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Many previous studies have demonstrated that gold compounds possess successful results in catalysis and in medicinal chemistry. The central aim of this dissertation is the design and synthesis of novel gold (I) acyclic diamino carbene complexes as a chemotherapeutic agent for triple-negative breast cancer (TNBC) and for catalysis. In this study, a series of chiral neutral and cationic gold (I) acyclic diamino carbene (ADC) complexes and neutral gold (I) bis- ADC complexes have been synthesized. As the chiral neutral gold (I) ADCs, four diastereomers of S binaphthyl L proline tertiary butyl ester gold (I) chloride, S binaphthyl D proline tertiary butyl ester gold (I) chloride, R binaphthyl L proline tertiary butyl ester gold (I) chloride, and R binaphthyl D proline tertiary butyl ester gold (I) chloride have been synthesized and characterized. Different chiral gold (I) ADC complexes with bulky chiral binaphthyl group and with different amine groups of morpholine, chiral proline methyl ester, and benzyl ester have been synthesized and characterized. After that four diastereomers of the nitrile adduct of cationic binaphthyl proline tertiary butyl ester nitrile and four diastereomers of the isonitrile versions of it have been synthesized and characterized. A series of gold (I) cationic bis ADC complexes have been synthesized and characterized. All these novel gold ADC complexes were tested for biological activity against TNBC cell line MDA-MB-231 and cationic S binaphthyl D proline ester isonitrile adduct, S binaphthyl D proline ester isonitrile adduct and R binaphthyl D proline ester isonitrile adduct gave promising inhibition rates. According to Lipinski's rule, lipophilicity determines the effectiveness of the drug absorption to the body through the lipid membrane. To determine the drug-likeness of the gold ADC complexes, log P values were calculated for some of the synthesized complexes using a modified shake flask method. Gold (I) ADC complexes have been renowned for their ability in catalysis, but enantioselective catalysis is not that well studied. A3 coupling reaction is a well-known reaction for the synthesis of propargyl amines. Here, A3 coupling reaction with a chiral amine has been performed using the previously synthesized four diastereomers of binaphthyl proline tertial butyl ester gold (I) ADCs (SL, RD, RL, SD) as the catalyst expecting four different diastereomers of the product. The reaction exhibited reasonable yields but with a low enantiomeric excess (ee%). However, it gave proof of the principle that asymmetric induction is possible with the synthesized novel chiral gold (I) ADC complexes.

Le cancer du sein est une maladie complexe et difficile à caractériser. Parmi les différents sous-types moléculaires, les tumeurs du sein Triple-Négatives (TN) sont particulièrement agressives et de mauvais pronostic. Elles sont caractérisées par une absence d’expression des récepteurs aux œstrogènes (ER), à la progestérone (PR), l’absence de surexpression du récepteur Human Epidermal growth factor 2 (HER2) et de fréquentes mutations sur les gènes BRCA1/2 (profil « BRCAness »). En absence de thérapies ciblées efficaces, de nombreux traitements ciblés notamment les inhibiteurs de poly-ADP-ribose polymérases (anti-PARPs) sont actuellement en cours de développement, en recherche préclinique et clinique. Basés sur le principe de létalité synthétique, les anti-PARPs ciblent les propriétés BRCAness des tumeurs TN. Dans ce contexte, ces travaux de recherche ont été orientés sur le développement d’outils diagnostics afin d’optimiser l’efficacité des anti-PARPs sur des tumeurs TN. Pour ce faire, dans un premier temps, des cultures cellulaires en 3D via la technique Liquid Overlay ainsi que des tests de cytotoxicités associés ont été développés, à partir des lignées cellulaires MDA-MB-231 et SUM1315 de phénotype TN. Ces deux modèles de sphéroïdes ont ensuite été optimisés/normalisés dans un milieu de culture synthétique intitulé OPTIPASS (BIOPASS). Dans un deuxième temps, l’efficacité d’un co-traitement combinant l’anti-PARP1 Olaparib à faibles et à fortes doses et la radiothérapie fractionnée (5x2 Gy) a été modélisée sur les deux lignées MDA-MB-231 et SUM1315, en conditions 2D et 3D. Ces expériences ont clairement mis en évidence un effet potentialisateur de l’Olaparib sur la radiothérapie (i) en présence de faibles doses de cet anti-PARP (5 µM ou inférieur) (ii) à long terme et (iii) en présence d’un fractionnement maximum (5x2 Gy). De plus, les lignées tumorales TN étudiées présentaient des différences de sensibilité vis-à-vis du co-traitement. Ainsi, une analyse transcriptomique in silico a mis en évidence des profils très différents de ces lignées hautement métastatiques et très agressives. Notamment, la lignée SUM1315 semblait présenter un engagement neuronal, suggérant son origine métastatique cérébrale. Ces résultats encourageants pourraient ouvrir de nouvelles perspectives pour le traitement des