Cell cycle deregulation in breast cancer subgroups and effects on proliferation, migration and tamoxifen resistance

Breast cancer is a heterogenous disease which can be divided in subgroups of distinct biology with disparate prognosis and response to treatment. The overall aim of this thesis was to delineate subgroup specific behaviour in breast cancer cells with focus on proliferation, migration and stem-like cell activity. In addition, we have studied the functional retinoblastoma tumour suppressor (RB) protein pathway and associations to tamoxifen response. Furthermore, the expression of yes-associated protein (YAP1), reported to have both oncogenic and tumour suppressive functions, was investigated in breast cancer subgroups and related to tamoxifen response. Two of the key processes in malignant behaviour, proliferation and migration, have previously been reported to act as two opposing events in a cancer cell. We found that siRNA-mediated reduction of cyclin D1, a protein expressed in the active cell cycle, resulted in a migratory increase in cell lines negative of estrogen receptor (ER) expression. Conversely, in ER positive cell lines, downregulation of cyclin D1 resulted in decreased migratory capacity and a reduced number of stem-like cells, as measured by the mammosphere assay. Two agents inhibiting the cell cycle machinery, currently undergoing clinical trials, were further evaluated. Results showed that use of these agents in ER negative cell lines increased the number of stem-like cells, whereas a decrease was observed in the ER positive cells. These results point to the disparate effects of cell cycle-targeting treatments on breast cancer cells and highlight the importance of subgroup analysis. The breast cancer therapy tamoxifen is widely used in patients with ER positive breast cancers; however resistance occurs in approximately one-third of patients. By analysing a breast cancer tumour material from a patient cohort randomised to receive tamoxifen or control treatment, we have found that a non-functional RB pathway in ER positive breast tumours was predictive of tamoxifen insensitivity. The non-functional RB pathway was however not correlated to prognosis, indicating that status of RB pathway holds purely treatment predictive information. In addition, YAP1 was analysed in the tamoxifen randomised patient cohort and tumours lacking YAP1 protein expression were correlated with resistance to tamoxifen. Furthermore, in ER negative breast tumours, higher YAP1 expression was associated with increased proliferation whereas in ER positive tumours, YAP1 was negatively correlated to proliferation and grade. In vitro experiments downregulating YAP1 resulted in increased levels of ER and progesterone receptor (PgR), indicating deregulated signalling of the ER pathway. Taken together, we have shown that the consequences of targeting cell cycle proteins may differ depending on ER expression. Furthermore, we have linked a non-functional pathway of the cell-cycle regulator RB and absent YAP1 protein expression to impaired tamoxifen response, identifying two potential biomarkers for predicting tamoxifen insensitivity

Arsonium-Containing Lipophosphoramides, Poly-Functional Nano-Carriers for Simultaneous Antibacterial Action and Eukaryotic Cell Transfection.

International audienceGene therapy of diseases like cystic fibrosis (CF) would consist of delivering a gene medicine towards the lungs via the respiratory tract into the target epithelial cells. Accordingly, poly-functional nano-carriers are required in order to overcome the various successive barriers of such a complex environment, such as airway colonization with bacterial strains. In this work, the antibacterial effectiveness of a series of cationic lipids is investigated before evaluating its compatibility with gene transfer into human bronchial epithelial cells. Among the various compounds considered, some bearing a trimethyl-arsonium headgroup demonstrate very potent biocide effects towards clinically relevant bacterial strains. In contrast to cationic lipids exhibiting no or insufficient antibacterial potency, arsonium-containing lipophosphoramides can simultaneously inhibit bacteria while delivering DNA into eukaryotic cells, as efficiently and safely as in absence of bacteria. Moreover, such vectors can demonstrate antibacterial activity in vitro while retaining high gene transfection efficiency to the nasal epithelium as well as to the lungs in mice in vivo. Arsonium-containing amphiphiles are the first synthetic compounds shown to achieve efficient gene delivery in the presence of bacteria, a property particularly suitable for gene therapy strategies under infected conditions such as within the airways of CF patients

Self-assembly of supramolecular triarylamine nanowires in mesoporous silica and biocompatible electrodes thereof

Biocompatible silica-based mesoporous materials, which present high surface areas combined with uniform distribution of nanopores, can be organized in functional nanopatterns for a number of applications. However, silica is by essence an electrically insulating material which precludes applications for electro-chemical devices. The formation of hybrid electroactive silica nanostructures is thus expected to be of great interest for the design of biocompatible conducting materials such as bioelectrodes. Here we show that we can grow supramolecular stacks of triarylamine molecules in the confined space of oriented mesopores of a silica nanolayer covering a gold electrode. This addressable bottom-up construction is triggered from solution simply by light irradiation. The resulting self-assembled nanowires act as highly conducting electronic pathways crossing the silica layer. They allow very efficient charge transfer from the redox species in solution to the gold surface. We demonstrate the potential of these hybrid constitutional materials by implementing them as biocathodes and by measuring laccase activity that reduces dioxygen to produce water

Decreased expression of Yes-associated protein is associated with outcome in the luminal A breast cancer subgroup and with an impaired tamoxifen response

Background: Yes-associated protein (YAP1) is frequently reported to function as an oncogene in many types of cancer, but in breast cancer results remain controversial. We set out to clarify the role of YAP1 in breast cancer by examining gene and protein expression in subgroups of patient material and by downregulating YAP1 in vitro and studying its role in response to the widely used anti-estrogen tamoxifen. Methods: YAP1 protein intensity was scored as absent, weak, intermediate or strong in two primary breast cancer cohorts (n = 144 and n = 564) and mRNA expression of YAP1 was evaluated in a gene expression dataset (n = 1107). Recurrence-free survival was analysed using the log-rank test and Cox multivariate analysis was used to test for independence. WST-1 assay was employed to measure cell viability and a luciferase ERE (estrogen responsive element) construct was used to study the effect of tamoxifen, following downregulation of YAP1 using siRNAs. Results: In the ER+ (Estrogen Receptor a positive) subgroup of the randomised cohort, YAP1 expression was inversely correlated to histological grade and proliferation (p = 0.001 and p = 0.016, respectively) whereas in the ER-(Estrogen Receptor a negative) subgroup YAP1 expression correlated positively to proliferation (p = 0.005). Notably, low YAP1 mRNA was independently associated with decreased recurrence-free survival in the gene expression dataset, specifically for the luminal A subgroup (p less than 0.001) which includes low proliferating tumours of lower grade, usually associated with a good prognosis. This subgroup specificity led us to hypothesize that YAP1 may be important for response to endocrine therapies, such as tamoxifen, extensively used for luminal A breast cancers. In a tamoxifen randomised patient material, absent YAP1 protein expression was associated with impaired tamoxifen response which was significant upon interaction analysis (p = 0.042). YAP1 downregulation resulted in increased progesterone receptor (PgR) expression and a delayed and weaker tamoxifen in support of the clinical data. Conclusions: Decreased YAP1 expression is an independent prognostic factor for recurrence in the less aggressive luminal A breast cancer subgroup, likely due to the decreased tamoxifen sensitivity conferred by YAP1 downregulation

Serrathor : analyse intermédiaire de la pharmacodynamique du bloc Serratus chez des patients opérés par vidéothoracoscopie

Médecine. Anesthésie réanimationL’étude décrit la pharmacocinétique de la ropivacaine (ROP) sur les 24 premières h après une injection au niveau du muscle serratus (SAPB). 40 patients opérés sous vidéothoracoscopie ont été randomisés (groupe placebo et ROP). Le bloc a été réalisé en post opératoire sous échoguidage avec une dose de ROP 3.75mg/ml d’un volume de 0,5 ml/kg. Les concentrations plasmatiques ont été déterminées par chromatographie liquide. La cinétique des concentrations montre une phase d’augmentation rapide suivi d’une phase de décroissance lente témoin d’une absorption lente et de l’élimination plasmatique. Le délai médian au pic était de 20 min avec une concentration maximale médiane de 1.65mg/L. La concentration maximale était de 2.59mg/L et survenait dès la 10ème minute. 24 h après, la concentration médiane était de 0.15mg/L. Aucun patient ne présentait de signes de toxicité. Après SAPB, l’absorption systémique de la ROP est rapide. Les taux plasmatiques restent en dessous des seuils de toxicité.A prospective pharmacocinetic study was undertaken to describe the pharmacocinetics of ropivacaine SAPB during 24hours. 40 patients were randomly allocated to two groups ( ROP 3.75mg/ml of a volume of 0.5ml/kg at the end of surgery, or 0.5ml/kg of NaCL 0.9% solution). Central arterial plasma samples were collected. Plasma concentration of ropivacaine was measured by high performance liquid chromatography. The mean (SD) peak total ropivacaine concentration occured 20 min post injection and was 1.65mg/L. The highest measured concentration was 2.59mg/L and occured at 10 min. 24 h later, the ropivacaine was still present at a median concentration of 0.15mg/L. No toxic effects were observed. After SAPB the absorption of ropivacaine shows a rapid absorption phase leading to relatively high arterial plasma concentrations followed by a slower absorption phase in the course of elimination phase. The arterial peak concentration of ROP stay below the allege toxic concentrations

Mise au point de bibliothèques combinatoires dynamiques pour l'application à des cibles biologiques

La chimie combinatoire dynamique (CCD) est un nouveau concept qui consiste à créer des bibliothèques à partir de composés liés entre eux par des connexions réversibles. La réversibilité confère au système des propriétés d'adaptation et d'évolution permettant ainsi une sélection darwinienne.Nous nous sommes intéressés dans un premier temps à la réversibilité des connexions de types imines, hydrazones, acylhydrazones et oximes. Afin d'envisager leur future utilisation dans la formation de bibliothèques combinatoires dynamiques, nous avons étudié la facilité de ces composés à se former et à s'échanger en milieu aqueux. Des expériences effectuées par RMN 1H ont permis de montrer qu'en milieu aqueux, seuls les hydrazides forment avec les aldéhydes des liaisons covalentes réversibles stables et s'échangent rapidement à température ambiante et à pH=7. L'étude a également mis en lumière l'importance de la structure de l'aldéhyde sur la vitesse d'échange des acylhydrazones.Par la suite, nous avons réalisé en utilisant les liaisons réversibles d'acylhydrazones, la synthèse de différentes unités d'organisation et groupes de reconnaissance ainsi que la génération de deux bibliothèques combinatoires dynamiques. Testées sur des cibles biologiques, telles que la catéchol-O-méthyl-transférase et la thrombine, ces bibliothèques ont permis d'identifier des composés ayant une affinité avec ces enzymes et de confirmer que les acylhydrazones sont adaptées pour générer de telles bibliothèques.Les principes de la chimie combinatoire dynamique ont également été appliqués aux systèmes de biopolymères classiques tels que les protéines pour créer des biopolymères dynamiques dérivés d'acides aminés. Nous avons ainsi réalisé une structure de polypeptide dynamique en solution aqueuse et induit une auto-polymérisation réversible en contrôlant le pH. L'étude des conditions de polymérisation et d'échange a été réalisée par différentes techniques analytiques (RMN 1H, SM-ES, SM-MALDI, Multidétection-GPC) afin de suivre l'évolution du système et de caractériser les espèces formées in situ.Dynamic combinatorial chemistry (DCC) is a new concept in which libraries are created from building blocks linked together by reversible connections. The reversibility confers on the system the properties of adaptation and evolution, thus enabling Darwinian-type selection.We were interested initially in the reversibility of covalent bonds such as imines, hydrazones, acylhydrazones and oximes. In order to determine their suitability for future use in the generation of dynamic combinatorial libraries, we studied the ability of these compounds to form and exchange in aqueous media. 1H NMR spectroscopy experiments revealed that in aqueous solution, only acylhydrazones form stable reversible covalent bonds and undergo rapid interchange at room temperature and pH=7. Additionally, we have shown the importance of the aldehyde structure on the rate of acylhydrazone exchange.We synthesised various organisation units and recognition groups and also generated two dynamic combinatorial libraries using acylhydrazone covalent bonds. Tested on biological targets, such as catechol-O-methyltransferase and thrombin, these libraries enabled the identification of a compounds having an affinity with these enzymes. These results confirm that acylhydrazones are suitable for generating dynamic combinatorial libraries.The principles of DCC were also applied to create dynamic biopolymers based on proteins and derived from amino acids. Dynamic polypeptide-like oligomers were attained in aqueous solution whose reversible auto-polymerisation could be controlled by pH. Polymerisation and exchange properties were studied using different analytical techniques (1H NMR, ES-MS, MALDI-MS, Multidetection-GPC) to follow the evolution of the system and to characterise the species formed in situ.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF