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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Bioimpedance presents a versatile, label-free means of monitoring biological cells and their responses to physical, chemical and biological stimuli. Breast cancer is the second most common type of cancer among women in the United States. Although significant progress has been made in diagnosis and treatment of this disease, there is a need for robust, easy-to-use technologies that can be used for the identification and discrimination of critical subtypes of breast cancer in biopsies obtained from patients. This dissertation makes contributions in three major areas towards addressing the goal. First, we developed miniaturized bioimpedance sensors using MEMS and microfluidics technology that have the requisite traits for clinical use including reliability, ease-of-use, low-cost and disposability. Here, we designed and fabricated two types of bioimpedance sensors. One was based on electric cell-substrate impedance sensing (ECIS) to monitor cell adhesion based events and the other was a microfluidic device with integrated microelectrodes to examine the biophysical properties of single cells. Second, we examined a panel of triple negative breast cancer (TNBC) cell lines and a hormone therapy resistant model of breast cancer in order to improve our understanding of the bioimpedance spectra of breast cancer subtypes. Third, we explored strategies to improve the sensitivity of the microelectrodes to bioimpedance measurements from breast cancer cells. We investigated nano-scale coatings on the surface of the electrode and geometrical variations in a branched electrode design to accomplish this. This work demonstrates the promise of bioimpedance technologies in monitoring diseased cells and their responses to pharmaceutical agents, and motivates further research in customization of this technique for use in personalized medicine. / Ph. D.

La protéine E4F1 découverte comme cible cellulaire de l'oncoprotéine adénovirale E1A est une protéine ubiquitaire agissant comme facteur de transcription et comme E3-ligase atypique. La protéine E4F1 interagit également directement avec plusieurs gènes suppresseurs de tumeurs et des oncoprotéines, suggérant son implication dans la tumorigénèse. Des travaux antérieurs du laboratoire, sur les fonctions cellulaires d’E4F1 dans les cellules cancéreuses ont montré que sa déplétion entraîne une mort cellulaire massive dans les Mefs transformés déficients en p53. De plus, E4F1 contrôle directement l'expression de 38 gènes, notamment impliqués dans le métabolisme cellulaire et les checkpoints du cycle cellulaire/Réponse aux dommages de l'ADN (DDR), tel que Chek1 qui code un composant majeur du checkpoint ATR/ATM. Conformément à ce rôle d’E4F1 dans la survie des cellules cancéreuses chez la souris, des patientes atteintes d'un cancer du sein triple négatif (TNBC) exprimant fortement E4F1 présentent une survie sans rechute (RFS) plus faible.Nous avons donc décidé d’étudier pour la première fois le programme transcriptionnel d’E4F1 dans les cellules humaines et d’explorer son rôle dans la survie des cellules de TNBC, avec une attention particulière pour son rôle dans la réponse aux agents de chimiothérapie.Les transcriptomes (RNAseq) de cellules SUM159 de TNBC montrent, lors de la déplétion d’E4F1, une diminution de l’expression de 147 des 276 gènes associés à la DDR. La combinaison de RNAseq et de ChIPseq révèle qu’E4F1 régule directement 57 gènes dans les cellules de TNBC humaines. Parmi ces gènes, E4F1 lui-même, CHEK1, mais aussi TTI2 et PPP5C codant pour des régulateurs post-transcriptionnels de l'axe ATM/ATR-CHK1, et définissant ainsi un "régulon" ATM/ATR-CHK1, encore inconnu et dépendant d’E4F1. TTI2 forme avec TELO2 et TTI1, le complexe TTT nécessaire au repliement correct et à la stabilité des protéines de la famille PIKK, telles qu’ATR et ATM. La phosphatase PPP5C est impliquée dans l'activation de la signalisation ATR-CHK1. Fait important, nous montrons qu’E4F1 se fixe sur et régule probablement ces trois gènes in vivo dans des tumeurs TNBC dérivées de patientes (PDTX). Dans la lignée SUM159 et les PDTX, le recrutement d’E4F1 sur ces gènes est augmenté lors du