistic effects on MCL-1 levels in the breast cancer cell lines (induction of MCL-1 degradation or no effect on MCL-1). In Noxa-sensitive cells, the finding could be used as a potential therapeutic strategy for the treatment of breast cancer.

L’apoptose ou mort cellulaire programmée joue un rôle prépondérant dans l’homéostasie cellulaire. Ce processus est très finement régulé par les protéines de la famille BCL-2 qui contrôlent la perméabilité de membrane mitochondriale externe et la libération du cytochrome c, deux événements majeurs précédant la mort cellulaire. Les protéines anti-apoptotiques de la famille BCL-2 contribuent à la tumorigenèse et sont impliquées dans la résistance des cancers aux molécules chimiothérapeutiques ; à ce titre, elles représentent des cibles importantes pour le développement de nouvelles thérapies. BCL2A1 est un membre anti-apoptotique de la famille BCL-2 impliqué dans la chimiorésistance de nombreuses tumeurs. La protéine BCL2A1 a pour caractéristique d’avoir une demi-vie courte due à sa dégradation constitutive par le système ubiquitine-protéasome. Ceci régule la stabilité et la fonction anti-apoptotique de BCL2A1 et représente un mécanisme suppresseur de tumeur majeur. Cependant, les enzymes qui contrôlent les modifications post-traductionnelles impliquées dans l’ubiquitination et la dégradation de BCL2A1 demeurent, à ce jour, inconnues. Dans la présente thèse, nous donnons un aperçu des acteurs et des mécanismes impliqués dans la régulation de l’ubiquitination de BCL2A1. Nous présentons des preuves que TRIM28 est une E3 ubiquitine-ligase pour BCL2A1. En effet, les protéines TRIM28 et BCL2A1 endogènes interagissent ensemble au niveau des mitochondries et la déplétion de TRIM28 diminue l’ubiquitination de BCL2A1. Nous montrons aussi que TRIM17 stabilise BCL2A1 en empêchant son interaction avec TRIM28 et son ubiquitination médiée par TRIM28, et que l’activité de GSK3 est impliquée dans l’inhibition de la dégradation de BCL2A1. Ainsi, BCL2A1 et son proche homologue MCL-1 sont régulés par des facteurs communs mais de façon opposé. Finalement, la surexpression de TRIM28 ou l’inactivation de TRIM17 diminue le niveau protéique de BCL2A1 et restaure la sensibilité des cellules de mélanomes aux thérapies utilisant des inhibiteurs de la kinase BRAF. Globalement, nos résultats décrivent un rhéostat moléculaire au sein duquel deux protéines de la famille TRIM régulent de façon antagoniste la stabilité de BCL2A1 et modulent ainsi la mort cellulaire. / Apoptosis or programmed cell death plays a crucial role in tissue homeostasis and is regulated by the Bcl-2 proteins, which control mitochondria membrane permeability and cytochrome c release, two events that precede cell demise. Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and cause resistance to anti-cancer regimens, therefore representing important targets for novel therapeutics. BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.Sommaire

Apoptosis is a cell death pathway that regulates tissue homeostasis, and is often altered in oncogenesis. Autophagy is a lysosome degradation pathway that mediates cellular adaptation in response to stresses. Altered autophagy pathways are proposed to be associated with pathogenesis of neurodegenerative diseases and oncogenesis. The goal of this work is to study the complex link between apoptosis and autophagy pathways, and their possible roles in the development of cancer. Using transgenic mice models, we found that impaired apoptosis by overexpression of a dominant negative form of Caspase-9 (Casp9DN) failed to accelerate T-cell lymphoma either by itself or in tumor-prone Bax overexpressing transgenic mice. Additionally, heterozygous disruption of Beclin 1, a central upstream autophagy regulator, failed to promote T-cell lymphoma in either Casp9DN or tumor-prone Bax overexpressing transgenic mice. However, caspase inhibition enhanced a unique form of selective mitochondrial autophagy, referred to as mitochondrial outer membrane permeabilization (MOMP)-induced mitophagy. Parkin, a protein mutated in early-onset Parkinson's disease, mediates mitophagy following protonophore (CCCP) treatment, suggesting that Parkin may also play a role in MOMP-induced mitophagy. Thus, two different types of mitochondrial stresses, MOMP and CCCP, cause mitochondrial depolarization and induce mitophagy. We therefore examined if there is a mechanistic link between two mitophagy pathways. Focusing on the roles of autophagy and apoptosis regulators using isogenic hematopoietic cell lines, our studies demonstrate that MOMP-induced mitophagy is dependent upon Bcl-2 family members, but independent of Parkin or ULK1 (an autophagy regulator). In contrast, CCCP-induced mitophagy is dependent upon Parkin and ULK1, but independent of Bcl-2 family members. However, we found that both pathways ultimately result in the following properties: reduced mitochondrial respiration rate, altered cellular metabolism, and high sensitivity to 2-DG (an inhibitor of glycolysis). Interestingly, 2-DG induced cell death in cells following Parkin-dependent mitophagy is independent of Bcl-2 and Bax/Bak. Overall, the work in this dissertation demonstrates that the two different mitochondrial stresses, MOMP and protonophore (CCCP) treatment, lead to two mechanistically distinct mitophagy pathways, but both alter mitochondrial respiration and cellular metabolism.

Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.