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

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

Quantitative analysis of ERG expression and its splice isoforms in formalin-fixed, paraffin-embedded prostate cancer samples: Association with seminal vesicle invasion and biochemical recurrence

© American Society for Clinical Pathology. Objectives: The proto-oncogene ETS-related gene (ERG) is consistently overexpressed in prostate cancer. Alternatively spliced isoforms of ERG have variable biological activities; inclusion of exon 11 (72 base pairs [bp]) is associated with aggressiveness and progression of disease. Exon 10 (81 bp) has also been shown to be alternatively spliced. Within this study, we assess whether ERG protein, messenger RNA (mRNA), and ERG splice isoform mRNA expression is altered as prostate cancer progresses. Methods: Detection of the TMPRSS2-ERG fusion was done using direct methods (reverse transcription polymerase chain reaction [PCR] and fluorescence in situ hybridization) and indirect methods for ERG mRNA and protein expression using quantitative PCR and immunohistochemistry, respectively. A linear equation method was used to quantitatively determine relative proportions of ERG variants (ERG72/Δ72, ERG81/Δ81) for each sample. Results: ERG mRNA and protein expression is increased in patients with advanced prostate cancer, with higher levels of ERG expression significantly associated with seminal vesicle invasion (stage pT3b) and biochemical recurrence. Genes involved in cell migration and invasiveness (matrix metalloproteinase 7, osteopontin, and septin 9) are increased in prostate cancers that overexpress ERG. In addition, there is a clear indication of increased retention of exons 10 and 11 in prostate cancer. Conclusions: Analysis of ERG and its variants may be valuable in determining prognosis and development of prostate cancer

Role of splice variants in the metastatic progression of prostate cancer

AS (alternative splicing) and its role in disease, especially cancer, has come to forefront in research over the last few years. Alterations in the ratio of splice variants have been widely observed in cancer. Splice variants of cancer-associated genes have functions that can alter cellular phenotype, ultimately altering metastatic potential. As metastases are the cause of approximately 90% of all human cancer deaths, it is crucial to understand how AS is dysregulated in metastatic disease. We highlight some recent studies into the relationship between altered AS of key genes and the initiation of prostate cancer metastasis. ©The Authors Journal compilation ©2012 Biochemical Society

Sex- and Diet-Specific Changes of Imprinted Gene Expression and DNA Methylation in Mouse Placenta under a High-Fat Diet

Changes in imprinted gene dosage in the placenta may compromise the prenatal control of nutritional resources. Indeed monoallelic behaviour and sensitivity to changes in regional epigenetic state render imprinted genes both vulnerable and adaptable

Implications of the polymorphism of HLA-G on its function, regulation, evolution and disease association

The HLA-G gene displays several peculiarities that are distinct from those of classical HLA class I genes. The unique structure of the HLA-G molecule permits a restricted peptide presentation and allows the modulation of the cells of the immune system. Although polymorphic sites may potentially influence all biological functions of HLA-G, those present at the promoter and 3′ untranslated regions have been particularly studied in experimental and pathological conditions. The relatively low polymorphism observed in the MHC-G coding region both in humans and apes may represent a strong selective pressure for invariance, whereas, in regulatory regions several lines of evidence support the role of balancing selection. Since HLA-G has immunomodulatory properties, the understanding of gene regulation and the role of polymorphic sites on gene function may permit an individualized approach for the future use of HLA-G for therapeutic purposes

A new murine model of osteoblastic/osteolytic lesions from human androgen-resistant prostate cancer

BACKGROUND: Up to 80% of patients dying from prostate carcinoma have developed bone metastases that are incurable. Castration is commonly used to treat prostate cancer. Although the disease initially responds to androgen blockade strategies, it often becomes castration-resistant (CRPC for Castration Resistant Prostate Cancer). Most of the murine models of mixed lesions derived from prostate cancer cells are androgen sensitive. Thus, we established a new model of CRPC (androgen receptor (AR) negative) that causes mixed lesions in bone. METHODS: PC3 and its derived new cell clone PC3c cells were directly injected into the tibiae of SCID male mice. Tumor growth was analyzed by radiography and histology. Direct effects of conditioned medium of both cell lines were tested on osteoclasts, osteoblasts and osteocytes. RESULTS: We found that PC3c cells induced mixed lesions 10 weeks after intratibial injection. In vitro, PC3c conditioned medium was able to stimulate tartrate resistant acid phosphatase (TRAP)-positive osteoclasts. Osteoprotegerin (OPG) and endothelin-1 (ET1) were highly expressed by PC3c while dikkopf-1 (DKK1) expression was decreased. Finally, PC3c highly expressed bone associated markers osteopontin (OPN), Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP) and produced mineralized matrix in vitro in osteogenic conditions. CONCLUSIONS: We have established a new CRPC cell line as a useful system for modeling human metastatic prostate cancer which presents the mixed phenotype of bone metastases that is commonly observed in prostate cancer patients with advanced disease. This model will help to understand androgen-independent mechanisms involved in the progression of prostate cancer in bone and provides a preclinical model for testing the effects of new treatments for bone metastases

Vers la compréhension du fonctionnement des consortia microbiens lignocellulolytiques : contribution des approches méta-omiques et biostatistiques

Lignocellulose, the main component of plant walls and a very abundant agricultural by-product, is one of the most promising renewable carbon sources for the production of energy, chemicals or biofuels. The use of lignocellulose for the production of carboxylates is based on the use of microbial consortia. To improve carboxylate production yields, this thesis work focused on i) characterizing the functioning of consortia selected for their lignocellulolytic potential from different natural ecosystems and ii) understanding their functional changes in the face of modifications in the physicochemical structure and composition of the substrate. For this, microbial ecology approaches and multi-omics tools were applied.Thus, consortia derived from bovine rumen or termite gut have been studied by 16S metabarcoding and label-free metaproteomics approaches. Their enzyme activities and taxonomic structure were evaluated using wheat straw pretreated by mechanical and / or chemical methods. This research has shed light on the role and complementarity of the different populations making up microbial consortia, as well as the effect of the substrate changes induced by the pretreatment on the regulation of the enzymes expression operating during the bioconversion of carbohydrates (CAZymes) and carboxylates. In addition, a consortium derived from bovine rumen and enriched on corn stover residues was also performed. The study of the dynamics of bacterial populations during the enrichment and bioconversion of lignocellulose has made it possible to shed light on the role of free microorganisms and those attached to the substrate, and of their enzymes. This thesis work provides an unprecedented insight of the functioning of lignocellulolytic communities in a bioreactor.La lignocellulose, principal composant des parois végétales et sous-produit agricole très abondant, est une des sources de carbone renouvelable les plus prometteuses pour la production d'énergie, de produits chimiques ou de biocarburants. L’utilisation de la lignocellulose pour la production de carboxylates repose sur l'utilisation de consortia microbiens. Pour améliorer les rendements de production de carboxylates, ces travaux de thèse se sont intéressés à i) caractériser le fonctionnement des consortia sélectionnés pour leur potentiel lignocellulolytique à partir de différents écosystèmes naturels et ii) à mieux comprendre leurs réponses fonctionnelles face à des changements dans la composition et la structure physicochimique du substrat. Pour cela, des approches d’écologie microbienne et des outils multi-omiques ont été appliquées.Ainsi, des consortia dérivés de rumen bovin ou d'intestin de termites ont été étudiés par des approches de métabarcoding 16S et de métaprotéomique comparative label-free. Leurs réponses fonctionnelles, enzymatiques et taxonomiques ont été évaluées en utilisant la paille de blée prétraitée par des méthodes mécaniques et/ou chimiques. Ces recherches ont permis de mettre en lumière le rôle et la complémentarité des différentes populations composant les consortia microbiens, ainsi que l’effet des modifications induites par le prétraitement du substrat sur la régulation de l’expression des enzymes impliquées dans la bioconversion des carbohydrates (CAZymes) en carboxylates. De plus, un consortium issu de rumen bovin et enrichi sur des résidus de maïs a également été étudié. L’étude de la dynamique des populations bactériennes au cours de l’enrichissement et de la bioconversion de la lignocellulose a permis de mettre en lumière le rôle des microorganismes libres et attachées au substrat, et de leurs enzymes, fournissant un aperçu inédit du fonctionnement des communautés lignocellulolytiques en bioréacteur

Vers la compréhension du fonctionnement des consortia microbiens lignocellulolytiques : contribution des approches méta-omiques et biostatistiques

Lignocellulose, the main component of plant walls and a very abundant agricultural by-product, is one of the most promising renewable carbon sources for the production of energy, chemicals or biofuels. The use of lignocellulose for the production of carboxylates is based on the use of microbial consortia. To improve carboxylate production yields, this thesis work focused on i) characterizing the functioning of consortia selected for their lignocellulolytic potential from different natural ecosystems and ii) understanding their functional changes in the face of modifications in the physicochemical structure and composition of the substrate. For this, microbial ecology approaches and multi-omics tools were applied.Thus, consortia derived from bovine rumen or termite gut have been studied by 16S metabarcoding and label-free metaproteomics approaches. Their enzyme activities and taxonomic structure were evaluated using wheat straw pretreated by mechanical and / or chemical methods. This research has shed light on the role and complementarity of the different populations making up microbial consortia, as well as the effect of the substrate changes induced by the pretreatment on the regulation of the enzymes expression operating during the bioconversion of carbohydrates (CAZymes) and carboxylates. In addition, a consortium derived from bovine rumen and enriched on corn stover residues was also performed. The study of the dynamics of bacterial populations during the enrichment and bioconversion of lignocellulose has made it possible to shed light on the role of free microorganisms and those attached to the substrate, and of their enzymes. This thesis work provides an unprecedented insight of the functioning of lignocellulolytic communities in a bioreactor.La lignocellulose, principal composant des parois végétales et sous-produit agricole très abondant, est une des sources de carbone renouvelable les plus prometteuses pour la production d'énergie, de produits chimiques ou de biocarburants. L’utilisation de la lignocellulose pour la production de carboxylates repose sur l'utilisation de consortia microbiens. Pour améliorer les rendements de production de carboxylates, ces travaux de thèse se sont intéressés à i) caractériser le fonctionnement des consortia sélectionnés pour leur potentiel lignocellulolytique à partir de différents écosystèmes naturels et ii) à mieux comprendre leurs réponses fonctionnelles face à des changements dans la composition et la structure physicochimique du substrat. Pour cela, des approches d’écologie microbienne et des outils multi-omiques ont été appliquées.Ainsi, des consortia dérivés de rumen bovin ou d'intestin de termites ont été étudiés par des approches de métabarcoding 16S et de métaprotéomique comparative label-free. Leurs réponses fonctionnelles, enzymatiques et taxonomiques ont été évaluées en utilisant la paille de blée prétraitée par des méthodes mécaniques et/ou chimiques. Ces recherches ont permis de mettre en lumière le rôle et la complémentarité des différentes populations composant les consortia microbiens, ainsi que l’effet des modifications induites par le prétraitement du substrat sur la régulation de l’expression des enzymes impliquées dans la bioconversion des carbohydrates (CAZymes) en carboxylates. De plus, un consortium issu de rumen bovin et enrichi sur des résidus de maïs a également été étudié. L’étude de la dynamique des populations bactériennes au cours de l’enrichissement et de la bioconversion de la lignocellulose a permis de mettre en lumière le rôle des microorganismes libres et attachées au substrat, et de leurs enzymes, fournissant un aperçu inédit du fonctionnement des communautés lignocellulolytiques en bioréacteur

Towards understanding the functioning of lignocellulolytic microbial consortia : contribution of meta-omics approaches and biostatistics

La lignocellulose, principal composant des parois végétales et sous-produit agricole très abondant, est une des sources de carbone renouvelable les plus prometteuses pour la production d'énergie, de produits chimiques ou de biocarburants. L’utilisation de la lignocellulose pour la production de carboxylates repose sur l'utilisation de consortia microbiens. Pour améliorer les rendements de production de carboxylates, ces travaux de thèse se sont intéressés à i) caractériser le fonctionnement des consortia sélectionnés pour leur potentiel lignocellulolytique à partir de différents écosystèmes naturels et ii) à mieux comprendre leurs réponses fonctionnelles face à des changements dans la composition et la structure physicochimique du substrat. Pour cela, des approches d’écologie microbienne et des outils multi-omiques ont été appliquées.Ainsi, des consortia dérivés de rumen bovin ou d'intestin de termites ont été étudiés par des approches de métabarcoding 16S et de métaprotéomique comparative label-free. Leurs réponses fonctionnelles, enzymatiques et taxonomiques ont été évaluées en utilisant la paille de blée prétraitée par des méthodes mécaniques et/ou chimiques. Ces recherches ont permis de mettre en lumière le rôle et la complémentarité des différentes populations composant les consortia microbiens, ainsi que l’effet des modifications induites par le prétraitement du substrat sur la régulation de l’expression des enzymes impliquées dans la bioconversion des carbohydrates (CAZymes) en carboxylates. De plus, un consortium issu de rumen bovin et enrichi sur des résidus de maïs a également été étudié. L’étude de la dynamique des populations bactériennes au cours de l’enrichissement et de la bioconversion de la lignocellulose a permis de mettre en lumière le rôle des microorganismes libres et attachées au substrat, et de leurs enzymes, fournissant un aperçu inédit du fonctionnement des communautés lignocellulolytiques en bioréacteur.Lignocellulose, the main component of plant walls and a very abundant agricultural by-product, is one of the most promising renewable carbon sources for the production of energy, chemicals or biofuels. The use of lignocellulose for the production of carboxylates is based on the use of microbial consortia. To improve carboxylate production yields, this thesis work focused on i) characterizing the functioning of consortia selected for their lignocellulolytic potential from different natural ecosystems and ii) understanding their functional changes in the face of modifications in the physicochemical structure and composition of the substrate. For this, microbial ecology approaches and multi-omics tools were applied.Thus, consortia derived from bovine rumen or termite gut have been studied by 16S metabarcoding and label-free metaproteomics approaches. Their enzyme activities and taxonomic structure were evaluated using wheat straw pretreated by mechanical and / or chemical methods. This research has shed light on the role and complementarity of the different populations making up microbial consortia, as well as the effect of the substrate changes induced by the pretreatment on the regulation of the enzymes expression operating during the bioconversion of carbohydrates (CAZymes) and carboxylates. In addition, a consortium derived from bovine rumen and enriched on corn stover residues was also performed. The study of the dynamics of bacterial populations during the enrichment and bioconversion of lignocellulose has made it possible to shed light on the role of free microorganisms and those attached to the substrate, and of their enzymes. This thesis work provides an unprecedented insight of the functioning of lignocellulolytic communities in a bioreactor