Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 104-114).

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

The dissemination of tumor cells to distant organs i.e. metastasis is an exceedingly complex process leading to 90% of all cancer deaths. Despite being so clinically important, little is known about this process that requires tumor cells to leave the primary tumor site, intravasate and transport through the blood stream, extravasate and colonize at secondary sites leading to distant metastases. Survivin, a member of the IAP (Inhibitor of Apoptosis) family with known functions in apoptosis and mitosis, is highly expressed in aggressive tumors and is associated with poor prognosis and adverse clinical outcome. But the mechanistic role of survivin in metastatic dissemination has not been investigated. In this study, we demonstrate an important and novel role of survivin in activating a broad gene expression program in tumor cells. Of particular importance is the upregulation of a distinct class of cell adhesion molecules, particularly fibronectin. This IAP mediated gene regulation requires synergistic intermolecular cooperation between survivin and its related cofactor molecule, XIAP that results in activation of NF-κB dependent fibronectin gene expression. The binding of fibronectin with its cognate cell surface receptors initiates outside–in signaling leading to the autocrine and paracrine activation of cell motility kinases, FAK and Src, in turn leading to enhanced tumor invasion and metastasis. The importance of survivin and XIAP in the process of metastasis has also been demonstrated in vivousing intrasplenic injections in mouse models. Overall this study is the first to place survivin upstream of transcriptional activation of gene expression particularly fibronectin. In addition, it also demonstrates the importance of survivin-XIAP complex in mediating NF-κB activation which in turn switches on the expression of various target genes involved in tumor metastasis. Hence this study dissects the upstream and downstream requirements of survivin- XIAP complex mediated tumor dissemination and metastasis. Significance of this Study The hallmark of end-stage cancer is metastasis, an incurable condition almost invariably associated with death from disease. Despite a better understanding of the metastatic process, and the identification of key gene expression requirements of this pathway, the development of anti-metastatic therapies has lagged behind, with no viable options being currently offered in the clinical setting. Our findings that Inhibitor of Apoptosis (IAP) proteins functions as metastasis-promoting genes independently of cell survival, but through activation of cell motility could have important ramifications for the broader application of IAP antagonists currently in early clinical trials, as novel anti-metastatic therapies.

Contexte. Les carcinomes des voies aérodigestives supérieures (VADS) arrivent en 6ème position parmi les cancers les plus fréquents au niveau mondial. La fonction du récepteur TLR3 dans les cellules de carcinomes des VADS est encore très mal comprise. Objectifs et méthodes. 1) Déterminer le niveau d’expression du récepteur TLR3 dans les lignées et les biopsies de carcinomes des VADS par western blot et par immunohistochimie. 2) Etudier le rôle de TLR3 dans la croissance tumorale de ces tumeurs, en utilisant notamment des lignées invalidées de façon conditionnelle pour TLR3. 3) Evaluer in vitro les effets cytotoxiques de ligands artificiels de TLR3 soit seuls, soit utilisés en combinaisons avec un inhibiteur d’IAP (inhibitor of apoptosis protein).Résultats. La protéine TLR3 est détectée à un niveau élevé en western blot dans les lignées de carcinomes des VADS étudiées, comparativement à un panel d’autres tumeurs épithéliales humaines. TLR3 est également constamment détecté en immunohistochimie dans les biopsies. TLR3 semble jouer un rôle dans la croissance tumorale des carcinomes des VADS : dans certaines conditions de culture (culture en hypoxie ou en milieu pauvre en SVF et en nutriments), la stimulation de TLR3 par un ligand exogène, le poly(A:U), favorise la croissance des cellules tumorales. Nous avons étudié l’effet de la stimulation de TLR3 sur le métabolisme glucidique dans ces mêmes cellules en utilisant un appareil de type Seahorse® qui mesure la consommation d’oxygène et la production de protons à partir de cellules cultivées en microplaques. Ces expériences montrent que la stimulation de TLR3 fait augmenter l’activité des voies du métabolisme cellulaire anaérobie (glycolyse extra-mitochondriale). Une étude métabolomique a mis en évidence des différences significatives dans le profil métabolique des cellules tumorales stimulées par le poly(A:U) comparativement aux cellules non traitées. Par ailleurs, nous avons montré que la stimulation de TLR3 permettait de détecter le facteur de transcription HIF1 en Western blot, même en conditions normoxiques. Sachant que des ARN libérés par des cellules en état de nécrose peuvent stimuler TLR3, il est tentant de penser que ce récepteur pourrait favoriser la survie des cellules malignes en zone hypoxique au voisinage de cellules nécrotiques. Néanmoins, l’expression de TLR3 représente aussi un facteur de vulnérabilité pour les cellules de carcinome des VADS : en effet les ligands artificiels de TLR3 utilisés en combinaison avec un inhibiteur d’IAP (Inhibitor of Apoptosis Protein) produisent des effets cytotoxiques sur les lignées de carcinomes des VADS étudiées. / Background. Head and Neck (HN) carcinomas are the 6th most frequent type of cancer worldwide. The role of the TLR3 receptor in HN carcinomas remains poorly understood.Objectives and Methods. 1) To assess the expression level of TLR3 in HN carcinoma cell lines and biopsies by Western blot and immunohistochemistry, respectively. 2) To study the role of TLR3 in tumour growth using specific cell lines with conditional knock-down of TLR3. 3). To assess in vitro the cytotoxic effects of artificial ligands of TLR3 used either alone or in combination with an IAP (inhibitor of apoptosis protein) inhibitor.Results. TLR3 protein was detected at a high level by Western blot analysis in HN carcinoma cell lines, by comparison with a panel of other human epithelial cancer cell lines. TLR3 was also consistently detected by immunohistochemistry in tumour biopsies. TLR3 seem to play a role in HN carcinoma cell growth: under certain culture conditions (hypoxic or low fetal calf serum/low nutrient culture conditions), TLR3 stimulation by a synthetic ligand, the poly(A:U), favours tumour cell growth. We investigated the effects of TLR3 stimulation on glucose metabolism using a Seahorse® analyzer, which measures the oxygen consumption and the proton production in living cells. Our results indicate that TLR3 stimulation induces an increase in anaerobic metabolism (extra-mitochondrial glycolysis). A metabolomic study revealed significant changes in the metabolic profile of cancer cells treated by poly(A:U) by comparison with untreated cells. We also showed that under TLR3 stimulation, HIF1 became detectable by Western blot analysis, even in normoxia. Given the fact that RNA fragments released by dying cells are able to trigger TLR3, one can assume that TLR3 might favour cancer cell survival in hypoxic areas located near the necrotic core of the tumour. However, TLR3 expression is also a factor of vulnerability for HN carcinoma cells: indeed, the combination of TLR3 artificial ligands with an IAP inhibitor has a strong cytotoxic effect on HN carcinoma cells in vitro.