Anne Flourens

Erg proteins, transcription factors of the Ets family, form homo, heterodimers and ternary complexes via two distinct domains.

The ets genes family encodes a group of proteins which function as transcription factors under physiological conditions. We report here that the Erg proteins, members of the Ets family, form homo and heterodimeric complexes in vitro. We demonstrate that the Ergp55 protein isoform forms dimers with itself and with the two other isoforms, Ergp49 and Ergp38. Using a set of Erg protein deletion mutants, we define two distinct domains independently involved in dimerization. The first one is located in the amino-terminal part of the protein containing the pointed domain (PNT), conserved in a subset of Ets proteins. The second one resides within the ETS domain, the DNA-binding domain. We also show that the Erg protein central region behaves as an inhibitory domain of dimerization and its removal enhances the Ergp55 transactivation properties. Furthermore, Ergp55 forms heterodimers with some other Ets proteins. Among the latter, we show that Fli-1, Ets-2, Er81 and Pu-1 physically interact with Erg. Finally, we show that the formation of the previously described ternary complex Ergp55/Fos/jun is mediated by ETS domain and Jun protein, while the ternary complex Ergp49/Fos/Jun is mediated by Fos protein.

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

Prostate 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.

Abnormal expression of the ERG transcription factor in prostate cancer cells activates osteopontin.

Osteopontin (OPN) is an extracellular matrix glycophosphoprotein that plays a key role in the metastasis of a wide variety of cancers. The high level of OPN expression in prostate cells is associated with malignancy and reduced survival of the patient. Recent studies on prostate cancer (PCa) tissue have revealed recurrent genomic rearrangements involving the fusion of the 5′ untranslated region of a prostate-specific androgen-responsive gene with a gene coding for transcription factors from the ETS family. The most frequently identified fusion gene is TMPRSS2:ERG, which causes ERG protein overexpression in PCa cells. ERG is a transcription factor linked to skeletogenesis. This study was designed to test whether ERG and the product of the TMPRSS2:ERG fusion gene modulate OPN gene expression in PCa cells. To characterize ERG and TMPRSS2:ERG transcriptional activity of OPN, we focused on ETS binding sites (EBS) localized in conserved regions of the promoter. Using in vitro and in vivo molecular assays, we showed that ERG increases OPN expression and binds to an EBS (nt −115 to −118) in the OPN promoter. Moreover, stable transfection of prostate tumor cell lines by TMPRSS2:ERG upregulates endogenous OPN expression. Finally, in human prostate tumor samples, detection of the TMPRSS2:ERG fusion gene was significantly associated with OPN overexpression. Taken together, these data suggest that OPN is an ERG-target gene in PCa where the abnormal expression of the transcription factor ERG, due to the TMPRSS2:ERG fusion, disturbs the expression of genes that play an important role in PCa cells and associated metastases. Mol Cancer Res; 9(7); 914–24. ©2011 AACR.

Isolation and characterization of a chicken homologue of the Spi-1/PU.1 transcription factor

Spi-1/PU.1 is a member of the Ets family of transcription factors important in regulation of hematopoiesis. We have isolated a chicken cDNA homologuous to the mammalian Spi-1/PU.1 gene with an open reading frame of 250 amino acids (aa). The chicken Spi-1/PU.1 protein is 14 aa and 16 aa shorter than its human and mouse counterparts but is extremely well conserved with 78.8% and 75.2% identity respectively. The carboxy terminal DNA binding region, or ETS binding domain, is 100% identical to that of human and mouse. Some differences with the mammalian homologues are seen in the N-terminal part of the protein and in the PEST connecting domain. However, the differences are mainly conservative and all the features underlying functional aspects seem preserved. The major discrepancy lies in a 12 aa deletion in an already poorly conserved part of the PEST sequence. Spi-1/PU.1 transcripts were detected at high levels in spleen and Fabricius bursa of chick embryos by Northern blot and in situ hybridization. Our results show that the chicken Spi-1/PU.1 protein behaves like a bonafide Spi-1/PU.1 transcription factor in its DNA binding and transactivating properties.

