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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.

INTRODUÇÃO: Os microRNAs (miRNAs) são uma classe de pequenas moléculas não codificadoras de proteínas que regulam a expressão gênica durante a etapa de tradução. Esta regulação é feita pelo pareamento de bases com o mRNA-alvo (RNA mensageiro), resultando na supressão da tradução ou na clivagem do mRNA. A depender se os miRNAs têm como alvo genes supressores de tumor ou oncogenes, eles podem atuar como supressores tumorais ou oncogenes. A imunoistoquímica triplo negativa, no câncer de mama, é, comumente, utilizada como substituto clínico para identificação dos tumores basaloides, que se caracterizam pela expressão de genes epiteliais basais, sendo associados a menores taxas de sobrevida livre de doença e sobrevida global. O câncer de mama triplo negativo faz com que seja necessária a descoberta de marcadores moleculares que possam servir de alvos terapêuticos ou, pelo menos, que sirvam como marcadores preditivos da resposta aos quimioterápicos. OBJETIVO: avaliar a expressão de microRNAs, por PCR em tempo real, no carcinoma mamário ductal invasivo (CDI) triplo negativo. MÉTODOS: Foram avaliados materiais em parafina de tumor de 31 pacientes com as seguintes características: carcinoma invasivo de mama, receptores de estrogênio e de progesterona negativos e HER 2 negativo, bem como tecido mamário histologicamente normal. Foram utilizados kit para extração de RNA de amostras fixadas e parafinadas - miRNeasy FFPE; kit para síntese de cDNA - miScript II RT; kit miScript SYBR Green PCR e miScript miRNA PCR Arrays para análise de 84 sequências de miRNA de câncer humano. Foram avaliados dados clínicos, como idade, paridade, amamentação, status menopausal; variáveis histológicas, como tamanho do tumor, status linfonodal, invasão linfática; características imunoistoquímicas, como expressão de Ki-67, EFGR e CK 5/6. O seguimento das pacientes buscou verificar a ocorrência e o tempo de aparecimento de recidiva loco regional, metástase à distância e óbito. Para análise estatística foi utilizado o software miScript miRNA PCR Array Data Analysis, que utiliza o método de quantificação relativa DeltaCt. RESULTADOS: A análise comparativa dos 31 casos de CDI triplo negativo com os 18 casos de parênquima mamário normal definiu microRNAs hiperexpressos, sendo eles: miR-96-5p (fold-regulation(FR) = 9,68, p = 0,000008), miR-21-5p (FR = 4,47, p = 0,00), miR-7-5p (FR = 5,8, p = 0,00137) , miR-182-5p (FR= 7,92, p = 0,000001), miR-210-3p (FR = 11,83, p = 0,000048), miR-18a-5p (FR = 9,51, p = 0,000034), miR-155-5p (FR= 4,40 , p = 0,00019) e miR-93-5p (FR= 4,15, p = 0,000023). Aponta, ainda, microRNAs com hipoexpressão, a saber: miR-204-5p (FR = -10,26, p = 0), miR-205-5p (FR= -4,07, p = 0,019822), miR-125b-5p (FR= -4,29, p=0) e let 7c-5p (FR= -4,91, p=0). CONCLUSÃO: a expressão de microRNAs no carcinoma ductal invasivo triplo negativo permite diferenciá-lo do tecido normal / INTRODUCTION: MicroRNAs (miRNAs) are a class of small non-coding protein molecules that regulate gene expression during the translation stage. This adjustment is made by base pairing with the mRNA (messenger RNA) target resulting in suppression of translation or cleavage of the mRNA. Depending on whether miRNAs target tumor suppressor genes or oncogenes, they can act as tumor suppressors or oncogenes. The triple negative immunohistochemistry in breast cancer is commonly used as a substitute for clinical identification of basaloid tumors, which are characterized by the expression of basal epithelial genes and are associated with lower rates of disease-free survival and overall survival. The triple negative breast cancer makes necessary the discovery of molecular markers that may serve as therapeutic targets or at least as predictive markers of response to chemotherapy. OBJECTIVE: evaluate the expression of microRNAs by RT-PCR in triple negative breast invasive ductal carcinoma (IDC). METHODS: Paraffin embedded tumor material from 31 patients with the following characteristics were evaluated: invasive breast carcinoma, negative estrogen and progesterone receptor, negative HER 2, and histologically normal breast tissue. Were used: Kit for RNA extraction from fixed and paraffin embedded samples - miRNeasy FFPE; cDNA synthesis kit - miScript II RT; miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays for analysis of 84 miRNA sequences of human cancer. Clinical data such as age, parity, breastfeeding, menopausal status; histological variables such as tumor size, lymph node status, lymphatic invasion; immunohistochemical characteristics, such as expression of Ki-67, EFGR and CK 5/6 were evaluated. The follow-up of patients aimed to verify the occurrence and time of appearance of loco regional recurrence, distant metastasis and death. For statistical analysis the miScript miRNA PCR Array Data Analysis software, which uses the method of relative quantification DeltaCt, was used. RESULTS: A comparative analysis of 31 cases of triple negative IDC with 18 cases of normal breast parenchyma defined microRNAs overexpressed, as follows: miR-96-5p (fold-regulation (FR) = 9.68, p = 0.000008), miR -21-5p (FR = 4.47, p = 0.00), 5p, miR-7 (FR = 5.8, p = 0.00137), miR-182-5p (FR = 7.92, p = 0.000001), miR-210-3p (FR = 11.83, p = 0.000048), miR-18a-5p (FR = 9.51, p = 0.000034), miR-155-5p (FR = 4.40, p = 0.00019) and miR-93-5p (FR = 4.15, p = 0.000023). Furthermore, microRNAs with reduced expression, as follows: miR-204-5p (FR = -10.26, p = 0), miR-205-5p (FR = -4.07, p = 0.019822), miR -125b-5p (FR = -4.29, p = 0) and Let-7c 5p (FR = -4.91, p = 0). CONCLUSION: the expression of microRNAs in triple negative invasive ductal carcinoma allows to differentiate it from normal tissue

INTRODUÇÃO: Os microRNAs (miRNAs) são uma classe de pequenas moléculas não codificadoras de proteínas que regulam a expressão gênica durante a etapa de tradução. Esta regulação é feita pelo pareamento de bases com o mRNA-alvo (RNA mensageiro), resultando na supressão da tradução ou na clivagem do mRNA. A depender se os miRNAs têm como alvo genes supressores de tumor ou oncogenes, eles podem atuar como supressores tumorais ou oncogenes. A imunoistoquímica triplo negativa, no câncer de mama, é, comumente, utilizada como substituto clínico para identificação dos tumores basaloides, que se caracterizam pela expressão de genes epiteliais basais, sendo associados a menores taxas de sobrevida livre de doença e sobrevida global. O câncer de mama triplo negativo faz com que seja necessária a descoberta de marcadores moleculares que possam servir de alvos terapêuticos ou, pelo menos, que sirvam como marcadores preditivos da resposta aos quimioterápicos. OBJETIVO: avaliar a expressão de microRNAs, por PCR em tempo real, no carcinoma mamário ductal invasivo (CDI) triplo negativo. MÉTODOS: Foram avaliados materiais em parafina de tumor de 31 pacientes com as seguintes características: carcinoma invasivo de mama, receptores de estrogênio e de progesterona negativos e HER 2 negativo, bem como tecido mamário histologicamente normal. Foram utilizados kit para extração de RNA de amostras fixadas e parafinadas - miRNeasy FFPE; kit para síntese de cDNA - miScript II RT; kit miScript SYBR Green PCR e miScript miRNA PCR Arrays para análise de 84 sequências de miRNA de câncer humano. Foram avaliados dados clínicos, como idade, paridade, amamentação, status menopausal; variáveis histológicas, como tamanho do tumor, status linfonodal, invasão linfática; características imunoistoquímicas, como expressão de Ki-67, EFGR e CK 5/6. O seguimento das pacientes buscou verificar a ocorrência e o tempo de aparecimento de recidiva loco regional, metástase à distância e óbito. Para análise estatística foi utilizado o software miScript miRNA PCR Array Data Analysis, que utiliza o método de quantificação relativa DeltaCt. RESULTADOS: A análise comparativa dos 31 casos de CDI triplo negativo com os 18 casos de parênquima mamário normal definiu microRNAs hiperexpressos, sendo eles: miR-96-5p (fold-regulation(FR) = 9,68, p = 0,000008), miR-21-5p (FR = 4,47, p = 0,00), miR-7-5p (FR = 5,8, p = 0,00137) , miR-182-5p (FR= 7,92, p = 0,000001), miR-210-3p (FR = 11,83, p = 0,000048), miR-18a-5p (FR = 9,51, p = 0,000034), miR-155-5p (FR= 4,40 , p = 0,00019) e miR-93-5p (FR= 4,15, p = 0,000023). Aponta, ainda, microRNAs com hipoexpressão, a saber: miR-204-5p (FR = -10,26, p = 0), miR-205-5p (FR= -4,07, p = 0,019822), miR-125b-5p (FR= -4,29, p=0) e let 7c-5p (FR= -4,91, p=0). CONCLUSÃO: a expressão de microRNAs no carcinoma ductal invasivo triplo negativo permite diferenciá-lo do tecido normal / INTRODUCTION: MicroRNAs (miRNAs) are a class of small non-coding protein molecules that regulate gene expression during the translation stage. This adjustment is made by base pairing with the mRNA (messenger RNA) target resulting in suppression of translation or cleavage of the mRNA. Depending on whether miRNAs target tumor suppressor genes or oncogenes, they can act as tumor suppressors or oncogenes. The triple negative immunohistochemistry in breast cancer is commonly used as a substitute for clinical identification of basaloid tumors, which are characterized by the expression of basal epithelial genes and are associated with lower rates of disease-free survival and overall survival. The triple negative breast cancer makes necessary the discovery of molecular markers that may serve as therapeutic targets or at least as predictive markers of response to chemotherapy. OBJECTIVE: evaluate the expression of microRNAs by RT-PCR in triple negative breast invasive ductal carcinoma (IDC). METHODS: Paraffin embedded tumor material from 31 patients with the following characteristics were evaluated: invasive breast carcinoma, negative estrogen and progesterone receptor, negative HER 2, and histologically normal breast tissue. Were used: Kit for RNA extraction from fixed and paraffin embedded samples - miRNeasy FFPE; cDNA synthesis kit - miScript II RT; miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays for analysis of 84 miRNA sequences of human cancer. Clinical data such as age, parity, breastfeeding, menopausal status; histological variables such as tumor size, lymph node status, lymphatic invasion; immunohistochemical characteristics, such as expression of Ki-67, EFGR and CK 5/6 were evaluated. The follow-up of patients aimed to verify the occurrence and time of appearance of loco regional recurrence, distant metastasis and death. For statistical analysis the miScript miRNA PCR Array Data Analysis software, which uses the method of relative quantification DeltaCt, was used. RESULTS: A comparative analysis of 31 cases of triple negative IDC with 18 cases of normal breast parenchyma defined microRNAs overexpressed, as follows: miR-96-5p (fold-regulation (FR) = 9.68, p = 0.000008), miR -21-5p (FR = 4.47, p = 0.00), 5p, miR-7 (FR = 5.8, p = 0.00137), miR-182-5p (FR = 7.92, p = 0.000001), miR-210-3p (FR = 11.83, p = 0.000048), miR-18a-5p (FR = 9.51, p = 0.000034), miR-155-5p (FR = 4.40, p = 0.00019) and miR-93-5p (FR = 4.15, p = 0.000023). Furthermore, microRNAs with reduced expression, as follows: miR-204-5p (FR = -10.26, p = 0), miR-205-5p (FR = -4.07, p = 0.019822), miR -125b-5p (FR = -4.29, p = 0) and Let-7c 5p (FR = -4.91, p = 0). CONCLUSION: the expression of microRNAs in triple negative invasive ductal carcinoma allows to differentiate it from normal tissue

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.

Les carcinomes mammaires invasifs sont des tumeurs solides, au sein desquelles le fort pouvoir de prolifération des cellules cancéreuses entraîne la formation de zones hypoxiques. Les mécanismes cellulaires d’adaptation à la diminution des apports en oxygène sont principalement déterminés par des facteurs de transcription hétérodimériques induits par l’hypoxie (HIF-1 et HIF-2, hypoxia inducible factor). Le monomère alpha de ces complexes est déstabilisé en situation de normoxie. Les récepteurs aux facteurs de croissance épidermique (EGFR) peuvent également augmenter la traduction de l’ARNm codant ces monomères indépendamment de l’oxygène. La première partie de la thèse repose sur le développement de deux clones cellulaires fluorescents stables et inductibles par l’hypoxie, à partir d’une lignée de carcinome mammaire invasif triple négatif surexprimant le récepteur EGFR (MDA- MB-231). Le modèle CA9-GFP permet de restituer l’activité transcriptionnelle des complexes HIFs et le modèle GFP-P564 traduit la stabilité de leur sous-unité alpha. La fluorescence des deux modèles cellulaires est fortement induite en situation d’hypoxie (1% O2) et en présence d’agents mimétiques de l’hypoxie (cobalt, desferrioxamine et diméthyl-oxalyl glycine). Ces modèles ont ensuite été utilisés pour évaluer l’effet de trois thérapies anti-EGFR sur les voies de réponses à l’hypoxie. Le cétuximab et le lapatinib n’ont aucun effet sur la fluorescence des cellules alors que le géfitinib diminue l’intensité du signal en situation d’hypoxie. Ces différences sont associées à un impact similaire sur la migration et la viabilité cellulaire traduisant ainsi une sensibilité différentielle à ces trois composés anti-EGFR. La deuxième partie de la thèse s’est ensuite orientée vers la quantification par RT-qPCR de 45 gènes inductibles par l’hypoxie et impliqués dans le développement du cancer du sein sur une série rétrospective portant sur 32 exérèses de carcinomes mammaires invasifs précoces. Une analyse comparative des données a été effectuée en fonction des critères anatomo-pathologiques (stade, grade, statut en récepteur HER2, rechute). Une surexpression coordonnée de l’ensemble des gènes est observée dans les tumeurs de haut grade, HER2+ et pour les sujets ayant récidivé. La comparaison des groupes « rechute » versus « non rechute » a permis de sélectionner six marqueurs de référence s’exprimant de manière significative. Elle permet en outre la réalisation d’un algorithme basique de classement des individus en fonction du risque de récidive. Celui-ci a été validé par la construction de courbes de survie (méthode de Kaplan-Meier et test du Mantel-Haenszel). L’utilisation combinée des deux modèles cellulaires fluorescents permet l’identification et la caractérisation dynamique de molécules agissant directement ou indirectement sur les réponses cellulaires à l’hypoxie tumorale. L’analyse de l’expression de gènes connus pour leur profil agressif et régulés par le microenvironnement tumoral, constitue un outil clinique d’aide au pronostic des cancers du sein. / The invasive breast cancers are solid tumors, wherein the proliferation of cells causes the formation of hypoxic zones. Cellular adaptive responses to reduced oxygen concentrations are mainly determined by heterodimeric transcription factors induced by hypoxia (HIF-1 & HIF-2, Hypoxia Inducible Factors). The alpha subunits of HIF are rapidly degraded under normoxic conditions. HIF complexes are also regulated by activation of epidermal growth-factor receptor (EGFR) signaling pathways in an oxygen-independent manner. The first part of this work was to develop stable fluorescent clones for examining responses to hypoxia from a metastatic triple-negative breast cancer cell line overexpressing EGFR (MDA-MB-231). The Ca9-GFP and GFP-P564 cell models allow to assess HIF activity and alpha subunits stability, respectively. In both models, fluorescence signals were strongly increased with hypoxia-mimicking reagents (cobalt, desferrioxamine and dimethyl-oxalyl glycine) and under hypoxia (1% O2). The impact of three EGFR inhibitors on HIF activity and stability was assessed. Cetuximab and lapatinib did not affect the signal induced by hypoxia, whereas gefitinib sharply reduced its intensity in both models. The differential effect of these three EGFR-targeted therapies on hypoxia responses was correlated with a similar impact on viability and cell migration. The second part of this work focused on the relative quantification using qPCR analysis of 45 genes involved in hypoxia responses and in the development of breast cancer, from a retrospective series of 32 tumor samples from patients with early- stage invasive breast cancer. A comparative analysis was performed according to anatomo-pathological criteria (stage, grade, HER2 status, and relapse). A coordinated overexpression of all genes was observed in high-grade and HER2+ tumors and in the group of patients that relapsed. The comparison of gene expression between "relapse" and "no relapse" groups was used to identify six reference markers. It also allowed to develop a basic algorithm to classify patients according to the risk of relapse. This algorithm was validated by constructing survival curves (Kaplan-Meier method and Mantel-Haenszel test). Combined use of two fluorescent cell models allows identification and dynamic characterization of compounds able to directly or indirectly inhibit cellular responses to tumor hypoxia. Analysis of gene expression known for their aggressive character and regulated by the tumor microenvironment is a clinical tool for assessing breast cancer prognosis.

