Breast epithelial oncogenesis and cytomegalovirus at 'high' and 'low' risk of transformation

Le cancer du sein est le cancer le plus diagnostiqué et la première cause de mortalité liée au cancer chez la femme. Le cancer du sein regroupe un ensemble de tumeurs malignes variées, ayant une expression génique différente expliquant son hétérogénéité. Cela explique les variations fondamentales au niveau moléculaire des tumeurs du sein, définissant ainsi une signature génique unique pour chaque sous-type de cancer. Les découvertes liées à l’oncogenèse du cancer du sein ont permis d’identifier les cellules épithéliales mammaires humaines (HMECs) comme le lieu d’origine de la grande majorité des carcinomes. Les facteurs étiologiques du cancer du sein sont divisés en facteurs de risque génétiques ou environnementaux, parmi lesquels les virus sont estimés comme participants à 15-20% de tous les cas de cancer. Par ailleurs, le cytomegalovirus humain (HCMV) est un pathogène ubiquitaire appartenant à la famille des Herpesviridae. Un faisceau de preuves grandissant reconnait le HCMV comme un potentiel virus oncogène. Le génome et/ou les antigènes du HCMV ont été détectés dans une multitude de cancers, incluant les glioblastomes, les cancers colorectaux, de la prostate, et plus particulièrement dans le cancer du sein, mettant en avant le paradigme de l’oncomodulation, le fait que le HCMV accélère l’évolution maligne d’une tumeur. Par ailleurs, mettant en avant un rôle direct oncogène du HCMV, des études ont précédemment démontré la capacité du HCMV à induire la transformation de fibroblastes humains embryonnaires de poumon et de HMECs in vitro. Dans notre laboratoire, la souche clinique de HCMV DB (KT959235) a transformé des HMECs en « CMV-Transformed HMECs » (CTH) avec des traits oncogéniques et résultant en l’appartion de tumeurs à croissance rapide dans des souris NSG.Au cours de cette thèse, plusieurs souches cliniques de HCMV ont été évaluées pour leur potentiel oncogène. Pour commencer, des HMECs ont été infectées avec six isolats cliniques de HCMV et l’induction différentielle d’un environnement pro-oncogénique a été étudiée pour un temps court d’infection. Deuxièmement, le potentiel transformant, ainsi que les caractéristiques génétiques, moléculaires et cellulaires des CTH résultantes de la transformation ont été analysée dans des cultures à long terme. La reprogrammation des HMECs matures à travers l’induction d’une dédifférentiation, l’apparition d’un phénotype de cellules cancéreuses polyploïdes géantes (PGCC), caractérisé par une croissance importante de cellules ressemblant à des blastomères, ce qui concorde avec l’acquisition de caractéristiques de cellules souches embryonnaires et d’une plasticité épithélio-mésenchymale, que nous avons évalué dans notre modèle de CTH. Pour finir, un virus compétent pour la réplication et sensible à la réactivation depuis un état de latence par traitement avec du TPA a été détecté dans les cultures de CTH, en parallèle d’une succession d’événements cellulaires et moléculaire, potentiellement résultant du processus de transformation. La corrélation entre la détection des PGCCs et de la présence du HCMV dans des tissus de cancer du sein valide notre hypothèse in vivo.Pris dans son ensemble, ce travail fourni une nouvelle vision du potentiel oncogène de certaines souches cliniques de HCMV pour la transformation des HMECs et correspond à un modèle d’oncogenèse « semblable aux blastomères », qui pourrait être pertinent dans la physiopathologie du cancer du sein et d’autres adénocarcinomes, en particulier ayant un mauvais pronostic.Breast cancer is the most diagnosed cancer and the first leading cause of cancer mortality amongst women. Breast cancer embodies a compilation of distinct malignancies, with divergent gene expression patterns deciphering its heterogeneity. This elucidates the fundamental variances of breast tumors at the molecular level, thus defining unique gene signature for intrinsic subtypes. Insights into breast oncogenesis have identified human mammary epithelial cells (HMECs) as the site from which the prevailing majority of carcinomas originate. Breast cancer etiological factors are assorted into genetic or environmental risk factors, of which viruses are estimated to attribute to 15 to 20% of all cancer cases. On the other hand, human cytomegalovirus (HCMV) is a ubiquitous pathogen belonging to Herpesviridae family. A growing body of evidence is recognizing HCMV as a potential oncogenic virus. HCMV genome and/or antigens have been detected in a multitude of malignancies including glioma, colorectal, prostate, and most importantly breast cancer, an exploration hypothetically defended by the oncomodulation paradigm. Going beyond the latter, previous studies demonstrated HCMV ability to induce transformation of human embryonal lung fibroblasts and HMECs in vitro. In our laboratory, the clinical HCMV strain DB (KT959235) transformed HMECs into CMV-Transformed HMECs (CTH) cells and induced the appearance of oncogenic traits and fast-growing breast tumors in NSG mice.In this thesis, several HCMV clinical strains were screened for their transforming potential. First, HMECs were infected with six HCMV clinical isolates and differential induction of pro-oncogenic environment was explored on the short-term infection. Second, the transforming potential, as well as the genetic, molecular, and cellular features of the resulting CTH cells in long-term cultures were analyzed. Reprogramming of mature HMECs through the induction of dedifferentiation, generation of a polyploid giant cancer cell (PGCC) phenotype characterized by sustained growth of blastomere-like cells, in concordance with the acquisition of embryonic stem cell characteristics and epithelial-mesenchymal plasticity were assessed in our CTH model. Finally, a replication-competent virus prone to reactivation from latency upon TPA treatment was detected in CTH cultures, in parallel to the succession of observed cellular and molecular events, potentially ensuing the transformation process. Correlation between PGCCs detection and HCMV presence in breast cancer tissues further validated our hypothesis in vivo. Taken together, this work provides insights into the potential of some clinical HCMV strains to transform HMECs and fit with a “blastomere-like” model of oncogenesis, which may be relevant in the pathophysiology of breast cancer and other adenocarcinoma, especially of poor prognosis

Control of viral infections by epigenetic-targeted therapy

Abstract Epigenetics is defined as the science that studies the modifications of gene expression that are not owed to mutations or changes in the genetic sequence. Recently, strong evidences are pinpointing toward a solid interplay between such epigenetic alterations and the outcome of human cytomegalovirus (HCMV) infection. Guided by the previous possibly promising experimental trials of human immunodeficiency virus (HIV) epigenetic reprogramming, the latter is paving the road toward two major approaches to control viral gene expression or latency. Reactivating HCMV from the latent phase (“shock and kill” paradigm) or alternatively repressing the virus lytic and reactivation phases (“block and lock” paradigm) by epigenetic-targeted therapy represent encouraging options to overcome latency and viral shedding or otherwise replication and infectivity, which could lead eventually to control the infection and its complications. Not limited to HIV and HCMV, this concept is similarly studied in the context of hepatitis B and C virus, herpes simplex virus, and Epstein-Barr virus. Therefore, epigenetic manipulations stand as a pioneering research area in modern biology and could constitute a curative methodology by potentially consenting the development of broad-spectrum antivirals to control viral infections in vivo

Tight Junction Protein Signaling and Cancer Biology

International audienceTight junctions (TJs) are intercellular protein complexes that preserve tissue homeostasis and integrity through the control of paracellular permeability and cell polarity. Recent findings have revealed the functional role of TJ proteins outside TJs and beyond their classical cellular functions as selective gatekeepers. This is illustrated by the dysregulation in TJ protein expression levels in response to external and intracellular stimuli, notably during tumorigenesis. A large body of knowledge has uncovered the well-established functional role of TJ proteins in cancer pathogenesis. Mechanistically, TJ proteins act as bidirectional signaling hubs that connect the extracellular compartment to the intracellular compartment. By modulating key signaling pathways, TJ proteins are crucial players in the regulation of cell proliferation, migration, and differentiation, all of which being essential cancer hallmarks crucial for tumor growth and metastasis. TJ proteins also promote the acquisition of stem cell phenotypes in cancer cells. These findings highlight their contribution to carcinogenesis and therapeutic resistance. Moreover, recent preclinical and clinical studies have used TJ proteins as therapeutic targets or prognostic markers. This review summarizes the functional role of TJ proteins in cancer biology and their impact for novel strategies to prevent and treat cancer

Tight Junction Protein Signaling and Cancer Biology

Tight junctions (TJs) are intercellular protein complexes that preserve tissue homeostasis and integrity through the control of paracellular permeability and cell polarity. Recent findings have revealed the functional role of TJ proteins outside TJs and beyond their classical cellular functions as selective gatekeepers. This is illustrated by the dysregulation in TJ protein expression levels in response to external and intracellular stimuli, notably during tumorigenesis. A large body of knowledge has uncovered the well-established functional role of TJ proteins in cancer pathogenesis. Mechanistically, TJ proteins act as bidirectional signaling hubs that connect the extracellular compartment to the intracellular compartment. By modulating key signaling pathways, TJ proteins are crucial players in the regulation of cell proliferation, migration, and differentiation, all of which being essential cancer hallmarks crucial for tumor growth and metastasis. TJ proteins also promote the acquisition of stem cell phenotypes in cancer cells. These findings highlight their contribution to carcinogenesis and therapeutic resistance. Moreover, recent preclinical and clinical studies have used TJ proteins as therapeutic targets or prognostic markers. This review summarizes the functional role of TJ proteins in cancer biology and their impact for novel strategies to prevent and treat cancer

Impact of glucocorticoids on systemic sirtuin 1 expression and activity in rats with adjuvant-induced arthritis

The class III histone deacetylase sirtuin 1 (SIRT1) plays a pivotal role in numerous biological and physiological functions, including inflammation. An association between SIRT1 and proinflammatory cytokines might exist. In addition to their important role in inflammation associated with rheumatoid arthritis (RA), proinflammatory cytokines mediate the development of systemic effects. Here, we evaluated systemic SIRT1 expression and enzymatic activity, in peripheral blood mononuclear cells (PBMCs) and in liver isolated from rats with adjuvant-induced arthritis (AIA), treated or not with low or high doses of glucocorticoids (GCs). We also measured the production of tumour necrosis factor alpha (TNF) and interleukin-1 beta (IL-1 beta) in PBMCs and liver. We found that SIRT1 expression and activity increased in PBMCs of AIA rats compared to healthy controls and decreased under GC treatment. Similarly, we observed an increased SIRT1 activity in the liver of AIA rats compared to healthy controls which decreased under high doses of GCs. We also found an increase in IL-1 beta and TNF levels in the liver of AIA rats compared to healthy controls, which decreased under high doses of GC. We did not observe a significant correlation between SIRT1 activity and proinflammatory cytokine production in PBMC or liver. In contrast, a strong positive correlation was found between the liver levels of TNF and IL-1 beta (rho=0.9503, p=7.5x10−21). Our results indicate that increased inflammation in AIA rats compared to healthy control is accompanied by an increased SIRT1 activity in both PBMCs and liver, which could be decreased under GC treatment

Resveratrol Inhibits HCoV-229E and SARS-CoV-2 Coronavirus Replication In Vitro

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19

Resveratrol Inhibits HCoV-229E and SARS-CoV-2 Coronavirus Replication In Vitro

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19

PROPHYLACTIC ADMINISTRATION OF DOXYCYCLINE REDUCES CENTRAL VENOUS CATHETER INFECTIONS IN PATIENTS UNDERGOING HEMATOPOIETIC CELL TRANSPLANTATION

Hematopoietic stem cells are usually transfused through a central venous catheter (CVC), which also facilitates administration of medications and intravenous fluids. We had observed high rate of catheter-related blood-stream infection (CR-BSI) at our Bone Marrow Transplantation (BMT) unit despite prescribing fluoroquinolones for anti-bacterial prophylaxis. Accordingly, we implemented prophylactic use of a relatively inexpensive broad spectrum antibiotic, namely doxycycline to address this problem. We wanted to investigate whether doxycycline prophylaxis reduces CR-BSI rate. Data was collected retrospectively on 54 consecutive patients, 26 of whom received doxycycline (doxycycline group), and we compared their outcomes to a previous cohort of 28 patients who did not receive doxycycline (comparison group). The groups were comparable in regards to age, gender, hematopoietic cell transplant type, and primary diagnosis. No CVC infection (0%) was observed in the doxycycline group, while 5 infection episodes (18.5%) occurred in 4 patients in the comparison group (p<0.001). Episodes of CR-BSI were due to: Escherichia-coli (EC)=1, coagulase-negative Staphylococcus-spp (CNSS)=2, both EC & CNSS=1. Our results demonstrate that CR-BSI was reduced significantly after introducing doxycycline. This finding suggests a beneficial role for systemic use of doxycycline prophylaxis to prevent CR-BSI in adult BMT patients. Nevertheless, a randomized controlled study is warranted to confirm these findings