Fra-1 et Fra-2 dans les cancers du sein triple négatifs : mécanismes transcriptionnels et identification de cibles thérapeutiques potentielles

Triple negative breast cancers (TNBC) are characterized by a poor prognosis and no targeted therapy is currently available. The identification of new diagnostic and therapeutic targets is crucial for the treatment of these cancers. Fra-1 and Fra-2, two members of the AP-1 transcriptional complex, are frequently overexpressed in TNBC, where they contribute to the tumorigenic phenotype. The panel of genes under the control of Fra-1 and/or Fra-2 in TNBC, as well as the molecular mechanisms by which they control their target gene expression are mostly unknown. The aim of my thesis was to identify the panel of genes controlled by Fra-1 and/or Fra-2 in TNBC and to characterize the binding sites of Fra-1 and Fra-2 on chromatin to select direct targets for further studies, by using transcriptomic and ChIP-seq approaches combined to RNAi in the model cell line MDA-MB231. The results allowed us to select target genes for transcriptional and functional studies. The study of the transcriptional mechanisms governed by Fra-1 and/or Fra-2 was carried out on the HMGA1 gene, already known for its crucial role in the aggressiveness of epithelial tumours. The fonctional study was focused on CD68, as its expression in highly induced by Fra-1 and Fra-2. CD68 encodes a transmembrane protein which cellular fonction is still not known and its potential role in tumorigenesis has not been studied yet.Les cancers du sein triple négatifs (TNBC) ne bénéficient d’aucune thérapie ciblée et restent incurables. L’identification de nouvelles cibles moléculaires diagnostiques et surtout, thérapeutiques constitue donc un enjeu majeur pour le traitement de ces cancers. Fra-1 et Fra-2, deux constituants du complexe transcriptionnel AP-1, sont surexprimés dans les TNBC où ils participent à l’agressivité tumorale et/ou la métastatisation. Les gènes cibles de Fra-1 et Fra-2, ainsi que les mécanismes moléculaires via lesquels ils gouvernent la transcription de leurs gènes cibles sont très peu caractérisés. Dans ce contexte, l’objectif général de ma thèse à été d’identifier les gènes codants sous contrôle de Fra-1 et/ou Fra-2 et de caractériser les sites de fixation de Fra-1 et Fra-2 sur la chromatine pour identifier des cibles directes, en combinant des approches transcriptomiques et génomiques combinées à l’interférence à l’ARN dans la lignée modèle MDA-MB231. Les résultats obtenus, associés à l’analyse de banques de données humaines, ont permis la sélection de gènes cibles pour les études mécanistiques et fonctionnelles. Le gène HMGA1, choisi en raison de son rôle maintenant bien établi dans l’agressivité des tumeurs épithéliales, a fait l’objet d’une étude portant sur les mécanismes transcriptionnels gouvernés par Fra-1 et/ou Fra-2 pour sa régulation. L’étude fonctionnelle a été focalisée sur CD68 dont l’expression est fortement induite par Fra-1 et Fra-2. CD68 code pour une protéine transmembranaire dont la fonction n’est pas clairement établie et dont l’implication dans le phénotype tumoral n’a jamais été étudiée

Fra-1 and Fra-2 in triple negative breast cancers : transcriptional mechanisms and identification of potential therapeutic targets

Les cancers du sein triple négatifs (TNBC) ne bénéficient d’aucune thérapie ciblée et restent incurables. L’identification de nouvelles cibles moléculaires diagnostiques et surtout, thérapeutiques constitue donc un enjeu majeur pour le traitement de ces cancers. Fra-1 et Fra-2, deux constituants du complexe transcriptionnel AP-1, sont surexprimés dans les TNBC où ils participent à l’agressivité tumorale et/ou la métastatisation. Les gènes cibles de Fra-1 et Fra-2, ainsi que les mécanismes moléculaires via lesquels ils gouvernent la transcription de leurs gènes cibles sont très peu caractérisés. Dans ce contexte, l’objectif général de ma thèse à été d’identifier les gènes codants sous contrôle de Fra-1 et/ou Fra-2 et de caractériser les sites de fixation de Fra-1 et Fra-2 sur la chromatine pour identifier des cibles directes, en combinant des approches transcriptomiques et génomiques combinées à l’interférence à l’ARN dans la lignée modèle MDA-MB231. Les résultats obtenus, associés à l’analyse de banques de données humaines, ont permis la sélection de gènes cibles pour les études mécanistiques et fonctionnelles. Le gène HMGA1, choisi en raison de son rôle maintenant bien établi dans l’agressivité des tumeurs épithéliales, a fait l’objet d’une étude portant sur les mécanismes transcriptionnels gouvernés par Fra-1 et/ou Fra-2 pour sa régulation. L’étude fonctionnelle a été focalisée sur CD68 dont l’expression est fortement induite par Fra-1 et Fra-2. CD68 code pour une protéine transmembranaire dont la fonction n’est pas clairement établie et dont l’implication dans le phénotype tumoral n’a jamais été étudiée.Triple negative breast cancers (TNBC) are characterized by a poor prognosis and no targeted therapy is currently available. The identification of new diagnostic and therapeutic targets is crucial for the treatment of these cancers. Fra-1 and Fra-2, two members of the AP-1 transcriptional complex, are frequently overexpressed in TNBC, where they contribute to the tumorigenic phenotype. The panel of genes under the control of Fra-1 and/or Fra-2 in TNBC, as well as the molecular mechanisms by which they control their target gene expression are mostly unknown. The aim of my thesis was to identify the panel of genes controlled by Fra-1 and/or Fra-2 in TNBC and to characterize the binding sites of Fra-1 and Fra-2 on chromatin to select direct targets for further studies, by using transcriptomic and ChIP-seq approaches combined to RNAi in the model cell line MDA-MB231. The results allowed us to select target genes for transcriptional and functional studies. The study of the transcriptional mechanisms governed by Fra-1 and/or Fra-2 was carried out on the HMGA1 gene, already known for its crucial role in the aggressiveness of epithelial tumours. The fonctional study was focused on CD68, as its expression in highly induced by Fra-1 and Fra-2. CD68 encodes a transmembrane protein which cellular fonction is still not known and its potential role in tumorigenesis has not been studied yet

Multiple Fra-1-bound enhancers showing different molecular and functional features can cooperate to repress gene transcription

Abstract Background How transcription factors (TFs) down-regulate gene expression remains ill-understood, especially when they bind to multiple enhancers contacting the same gene promoter. In particular, it is not known whether they exert similar or significantly different molecular effects at these enhancers. Results To address this issue, we used a particularly well-suited study model consisting of the down-regulation of the TGFB2 gene by the TF Fra-1 in Fra-1-overexpressing cancer cells, as Fra-1 binds to multiple enhancers interacting with the TGFB2 promoter. We show that Fra-1 does not repress TGFB2 transcription via reducing RNA Pol II recruitment at the gene promoter but by decreasing the formation of its transcription-initiating form. This is associated with complex long-range chromatin interactions implicating multiple molecularly and functionally heterogeneous Fra-1-bound transcriptional enhancers distal to the TGFB2 transcriptional start site. In particular, the latter display differential requirements upon the presence and the activity of the lysine acetyltransferase p300/CBP. Furthermore, the final transcriptional output of the TGFB2 gene seems to depend on a balance between the positive and negative effects of Fra-1 at these enhancers. Conclusion Our work unveils complex molecular mechanisms underlying the repressive actions of Fra-1 on TGFB2 gene expression. This has consequences for our general understanding of the functioning of the ubiquitous transcriptional complex AP-1, of which Fra-1 is the most documented component for prooncogenic activities. In addition, it raises the general question of the heterogeneity of the molecular functions of TFs binding to different enhancers regulating the same gene

AP-1 Signaling by Fra-1 Directly Regulates HMGA1 Oncogene Transcription in Triple-Negative Breast Cancers

International audienc

Characterization of Resident B Cells of Vascular Walls in Human Atherosclerotic Patients

International audienceAnimal models of atherosclerosis suggest that B cells have contradictory protective or proatherogenic effects that are also subset and context dependent. To further understand the pathophysiology of human atheroma, we characterized local Ig production and functional properties of resident B cells in human arterial lesions. Ig repertoires were analyzed by RT-PCR in carotid endarterectomy samples. Cytokine, differentiation marker and transcription factor mRNA expression was studied on arterial wall lymphocytes isolated by laser capture microdissection. Ig sequence analysis revealed that individual samples each contained a limited number of B cell clones. Functional α and γ mRNAs made up the majority of H chain mRNAs in the adventitia. Clonal evolution of Ig V regions, expression of activation-induced cytidine deaminase, clonal H chain switch, and an inverted λ/κ ratio of Ig L chain usage indicated that a local differentiation process was taking place in arterial walls. Clonotypic markers revealed different plaque and adventitia Ig repertoires and a B cell recirculation between adventitia and draining lymph nodes. Microdissected mononuclear cells had an activated phenotype expressing IL-6, GM-CSF, and TNF-α, whereas IL-2, IL-4, IL-10, M-CSF, and IFN-γ were not detected. Adventitial oligoclonal resident B cells of atherosclerotic patients are mainly mature B2 (conventional) CD20⁻ plasmablasts lacking markers of terminal differentiation to plasma cell (CD138 and Blimp-1). They present hallmarks of Ag-driven maturation and could act on inflammation and disease progression directly or by promoting polarization of other immune cells

Characterization of Resident B Cells of Vascular Walls in Human Atherosclerotic Patients

International audienceAnimal models of atherosclerosis suggest that B cells have contradictory protective or proatherogenic effects that are also subset and context dependent. To further understand the pathophysiology of human atheroma, we characterized local Ig production and functional properties of resident B cells in human arterial lesions. Ig repertoires were analyzed by RT-PCR in carotid endarterectomy samples. Cytokine, differentiation marker and transcription factor mRNA expression was studied on arterial wall lymphocytes isolated by laser capture microdissection. Ig sequence analysis revealed that individual samples each contained a limited number of B cell clones. Functional α and γ mRNAs made up the majority of H chain mRNAs in the adventitia. Clonal evolution of Ig V regions, expression of activation-induced cytidine deaminase, clonal H chain switch, and an inverted λ/κ ratio of Ig L chain usage indicated that a local differentiation process was taking place in arterial walls. Clonotypic markers revealed different plaque and adventitia Ig repertoires and a B cell recirculation between adventitia and draining lymph nodes. Microdissected mononuclear cells had an activated phenotype expressing IL-6, GM-CSF, and TNF-α, whereas IL-2, IL-4, IL-10, M-CSF, and IFN-γ were not detected. Adventitial oligoclonal resident B cells of atherosclerotic patients are mainly mature B2 (conventional) CD20⁻ plasmablasts lacking markers of terminal differentiation to plasma cell (CD138 and Blimp-1). They present hallmarks of Ag-driven maturation and could act on inflammation and disease progression directly or by promoting polarization of other immune cells