Breast cancers with overexpression or amplification of the HER2 tyrosine kinase receptor are more aggressive, resistant to chemotherapy, and associated with a worse prognosis. Currently, these breast cancers are treated with the monoclonal antibody trastuzumab (Herceptin®). Unfortunately, not all patients respond to trastuzumab drug therapy; some patients show de novo resistance, while others acquire resistance during treatment. This thesis describes our RNAi studies to identify novel regulators of the HER2 signaling pathway in breast cancer. Three kinome-wide siRNA screens were performed on five HER2 amplified and seven HER2 non-amplified breast cancer cell lines, two normal breast cell lines, as well as two HER2-positive breast cancer cell lines with acquired trastuzumab resistance and their isogenic trastuzumab-sensitive controls. To understand the main kinase drivers of HER2 signaling, we performed a comprehensive screen that selected against growth inhibitors of the non-HER2 amplified breast cancer cell lines. This screen identified the loss of the HER2/HER3 heterodimer as the most prominent selective inhibitor of HER2-amplified breast cancers. In a trastuzumab sensitization screen on five trastuzumab-treated breast cancer cell lines, we identified several siRNA against the PI3K pathway as well as various other signaling pathways that inhibited proliferation. Finally, in a screen for acquired trastuzumab resistance, PKCη and its downstream targets were identified. Loss of PKCη resulted in a decrease in G1/S transition and upregulation of the cyclin dependent kinase inhibitor p27. Initial data suggest that PKCη promotes p27 ubiquitination and degradation. Taken together, these studies provide novel insight into the complex signaling of HER2-positive breast cancers and the mechanisms of resistance to trastuzumab therapy. This work describes how various kinases can modulate cell proliferation, and points to possible novel drug targets for the treatment of HER2-positive breast cancers.

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide. In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection. Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules. / QC 20110922

L’utilisation de thérapies ciblées afin d’augmenter la fenêtre thérapeutique des traitements, notamment en oncologie, se développe de plus en plus. Les immunoconjugués font partie de l’arsenal de ces thérapies ciblées. En effet, il s’agit de vectoriser, grâce à un anticorps ciblant un antigène surexprimé sur les cellules cancéreuses, une molécule hautement cytotoxique comme les dérivés d’auristatines. L’espèce résultant est appelée antibody-drug conjugate (ADC). Ce faisant, l’efficacité du composé est augmentée et les potentiels effets secondaires sont limités. Plusieurs technologies permettant l’accroche de l’agent cytotoxique à l’anticorps existent avec différents dégrés d’homogénéité du produit final et différents niveaux de difficultés de mise en œuvre. De nos jours, les avancées technologiques permettent de concevoir des immunoconjugués (ADCs) présentant un chargement en molécule cytotoxique (DAR, drug-to-antibody ratio) contrôlé du point de vue du nombre et de la position (régio-spécifique). / The use of targeted therapies in order to increase the therapeutic window of treatments, especially in oncology, is growing. Immunoconjugates are part of the arsenal of these targeted therapies. Indeed, they aim at delivering, thanks to an antibody directed against overexpressed cancer cell antigens, a highly cytotoxic molecule like auristatins derivatives. The resulting species is called an antibody-drug-conjugate (ADC). By doing so, efficacy of the compound is increased and potential side-effects are limited. Several technologies for grafting the cytotoxic agent to the antibody exist with different levels of homogeneity of the final compound and different levels of difficulty of implementation. Nowadays, technological breakthroughs permit the design of immunoconjugates (ADCs) displaying a controlled drug-to-antibody ratio (DAR) in terms of quantity and position (site-specific).

L’imagerie en oncologie a fait des progrès considérables ces dernières années avec l’introduction du CT scan spiralé, de la résonance magnétique, de la mammographie digitalisée et du PET scan. Des combinaisons de différentes techniques ont vu le jour, telles que le PET/CT, et améliorent encore les possibilités de stadification de la maladie cancéreuse ainsi que le monitoring de son évolution dans le temps, et notamment sous traitement.Parallèlement, de grands progrès thérapeutiques ont étés réalisés en oncologie, en particulier le développement de médicaments « ciblés » dont l’efficacité dépend de l’expression par la cellule tumorale d’une molécule cible jouant un rôle important dans sa survie et/ou sa prolifération. L’expression de la molécule cible est une condition nécessaire mais pas suffisante pour observer une réponse au traitement ciblé :l’échec de ce dernier peut aussi s’expliquer par des altérations moléculaires en amont ou en aval de la « cible ».Le cancer du sein dit « HER2 positif » représente 20 à 25% des cancers du sein. Celui-ci est caractérisé par l’expression membranaire, en quantités importantes, d’une protéine, appelée HER2, qui lui confère une biologie agressive et un mauvais pronostic. L’expression de HER2 au niveau de la tumeur, déterminée en routine clinique par immunohistochimie et/ou par hybridation in situ en fluorescence, est le seul biomarqueur validé aujourd’hui dans le cancer du sein HER2 positif pour prédire l’efficacité des traitements ciblés anti-HER2. Cette prédiction est toutefois peu satisfaisante en termes de valeur prédictive positive (50% environ). Après une revue de la litérature sur les études d’imagerie fonctionnelle, peu nombreuses, réalisées dans le cancer du sein HER2 positif, nous avons décidé d’explorer le rôle de l’imagerie moléculaire avec la technologie PET/CT dans l’individualisation de la prise en charge du cancer du sein HER2 positif avec deux radio traceurs (FDG et zirconium89-trastuzumab), et ce, dans deux contextes cliniques distincts :dans la maladie précoce soumise à un traitement neoadjuvant et dans le contexte métastatique, en cas de traitement par le T-DM1. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI- and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein...