métastases cérébrales de tumeurs mammaires TN, très fréquentes chez ce sous-type. Dans un troisième temps, afin de mieux caractériser le mode d’action de l’Olaparib sur ces modèles de sphéroïdes, un dérivé fluorescent de l’Olaparib, l’Ola-FL, a été synthétisé et caractérisé. L’analyse de la pénétration et de la distribution de l’Ola-FL au sein des sphéroïdes MDA-MB-231 et SUM1315 a mis en évidence une distribution rapide et homogène du composé ainsi que sa persistance après 3h d’incubation, dans toute la profondeur des sphéroïdes et notamment dans les zones hypoxiques centrales. Enfin, l’analyse de la co-expression de deux pompes Multidrug Resistance (MDR) majeures, la MRP7 et la P-gp après le traitement des deux lignées TN avec l’Olaparib, a mis en évidence sur les cultures 2D, une expression de type relai de la MRP7 et la P-gp. Sur les sphéroïdes traités avec une faible dose d’Olaparib à long terme, une expression basale de la MRP7 et une surexpression de la P-gp ont été détectées, au sein des cellules résiduelles périphériques des sphéroïdes. Ces résultats mettent clairement en évidence l’implication des pompes d’efflux dans les mécanismes de résistances à l’Olaparib, dans ces tumeurs agressives. L’ensemble des résultats issus de la modélisation de l’action de l’Olaparib sur des sphéroïdes MDA-MB-231 et SUM1315 laissent supposer sa plus grande efficacité à faible dose et à long-terme, notamment dans les zones hypoxiques des sphéroïdes, probablement aussi à l’origine de son effet potentialisateur avec la radiothérapie. / Breast cancer is a very complex and heterogeneous disease. Among the different molecular subtypes, Triple-Negative (TN) breast cancers are particularly aggressive and of poor prognosis. TN tumours are characterized by a lack of estrogen receptors expression (ER), progesterone receptors expression (PR), the absence of Human Epidermal growth factor receptor 2 overexpression (HER2) of the frequent mutations on BRCA1 / 2 genes ("BRCAness" phenotype). In the absence of effective targeted therapies, many targeted therapies including poly-ADP-ribose polymerase inhibitors (anti-PARPs) are currently under development in preclinical and clinical studies. Based on the synthetic lethality concept, the anti-PARPs specifically target the BRCAness properties of TN tumors. In this context, these works were focused on the development of diagnostic tools for the optimization of TN tumours treatment with anti-PARPs. For this, firstly, 3D cell cultures formed with the Liquid Overlay technique as well as associated cytotoxicity tests were developed, from the TN breast cancer cell lines MDA-MB-231 and SUM1315. These two spheroid models were then optimized and standardized in a synthetic culture medium called OPTIPASS (BIOPASS). Secondly, the efficacy of a co-treatment combining anti-PARP1 Olaparib at low and high doses and fractioned radiotherapy (5x2 Gy) was analyzed on the two cell lines MDA-MB-231 and SUM1315 cultured in 2D and 3D conditions. These experiments clearly demonstrated a potentiating effect of Olaparib on radiotherapy (i) in presence of low doses of this anti-PARP (5 μM or inferior) (ii) at long term and (iii) in presence of the maximum fractionation (5x2 Gy). In addition, these two TN cell lines showed a heterogeneous sensitivity to the co-treatment. Thus, an in silico transcriptomic analysis revealed very different profiles of these highly metastatic and highly aggressive cell lines. Notably, the SUM1315 cell line presented a neuronal commitment, suggesting its cerebral metastatic origin. These promising results could open up new perspectives for the treatment of TN tumours brain metastases, which are very common in this subtype. Thirdly, in order to better characterize the mode of action of Olaparib on these spheroid models, a fluorescent derivative of Olaparib, Ola-FL, was synthesized and characterized. The analysis of Ola-FL penetration and distribution in MDA-MB-231 and SUM1315 spheroids showed a rapid and homogeneous distribution of the compound as well as its persistence after 3h of incubation, in all the depth of the spheroids and especially in the central hypoxic zones. Finally, the analysis of the co-expression of two major Multidrug Resistance (MDR) pumps, MRP7 and P-gp after the treatment of the two TN lines with Olaparib, revealed on 2D cultures, a relay type expression of the MRP7 and the P-gp. On spheroids treated with a low dose of Olaparib art long term (10 days), a basal expression of MRP7 and an overexpression of P-gp were detected in the peripheral residual cells of the spheroids. These results clearly highlighted the involvement of these efflux pumps in Olaparib resistance mechanisms, in these aggressive tumors. All the results resulting from the modeling of the action of Olaparib on MDA-MB-231 and SUM1315 spheroids suggest its greater efficacy at low dose and at long-term, especially in the hypoxic zones of the spheroids. This parameter might be probably at the origin of its potentiating effect with radiotherapy.

