Les récepteurs de la famille HER jouent un rôle majeur dans le développement du cancer. Alors qu’EGFR, HER2 et HER3 sont très bien étudiés et ciblés par des anticorps thérapeutiques, le dernier récepteur de cette famille, HER4, n’est que peu étudié et son implication dans la cancérogénèse est controversée. Il n’existe à ce jour pas d’anticorps thérapeutique anti-HER4 sur le marché ou en phase clinique. Ce récepteur est présent à la surface en quatre isoformes (JMa/CYT1 ; JMa/CYT2 ; JMb/CYT1 ; JMb/CYT2). Les isoformes JMa sont activées par clivage du récepteur, contrairement aux deux isoformes JMb. Le clivage de ces isoformes conduit à la libération de la partie intracellulaire du récepteur, appelée 4ICD. Ce fragment peut être dirigée au noyau ou dans d’autres compartiments cellulaires, impliquant HER4 dans des signalisations oncogéniques ou suppresseurs de tumeur. La littérature décrivant une activité pro-apoptotique de 4ICD et de la NRG1, le principal ligand de HER4, nous avons étudié la signalisation de ces isoformes afin de déterminer leurs rôles au niveau tumoral. Nos résultats indiquent que la NRG1 induit une signalisation suppresseur de tumeur via JMa/CYT1 et une signalisation oncogénique via JMa/CYT2. Sur la base de ces résultats, nous avons développé un criblage original d’anticorps anti-HER4 par phage display, sur des cellules exprimant l’isoforme JMa/CYT1 et stimulées par la NRG1. Nous avons caractérisés quatre anticorps anti-HER4, dont l’activité et les signalisations de certains sont modulées par la NRG1. Deux de ces anticorps, caractérisés comme étant des agonistes du récepteur HER4, induisent la mort des cellules tumorales par des mécanismes que nous sommes en train d’élucider. De manière similaire a la NRG1, un des anticorps induit la relocalisation de 4ICD-CYT1 a la mitochondrie pour induire la mort cellulaire. Ces résultats prometteurs ouvrent la voie à un ciblage thérapeutique du récepteur HER4 a l’aide d’anticorps agonistes pour le traitement des cancers / HER family is composed by four members which play a major role in cancer development. EGFR, HER2 and HER3 are well described and targeted with therapeutic monoclonal antibodies. HER4, the last one, is poorly described with a contentious role in cancerogenesis. Nowadays, there is no therapeutic antibody targeting HER4 in clinic. Four isoforms of the receptor are addressed to the plasma membrane and are called JMa/CYT1; JMa/CYT2; JMb/CYT1 and JMb/CYT2. JMa isoforms are activated by cleavage, but not JMb isoforms. Following their activation, JMa isoform cleavage releases the intracellular part of the receptor called 4ICD. This part can be directed to the nucleus or others subcellular compartments, involving HER4 in oncogenic or tumor suppressor signalling. Because a pro-apoptotic activity of 4ICD and its main ligand NRG1 have been described, we studied JMa isoforms signaling to determine their roles in cancer. We demonstrated that NRG1 induce a tumor suppressor signalling from JMa/CYT1 and an oncogenic signalling from JMa/CYT2. Based on these results, we developed an innovative screening for anti-HER4 antibodies by whole cell panning with phage display. To this end, we used NRG1- stimulated cells expressing JMa/CYT1 isoforms. We characterized four anti-HER4 antibodies and functions of some of them are affected and modulated by NRG1. Two antibodies were characterized as agonistic anti-HER4 antibodies and induce cell death of cancer cells by different mechanisms. Like NRG1, one of them induce mitochondrial localization of 4ICD-CYT1 to induce cell death. These promising results pave the way to a therapeutic targeting of HER4 receptor with agonistic antibodies to treat cancer

Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for embryonic retinal neurogenesis to proceed correctly, systems must be in place that restrict subsets of progenitor cells from differentiation. Pools of actively proliferating retinal progenitor cells are maintained to fill the needs of developmental processes and normal growth of the retina. In addition, subsets of radial glia in the retina retain the ability to de-differentiate into proliferating progenitor cells to meet the demands of the regenerating retina. All of these processes rely on the tight coordination of extrinsic and intrinsic cues, as well as regulation of gene expression by transcription factors. Although a considerable amount of work has been conducted to identify key regulators of retinal development and regeneration, many gene regulatory networks which include both master signaling pathways as well as individual transcription factors remain poorly characterized. Some of these factors implicated in retinal development and regeneration are members of the Hairy/Enhancer of Split (Hes) superfamily of genes, including the Hairy-related (Her) factors Her4 and Her9. Her transcription factors are basic-helix-loop-helix-orange (bHLH-O) transcription factors that bind to palindromic E- and N-box canonical sequences in the promoters of target genes. Her factors have been previously shown to play roles in a diverse array of developmental and neurogenic processes, including neural tube closure, floor plate development, somitogenesis, and development of various components of the central nervous system as well as the cranial sensory placodes. The roles of her4 and her9 in retinogenesis, however, remain undefined. To determine the possible roles of her4 and her9 factors in the retina, I characterized the expression patterns of these factors during developmental retinal neurogenesis and/or regeneration, examined loss of function phenotypes, and identified signaling pathways that modulate expression of these factors. Chapter 1 of this dissertation provides an overview of vertebrate retina and retinal development, the known functions of her4 in other tissues, and the Notch-Delta signaling pathway. Chapter 2 provides evidence that her4 is a primary effector of the Notch pathway during retinal development, and examines the role of her4 expressing cells during regeneration of the mature zebrafish retina within the context of both chronic and acute photoreceptor damage paradigms. In addition, generation and validation of the transgenic her4:Kaede zebrafish which was used to identify the lineage of her4-expressing cells is described. Characterization of her9 during retinal development, identification of the retinoic acid signaling pathway as a regulator of her9 expression in the retina, and the role her9 plays during retinal vasculogenesis are discussed in Chapter 3. Chapter 4 discusses the generation of her9 knock-out zebrafish lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterization of mutant phenotypes in mosaic her9 mutant F0 fish. In addition, in Chapter 4 I also discuss the screening processes used to identify and characterize genetic lesions in the her9 allele and establish various lines that stably transmit deleterious her9 alleles in the germline, and provide preliminary data of the her9 mutant phenotype. Finally, in Chapter 5 I discuss conclusions from the data generated from this dissertation, additional studies that would expand upon this work, and the implications of these results on the broader understanding of retinal development and regeneration. My dissertation incorporates reverse genetic analysis in zebrafish, biochemical analysis, transgenesis, and various molecular approaches to help better understand the roles of her4 and her9 during retinal neurogenesis. Moreover, these studies may also contribute to a better understanding of retinal development, and disease pathogenesis. It is my hope that this work could also ultimately contribute, even if in some small way, to the goal of enabling human patients who have suffered from vision loss a means by which a damaged retina could be regenerated and functional vision restored.

<p>Funding: Magnus Bergvall Cancer Foundation; Percy Falk foundation for research in breast and prostate cancer; Nyckelfonden; Örebro University Hospital; Lions cancer research foundation, Region Uppsala-Örebro</p>

Background Trastuzumab resistance imposes a major limitation to the successful treatment of HER2 positive breast cancer. The expression of HER4 and its prognostic value is controversial in breast cancer. Furthermore, its role in trastuzumab treatment and resistance in HER2 positive breast cancer has not been reported. Methods The effects of trastuzumab on HER4 cleavage and its localisation were studied in both parental and trastuzumab-resistant SKBR3 and BT474 cells using western blot, RT-PCR, nuclear fractionation and confocal microscopy. Tissue microarrays consisting of a cohort of HER2 positive breast cancer patients were stained for HER4 by immunohistochemistry and the results were correlated with patients’ outcome. This study also assessed HER4 expression in the tumor samples from a window study of trastuzumab alone or in combination with neoadjuvant chemotherapy in HER2 positive breast cancer patients. Results Trastuzumab treatment upregulated HER4 mRNA, and increased expression of both 80 and 180 kDa HER4 protein isoforms, and induced nuclear translocation of 80kDa HER4 protein isoforms, which the results similar to heregulin stimulation. This was also seen in trastuzumab resistant cells although HER4_80kDa and nuclear HER4 decreased upon overnight withdrawal of trastuzumab in resistant cell lines. In addition, knockdown of HER4 protein expression by specific siRNAs increased trastuzumab sensitivity and reversed trastuzumab resistance in SKBR3 and BT474 cells, confirming the importance of HER4 in trastuzumab response. This study also showed that trastuzumab-induced HER4 nuclear translocation is due to HER4 activation and cleavage since &gamma;-secretase inhibitor (GSi) and neratinib prevented the process when combined with trastuzumab treatment, correlating with an increased apoptosis and decreased cell viability. There was also increased nuclear HER4 expression in tumors from both BT474 xenografts and from patients with breast cancer treated with trastuzumab monotherapy. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was a poor prognostic factor in HER2 positive breast cancer. Conclusions This study suggests HER4 activation, cleavage and nuclear translocation play a key role in trastuzumab resistance in HER2 positive breast cancer. Nuclear HER4 could be a novel predictive and prognostic biomarker in HER2 positive breast cancer patients.