traitement avec la Gemcitabine, un agent de chimiothérapie bloquant la réplication de l’ADN. Étonnamment, nous avons révélé qu’E4F1 contrôle aussi indirectement l'expression de TELO2, un second membre du complexe TTT. Par conséquent, dans les cellules TNBC déplétées en E4F1, les taux de protéines des CHK1, TTI2, TELO2 mais aussi des kinases ATM/ATR, sont fortement diminués, entraînant une déficience de la DDR. Ainsi, les cellules SUM159 déplétées en E4F1 ne parviennent pas à s'arrêter en phase S lors du traitement à la Gemcitabine et sont hautement sensibilisées à cet agent de chimiothérapie, ainsi qu'à d'autres agents endommageant l'ADN comme le Cisplatine. Dans leur ensemble, mes travaux de thèse révèlent que la voie de signalisation ATM/ATR-CHK1, et la réponse au stress / dommages de l'ADN sont étroitement contrôlées aux niveaux transcriptionnel et post-transcriptionnel par E4F1. E4F1 apparait donc comme un acteur central dans la survie cellulaire des cellules TNBC, en particulier lorsqu'elles sont exposées à des agents endommageant l'ADN ou à des agents de chimiothérapie. Ainsi E4F1 pourrait représenter un marqueur pronostique de réponse à la chimiothérapie et une cible thérapeutique potentielle. / The E4F1 protein discovered as the cellular target of the adenoviral oncoprotein E1A is a ubiquitous protein acting both as a transcription factor and as an atypical E3-ligase. E4F1 protein also interacts directly with several cellular tumor suppressors and oncoproteins, suggesting its involvement in tumorigenesis. Previous laboratory work on the cellular functions of E4F1 in cancer cells has shown that its depletion leads to massive cell death in transformed Mefs deficient in p53. In addition, E4F1 directly controls the expression of 38 genes, including genes involved in cell metabolism and cell cycle checkpoints/DNA Damage Response (DDR), such as Chek1 that encodes a major component of the ATR/ATM checkpoint. Consistent with this role of E4F1 in cancer cell survival in mice, patients with triple-negative breast cancer (TNBC) with high E4F1 expression exhibit a poorer relapse free survival (RFS).We therefore aimed to study for the first time the transcriptional program of E4F1 in human cells and explore its role in the survival of TNBC cells, with particular focus on its role in the response to chemotherapy agents.Transcriptomes (RNAseq) of SUM159 TNBC cells show, when E4F1 is depleted, a decrease in expression of 147 out of 276 DDR-associated genes. The combination of RNAseq and ChIPseq shows that E4F1 directly regulates 57 genes in human TNBC cells. Among these genes, E4F1 itself, CHEK1, but also TTI2 and PPP5C coding for post-transcriptional regulators of the ATM/ATR-CHK1 axis, and thus defining an ATM/ATR-CHK1 "regulon", undescribed and E4F1-dependent. TTI2 composes with TELO2 and TTI1, the TTT complex required for the correct folding and stability of PIKK family proteins, such as ATR and ATM. PPP5C phosphatase is involved in the activation of ATR-CHK1 signaling. Importantly, we show that E4F1 binds to and probably regulates these three genes in vivo in Patient Derived TNBC Xenografts (PDTX). In both SUM159 cells and PDTX, the recruitment of E4F1 on these genes is increased upon Gemcitabine treatment, a chemotherapy agent that impairs DNA replication. Surprisingly, we found that E4F1 also indirectly controls the expression of TELO2, a second member of the TTT complex. Consequently, in TNBC cells depleted of E4F1, the protein levels of CHK1, TTI2, TELO2 but also ATM/ATR kinases, are significantly decreased, leading to DDR deficiency. Thus, SUM159 cells depleted of E4F1 fail to stop in phase S during Gemcitabine treatment and are highly sensitized to this chemotherapy agent, as well as other DNA damaging agents such as Cisplatin. Altogether, my thesis results demonstrate that the ATM/ATR-CHK1 signaling pathway, and the response to stress / DNA damage are tightly controlled at the transcription and post-transcription levels by E4F1. E4F1 therefore appears to be a central actor in the cellular survival of TNBC cells, particularly when exposed to DNA-damaging agents or chemotherapy agents. Thus, E4F1 could represent a prognostic marker for chemotherapy response and a potential therapeutic target.