[Show abstract][Hide abstract] ABSTRACT: Protein arginine methylation is a common post translational modification that regulates protein properties. This modification is carried out by a family of nine arginine methyltransferases (PRMTs). Arginine methylation has already been linked to tumourigenesis as overexpression of these enzymes was associated with various cancers, notably in breast cancers. Since the Jumonji Domain Containing 6 protein (JMJD6) possesses an arginine demethylase activity able to remove the methyl mark, we wanted to assess its potential role in breast tumourigenesis.
The expression of the protein by tissue microarray immunohistochemical staining was performed on a cohort of 133 breast tumours. Using cell lines stably overexpressing or knocked down for JMJD6, we evaluated its role on cell proliferation, cell migration, colony formation and mice tumour xenografts.
The analysis of JMJD6 expression in a cohort of breast tumour samples indicates that JMJD6 was highly expressed in aggressive breast tumours. Moreover, high expression of JMJD6 was associated with poor disease-free survival of patients in this cohort. JMJD6 silencing in breast tumoural cells promotes certain characteristics of tumourigenesis including proliferation, migration in vitro, and tumour growth in vivo. These effects are dependent on its demethylase activity as an enzymatic dead mutant lost these properties.
Although JMJD6 displays anti-tumoral properties in cell lines, its expression in breast tumours may be a marker of poor prognosis, suggesting that its function could be altered in breast cancer.
[Show abstract][Hide abstract] ABSTRACT: IntroductionIncreasing evidence indicates micro RNAs (miRNAs) being important players in oncogenesis. Considering the wide-spread use of aromatase inhibitors (AIs) in endocrine therapy as a first-line treatment for post-menopausal endocrine receptor ¿-positive breast cancer patients, identifying deregulated expression levels of miRNAs in association with AI resistance is of utmost importance.Methods
To gain further insight into the molecular mechanisms underlying the AI resistance, we performed miRNA microarray experiments using a new model of acquired resistance to letrozole (Res-Let cells), obtained by long-term exposure of aromatase-overexpressing MCF-7 cells (MCF-7aro cells) to letrozole, and a model of acquired anastrozole resistance (Res-Ana cells). Three miRNAs (miR-125b, miR-205 and miR-424) similarly deregulated in both AI-resistant cell lines were then investigated in terms of their functional role in AI resistance development, breast cancer cell aggressiveness and their clinical relevance using a cohort of 65 primary breast tumor samples.ResultsWe identified the deregulated expression of 33 miRNAs in Res-Let cells and of 18 miRNAs in Res-Ana cells compared to the sensitive MCF-7aro cell line. The top-ranked Kyoto Encyclopedia of Genes and Genomes pathways delineated by both miRNA signatures converged on the AKT pathway, which was found constitutively activated in both AI-resistant cell lines. We report for the first time that ectopic overexpression of either miR-125b or miR-205, or the silencing of miR-424 expression in the sensitive MCF-7aro cell line were sufficient to confer resistance to letrozole and to anastrozole, to target and to activate the AKT/mTOR pathway, and to increase formation capacity of cancer-initiating-like cells possessing self-renewing properties. Increasing miR-125b expression levels was also sufficient to confer estrogen-independent growth properties to the sensitive MCF-7aro cell line. Finally, elevated miR-125b expression levels were a novel marker for poor prognosis in breast cancer, and targeting miR-125b in Res-Let cells overcame letrozole resistance.Conclusion
This study highlights that acquisition of specific deregulated miRNAs is a newly discovered alternative mechanism developed by AI-resistant breast cancer cells to achieve constitutive activation of the AKT/mTOR pathway and develop AI resistance. It also highlights that miR-125b is a new biomarker of poor prognosis and a candidate therapeutic target of AI-resistant breast cancers.
Full-text · Article · Jan 2015 · Breast cancer research: BCR
[Show abstract][Hide abstract] ABSTRACT: The CCR4–NOT complex is a multi-subunit protein complex evolutionarily conserved across eukaryotes which regulates several aspects of gene expression. A fascinating model is emerging in which this complex acts as a regulation platform, controlling gene products ‘from birth to death’ through the coordination of different cellular machineries involved in diverse cellular functions. Recently the CCR4–NOT functions have been extended to the control of the innate immune response through the regulation of interferon signaling. Thus, a more comprehensive picture of how CCR4–NOT allows the rapid adaptation of cells to external stress, from transcription to mRNA and protein decay, is presented and discussed here. Overall, CCR4–NOT permits the efficient and rapid adaptation of cellular gene expression in response to changes in environmental conditions and stimuli.For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.
