Implication of Zonula Occludens-1 (ZO-1) in the metastatic progression of breast tumor cells

Durant l’étape d’invasion tumorale, les cellules cancéreuses d’origine épithéliale acquièrent des propriétés migratoires et invasives qui leur permettent d’envahir les tissus sous-jacents. L’expression de telles propriétés implique de nombreux changements phénotypiques représentatifs des processus de Transition Epithélio-Mésenchymateuse (TEM). Parmi ces modifications, on observe notamment une diminution de la cohésion intercellulaire, une augmentation des capacités à dégrader la matrice extracellulaire et une motilité accrue. Au niveau moléculaire, ces caractéristiques se traduisent par une réorganisation des complexes jonctionnels intercellulaires, et une expression élevée de molécules pro-invasives, telles que les chémokines ou les métalloprotéinases matricielles. Au cours de ce travail, nous avons étudié l’implication d’une protéine des jonctions serrées, ZO-1, dans la régulation de gènes pro-invasifs, et dans la dissémination métastatique des cellules tumorales mammaires. Nos résultats démontrent qu’une relocalisation de ZO-1 en dehors des jonctions intercellulaires vers les compartiments cytoplasmiques/nucléaires, corrèle avec l’acquisition d’un potentiel migratoire et invasif élevé par les cellules tumorales mammaires, aussi bien in vivo qu’in vitro. Par ailleurs, nous avons montré que la relocalisation intracellulaire de ZO-1 est impliquée dans la régulation de gènes pro-tumoraux, tels que l’IL-8 dans les cellules tumorales mammaires. La régulation de cette chémokine par ZO-1 n’implique pas la voie de la caténine beta;, comme des données de la littérature amenaient à le penser, mais pourrait faire intervenir la signalisation de NFκ;B. En utilisant la technologie des shRNA, nous avons également étudié l’implication potentielle de ZO-1 dans la progression métastatique in vivo. Ainsi, l’inhibition stable de ZO-1 dans deux lignées cellulaires modifie leur comportement métastatique d’une manière dépendante du contexte cellulaire. En conclusion, nos observations suggèrent que ZO-1 subit une relocalisation cytoplasmique diffuse lors des processus de TEM associée à la migration et l’invasion cellulaires. Cette relocalisation régule positivement l’expression de gènes pro-invasifs, tels que celui de l’IL-8, et serait impliquée dans la modulation du comportement métastatique des cellules tumorales mammaires./During tumor invasion, epithelial tumor cells acquire migratory and invasive properties allowing them to invade underlying tissues. The expression of such properties involves many phenotypic changes representative of Epithelial to Mesenchymal Transition (EMT) processes. Among these modifications, one can notably observe a decrease of intercellular cohesion, an enhanced ability to degrade the extracellular matrix and an increased motility. At the molecular levels, these characteristics are related to a reorganization of cell adhesion complexes, and an increased expression of pro-invasive molecules, such as chemokines or matrix metalloproteinases.In the present work, we have investigated the implication of a tight junction protein, ZO-1, in the regulation of pro-invasive genes, and in the metastatic dissemination of breast tumor cells. Our results demonstrate that a relocalization of ZO-1 out of intercellular junctions towards cytoplasmic/nuclear compartments, correlate with the acquisition of increased migratory and invasive potential by breast tumor cells, both in vivo and in vitro. Moreover, we have demonstrated that this intercellular relocalization of ZO-1 modulate the expression of pro-invasive genes, such as IL-8, in breast tumor cells. The upregulation of IL-8 by ZO-1 does not involve beta; catenin signaling pathway, as hypothetized from the literature data, but could implicate the signaling pathway of NFκ;B. Using shRNA technology, we have examined the potential implication of ZO-1 in the metastatic progression in vivo. Thus, the stable downregulation of ZO-1 in two cell lines modifies their metastatic behavior in a cell context-dependent manner.In conclusion, our observations suggest that ZO-1 undergoes a diffuse cytoplasmic relocalization during EMT processes associated with cellular migration and invasion. This relocalization positively regulates the expression of pro-invasive genes, such as IL-8, and would be involved in the modulation of the metastatic behavior of breast tumor cells

Epithelial-to-mesenchymal transitions and circulating tumor cells.

Epithelial-to-mesenchymal transition (EMT) phenomena endow epithelial cells with enhanced migratory and invasive potential, and as such, have been implicated in many physiological and pathological processes requiring cell migration/invasion. Although their involvement in the metastatic cascade is still a subject of debate, data are accumulating to demonstrate the existence of EMT phenotypes in primary human tumors, describe enhanced metastatic potential of EMT derivatives in animal models, and report EMT attributes in circulating tumor cells (CTCs). The relationships between EMT and CTCs remain largely unexplored, and we review here in vitro and in vivo data supporting a putative role of EMT processes in CTC generation and survival

Regulation of CXCL8/IL-8 expression by Zonula Occludens-1 in human breast cancer cells.

