Influence of BRCA1-mutated stroma on the early steps of the tumoral transformation in the breast cancer model.

L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires.The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers

Etude de l’influence du stroma BRCA1 muté sur les étapes précoces de transformation tumorale dans le modèle du cancer du sein

The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers.L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires

Host glycosylation pathways and the unfolded protein response contribute to the infection by Francisella

International audienc

Endocan, sepsis, pneumonia, and acute respiratory distress syndrome

Abstract Acute respiratory distress syndrome (ARDS) and hospital-acquired pneumonia (HAP) are major problems of public health in intensive care units (ICUs), occurring in 15% of critically ill patients. Among the factors explaining ARDS development, sepsis is known as a frequent cause. Sepsis, ARDS, and HAP increase morbidity, mortality, length of stay in the ICU, and the overall costs of healthcare. The major challenge remains to identify accurately among critically ill patients those at risk of poor outcomes who could benefit from novel therapies. Endocan is released by the pulmonary endothelium in response to local or systemic injury. It inhibits mainly leukocyte diapedesis rather than leukocyte rolling or adhesion to the endothelial cells both in vitro and in vivo. Endocan was evaluated in 25 clinical reports, including 2454 critically ill patients and 452 healthy controls. The diagnostic value of endocan for sepsis or sepsis severity was equal to procalcitonin but its prognostic value was better. A predictive value for postoperative pneumonia was evidenced in two studies, and a predictive value for ARDS in four studies from three independent centers. This review presents an overview of the structure, expression, and functions of endocan. We also hereby summarize the potential applications of endocan in the prediction and prognosis of ARDS and HAP, as well as in the prognosis of sepsis

Dosing time dependent in vitro pharmacodynamics of Everolimus despite a defective circadian clock

Everolimus (EV), a rapamycin analogue mTOR inhibitor, is used in the clinic to treat Estrogen positive (ER+) breast cancer in order to avoid the resistance to hormonotherapy. Here, we investigated whether EV efficacy varied according to administration timing by using the ER+ breast cancer cell line MCF-7 as model system. Our results showed that instead of apoptosis, EV induced a G0/G1 phase blockage of MCF-7 cells. Following serum shock, MCF-7 cells displayed a statistically significant 24h rhythm of mammalian target of Rapamycin (mTOR) activity, but perturbed circadian clock genes oscillations. Interestingly, the different delivery schedule of EV presented different efficacy in G0/G1 phase blockage in serum shocked MCF-7 cells. Moreover, serum shock induced also a circadian-like oscillation in expression or activity of several important G1 phase progression proteins, such as Cyclin D1 and phosphorylated Retinoblastoma protein (RB). Inhibition mTOR activity by EV reduced Cyclin D1 and Cyclin D3 protein level as well as RB phosphorylation level. Taken together, the results indicated that serum shock synchronization induced a circadian oscillation in mTOR activity in MCF-7 cells, which rhythmically regulated the synthesis or phosphorylation of key G1 progression proteins, such as Cyclin D1 and phosphorylated RB, ultimately resulting in different G0/G1 blockage efficiency according to different EV administration timing

Generation of induced pluripotent stem cell (iPSC) line from a patient with triple negative breast cancer with hereditary exon 17 deletion of BRCA1 gene

BRCA1 germline mutation confers hereditary predisposition for breast and ovarian cancer. To understand the physiopathology of mammary and ovarian epithelial cancer transformation, and to identify early driver molecular events, we have generated an iPSC line from a patient carrying a germline exon 17 deletion in BRCA1 gene (BRAC1Ex17 iPSC) in a high-risk family context. Blood cells were reprogrammed used non-integrative virus of Sendaï. The BRCA1-deleted iPSC had normal karyotype, harboured a deletion in the exon 17 of the BRCA1 gene, expressed pluripotent hallmarks and had the differentiation capacity into the three germ layers

iPSC-Derived Hereditary Breast Cancer Model Reveals the BRCA1-Deleted Tumor Niche as a New Culprit in Disease Progression

International audienceTumor progression begins when cancer cells recruit tumor-associated stromal cells to produce a vascular niche, ultimately resulting in uncontrolled growth, invasion, and metastasis. It is poorly understood, though, how this process might be affected by deletions or mutations in the breast cancer type 1 susceptibility (BRCA1) gene in patients with a lifetime risk of developing breast and/or ovarian cancer. To model the BRCA1-deleted stroma, we first generated induced pluripotent stem cells (iPSCs) from patients carrying a germline deletion of exon 17 of the BRCA1 gene (BRCA1+/− who, based on their family histories, were at a high risk for cancer. Using peripheral blood mononuclear cells (PBMCs) of these two affected family members and two normal (BRCA1+/+) individuals, we established a number of iPSC clones via non-integrating Sendai virus-based delivery of the four OCT4, SOX2, KLF4, and c-MYC factors. Induced mesenchymal stem cells (iMSCs) were generated and used as normal and pathological stromal cells. In transcriptome analyses, BRCA1+/− iMSCs exhibited a unique pro-angiogenic signature: compared to non-mutated iMSCs, they expressed high levels of HIF-1α, angiogenic factors belonging to the VEGF, PDGF, and ANGPT subfamilies showing high angiogenic potential. This was confirmed in vitro through the increased capacity to generate tube-like structures compared to BRCA1+/+ iMSCs and in vivo by a matrigel plug angiogenesis assay where the BRCA1+/− iMSCs promoted the development of an extended and organized vessel network. We also reported a highly increased migration capacity of BRCA1+/− iMSCs through an in vitro wound healing assay that correlated with the upregulation of the periostin (POSTN). Finally, we assessed the ability of both iMSCs to facilitate the engraftment of murine breast cancer cells using a xenogenic 4T1 transplant model. The co-injection of BRCA1+/− iMSCs and 4T1 breast cancer cells into mouse mammary fat pads gave rise to highly aggressive tumor growth (2-fold increase in tumor volume compared to 4T1 alone, p = 0.01283) and a higher prevalence of spontaneous metastatic spread to the lungs. Here, we report for the first time a major effect of BRCA1 haploinsufficiency on tumor-associated stroma in the context of BRCA1-associated cancers. The unique iMSC model used here was generated using patient-specific iPSCs, which opens new therapeutic avenues for the prevention and personalized treatment of BRCA1-associated hereditary breast cancer

Evaluation of Endocan as a Treatment for Acute Inflammatory Respiratory Failure

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening condition resulting from acute pulmonary inflammation. However, no specific treatment for ARDS has yet been developed. Previous findings suggest that lung injuries related to ARDS could be regulated by endocan (Esm-1). The aim of this study was to evaluate the potential efficiency of endocan in the treatment of ARDS. Methods: We first compared the features of acute pulmonary inflammation and the severity of hypoxemia in a tracheal LPS-induced acute lung injury (ALI) model performed in knockout (Esm1−/−) and wild type (WT) littermate C57Bl/6 mice. Next, we assessed the effects of a continuous infusion of glycosylated murine endocan in our ALI model in Esm1−/− mice. Results: In our ALI model, we report higher alveolar leukocytes (p p p p Esm1−/− mice compared to WT mice. Continuous delivery of glycosylated murine endocan after LPS-induced ALI resulted in decreased alveolar leukocytes (p = 0.012) and neutrophils (p = 0.012), higher blood oxygenation levels (p p = 0.04), compared to mice treated with PBS. Conclusions: Endocan appears to be an effective treatment in an ARDS-like model in C57Bl/6 mice