Identification of novel TMPRSS2:ERG mechanisms in prostate cancer metastasis: involvement of MMP9 and PLXNA2

International audienceProstate cancer (PCa) is one of the major public health problems in Western countries. Recently, the TMPRSS2:ERG gene fusion, which results in the aberrant expression of the transcription factor ERG, has been shown to be the most common gene rearrangement in PCa. Previous studies have determined the contributions of this fusion in PCa disease initiation and/or progression in vitro and in vivo. In this study on TMPRSS2:ERG regulation in PCa, we used an androgen receptor and TMPRSS2:ERG fusion double-negative PCa cell model: PC3c. In three cell clones with different TMPRSS2:ERG expression levels, ectopic expression of the fusion resulted in significant induction of cell migration and invasion in a dose-dependent manner. In agreement with this phenotype, high-throughput microarray analysis revealed that a set of genes, functionally associated with cell motility and invasiveness, were deregulated in a dose-dependent manner in TMPRSS2:ERG-expressing cells. Importantly, we identified increased MMP9 (Metalloproteinase 9) and PLXNA2 (Plexin A2) expression in TMPRSS2:ERG-positive PCa samples, and their expression levels were significantly correlated with ERG expression in a PCa cohort. In line with these findings, there was evidence that TMPRSS2:ERG directly and positively regulates MMP9 and PLXNA2 expression in PC3c cells. Moreover, PLXNA2 upregulation contributed to TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. Furthermore, and importantly, PLXNA2 expression was upregulated in metastatic PCa tumors compared with localized primary PCa tumors. This study provides novel insights into the role of the TMPRSS2:ERG fusion in PCa metastasis

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy ( Necturus maculosus ) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus . Strikingly, we found that Necturus TRΑ and TRΒ genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRΑ and TRΒ are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75694/1/j.1525-142X.2006.00099.x.pd

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenylamidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationshipsfrom comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site

Estrogen related receptor alpha in castration-resistant prostate cancer cells promotes tumor progression in bone

Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFβ1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated with ERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events

ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases

The interplay between mineral metabolism, vascular calcification and inflammation in Chronic Kidney Disease (CKD): challenging old concepts with new facts

Chronic kidney disease (CKD) is one of the most powerful predictors of premature cardiovascular disease (CVD), with heightened susceptibility to vascular intimal and medial calcification associated with a high cardiovascular mortality. Abnormal mineral metabolism of calcium (Ca) and phosphate (P) and underlying (dys)regulated hormonal control in CKD-mineral and bone disorder (MBD) is often accompanied by bone loss and increased vascular calcification (VC). While VC is known to be a multifactorial process and a major risk factor for CVD, the view of primary triggers and molecular mechanisms complexity has been shifting with novel scientific knowledge over the last years. In this review we highlight the importance of calcium-phosphate (CaP) mineral crystals in VC with an integrated view over the complexity of CKD, while discuss past and recent literature aiming to highlight novel horizons on this major health burden. Exacerbated VC in CKD patients might result from several interconnected mechanisms involving abnormal mineral metabolism, dysregulation of endogenous calcification inhibitors and inflammatory pathways, which function in a feedback loop driving disease progression and cardiovascular outcomes. We propose that novel approaches targeting simultaneously VC and inflammation might represent valuable new prognostic tools and targets for therapeutics and management of cardiovascular risk in the CKD population.Portuguese Foundation for Science and Technology (UID/Multi/04326/2019); Portuguese Society of Nephrology (SPN)info:eu-repo/semantics/publishedVersio

Structure et expression du proto-oncogene c-ets-1 (poulet)

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Mécanismes moléculaires du recrutement de l'hétérodimère FOS/JUN par la protéine ERG, membre des facteurs de transcription de la famille ETS

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Les protéines ERG, membres de la famille ETS (étude de la coopération fonctionnelle avec le complexe AP1 et recherche de gènes cibles)

Les protéines Ets appartiennent à une vaste famille de facteurs de transcription, qui comprend à ce jour plus d'une trentaine de membres impliqués dans de nombreux processus physiologiques, comme la différenciation et la prolifération cellulaire, ainsi que dans les phénomènes de cancérisation. Au sein de cette famille, nous nous sommes particulièrement intéressés aux protéines Erg (Ets Related Gene) afin d'étudier leurs spécificités fonctionnelles. Nous savons que celles-ci, faibles transactivateurs, sont capables d'établir une coopération fonctionnelle avec l'hétérodimère Jun/Fos. Cette synergie nécessite la formation d'un complexe quaternaire Erg/Jun/Fos/ADN impliquant une interaction physique entre le domaine de liaison à l'ADN des protéines Erg et le domaine basique de Jun. Dans ce contexte, nous avons visualisé dans les cellules vivantes par les techniques de pbFRET (photobleaching Fluorescence Resonance Energy Transfer) et de FLIM (Fluorescence Lifetime Imaging Microscopy) l'interaction physique entre Erg et Jun et ainsi confirmé le rôle essentiel de la tyrosine 371 de la protéine Erg dans cette interaction. Cette étude nous a également permis de mettre en évidence la localisation nucléaire stricte des protéines Erg et de montrer que leur co-expression avec les protéines Jun n'entraîne pas de délocalisation de l'un ou l'autre de ces facteurs transcriptionnels. Dans la deuxième partie de notre travail, nous avons élargi notre étude à la protéine de fusion EWS-Erg, impliquée dans les sarcomes d'Ewing. Cette protéine oncogénique résulte, suite à la translocation chromosomique t(21 ; 22), de la fusion de la partie N-terminale de la protéine EWS avec la région C-terminale de la protéine Erg. Nous avons montré que cette protéine est capable de transactiver et de fonctionner en synergie avec le dimère Jun/Fos sur l'enhancer du virus du Polyome mais elle semble cependant avoir une activité répressive sur le promoteur de la collagénase1. Ainsi, la protéine EWS-Erg est susceptible de réguler différemment les gènes cibles de Erg, ce qui pourrait en partie expliquer ses propriétés oncogéniques. Ces résultats nous ont amenés à rechercher, par une méthode différentielle, les gènes cibles des protéines Erg et en particulier, à discriminer ceux régulés ou non en synergie avec le complexe AP1. Un gène cible putatif, le gène de la fibronectine1 codant une glycoprotéine de la matrice extracellulaire, s'avère être activé par la protéine Erg. L'ensemble de ce travail nous a permis de mieux comprendre les mécanismes moléculaires avec lesquels les protéines Erg exercent leurs fonctions physiologiques. Ces dernières ne sont pas connues précisément à l'heure actuelle mais la recherche des gènes cibles de Erg permettra certainement de les approfondir.The Ets proteins belong to a large family of transcription factors, which to date includes more than thirty members involved in many physiological processes, such as differentiation, cellular proliferation, and cancerisation mechanisms. Within this family, we have a particular interest in the functional specificities of the Erg (Ets Related Gene) proteins. We know that these weak transcriptional activators are able to establish a functional cooperation with the Jun/Fos heterodimer and that this synergy requires the formation of an Erg/Jun/Fos/DNA quaternary complex, therefore implying a physical interaction between the DNA binding domain of the Erg proteins and the basic domain of Jun. In this context, we used pbFRET (photobleaching Fluorescence Resonance Energy Transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy) analyses, and we were able to visualize the physical interaction between Erg and Jun in living cells and to confirm the essential role of tyrosine 371 of the Erg protein in this interaction. This study also allowed us to observe the strict nuclear localisation of the Erg proteins and to show that their co expression with the Jun proteins does not involve delocalisation of one or the other of these transcriptional factors. In the second part of our work, we extended our study to the EWS-Erg fusion protein involved in Ewing's sarcomas. This oncogenic protein results of the fusion of the N-terminal part of the EWS protein with the C-terminal part of the Erg protein further to the t(21; 22) chromosomal translocation. We showed that this protein is able to transactivate and to function in synergy with the Jun/Fos dimer on the Polyomavirus enhancer but it seems to have a repressive activity on the collagenase1 promoter. Thus, the EWS-Erg protein might differently control the target genes of Erg, which could partly explain its oncogenic properties. These results led us to seek, by a differential method, the target genes of the Erg proteins and in particular, to discriminate those controlled or not in synergy with the AP1 complex. A putative target gene, the fibronectin1 gene coding a glycoprotein of the extracellular matrix, proved to be activated by the Erg protein. The whole of this work enabled to better understand the molecular mechanisms with which the Erg proteins exert their physiological functions. Although the latter are not precisely known at the present time, the research of the Erg target genes will certainly allow deepening them in the near future.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Vers la compréhension de la formation et du vieillissement du cartilage à partir de deux modèles (souris transgéniques et remplacement trachéal)

La première partie de mon travail de thèse concerne le gène Erg, appartenant à la famille des gènes Ets, et codant des facteurs de transcription impliqués dans de nombreux processus physiologiques. L'expression du gène Erg, précoce et transitoire chez l'embryon, est associée au mésenchyme. Erg s'exprime dès les premières étapes de mise en place du cartilage chez l'embryon puis se restreint au cartilage articulaire. Afin d'étudier le rôle du gène Erg in vivo, nous avons établi des lignées de souris transgéniques qui expriment une protéine Erg tronquée à effet transdominant négatif sur les protéines endogènes. Cette expression est ciblée au niveau de la mise en place du cartilage par l'utilisation du promoteur du gène du collagèneII 1. Les phénotypes des souris transgéniques obtenues s'apparentent à un vieillissement précoce du squelette. Comparativement aux souris de type sauvage au même âge, elles développent, dans les six premiers mois de leur vie, une hyperlordose et un blocage articulaire. Des expériences de radiologie et de scintigraphie révèlent une perte générale de densité osseuse. Un suivi histologique de l'apparition du phénotype a été effectué à partir de coupes à différents stades du développement. Parallèlement, des cultures de chondrocytes embryonnaires ont été réalisées. Dans une seconde partie, nous avons collaboré à la mise en place d'un modèle de remplacement trachéal. Lorsqu'il existe une indication tymorale ou d'origine malformative, de nombreux modèles de greffe trachéale ont été rapportés dans la littérature sans résultats probants. Une solution originale a récemment été mise au point sur un modèle animal, la brebis, pour palier aux résections trachéales étendues : il s'agit d'allogreffes aortiques. Plus de 50 brebis ont été opérées avec cette technique et les résultats sont très satisfaisants. Quelques mois après la greffe, on constate une modification de la structure du greffon aortique caractérisée par une transformation tissulaire en trachée " néo-induite ". Cette étude a également été effectuée sur un autre modèle animal, le mini-porc, afin de pouvoir confirmer les premiers résultats obtenus, et valider la technique opératoire, même si nous avons été confrontés à des difficultés d'ordre anatomiques. Nous avons effectué des analyses histologiques et de suivi d'expression de gènes par hybridation in situ sur les anneaux de cartilage " néo-formés ". Nous avons également souhiaté comprendre les mécanismes cellulaires sous-jacents à cette transformation, et réalisé des allogrefes de béliers sur des brebis ou de mini-porcs femelles sur des mâles, afin d'utiliser le chromosome Y comme marqueur de l'origine cellulaire. Nous avons ainsi recherhcé la présence du gène SRY dans les greffons par PCR et mis en évidence l'existence d'une colonisation du greffon à partir de la trachée native.The first part of my thesis concerns the Erg gene. Erg belongs to the Ets family encoding a class of transcription factors. In vivo expression of the Erg gene has been previously delineated. Throughout the murine embryogenesis, the Erg gene expression is precocious and transient, and predominates in mesodermal tissues including pre-cartilaginous. Erg expression in the cartilage is restricted to the early stages of differentiation. We performed further analyses to study the function of the Erg gene in vivo. We employed transgenic mice approach to induce an over-expression of the Erg protein in a model over-expressing a dominant-negative protein restricted to the ETS domain. The transgene construct was prepared under the control of a mouse colII 1 specific promoter. We were able to identify clinical abnormalities by comparison with age-matched littermates. The mice over-expressing the dominant-negative protein gradually developed early-ageing associated phenotypes including hyperlordosis, hyperkyphosis and reduced mobility. Skeletal x-rays and osteodensitometry confirmed the clinical findings, showing osteopenia and arthritis. Histological studies at different stages were also performed. In parallel, we analysed embryonic chondrocytes cultures. In the second part, we present a collaborative work on a tracheal replacement model. When a tumour or a malformation occurs, all kinds of attempts with different tracheal substitutes have been performed and reported in the literature without success. An original solution has recently been proposed on a sheep model to allow large tracheal replacement with aortic graft. More than fifty sheep were operated with successful results. Histological examinations showed a progressive transformation of the aortic graft into a tissue with tracheal epithelium and newly formed rings of cartilage. These preliminary results were confirmed on a second animal model, the mini-pig, despite unexpected surgical difficulties. We have performed histological studies and followed genes expression by in situ hybridisation on the newly formed cartilage rings. To allow a better understanding of this cellular transformation, we have performed aortic allograft from male sheep to female sheep trachea, and from female mini-pigs to male mini-pigs. Indeed, we have used a PCR technique of the SRY gene to analyse the origin of the cellular transformation. We have searched the SRY gene amplification in the grafts, and suggested that the underlying mechanism could be a colonisation of the graft by the native trachea.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocPARIS-Académie Médecine (751065201) / SudocSudocFranceF