Le cancer du sein est le cancer le plus fréquent chez la femme et malgré une baisse de la mortalité ces dernières années, attribuable à une détection précoce et une meilleure prise en charge, il reste la première cause de mortalité par cancer et demeure donc un problème majeur de santé publique. La mortalité par cancer du sein s’avère être plus élevée dans le cas des cancers dits agressifs ou de mauvais pronostic tels que le cancer du sein de la femme jeune et le cancer du sein triple négatif (CSTN). Mes travaux de thèse portent sur ces deux types de cancer du sein avec comme objectifs l’étude des conséquences de la ménopause chimio-induite (MCI) chez les femmes jeunes atteintes d’un cancer du sein non métastatique d’une part et l’analyse des facteurs pronostiques de la rechute du cancer du sein triple négatif d’autre part. Le premier axe de ma thèse concerne l’étude clinique MENOCOR dont le but est d’évaluer chez les femmes jeunes atteintes d’un cancer du sein non métastatique l’impact de la MCI sur la qualité de vie (QdV). L’incidence de la MCI et l’étude des variations hormonales font également partie des objectifs secondaires. Dans le cadre de cette thèse, l’analyse intermédiaire, prévue dans le protocole, a été réalisée sur 59 patientes avec un recul de 18 mois post-chimiothérapie. Ces premiers résultats ont tendance à montrer une altération de la QdV évaluée par le QLQ-BR23 chez les patientes ménopausées vs les non ménopausées (p=0.17). A ce stade de l’essai, le QLQ-BR23 semble donc être plus adapté que le QLQ-C30 pour évaluer la QdV dans le contexte de cette étude. Nous avons également observé une prédiction possible de la MCI par le dosage de l’AMH initial et l’âge des patientes. Ainsi, ces résultats doivent être comparés à ceux de l’analyse finale et nous supposons qu’avec une puissance plus importante (effectif total prévu de 240 patientes et suivi de 30 mois post-traitement) les résultats concernant la QdV pourraient être significatifs.Le deuxième axe de ma thèse a consisté à la création d’une base de données sur le CSTN. Cette étude rétrospective s’inscrit dans la continuité des travaux réalisés au Centre Jean PERRIN et a pour but d’évaluer la dynamique et les facteurs prédictifs de la rechute du CSTN. Les analyses ont montré que l’atteinte ganglionnaire, la présence d’emboles et la taille tumorale sont les principaux facteurs pronostiques de la rechute. Les résultats présentés dans cette thèse confirment également l’hétérogénéité des CSTN avec l’existence d’une disparité des réponses à la CTNA (allant de la réponse complète à la chimiorésistance) et d’une diversité des rechutes (précoces (< 1 an), standards (1 à 5 ans) et tardives (> 5 ans)) soulignant la nécessité de poursuivre la recherche de nouveaux biomarqueurs pouvant prédire la réponse ou encore permettant de prévenir les rechutes afin d’améliorer la prise en charge des patientes. En conclusion, ces résultats permettent d’ouvrir de nombreuses perspectives de recherches. Nous prévoyons de comparer les résultats finaux de l’étude MENOCOR à ceux de l’analyse intermédiaire présentés dans cette thèse. A l’issu de l’essai, nous espérons avoir une meilleure compréhension de la MCI, de son impact sur la QdV et du rôle de l’AMH dans la prédiction de la ménopause. Concernant le CSTN, il est prévu de mener une autre étude rétrospective sur une cohorte plus large, avec cette fois-ci, l’analyse des paramètres hématologiques. En fonction des résultats, une étude prospective pourra être mise en place afin d’étudier le rôle du microenvironnement tumoral ainsi que des paramètres hématologiques et génétiques dans la rechute des CSTN. / Breast cancer is the most common cancer among women and despite a decline in mortality in recent years, due to early detection and better treatment management, it remains a major public health issue. Breast cancer mortality has been found to be higher in the case of aggressive or poor prognostic cancers such as breast cancer in young women and the triple negative subtype of breast cancer (TNBC).This PhD focuses on these two types of breast cancer and aims to study the consequences of chemotherapy-induced menopause (CIM) in young women with non metastatic breast cancer and evaluate the prognostic factors of TNBC relapse. One of the aspect of my PhD is based on the clinical study MENOCOR. The