Accumulating data now suggest that ZO-1, once delocalized from tight junctions, could be implicated in the regulation of tumor promoting genes. Because of their major implication in different steps of tumor progression, we investigated here the influence of ZO-1 on chemokines expression in breast cancer cells. Using GeneArray analysis to compare chemokine mRNA expression in breast tumor cells transfected with a siRNA against ZO-1, we identified CXCL-8/IL-8 as a major potential target of ZO-1 signaling, being strongly downregulated following ZO-1 siRNA transfection. Examining further the relationship between ZO-1 and IL-8, we first demonstrated that CXCL8/IL-8 expression correlates with a relocalization of ZO-1 in several breast cancer cell lines. Moreover, CXCL8/IL-8 is downregulated in invasive BT549 cells transfected with 3 different ZO-1 siRNA and overexpressed in non-invasive BT20 and SKBR3 cells transfected with vectors expressing ZO-1. We also provide evidence for an activation of the CXCL8/IL-8 promoter by ZO-1. Finally, we demonstrate that the regulation of CXCL8/IL-8 by ZO-1 is independent of the beta-catenin pathway. Our results thus clearly demonstrate an implication of ZO-1 in CXCL8/IL-8 regulation. Because of the major implications of CXCL8/IL-8 in tumor invasion, such a regulation could play an important role in breast cancer progression

Incidents in Molecular Pathology: Frequency and Causes During Routine Testing.

CONTEXT.— Errors in laboratory medicine could compromise patient safety. Good laboratory practice includes identifying and managing nonconformities in the total test process. Varying error percentages have been described in other fields but are lacking for molecular oncology. OBJECTIVES.— To gain insight into incident causes and frequency in the total test process from 8 European institutes routinely performing biomarker tests in non-small cell lung cancer and colorectal cancer. DESIGN.— All incidents documented in 2018 were collected from all hospital services for pre-preanalytical entries before the biomarker test, as well as specific incidents for biomarker tests. RESULTS.— There were 5185 incidents collected, of which 4363 (84.1%) occurred in the pre-preanalytical phase (all hospital services), 2796 of 4363 (64.1%) related to missing or incorrect request form information. From the other 822 specific incidents, 166 (20.2%) were recorded in the preanalytical phase, 275 (33.5%) in the analytical phase, and 194 (23.6%) in the postanalytical phase, mainly due to incorrect report content. Only 47 of 822 (5.7%) incidents were recorded in the post-postanalytical phase, and 123 (15.0%) in the complete total test process. For 17 of 822 (2.1%) incidents the time point was unknown. Pre-preanalytical incidents were resolved sooner than incidents on the complete process (mean 6 versus 60 days). For 1215 of 5168 (23.5%) incidents with known causes a specific action was undertaken besides documenting them, not limited to accredited institutes. CONCLUSIONS.— There was a large variety in the number and extent of documented incidents. Correct and complete information on the request forms and final reports are highly error prone and require additional focus

Rapid Whole Genome Sequencing Diagnoses and Guides Treatment in Critically Ill Children in Belgium in Less than 40 Hours

Rapid Whole Genome Sequencing (rWGS) represents a valuable exploration in critically ill pediatric patients. Early diagnosis allows care to be adjusted. We evaluated the feasibility, turnaround time (TAT), yield, and utility of rWGS in Belgium. Twenty-one unrelated critically ill patients were recruited from the neonatal intensive care units, the pediatric intensive care unit, and the neuropediatric unit, and offered rWGS as a first tier test. Libraries were prepared in the laboratory of human genetics of the University of Liège using Illumina DNA PCR-free protocol. Sequencing was performed on a NovaSeq 6000 in trio for 19 and in duo for two probands. The TAT was calculated from the sample reception to the validation of results. Clinical utility data were provided by treating physicians. A definite diagnosis was reached in twelve (57.5%) patients in 39.80 h on average (range: 37.05–43.7). An unsuspected diagnosis was identified in seven patients. rWGS guided care adjustments in diagnosed patients, including a gene therapy, an off-label drug trial and two condition-specific treatments. We successfully implemented the fastest rWGS platform in Europe and obtained one of the highest rWGS yields. This study establishes the path for a nationwide semi-centered rWGS network in Belgium

Frequency of Participation in External Quality Assessment Programs Focused on Rare Diseases: Belgian Guidelines for Human Genetics Centers.

BACKGROUND: Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality. OBJECTIVE: The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases. METHODS: A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance. RESULTS: The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls. CONCLUSIONS: These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics