PRMT1, a Key Modulator of Unliganded Progesterone Receptor Signaling in Breast Cancer.

The progesterone receptor (PR) is a key player in major physiological and pathological responses in women, and the signaling pathways triggered following hormone binding have been extensively studied, particularly with respect to breast cancer development and progression. Interestingly, growing evidence suggests a fundamental role for PR on breast cancer cell homeostasis in hormone-depleted conditions, with hormone-free or unliganded PR (uPR) involved in the silencing of relevant genes prior to hormonal stimulation. We herein identify the protein arginine methyltransferase PRMT1 as a novel actor in uPR signaling. In unstimulated T47D breast cancer cells, PRMT1 interacts and functions alongside uPR and its partners to target endogenous progesterone-responsive promoters. PRMT1 helps to finely tune the silencing of responsive genes, likely by promoting a proper BRCA1-mediated degradation and turnover of unliganded PR. As such, PRMT1 emerges as a key transcriptional coregulator of PR for a subset of relevant progestin-dependent genes before hormonal treatment. Since women experience periods of hormonal fluctuation throughout their lifetime, understanding how steroid receptor pathways in breast cancer cells are regulated when hormones decline may help to determine how to override treatment failure to hormonal therapy and improve patient outcome.

Reduced menin expression leads to decreased ERα expression and is correlated with the occurrence of human luminal B-like and ER-negative breast cancer subtypes.

PURPOSE: Menin, encoded by the MEN1 gene, was recently reported to be involved in breast cancers, though the underlying mechanisms remain elusive. In the current study, we sought to further determine its role in mammary cells. METHODS: Menin expression in mammary lesions from mammary-specific Men1 mutant mice was detected using immunofluorescence staining. RT-qPCR and western blot were performed to determine the role of menin in ERα expression in human breast cancer cell lines. ChIP-qPCR and reporter gene assays were carried out to dissect the action of menin on the proximal ESR1 promoter. Menin expression in female patients with breast cancer was analyzed and its correlation with breast cancer subtypes was investigated. RESULTS: Immunofluorescence staining revealed that early mammary neoplasia in Men1 mutant mice displayed weak ERα expression. Furthermore, MEN1 silencing led to both reduced ESR1 mRNA and ERα protein expression in MCF7 and T47D cells. To further dissect the regulation of ESR1 transcription by menin, we examined whether and in which way menin could regulate the proximal ESR1 promoter, which has not been fully explored. Using ChIP analysis and reporter gene assays covering - 2500 bp to + 2000 bp of the TSS position, we showed that the activity of the proximal ESR1 promoter was markedly reduced upon menin downregulation independently of H3K4me3 status. Importantly, by analyzing the expression of menin in 354 human breast cancers, we found that a lower expression was associated with ER-negative breast cancer (P = 0.041). Moreover, among the 294 ER-positive breast cancer samples, reduced menin expression was not only associated with larger tumors (P = 0.01) and higher SBR grades (P = 0.005) but also with the luminal B-like breast cancer subtype (P = 0.006). Consistent with our clinical data, we demonstrated that GATA3 and FOXA1, co-factors in ESR1 regulation, interact physically with menin in MCF7 cells, and MEN1 knockdown led to altered protein expression of GATA3, the latter being a known marker of the luminal A subtype, in MCF7 cells. CONCLUSION: Taken together, our data provide clues to the important role of menin in ERα regulation and the formation of breast cancer subtypes.

LKB1 regulates PRMT5 activity in breast cancer.

Protein arginine methyltransferase 5 (PRMT5) is the main enzyme responsible for the symmetrical dimethylation of arginine residues on target proteins in both the cytoplasm and the nucleus. Though its activity has been associated with tumor progression in various cancers, the expression pattern of this oncoprotein has been scarcely studied in breast cancer. In the current work, we analyzed its expression in a large cohort of breast cancer patients, revealing higher nuclear PRMT5 levels in ERα-positive tumors and an association with prolonged disease free and overall survival. Interestingly, high PRMT5 nuclear expression was also associated with higher nuclear liver kinase B1 (LKB1), suggesting that a functional relationship may occur. Consistently, several approaches provided evidence that PRMT5 and LKB1 interact directly in the cytoplasm of mammary epithelial cells. Moreover, although PRMT5 is not able to methylate LKB1, we found that PRMT5 is a bona fade substrate for LKB1. We identified T132, 139 and 144 residues, located in the TIM-Barrel domain of PRMT5, as target sites for LKB1 phosphorylation. The point mutation of PRMT5 T139/144 to A139/144 drastically decreased its methyltransferase activity, due probably to the loss of its interaction with regulatory proteins such as MEP50, pICln and RiOK1. In addition, modulation of LKB1 expression modified PRMT5 activity, highlighting a new regulatory mechanism that could have clinical implications.

Alternative splicing of CNOT7 diversifies CCR4-NOT functions.

The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions.

hCAF1/CNOT7 regulates interferon signalling by targeting STAT1.

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.

Regulation of estrogen rapid signaling through arginine methylation by PRMT1.

Evidence is emerging that estrogen receptor alpha (ERalpha) is central to the rapid transduction of estrogen signaling to the downstream kinase cascades; however, the mechanisms underlying this nongenomic function are not fully understood. Here we report a paradigm of ERalpha regulation through arginine methylation by PRMT1, which transiently methylates arginine 260 within the ERalpha DNA-binding domain. This methylation event is required for mediating the extranuclear function of the receptor by triggering its interaction with the p85 subunit of PI3K and Src. Furthermore, we find that the focal adhesion kinase (FAK), a Src substrate involved in the migration process, is also recruited in this complex. Our data indicate that the methylation of ERalpha is a physiological process occurring in the cytoplasm of normal and malignant epithelial breast cells and that ERalpha is hypermethylated in a subset of breast cancers.

MicroRNA-125b upregulation confers aromatase inhibitor resistance and is a novel marker of poor prognosis in breast cancer.

INTRODUCTION: Increasing evidence indicates that microRNAs (miRNAs) are important players in oncogenesis. Considering the widespread use of aromatase inhibitors (AIs) in endocrine therapy as a first-line treatment for postmenopausal estrogen receptor α-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 and breast cancer cell aggressiveness and their clinical relevance using a cohort of 65 primary breast tumor samples. RESULTS: We identified the deregulated expression of 33 miRNAs in Res-Let cells and of 18 miRNAs in Res-Ana cells compared with 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/mTOR pathway, which was found to be constitutively activated in both AI-resistant cell lines. We report for the first time, to our knowledge, that ectopic overexpression of either miR-125b or miR-205, or the silencing of miR-424 expression, in the sensitive MCF-7aro cell line was sufficient to confer resistance to letrozole and anastrozole, to target and activate the AKT/mTOR pathway and to increase the formation capacity of stem-like and tumor-initiating 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. We also found that elevated miR-125b expression levels were a novel marker for poor prognosis in breast cancer and that 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 to develop AI resistance. It also highlights that miR-125b is a new biomarker of poor prognosis and a candidate therapeutic target in AI-resistant breast cancers.

LKB1 when associated with methylatedERα is a marker of bad prognosis in breast cancer.

Although the presence of nuclear estrogen receptor is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signaling. Recently, we have shown that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy. Here, we identified LKB1 as a partner of ERα and we explored its potential role in estrogen nongenomic signaling. The associations between LKB1 expression and the actors of this pathway, namely the methylated form of ERα (metERα), Src and PI3K, have been analyzed both in cultured cells and in 154 primary breast tumor samples. We found that LKB1 is a component of the cytoplasmic signaling complex in breast cell lines as well as in primary breast tumors. Moreover, an inverse correlation between the localization of LKB1 in nuclear and cytoplasmic compartments is observed. Importantly, high expression of cytoplasmic LKB1 is an independent marker of poor prognosis, associated with reduced overall survival (OS) and disease free survival (DFS). Conversely, the presence of nuclear LKB1 associates with increased OS and DFS. In conclusion, our results highlight that LKB1 expression in breast cancer appears to have opposite effects depending on its subcellular localization and may be used as a new prognostic biomarker.

Molecular characterization of anastrozole resistance in breast cancer: pivotal role of the Akt/mTOR pathway in the emergence of de novo or acquired resistance and importance of combining the allosteric Akt inhibitor MK-2206 with an aromatase inhibitor.

Acquisition of resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of estrogen receptor alpha (ERα)-positive breast cancers. The Res-Ana cells, a new model of acquired resistance to anastrozole, were established by long-term exposure of aromatase-overexpressing MCF-7 cells to this drug. These resistant cells developed ER-independent mechanisms of resistance and decreased sensitivity to the AI letrozole or to ERα antagonists. They also displayed a constitutive activation of the PI3K/Akt/mTOR pathway and a deregulated expression of several ErbB receptors. An observed increase in the phospho-Akt/Akt ratio between primary and matched recurrent breast tumors of patients who relapsed under anastrozole adjuvant therapy also argued for a pivotal role of the Akt pathway in acquired resistance to anastrozole. Ectopic overexpression of constitutively active Akt1 in control cells was sufficient to induce de novo resistance to anastrozole. Strikingly, combining anastrozole with the highly selective and allosteric Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings lead to us proposing a model of anastrozole-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to anastrozole and sensitivity to MK-2206. Altogether, our work demonstrated that the Akt/mTOR pathway plays a key role in resistance to anastrozole and that combining anastrozole with Akt/mTOR pathway inhibitors represents a promising strategy in the clinical management of hormone-dependent breast cancer patients.

