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Les ARN polymérases sont des acteurs indispensables de la transcription. Chez les eucaryotes il existe trois ARN polymérases (I, II et III). La ARN polymérase III (Pol III) possède 17 sous-unités, dont une qui existe sous deux formes: RPC32α et RPC32β. Seulement une des deux formes peut être intégrée dans la Pol III, créant ainsi deux polymérases différentes Pol IIIα et Pol IIIβ. Alors que RPC32β est présent dans les cellules somatiques, RPC32α est exprimé surtout dans des cellules souches et des cellules tumorales. Aujourd’hui rien n’est connu sur leurs rôles respectifs. Le cancer du sein est un problème majeur de santé publique car c’est le cancer féminin le plus fréquent. Plusieurs types de cancer du sein sont identifiés selon la présence ou absence de certains récepteurs hormonaux. Des cancers qui testent négative pour le récepteur d’oestrogène et de progestérone et qui ne surexpriment pas le récepteur pour les facteurs de croissance épidermiques humains 2 (HER2) sont appelés triple-négative. Ils ont un pronostique peu favorable, due à l’agressivité de ce type de cancer et un manque de thérapie cibles. Pour étudier le rôle de RPC32α il fallait identifier un model tumorale. En collaboration avec Jean-Paul Feugeas (INSERM UMR 1098) une étude transcriptomique a été fait sur 2627 échantillons cliniques de tissus de sein. L’étude montre que RPC32α est surexprimé dans les cancers triple-négative, alors que son homologue RPC32β est surexprimé dans les tissues normaux. Une analyse sur six lignées de cancer du sein et une ligné non-tumorale ont pu confirmer les résultats de l’analyse transcriptomique. Le modèle de cancer du sein a donc été validé. Une caractérisation des différentes lignées de cancer du sein a démontré que d’autres sousunités de la Pol III n’étaient pas surexprimées dans les cancers triple-négative. La surexpression de RPC32α n’était donc pas une conséquence d’une hyperactivité de la Pol III. Une analyse des transcrits synthétisé par la Pol III a montré que en générale les transcrits de la Pol III étaient plus fortement exprimé dans les cancers triple-négative que dans d’autres cancers. Afin d’étudier l’implication de RPC32α dans les phénomènes de tumorisation, plusieurs lignées cellulaires dépourvues de RPC32alpha ont été créé utilisant la technique CRISPRCAS9. L’absence de RPC32α n’a pas induit une augmentation de transcription ni de l’ARN de 4 RPC32α, ni de celle de RPC32β. Il n’existe donc pas de boucle de rétroaction pour RPC32α et les deux homologues ne sont pas co-régulés. Plusieurs, mais pas tous les transcrits synthétisé par la Pol III ont une expression fortement baissé dans les lignées mutants. Le fait que pas tous les transcrits ne soit affectés par la perte de RPC32α, indique qu’il existe une spécificité de transcription pour Pol IIIα et Pol IIIβ. Les cellules des linges mutants ne présentaient pas de phénotype différent des cellules mères et la croissance était la même dans toutes les lignées. Par contre les tests de croissance en agar-mou ont révélé que les lignées mutants formaient 85% de moins de colonies, indiquant que RPC32α est nécessaire pour la croissance tumorigénique in vitro. Pour tester l’effet de la perte de RPC32α sur la croissance tumorigénique in vivo, des cellules mutants et des cellules mères ont été injecté dans des souris. Les souris greffées avec des cellules mutantes montrent un départ de tumorisation retardé. Au bout de six semaines elles avaient de tumeurs deux fois plus petit que les souris avec des cellules mères. Après ablation de la tumeur primaire, les souris ont été surveillées pour l’apparition de métastases. Quatre semaines plus tard les souris greffées avec des cellules mutantes avaient 100 fois moins de métastases que les souris contrôles. Ces résultats montrent que RPC32α est nécessaire pour la tumorisation in vitro et in vivo. La protéine semble surtout jouer un rôle dans la formation des métastases, qui sont un des problèmes majeurs dans le traitement des cancers. / The RNA polymerases are key players of transcription. Eukaryotes have three RNA polymerases (I, II and III). The RNA polymerase III (Pol III) has 17 subunits, one of which exists in two alternative forms: RPC32α and RPC32β. Only one of the two forms can be integrated into the enzymes, thus generating either Pol IIIα or Pol IIIβ. While RPC32β is found in all somatic cells, RPC32α is expressed in stem cells and tumor cells. To date nothing is known of their respective roles. Breast cancer is one of the major public health problems, as it is the most common cancer in women. Several types of breast cancers are distinguished, according to the presence or absence of hormonal receptors. Cancers that test negative for estrogen receptors, progesterone receptors and that do not overexpress the human epidermal growth factor receptor 2, are called triple-negative breast cancers. They tend to have a poor prognosis, due to the aggressive nature of the cancer and the lack of targeted therapies. To study the role of RPC32α, a tumor model needed to be identified. In collaboration with Jean-Paul Feugeas (INSERM UMR 1098) a transcriptomic study was performed on 2627 clinical breast tissue samples. The study showed that RPC32α was overexpressed in triplenegative breast cancer, whereas RPC32β was overexpressed in normal tissue. A study on six breast cancer cell lines and one non-tumorigenic line confirmed the results of the transcriptomic study. The breast cancer model was thus validated. A characterization of different breast cancer cell lines showed that other Pol III subunits were not overexpressed in triple-negative breast cancer. The overexpression of RPC32α was therefore not a mere consequence of a Pol III hyperactivity. An analysis of the transcripts synthesized by Pol III showed that overall the Pol III transcript levels were elevated in triplenegative breast cancer compared to other breast cancer subtypes. In order to study the role of RPC32α in tumorigenesis, several RPC32α knock-out cell lines were created using CRISPR-Cas9. The loss of RPC32α did not induce an increase in transcription of the RNAs of RPC32α or RPC32β. This shows that no feed-back loop exists for RPC32α and that the two homologues are not co-regulated. Various Pol III transcripts showed decreased expression levels in the knock-out cell lines. Yet not all transcripts were reduced in the absence of RPC32α. This indicates that some sort of transcription specificity must exist for Pol IIIα and Pol IIIβ. The knock-out cell lines did not show any alterations in their phenotype or growth rates. However, in soft agar assays the knock-out cell lines produced 85% less colonies than the mother cell line. This proves that RPC32α is necessary for tumorigenic growth in vitro. To find out if RPC32α was also necessary for tumorigenic growth in vivo, knock-out and wild type cells were injected into mice. The mice grafted with knock-out cells showed a slowed onset of tumor growth. After six weeks, the mice injected with knock-out cells had tumors half the size of the mice injected with wild type cells. The primary tumor was ablated and mice were tracked for metastasis. Four weeks later, mice injected with RPC32α knock-out cells had 100 times less metastasis than the control group. These results show that RPC32α is necessary for tumorigenic growth in vitro and in vivo. The protein seems also to be implicated in the formation of metastasis, which are one of the greatest problems in cancer treatment today.

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Devant le grand nombre de tumeurs du sein triple négatif résistant aux traitements, il est essentiel de comprendre les mécanismes de résistance et de trouver de nouvelles molécules efficaces. En premier lieu, nous analysons deux ensembles de données pharmacogénomiques à grande échelle. Nous proposons une nouvelle classification basée sur des profils transcriptomiques de lignées cellulaires, selon un processus de sélection de gènes basé sur des réseaux biologiques. Notre classification moléculaire montre une plus grande homogénéité dans la réponse aux médicaments que lorsque l’on regroupe les lignées cellulaires en fonction de leur tissu d'origine. Elle permet également d’identifier des profils similaires de réponse aux traitements. Dans un second travail, nous étudions une cohorte de patients atteints d’un cancer du sein triple négatif ayant résisté à la chimiothérapie néoadjuvante. Nous effectuons des analyses moléculaires complètes basées sur du RNAseq et WES. Nous constatons une forte hétérogénéité moléculaire des tumeurs avant et après traitement. Bien que nous observons une évolution clonale sous traitement, aucun mécanisme récurrent de résistance n’a pu être identifié. Nos résultats suggèrent fortement que chaque tumeur a un profil moléculaire unique et qu'il est important d'étudier de grandes séries de tumeurs. Enfin, nous améliorons une méthode pour tester la surreprésentation de motifs connus de protéines de liaison à l'ARN, dans un ensemble donné de séquences régulées. Cet outil utilise une approche innovante pour contrôler la proportion de faux positifs qui n'est pas réalisé par l'algorithme existant. Nous montrons l'efficacité de notre approche en utilisant deux séries de données différentes. / Given the large number of treatment-resistant triple-negative breast cancers, it is essential to understand the mechanisms of resistance and to find new effective molecules. First, we analyze two large-scale pharmacogenomic datasets. We propose a novel classification based on transcriptomic profiles of cell lines, according to a biological network-driven gene selection process. Our molecular classification shows greater homogeneity in drug response than when cell lines are grouped according to their original tissue. It also helps identify similar patterns of treatment response. In a second analysis, we study a cohort of patients with triple-negative breast cancer who have resisted to neoadjuvant chemotherapy. We perform complete molecular analyzes based on RNAseq and WES. We observe a high molecular heterogeneity of tumors before and after treatment. Although we highlighted clonal evolution under treatment, no recurrent mechanism of resistance could be identified Our results strongly suggest that each tumor has a unique molecular profile and that that it is increasingly important to have large series of tumors. Finally, we are improving a method for testing the overrepresentation of known RNA binding protein motifs in a given set of regulated sequences. This tool uses an innovative approach to control the proportion of false positives that is not realized by the existing algorithm. We show the effectiveness of our approach using two different datasets.

Cancer is the second leading cause of death in the US and worldwide, accounting for 16% of deaths worldwide in 2015. Of more than 100 types of cancers affecting humans, breast cancer is the most common cancer among women and is the second leading cause of death in women. Triple negative breast cancer (TNBC) is a subtype of breast carcinomas defined by the lack of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2 /neu). The prognosis and survival of TNBC patients remains the poor due to the lack of effective targeted therapy. Nanotechnology-based drug delivery systems, such as liposomes, are widely investigated to enhance anticancer efficacy by concentrating the drug molecules in the tissues of interest and by altering the pharmacokinetic profile. Taking advantage of the pH gradient in the tumor microenvironment, pH-triggered release is a promising strategy to enhance the anticancer efficacy of drug delivery systems against TNBC. Previously, a strategy in our lab has been developed to render saturated and pegylated liposomes pH-sensitive: protonation-induced conformational switch of lipid tails, using trans-2-aminocyclohexanol lipids (TACH, flipids) as a molecular trigger. Based on previous work in our lab, pH-sensitive liposomes (fliposomes) composed of C-16 flipids with amine group of morpholine (MOR) and azetidine (AZE) demonstrated optimized triggered release in response to the tumor’s low pH microenvironment. In this study, different preparation methods were developed and optimized to produce viable fliposomes with high doxorubicin (DOX) encapsulation efficiency. In vitro release assays were established and validated to accurately reflect pH-triggered release of fliposomes. The physicochemical properties of DOX-loaded fliposomes were characterized and their pH-dependent release were investigated. Factors influencing the desirable attributes of liposomes, such as size, pH-sensitivity, stability and drug-loading capacity were explored. Based on these characterizations, central composite design (CCD) was utilized to optimize the formulation of fliposome with two critical factors, flipids and cholesterol. Cell viability assays on traditional monolayer and innovative three-dimensional multicellular spheroids (3D MCS) of TNBC cell lines were conducted to evaluate the anticancer efficacy of the resultant fliposomes in vitro. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells, as well as acidity inside the 3D MCS based on confocal microscopic imaging studies. The distribution and penetration of DOX-loaded fliposomes into 3D MCS was imaged by confocal microscopy in comparison to DOX-loaded non pH-sensitive liposomes and free DOX. As a result, fliposome manifested superior anticancer activity against TNBC 3D MCS by efficient penetration into 3D MCS, followed by tuning up the release rate of the anticancer agent DOX. A TNBC orthotopic xenograft model was established by transplanting TNBC into the murine mammalian fat pad, which maintains the organ-specific tumor microenvironment of the original organ . A pilot pharmacokinetic study was conducted in order to correlate the pH response and stability properties with the in vivo stability of the optimized AZE-C16 fliposome. The antitumor efficacy was comparable between free DOX and DOX-loaded stealth liposome with tumor volumes of ~ 80-90% of the control treatment 32 days post first dose. In contrast, the DOX-loaded fliposome, especially MOR-C16 fliposome, exhibited a significantly higher antitumor efficacy and delayed progression compared to free DOX and stealth liposome treatments. Taken together, DOX-loaded fliposomes were successfully prepared and optimized for in vivo application. They were able to achieve superior activity against TNBC in vitro and in vivo, facilitated by enhanced release of the anticancer drug DOX after penetration inside TNBC tumor.

