The ubiquitin-proteasome pathway plays vital roles in multiple cellular processes including protein turnover and transcription regulation. The fate of a ubiquitinated protein is determined by the number of ubiquitin molecules added and the site to which they are added. Monoubiquitinated proteins are stabilized and often activated, while polyubiquitinated proteins are rapidly targeted for degradation. Major histocompatibility complex class II (MHC II) molecules are a vital part of the immune response and are responsible for presenting antigens to CD4+ T cells. The class II transactivator (CIITA) is the master regulator of MHC II transcription and has been shown to have increased transactivity when monoubiquitinated. The focus of this thesis is on the impact of ubiquitination on CIITA stability and MHC II gene expression through the identification of an E3 ligase that targets and ubiquitinates CIITA.

The histone acetyltransferase protein, Tip60, has many important functions in epigenetic regulation of gene expression and DNA repair. Our objective is to design, synthesize, and evaluate potent inhibitors for Tip60. Full-length Tip60 (fl-Tip60) and catalytic domain of Tip60 (cat-Tip60) were expressed in E. coli and purified with nickel affinity chromatography. Quantitative analysis of enzyme activities demonstrated that both enzyme forms had very high activity with the substrate H4. To create new Tip60 inhibitors, various histone H3 peptides conjugated with CoA were synthesized using the Fmoc solid-phase peptide synthesis and solution phase synthesis protocols. The results from the inhibition radioactive assay showed that the synthetic H3-CoA conjugates inhibited effectively the enzymatic activity of both fl-Tip60 and cat-Tip60; and the addition of methyl groups to the Lys-4 or Lys-9 residue of H3 aided in a 7-9 fold enhancement in potency in comparison to nascent H3-CoA inhibitor.

Les cellules souches de gliomes (CSG), une sous-population de cellules tumorales, seraient en partie responsables de l’échec des traitements des gliomes de par leur résistance et leur capacité régénérative. Le mécanisme alternatif (ALT) de maintenance des télomères, basé sur la recombinaison homologue et non pas sur la télomérase, est détecté dans environ 30% des gliomes humains suggérant que des stratégies thérapeutiques spécifiquement dirigées contre ALT pourraient avoir un intérêt thérapeutique. Dans ce travail, nous avons poursuivi la caractérisation du premier exemple de CSG humaines ayant un phénotype ALT, les cellules TG20. Nous avons montré que malgré leur très fort taux de recombinaison, les télomères de ces cellules continuaient à assurer leur fonction de protection des chromosomes. Nous avons vérifié que les cellules TG20 conservaient leur capacité à générer des tumeurs intracérébrales après des transplantations sériées chez les souris immunodéprimées tout en gardant un phénotype ALT. Ces résultats confirment à la fois les propriétés de cellules souches cancéreuses des cellules TG20 et la capacité de ALT à assurer la maintenance des télomères nécessaire à l’autorenouvellement et au fort taux de prolifération des CSG in vivo. La greffe intracérébrale de cellules TG20 chez des souris immunodéprimées représente donc un bon modèle d’étude préclinique des gliomes ALT. Nous avons ainsi montré qu’un traitement précoce par un ligand des G-quadruplexes télomériques, 360B, juste après la greffe de cellules TG20, était capable d’inhiber le développement tumoral suggérant l’intérêt de l’utilisation de ligands des G-quadruplexes pour cibler spécifiquement les CSG ALT. Une étude des profils d'expression transcriptomique des cellules TG20 et de plusieurs lignées de CSG humaines exprimant la télomérase, nous a conduit à nous intéresser aux rôles de deux lysine acétyl transférases homologues, PCAF (P300/CBP Associated Factor) et GCN5 (General Control Nonderepressible 5) dans la régulation de la recombinaison télomérique des cellules ALT. Nous avons montré que les inhibitions de ces deux protéines ont des effets opposés sur le mécanisme ALT. Nous proposons qu’une balance d’expression de PCAF et GCN5 régule la maintenance des télomères dans les cellules ALT via le contrôle du turnover de TRF1 ce qui pourrait constituer la base d’une nouvelle stratégie thérapeutique vis-à-vis des gliomes ayant un phénotype ALT. / Glioma stem cells (GSC), a subpopulation of tumor cells, are partly responsible for the failure of treatment of gliomas because of their resistance and regenerative capacity. The mechanism of alternative lengthening of telomere (ALT), based on homologous recombination, is detected in approximately 30 % of human gliomas. Therefore, therapeutic strategies directed specifically against ALT may have a therapeutic value. In this work, we further characterized the first model of human ALT GSC, the TG20 cells. We showed that despite their very high rate of recombination, the telomeres were still capable of fulfilling their protective function of chromosomes. We verified that the TG20 cells retained their ability to generate intracerebral tumors after serial transplantations in immunocompromised mice, while preserving an ALT phenotype. These results confirm the cancer stem properties of TG20 cells and the ability of ALT to ensure telomeres maintenance, which is required for the self-renewal and the high proliferation rate of GSC in vivo. Intracerebral grafts of TG20 cells in immunocompromised mice represent thus a good preclinical model for studying ALT gliomas. We have shown that treatment with a ligand of telomeric G-quadruplexes, the 360B, at an early stage of TG20 tumor engraftment, was able to inhibit tumor growth, showing the interest of the use of G-quadruplex ligands to specifically target ALT GSC. Transcriptomic profiling of TG20 cells and several other GSC telomerase-positive lines, incited us to study the roles of two homologous lysine acetyl transferases, PCAF (p300/CBP Associated Factor) and GCN5 (General Control Nonderepressible 5), in the regulation of telomeric recombination in ALT cells. We showed that the inhibition of these two proteins has opposite effects on the ALT mechanism. We propose that a balance of expression of PCAF and GCN5 regulates the telomere maintenance in ALT cells by controlling the turnover of TRF1. This model could serve for the development of new therapeutic strategies targeting ALT gliomas.