Quatorze femmes canadiennes meurent chaque jour du cancer du sein. Le cancer du sein triple-négatif (CSTN) détient un mauvais pronostic De nombreux efforts sont fournis afin d'offrir à ces patientes des traitements ciblés, comme les inhibiteurs de poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) afin d’améliorer leur survie et de minimiser la toxicité liée à la chimiothérapie. Le sous-groupe de CSTN qui pourrait bénéficier des PARPi reste à être identifié. De plus, différentes stratégies d'administration des PARPi et de la chimiothérapie pourraient améliorer leur efficacité thérapeutique tout en diminuant la toxicité. Nous avons précédemment dérivé une signature génétique de 63 gènes prédisant la réponse aux PARPi avec une précision globale élevée. Nos objectifs sont 1) d'évaluer les implications cliniques de la signature génétique; et 2) de déterminer la séquence optimale d'administration du talazoparib et du carboplatin in vivo en cancer du sein triple-négatif BRCAWT. D'abord, nous avons évalué la fréquence mutationnelle des 63 gènes dans différents contextes cliniques. Deux bases de données publiques furent utilisées. Puis, nous avons comparé trois cohortes de xénogreffes orthotopiques: A) talazoparib en premier, combiné au carboplatin le jour 3; carboplatin en premier suivi du talazoparib B) un jour après; et C) sept jours après. La fréquence mutationnelle des 63 gènes était élevée chez les tumeurs luminales B et celles de mauvais pronostic. Les patientes luminales B mutées avaient une moindre survie que les patientes non mutées. Aussi, l'inhibition tumorale et métastatique était similaire pour les cohortes A et B, cependant la cohorte B avait moins de toxicité. Les PARPi pourraient avoir un rôle chez les tumeurs luminales B et celles de mauvais pronostic. Deuxièmement, le prétraitement avec le carboplatin semble améliorer la sensibilité au talazoparib et diminuer la toxicité. / Fourteen Canadian women die every day from breast cancer. Triple-negative breast cancer (TNBC) has a poor prognosis. Numerous efforts are made to offer these patients targeted therapies such as poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) to improve survival and minimize chemotherapy-related toxicity. It is not well understood which subset of TNBC patients will benefit from PARPi; and if different sequencing strategies of PARPi and chemotherapy can improve therapeutic efficacy and decrease toxicity. We previously derived a 63-gene signature predicting response to PARPi with a high overall accuracy. Our objectives are 1) to evaluate the clinical implications of the 63-gene signature; and 2) to determine the optimal sequence of administration of talazoparib and carboplatin in vivo in BRCAWT TNBC. First, we evaluated the mutational frequency of the 63 genes in different clinical settings using two publically-available datatsets. Second, we compared three cohorts of orthotopic xenografts: A) talazoparib first, combined with carboplatin on day 3; carboplatin first, followed by talazoparib B) one day later; and C) seven days later. We found that the mutational frequency was high in breast cancer subtypes of poor prognosis. Mutated luminal B patients had a lower survival than non-mutated patients. We also found that tumoral and metastatic inhibition were similar between cohorts A and B, but cohort B had less toxicity. In conclusion, there is potential for PARPi efficacy in luminal B and poor prognosis tumors. Second, pretreatment with carboplatin may be an effective approach with less toxicity.

<p>With 2.1 million new cases, breast cancer is the most common cancer in women. Triple negative breast cancer (TNBC), which is 15-20% of these breast cancer cases is clinically negative for expression of estrogen and progesterone receptors (ER/PR) and human epidermal growth factor receptor 2 (HER2) receptors<a>.</a> It is characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis, and lack of targeted therapies. TNBCs utilize glycolysis for growth, proliferation, invasiveness, chemotherapeutic resistance and hence has poor therapeutic response. There is an urgent need for novel/alternate therapeutic strategies beyond current standard of treatment for this subset of high-risk patients. Electrical pulse-based chemotherapy, known as electrochemotherapy (ECT) could be a viable option for TNBC therapy. ECT involves the local application of precisely controlled electrical pulses to reversibly permeabilize the cell membrane for enhanced uptake. ECT can increase the cytotoxicity of the chemotherapeutics up-to 1000 times, facilitating a potent local cytotoxic effect. </p> <p>The high cost and severe side-effects of conventional chemotherapeutics motivate the application of effective natural compounds. Combining electrical pulses with natural compounds will enhance the treatment efficacy. This dissertation focuses on curcumin, the yellow pigment of natural herb turmeric, that has been used for over 5000 years for its excellent anticancer properties. Previous studies have demonstrated the effectiveness of curcumin for treating multiple cancers, including TNBC, with limited side effects. The potency of curcumin can be enhanced further by combining it with ECT to provide an attractive and cost-effective alternative for TNBC treatment. </p> <p>Towards this we studied the effect of ECT with curcumin on MDA-MB-231 cell line, a human adenocarcinoma epithelial TNBC cell line. We performed various assays, including cell viability, colony forming, cell cycle, apoptosis, H₂O₂ reactive oxygen species (ROS), immunoblotting, real time quantitative PCR (qPCR), and cellular metabolites detection to study the impact of ECT with curcumin on MDA-MB-231 cells. In addition, to better understand the underlying mechanisms, we used high throughput, label-free quantitative proteomics. While several studies have attempted to define the mechanism of action of curcumin on cancer cells, little is known on the action mechanism of the curcumin delivered with electrical pulses. This work unravels the molecular mechanism behind the enhanced effects observed under the ECT-based curcumin therapy in TNBC cells, employing a high-throughput, quantitative, label-free mass spectroscopy-based proteomics approach. The proteomics approach provides information on the thousands of cellular proteins involved in the cellular process, allowing a comprehensive understanding of the electro-curcumin-therapy mechanism. Similar studies were also performed for ECT with cisplatin to compare the efficacy of the electro-curcumin-therapy to the standard stand-alone cisplatin-based therapy.</p> <p>Our results revealed a switch in the metabolism from glycolysis to mitochondrial metabolic pathways. This metabolic switch caused an excessive production of H₂O₂ ROS to inflict apoptotic cell death in MDA-MB-231 cells, demonstrating the potency of this ECT based curcumin therapy. These results encourage further studies to extend the application of ECT for clinical practice.</p>

Trippelnegativ bröstcancer (TNBC) är den mest maligna formen av bröstcancer utan någon framträdande behandlingsbar biomarkör. Således har den djupa återfallsfrekvensen och bristen på behandlingsalternativ öppnat behovet av att förstå TNBC: s etiopatogenes och molekylära mekanism. Huvudsyftet är att kartlägga det genomomfattande differentiella uttrycket av den förmodade cancergenen (en prenyleringsgen) och dess betydelse vid trippelnegativ bröstcancer. Hi-Cap (Capture Hi-C) är en teknik som genererar högupplösta promotor-förstärkare interak- tioner med nästan en-förstärkare upplösning.Vi arbetade med cancercellinjer, MDA-MB_231, med och utan den förmodade genen. Hi-C-tekniken optimerades i enlighet därmed för cancer- cellinjen för att generera en högupplöst berikad region. Resultaten kan vidare användas för att utföra biblioteksförberedelser och bioinformatikanalys. Dessa fynd kommer att ägnas åt att upptäcka nya vägar involverade i prenylering och TNBC. / Triple-negative Breast cancer (TNBC) is the most malignant form of breast cancer with no prominent treatable biomarker. The profound recurrence rate and lack of treatment options have opened the need to understand the etiopathogenesis and molecular mechanism of TNBC. The main objective of this study is to map the genome-wide differential expression of the putative cancer gene (a prenylation gene) and its importance in Triple-Negative Breast cancer. Hi-Cap (Capture Hi-C) is a technique which generates high-resolution promoter-enhancer interactions with almost single-enhancer resolution. We worked with cancer cell lines, MDA-MB_231, with and without the putative gene. The Hi-C technique was optimized accordingly for the cancer cell line to generate a high-resolution enriched region. The results can be further used to perform library prep and bioinformatics analysis. These findings will devote to discovering novel pathways involved with prenylation and TNBC.