[Show abstract][Hide abstract] ABSTRACT: In situ proximity ligation assay (PLA) is a powerful method for detection, localization, and quantification of proteins, protein-protein interactions, and posttranslational modifications. Proteins detected by two specific antibodies are recognized by proximity probes conjugated with complementary oligonucleotides to allow the formation of circular DNA probes when bound in close proximity. Subsequent amplification of this DNA can then be visualized. Here, we describe the in situ PLA method for the detection of the ERα/Src/PI3K complex in breast cancer. We used two different techniques for detecting the signals: fluorescent detection for cell line analysis and bright-field revelation, which is better suited to clinical analysis of patient samples.
No preview · Article · Sep 2014 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: We aimed at highlighting the role of ZNF217, a Krüppel-like finger protein, in Estrogen Receptor-α (ERα)-positive (ER+) and luminal breast cancers. Here we report for the first time that ZNF217 and ERα proteins bind to each other in both breast cancer cells and breast tumour samples, via the ERα hinge domain and the ZNF217 C-terminal domain. ZNF217 enhances the recruitment of ERα to its estrogen response elements (ERE) and the ERα-dependent transcription of the GREB1 estrogen-regulated gene. The prognostic power of ZNF217 mRNA expression levels is most discriminatory in breast cancers classified with a "good prognosis", particularly the Luminal-A subclass. A new immunohistochemistry ZNF217 index, based on nuclear and cytoplasmic ZNF217 staining, also allowed the identification of intermediate/poor relapse-free survivors in the Luminal-A subgroup. ZNF217 confers tamoxifen resistance in ER+ breast cancer cells and is a predictor of relapse under endocrine therapy in patients with ER+ breast cancer. ZNF217 thus allows the re-stratification of patients with ER+ breast cancers considered as cancers with good prognosis where no other biomarkers are currently available and widely used. Here we propose a model in ER+ breast cancer where ZNF217-driven aggressiveness incorporates ZNF217 as a positive enhancer of ERα direct genomic activity and where ZNF217 possesses its highest discriminatory prognostic value.
Full-text · Article · Jun 2014 · Molecular Oncology
[Show abstract][Hide abstract] ABSTRACT: ERα functions are tightly controlled by numerous post-translational modifications including arginine methylation, which is required to mediate the extranuclear functions of the receptor. We report that upon oestrogenic stimulation, JMJD6, the only arginine demethylase described so far, interacts with and regulates methylated ERα (metERα) function. Moreover, by combining the silencing of JMJD6 with demethylation assays, we show that metERα is a new substrate for JMJD6. We propose that the demethylase activity of JMJD6 is a decisive regulator of the rapid physiological responses to oestrogen.
[Show abstract][Hide abstract] ABSTRACT: Stringent regulation of the interferon (IFN) signalling pathway is essential for maintaining the immune response to pathogens and tumours. The transcription factor STAT1 is a crucial mediator of this response. Here, we show that hCAF1/CNOT7 regulates class I and II IFN pathways at different crucial steps. In resting cells, hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistently, hCAF1 silencing enhances STAT1 basal promoter occupancy associated with increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradation of some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activation is associated with immune disorders and cancer, hCAF1 could play a major role in innate immunity and oncogenesis, contributing to tumour escape.
[Show abstract][Hide abstract] ABSTRACT: Estrogen receptors (ERs) are ligand-activated transcription factors involved in many physiological and pathological processes, including breast cancer. Their activity is fine-tuned by posttranslational modifications, notably sumoylation. In the present study, we investigated the role of the small ubiquitin-related modifier (SUMO) protease, SUMO1/sentrin/suppressor of Mif 2-specific peptidase 2 (SENP2), in the regulation of ERα activity. We first found SENP2 to significantly repress estradiol-induced transcriptional activity in breast cancer cells (MCF7 and T47D). This effect was observed with a reporter plasmid and on endogenous genes such as TFF1 and CTSD, which were shown to recruit SENP2 in chromatin immunoprecipitation experiments. Using glutathione-S-transferase pull-down, coimmunoprecipitation and proximity ligation assays, SENP2 was found to interact with ERα and this interaction to be mediated by the amino-terminal region of the protease and the hinge region of the receptor. Interestingly, we demonstrated that ERα repression by SENP2 is independent of its SUMO protease activity and requires a transcriptional repressive domain located in the amino-terminal end of the protease. Using small interfering RNA assays, we evidenced that this domain recruits the histone deacetylase, histone deacetylase-3, to be fully active. Furthermore, using both overexpression and knockdown strategies, we showed that SENP2 robustly represses estrogen-dependent and independent proliferation of MCF7 cells. We provided evidence that this effect requires both the proteolytic and transcriptional activities of SENP2. Altogether, our study unravels a new property for a SUMO protease and identifies SENP2 as a classical transcription coregulator.
Preview · Article · Jan 2013 · Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ERα is required for the formation of the ERα/Src/PI3K complex and that ERα is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumours. Of particular interest, the analysis of 175 breast tumours showed that overexpression of this complex in a subset of breast tumours correlates to the activation of the downstream effector Akt. Survival analysis revealed that high expression of this complex is an independent marker of poor prognosis and associated with reduced disease-free survival. Our data introduces the new concept that the rapid oestrogen pathway is operative in vivo. It also provides a rationale for patient stratification defined by the activation of this pathway and the identification of target therapies.
Full-text · Article · Nov 2012 · EMBO Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: The Krüppel-like zinc finger protein ZNF217 is a candidate oncogene in breast cancer. In this study, we showed that high levels of expression of ZNF217 mRNA are associated with poor prognosis and the development of metastases in breast cancer. Overexpression of ZNF217 in breast cancer cells stimulated migration and invasion in vitro and promoted the development of spontaneous lung or node metastases in mice in vivo. ZNF217 also promoted epithelial-mesenchymal transition (EMT) in human mammary epithelial cells, and the TGF-β-activated Smad signaling pathway was identified as a major driver of ZNF217-induced EMT. In addition, a TGF-β autocrine loop sustained activation of the TGF-β pathway in ZNF217-overexpressing mammary epithelial cells, most likely because of ZNF217-mediated direct upregulation of TGFB2 or TGFB3. Inhibition of the TGF-β pathway led to the reversal of ZNF217-mediated EMT. Together, our findings indicate that ZNF217 mRNA expression may represent a novel prognostic biomarker in breast cancer. Therapeutic targeting of ZNF217 of the TGF-β signaling pathway may benefit the subset of patients whose tumors express high levels of ZNF217.
[Show abstract][Hide abstract] ABSTRACT: Estrogen signaling pathways, because of their central role in regulating the growth and survival of breast tumor cells, have been identified as suitable and efficient targets for cancer therapies. Agents blocking estrogen activity are already widely used clinically, and many new molecules have entered clinical trials, but intrinsic or acquired resistance to treatment limits their efficacy. The basic molecular studies underlying estrogen signaling have defined the critical role of estrogen receptors (ER) in many aspects of breast tumorigenesis. However, important knowledge gaps remain about the role of posttranslational modifications (PTM) of ER in initiation and progression of breast carcinogenesis. Whereas major attention has been focused on the phosphorylation of ER, many other PTM (such as acetylation, ubiquitination, sumoylation, methylation, and palmitoylation) have been identified as events modifying ER expression and stability, subcellular localization, and sensitivity to hormonal response. This article will provide an overview of the current and emerging knowledge on ER PTM, with a particular focus on their deregulation in breast cancer. We also discuss their clinical relevance and the functional relationship between PTM. A thorough understanding of the complete picture of these modifications in ER carcinogenesis might not only open new avenues for identifying new markers for prognosis or prediction of response to endocrine therapy but also could promote the development of novel therapeutic strategies.
Full-text · Article · Jun 2011 · Endocrine reviews
[Show abstract][Hide abstract] ABSTRACT: Regulation of the proteome by post-translational modifications (PTM) emerges as a major contributing factor to the functional diversity in biology regulating cellular processes. Because PTM are key to the physiologic functions of the proteins involved, it is imperative that we understand the < coding > that these modifications impart to regulate diverse activities. As estrogen signalling mediates a plethora of PTM not only on the receptors themselves but also on their coregulators, we investigate to < crack > the ER code. Besides the long-known phosphorylation, other covalent additions such as acetylation, ubiquitination, sumoylation and methylation have been described for estrogen receptors in recent years. These modifications affect receptor stability and activity, and provide potential mechanisms for cell- or-gene-specific regulation. A better understanding of the impact of these PTMs on estrogen receptor should help in the identification of new drugs for breast cancer treatments.
No preview · Article · Jan 2011 · Medecine sciences: M/S
[Show abstract][Hide abstract] ABSTRACT: Estrogen receptor alpha (ERalpha) is a member of a large conserved superfamily of steroid hormone nuclear receptors which regulates many physiological pathways by acting as a ligand-dependent transcription factor. Evidence is emerging that estrogens also induce rapid signaling to the downstream kinase cascades; however the mechanisms underlying this nongenomic function remain poorly understood. We have recently shown that ERalpha is methylated specifically by the arginine methyltransferase PRMT1 at arginine 260 in the DNA-binding domain of the receptor. This methylation event is required for mediating the extra-nuclear function of the receptor which would thereby interact with Src/FAK and p85 and propagate the signal to downstream transduction cascades that orchestrate cell proliferation and survival. Of particular interest, a possible role of methylated ERalpha in mammary tumorigenesis is also evident by the fact that, as demonstrated by immunohistochemical studies on a cohort of breast cancer patients, ERalpha is methylated in normal epithelial breast cells and is hypermethylated in a subset of breast cancers. Hypermethylation of ERalpha in breast cancer might cause hyperactivation of cellular kinase signaling, notably of Akt, described as a selective survival advantage for primary tumor cells even in the presence of anti-estrogens. A detailed understanding of the molecular mechanisms that control estrogen signaling in breast cancer is a crucial step in identifying new effective therapies.