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Breast cancer (BCa) is a leading cause of cancer-related female deaths worldwide and is a complex disease consisting of many different subtypes with varying clinical course and outcomes. Triple negative breast cancer (TNBC), an aggressive and highly metastatic subtype, is most prevalent in women of African ancestry (WAA) but the causes of this disparity are not fully understood. The goal of this study was to investigate the epidemiological and genetic profiles in ancestrally-related WAA in Barbados and Nigeria to advance knowledge and lay the foundation for development of improved or novel BCa therapeutics. To gain insight about TNBC across the African continent, a systematic review and meta-analysis was conducted. TNBC frequencies on average across Africa were estimated at 26.8% but were highest in West African countries (46.0%). We also sought to identify the epidemiological profile of BCa in Barbados—a Caribbean island with significant West African ancestry. We reviewed pathological reports for BCa from the sole public hospital in Barbados and compared those data with USA population-based data. We found a high prevalence of high prevalence of TNBC amongst women diagnosed with breast cancer in Barbados (25%), compared to 21% in non-Hispanic Black and 10% in non-Hispanic White women in the USA for the 2010-2016 period. We also investigated the somatic mutational profile of WAA with TNBC in Barbados and Nigeria using whole exome sequencing (WES) of formalin-fixed paraffinembedded TNBC tissues. This investigation revealed novel and pathogenic variants in well-known cancer-associated genes such as TP53, BRCA1 and MDC1. The somatic mutation signature in Nigerian tissues correlated with aflatoxin signature, implying a role for environmental factors influencing the genomics profile in this cohort. Copy number variants were revealed at high frequencies for PIK3CA, FGFR2 and HIF1AN genes. Collectively, these findings uncovered novel epidemiological and genetic trends in WAA with high prevalence of the aggressive TNBC subtype / Thesis / Doctor of Philosophy (PhD) / Breast cancer (BCa) is a leading cause of cancer-related death in women worldwide. Although Caucasian women are diagnosed with BCa more than women of African ancestry (WAA), more WAA unfortunately die from BCa. The reasons for this disparity are currently unknown, however, a higher proportion of WAA are diagnosed with an aggressive type of BCa called triple negative breast cancer (TNBC). This might partially explain the high cancer death rate in WAA. To understand this disparity in BCa incidence and outcomes, we investigated TNBC disease trends across the African continent and in Barbados (a Caribbean island with predominantly African ancestry) and found a high proportion of TNBC diagnoses in Barbados and West African countries. We also discovered a novel genetic profile within these groups that may be useful to develop new cancer therapies that would decrease TNBC aggressiveness and death in these populations.

In breast cancer, the genetic profiling of circulating cell-free DNA (cfDNA) from blood plasma was shown to have good potential for clinical use. In contrast, only a few studies were performed investigating urinary cfDNA. In this pilot study, we analyzed plasma-derived and matching urinary cfDNA samples obtained from 15 presurgical triple-negative breast cancer patients. We used a targeted next-generation sequencing approach to identify and compare genetic alterations in both body fluids. The cfDNA concentration was higher in urine compared to plasma, but there was no significant correlation between matched samples. Bioinformatical analysis revealed a total of 3339 somatic breast-cancer-related variants (VAF ≥ 3%), whereof 1222 vs. 2117 variants were found in plasma-derived vs. urinary cfDNA, respectively. Further, 431 shared variants were found in both body fluids. Throughout the cohort, the recovery rate of plasma-derived mutations in matching urinary cfDNA was 47% and even 63% for pathogenic variants only. The most frequently occurring pathogenic and likely pathogenic mutated genes were NF1, CHEK2, KMT2C and PTEN in both body fluids. Notably, a pathogenic CHEK2 (T519M) variant was found in all 30 samples. Taken together, our results indicated that body fluids appear to be valuable sources bearing complementary information regarding the genetic tumor profile.

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