main objective of this study is to evaluate the impact of CIM on the quality of life (QoL) of young women with non metastatic breast cancer. The incidence of CIM and the study of hormonal variations are also a part of the secondary objectives. As a part of this PhD work, the interim analysis planned in the study protocol was conducted on 59 patients with a follow-up of 18 months post-chemotherapy. These first results tend to show a QoL decrease, evaluted by the QLQ-BR23, in menopausal women vs non menopausal women (p=0.17). At this stage of the trial, the QLQ-BR23 seems to be more appropriate than the QLQ-C30 to evaluate QoL. We also underline the possible prediction of CIM by the inital AMH level and age. Thus, these results should be compared to the final analysis and we expect that with a greater power (240 patients and a follow-up of 30 months post-chemotherapy) the QoL results will reach significance.The second axis consists of a database on TNBC. This retrospective study follows the work carried out in Jean PERRIN Cancer Center and aims to evaluate dynamics and pronostic factors of TNBC relapse. The analysis showed that the main pronostic factors are the node metastasis, the presence of emboli and the tumor size. The variability of response to chemotherapy (pCR to chemoresistance) and the diversity of relapses (early relapse (<1 year), standard relapse (1 to 5 years) and late relpase (> 5 years)) confirmed the heterogeneity of TNBC highlighting the need to continue the research of new biomarkers. In conclusion, these results open up many research perspectives. We plan to compare the final results of the MENOCOR study with those of the interim analysis presented in this PhD work. At the end of the study, we expect to have a better knowledge of CIM, its impact on QoL and the role of AMH in predicting menopause. Regarding the TNBC, it is planned to conduct another larger retrospective study focused in hematologic parameters evaluating the role of hematologic and genetic parameters. Depending on the results, a prospective study could be promoted in order to evaluate the role of tumoral micro-environment, and hematologic and genetic parameters in TNBC relapse.

The rationale behind targeted molecular therapy in cancer, oncogene addiction, is that tumors rely on driver oncogenes to control their proliferation and survival. Therefore, an efficacious targeted therapy should induce a dual, detrimental response to the tumor. While there have been clinical success stories using targeted therapies, even tumors that are initially sensitive invariably develop resistance. In the case of triple negative breast cancer (TNBC), despite extensive evidence pointing to its driver oncogene status, inhibitors of the Epidermal Growth Factor Receptor (EGFR) are considered clinically inefficacious. Resistance to EGFR inhibition has been predominantly described as due to genetic alterations. Yet it remains unclear why patients exhibiting the same dysregulated status of a driver oncogene react to targeted therapy, as in the case of EGFR-mutant non-small cell lung cancer, while others do not at all (i.e., TNBC). Furthermore, not all of resistance can be described by genetic alterations to EGFR, to its pathway effectors, or to compensatory pathways. Emerging data reveals that drugs can induce resistance by rewiring epigenomic, transcriptional, and translational regulatory mechanisms. Unfortunately, a major limitation in designing efficacious treatments is our inability to predict whether cell types can rewire in response to drug exposure. Therefore, it is necessary to elucidate mechanisms of growth and survival in cells that have undergone rewiring. This study characterized intrinsic resistance to EGFR inhibition in TNBC. We found that EGFR inhibition induces rewiring, which results in a resistant growth state that bypasses the EGFR-MAPK pathway as a whole. Additionally, we found that a tRNA-modifying complex masks the oncogene addiction status of EGFR in TNBC by stabilizing the protein abundance of a pro-survival protein. Importantly, this happens solely in the context of EGFR inhibition. Taken together, this study highlights potential therapeutic strategies for TNBC and strategies that can be used to improve our understanding of targeted therapy resistance, especially intrinsic resistance.