The use of peptides in drug development has been hampered by their poor pharmaceutical properties, most notably their inability to reliably permeate biological membranes and lack of targeting. To overcome these disadvantages, the AMino acid Intracellular Delivery SysTem (AMIDST) was developed. This modular peptide-based delivery system confers cellular permeability and organelle-specific targeting for therapeutic peptides. As demonstrated in this study, the delivery of a HER2-binding peptide to the secretory organelles of breast cancer cells resulted in intracellular sequestration, a reduction in downstream signalling, and reduced viability compared to the delivery of a control peptide. Given its modular design and ease of production, AMIDST has the potential to enhance the use of peptides as therapeutic agents.

Synthetic xenoestrogens have differential estrogenic properties. Research has shown that exposures to xenoestrogens could promote breast cancer by disrupting normal function of the human epidermal growth factor receptor 2 (Her2) gene. Although animal models demonstrated a connection between xenoestrogen exposure and Her2 activity, no study using human cells has systematically examined their carcinogenic potential influencing the Her2 gene expression. Furthermore, breast cancer cells are phenotypically disparate (ER+, Her2+), with some phenotypes (Her2+), leading to more aggressive disease. This study aimed to dosimetrically assess the carcinogenic potential of commonly used xenoestrogens influencing Her2 gene expression, and delineate cellular phenotypes at greater risk of more aggressive disease. The study assessed whether the composition, concentrations, and exposure duration of BPA, EE, NPH, and DDT significantly altered Her2 copy numbers in estrogen and Her2 receptor positive or negative breast cancer lines. Each line was randomly assigned to cases (exposed) and control (unexposed) groups using a randomized block design. Fluorescent in-situ hybridization measured Her2 gene copies. Mann Whitney, Kruskal Wallis, and Incidence Rate Ratios revealed Her2 copy gains in all 4 xenoestrogens and receptor types with persistent exposures. A 44% increase in Her2 was observed in the normal ER and Her2 line, marking a shift in its Her2 status, and a 30-times greater risk was noted in the Her2+ lines. These findings promote positive social change by revealing all 4 xenoestrogens as risk factors for breast cancer. This information can be used by breast cancer advocacy groups, health educators, and steering committees to educate women and formulating policies.

Chemotherapy is one of the basic therapeutic procedures of breast cancer (BC) which may precede and/or follow the surgical resection of a tumor as a part of neoadjuvant or adjuvant therapy. However, the selective pressure of chemotherapy on tumor cells may change their molecular and expression profile and thus also their chemosensitivity. The aim of our work was to document the expression changes of selected markers in BC after neoadjuvant chemotherapy, which may contribute to the understanding of the role of these proteins and genes in tumor response to chemotherapy and the development of chemoresistance. Immunohistochemical analysis of expression of standard BC markers [estrogen (ER) and progesterone receptors (PR), HER2 and proliferation activity (Ki67)] and intercellular junction proteins (claudin 1, 3 and 4, E- and N-cadherin) before and after neoadjuvant chemotherapy revealed a decrease of PR, Ki67 and claudin 3 expression and an increase of claudin 1 expression. The expression of ER, HER2, claudin 4, E- and N-cadherin proved to be stable. Assessment of standard BC markers is performed routinely during a bioptic investigation as a necessary factor for therapy indication. Our results support the current recommendations for the re-examination before indication of adjuvant chemotherapy. Claudins...

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Breast cancer is the most common cancer in a woman with a five-year survival of patients with metastatic disease is estimated at 23%. Ado-trastuzumab emtansine (T-DM1) is a HER2-antibody drug conjugate currently approved for the treatment of HER2-positive pre-treated metastatic breast cancer (BC). We report a case of recurrent metastatic breast cancer with unusually lengthy progression-free survival (PFS) on T-DM1 chemotherapy. She was diagnosed with Triple Positive Stage IIIC multifocal invasive ductal carcinoma of the left breast. After completing neoadjuvant chemotherapy, she underwent a bilateral mastectomy. Final pathology showed partial response. Postoperatively, she received adjuvant chemotherapy and radiation therapy. She was started on Q21 days trastuzumab following completion of adjuvant chemotherapy. Systemic imaging showed liver lesions and biopsy confirmed recurrence. She was started on T-DM1, endocrine therapy with anastrozole was continued. She is currently status post 45 cycles. T-DM1 was approved for the treatment (single-agent) of HER2-positive, metastatic BC based on phase III data from the EMILIA and TH3RESA study. Median PFS in the T-DM1 arm was 9.6 months. Herein, we present a case of a woman with recurrent triple positive metastatic BC with a lengthy progression-free survival on T-DM1 chemotherapy.