TMPRSS2-ERG fusion promotes prostate cancer metastases in bone

Bone metastasis is the major deleterious event in prostate cancer (PCa). TMPRSS2-ERG fusion is one of the most common chromosomic rearrangements in PCa. However, its implication in bone metastasis development is still unclear. Since bone metastasis starts with the tropism of cancer cells to bone through specific migratory and invasive processes involving osteomimetic capabilities, it is crucial to better our understanding of the influence of TMPRSS2-ERG expression in the mechanisms underlying the bone tropism properties of PCa cells. We developed bioluminescent cell lines expressing the TMPRSS2-ERG fusion in order to assess its role in tumor growth and bone metastasis appearance in a mouse model. First, we showed that the TMPRSS2-ERG fusion increases cell migration and subcutaneous tumor size. Second, using intracardiac injection experiments in mice, we showed that the expression of TMPRSS2-ERG fusion increases the number of metastases in bone. Moreover, TMPRSS2-ERG affects the pattern of metastatic spread by increasing the incidence of tumors in hind limbs and spine, which are two of the most frequent sites of human PCa metastases. Finally, transcriptome analysis highlighted a series of genes regulated by the fusion and involved in the metastatic process. Altogether, our work indicates that TMPRSS2-ERG increases bone tropism of PCa cells and metastasis development.

Identification of two ets related genes in a marine worm, the polychaete annelid Nereis diversicolor.

The ETS family includes a growing number of transcription factors with a highly conserved DNA‐binding domain, the ETS domain. We have used PCR amplification with degenerated oligonucleotides to isolate two putative ETS DNA‐binding coding domains in a primitive form of cœlomate, the polychaete annelid Nereis diversicolor. These sequences are highly related to the ETS and ERG groups of the ets gene family. For the erg sequence an adjacent region encoding for 91 amino acids has been characterized after library screening, and we show an expression in cells isolated from the cœlomic cavity of the animal. A phylogenic analysis confirms that the ets‐1/ets‐2 and the erg/fli dichotomy arose specifically in the vertebrate lineage.

TMPRSS2:ERG gene fusion expression regulates bone markers and enhances the osteoblastic phenotype of prostate cancer bone metastases

Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells.

Abstract 1691: TMPRSS2:ERG fusion enhances osteoblastic phenotype of prostate cancer bone metastases

Background: Bone metastases are the major cause of morbidity and mortality in prostate cancer (PCa) patients. Recently, TMPRSS2:ERG gene fusions produced by rearrangements along chromosome 21 was found in more than 50% of PCa samples, resulting in androgen-dependent aberrant expression of the functional ERG transcription factor. Interestingly, ERG transcription factor has been previously shown to be involved in bone development. This study is therefore focused on investigating whether TMPRSS2:ERG gene fusions are involved in PCa bone metastasis development. Methods: We previously established cell clones overexpressing TMPRSS2:ERG from two PCa cell lines, PC3 and PC3c. We first studied induced bone lesion phenotype using in vivo intra-tibial injection models. Secondly, we analyzed transcriptional changes induced by ERG transcription factors in PC3c clones to identify potential target genes. Then, direct target genes were further validated and investigated in vitro using RT-qPCR, ELISA, siRNA and ChIP techniques. Importantly, using a cohort of prostate carcinoma samples, we validated the expression correlation between ERG and its target target genes expression in human pathology. Results: Bone lesions induced in vivo by intra-tibial injections of PC3 or PC3c cells are known to be osteolytic or mixed (osteoblastic/osteolytic) respectively. Interestingly, intra-tibial injections of PC3c clones expressing the fusion showed a statistically significant increase of osteoblastic phenotype compared to control cells. Furthermore, intra-tibial injections of PC3 clones expressing the fusion showed a strong decrease of osteolytic phenotype, reinforcing our previous result in PC3c clones. Among the genes identified by transcriptomic study and dysregulated in PC3c clones expressing the fusion, we have identified the ERG candidate target Endothelin-1 (ET-1), which is known to be involved in osteoblast proliferation and in osteoblastic metastasis formation in PCa. Indeed, we found that ET-1 expression was up-regulated in PC3c-TMPRSS2:ERG clones, and the up-regulation was dependent on ERG expression levels. Importantly, silencing of ERG resulted in decreased expression of ET-1. In silico analysis of the promoter of ET-1 revealed the presence of several potential binding sites of ERG. ChIP experiments followed by qPCR demonstrated a direct binding to one of them. Moreover, in human PCa samples, there was a significant expression correlation between ET-1 and fusion gene TMPRSS2:ERG, reinforcing the direct functional link between ET-1 and the fusion in PCa. Conclusion: Taken together, these results strongly suggest that the TMPRSS2:ERG gene fusion contributes to the osteoblastic phenotype of PCa bone metastases and ET-1 is one of the involved factors, directly regulated by the transcription factor ERG. Citation Format: Carine Delliaux, Tian V. Tian, Mathilde Bouchet, Anais Fradet, Nathalie Vanpouille, Anne Flourens, Rachel Deplus, Xavier Leroy, Yvan de Launoit, Edith Bonnelye, Martine Duterque-Coquillaud. TMPRSS2:ERG fusion enhances osteoblastic phenotype of prostate cancer bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1691.