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.

Posttranslational modifications are of great importance in the mediation of transcriptional effects, necessary for signalling in cancer. A characteristic example of such modifications is acetylation of the p53 tumour suppressor, a transcription factor involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 is stabilised under hypoxic and DNA damaging-conditions. However, only in the latter scenario is p53 fully capable of inducing the expression of its proapoptotic targets through acetylation. The hypoxia inducible factor 1 (HIF-1) transcription factor is stabilised at low oxygen levels to mediate a cellular adaptive response under these conditions, promoting cell survival. As these two opposing transcription factors share a common transcriptional regulator, p300/CBP, this study focused on deciphering the p300/CBP complex components under differential stress to determine its composition required for cellular responses elicited in response to DNA damage or hypoxia, in an effort to investigate a possible link between differential posttranslational modifications and the resulting cell fate. Hence, the aim of this study was to investigate the roles of p300/CBP components in dictating transcriptional regulation of both HIF-1 and p53 in hypoxic conditions. To carry out this study, the proapoptotic BID gene was the system used, as its promoter contains a p53 response element and a HIF-1 response element (HRE). The p300/CBP associated factors PCAF and Strap were appointed as potent candidates for posttranslational modifications under differential conditions, as they are stress-responsive cofactors. Under DNA damage, PCAF acetylates p53 at K320 and Strap augments p300 binding to p53, both of which amplify the p53 response. Evidence from this study demonstrates that under hypoxia-mimicking conditions PCAF-mediated p53 acetylation at K320 is reduced to a greater extent compared to p300/CBP acetylation at K382. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the cell cycle arrest p21WAF-1/CIP-1 gene that appears to be unaffected by hypoxia, but fail to be recruited to the BID promoter, rendering p53 incapable of upregulating proapoptotic BID in hypoxic conditions. In addition, under the same conditions, PCAF was found to acetylate, and direct HIF-1 to a particular subset of its targets, leading to alterations in the net physiological effect. Moreover, the intrinsic acetyl transferase activity of PCAF was shown to increase the stability of HIF-1. An additional role was attributed to PCAF in relation to apoptosis, albeit from another angle. BID protein translocation to the cytoplasm in hypoxic conditions was facilitated by ectopically expressed PCAF.Strap was found to be preferentially recruited to the HRE of the BID promoter in hypoxic conditions, and to exert a transrepression effect that appeared to be p53-dependent. Strap also interacted with specific PCAF isoforms depending on the type of cellular stress. Contrary to PCAF, ectopically expressed Strap did not have any effect on BID subcellular distribution. This study has provided additional insight in the mechanisms by which cofactors are involved in cell fate, either by affecting activity and stability of HIF-1 and p53, or having a direct effect on Bcl-2 member subcellular distribution.

Energy production is a very important function for the cells to maintain homeostasis, survive and proliferate. Cellular energy can be produced either through oxidative phosphorylation (OXPHOS) in the presence of oxygen or glycolysis in its absence. Cancer cells, even in the presence of oxygen prefer to produce energy through glycolysis and this confers them a survival advantage. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. For that purpose the role of various transcription factors in determining the pathway of energy production has been investigated extensively and there is evidence to suggest that oncogenic transcription factors promote glycolysis whereas tumour suppressors demote it. In line with this notion, the master regulator of cellular response to hypoxia, the Hypoxia Inducible Factor 1 (HIF-1) has been shown to induce the expression of a variety of genes encoding enzymes involved in glucose metabolism as well as OXPHOS favouring energy production through glucose metabolism in hypoxic cells. The tumour suppressor p53 on the other hand inhibits glycolysis and stimulates OXPHOS. One of the pathways through which p53 exerts these effects, is by inducing the inhibitor of glycolysis TIGAR and the cytochrome c oxidase assembly factor SCO2 gene expression under DNA damage conditions. However, the regulation of the expression of these genes in hypoxic conditions has been only partially elucidated. We hypothesised that under hypoxic conditions, TIGAR and SCO2 gene expression might be differentially regulated in cells bearing mutated p53 and in these cells the involvement of HIF-1 could be crucial. Indeed under hypoxia mimicking conditions, the TIGAR and SCO2 protein and mRNA levels were found to be modulated differentially in p53 wild type and mutant cell lines. The bioinformatics analysis revealed the presence of hypoxia responsive elements (HREs) within the regulatory region of the promoters of TIGAR and SCO2 genes. Firefly reporter assays and chromatin immunoprecipitation (ChIP) assays have indicated that HIF-1 plays a crucial role in the regulation of TIGAR gene expression. The direct involvement of HIF-1 in the regulation of SCO2 gene expression requires further investigation. We and others have recently reported that PCAF is a common cofactor for p53 and HIF-1α, regulating the protein stability and transcription target selectivity of both transcription factors thereby orchestrating the balance between life and death in cancer cells. We hypothesised that PCAF plays a similar role in the regulation of cellular energy metabolism by differentially targeting HIF-1α and p53 to the promoter of TIGAR and SCO2 genes. In this study we present evidence to support the notion that PCAF plays an import role in the regulation of TIGAR and SCO2 gene expression under hypoxic mimicking conditions. This conclusion was supported by assessing the functional consequences of PCAFwt and PCAFΔHAT overexpression on the intracellular lactate production, cellular oxygen consumption, NAD+/NADH ratio and ROS generation in cells under hypoxia mimicking conditions.

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