[Post-translational modifications modulate estrogen receptor alpha activity in breast tumors]. / Les modifications post-traductionnelles orchestrent l'action du récepteur des oestrogènes ERalpha dans les tumeurs mammaires.

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.

PRMT1 Is Critical for the Transcriptional Activity and the Stability of the Progesterone Receptor.

The progesterone receptor (PR) is an inducible transcription factor that plays critical roles in female reproductive processes and in several aspects of breast cancer tumorigenesis. Our report describes the type I protein arginine methyltransferase 1 (PRMT1) as a cofactor controlling progesterone pathway, through the direct methylation of PR. Mechanistic assays in breast cancer cells indicate that PRMT1 methylates PR at the arginine 637 and reduces the stability of the receptor, thereby accelerating its recycling and finally its transcriptional activity. Depletion of PRMT1 decreases the expression of a subset of progesterone-inducible genes, controlling breast cancer cells proliferation and migration. Consistently, Kaplan-Meier analysis revealed that low expression of PRMT1 predicts a longer survival among the subgroup with high PR. Our study highlights PR methylation as a molecular switch adapting the transcription requirement of breast cells during tumorigenesis.

The arginine methyltransferase PRMT1 regulates IGF-1 signaling in breast cancer.

Aside from its well-known nuclear routes of signaling, estrogen also mediates its effects through cytoplasmic signaling. Estrogen signaling involves numerous posttranslational modifications of its receptor ERα, the best known being phosphorylation. Our research group previously showed that upon estrogen stimulation, ERα is methylated on residue R260 and forms the mERα/Src/PI3K complex, central to the rapid transduction of nongenomic estrogen signals. Regulation of ERα signaling via its phosphorylation by growth factors is well recognized, and we wondered whether they could also trigger ERα methylation (mERα). Here, we found that IGF-1 treatment of MCF-7 cells induced rapid ERα methylation by the arginine methyltransferase PRMT1 and triggered the binding of mERα to IGF-1R. Mechanistically, we showed that PRMT1 bound constitutively to IGF-1R and that PRMT1 became activated upon IGF-1 stimulation. Moreover, we found that expression or pharmacological inhibition of PRMT1 impaired mERα and IGF-1 signaling. Our findings were substantiated in a cohort of breast tumors in which IGF-1R expression was positively correlated with ERα/Src and ERα/PI3K expression, hallmarks of nongenomic estrogen signaling, reinforcing the link between IGF-1R and mERα. Altogether, these results provide a new insight into ERα and IGF-1R interference, and open novel perspectives for combining endocrine therapies with PRMT1 inhibitors in ERα-positive tumors.

Identification of NF-kappaB responsive elements in follistatin related gene (FLRG) promoter.

Follistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP/follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFbeta) superfamily such as activin A and myostatin. We have previously shown that TGFbeta and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFalpha) could regulate FLRG expression since TNFalpha plays a critical role in hematopoietic malignancies. We demonstrate that TNFalpha activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats, which are evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not common among non-coding sequences. Each DNA repeat contains one TNFalpha responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-kappaB) transcription factors. We also show that TGFbeta, through the Smad proteins, potentates the effect of TNFalpha on FLRG expression. This cooperation is unexpected since TGFbeta and TNFalpha usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis.

Sumoylation of the estrogen receptor alpha hinge region regulates its transcriptional activity.

The steroid hormone 17beta-estradiol (estrogen) plays a significant role in the normal physiology of the mammary gland and breast cancer development primarily through binding to its receptor, the estrogen receptor alpha (ERalpha). ERalpha is a nuclear transcription factor undergoing different types of posttranslational modifications, i.e. phosphorylation, acetylation, and ubiquitination, which regulate its transcriptional activation and/or stability. Here we identify ERalpha as a new target for small ubiquitin-like modifier (SUMO)-1 modification in intact cells and in vitro. Moreover, ERalpha sumoylation occurs strictly in the presence of hormone. SUMO-1 appears to regulate ERalpha-dependent transcription. Using a series of mutants, we demonstrated that ERalpha is sumoylated at conserved lysine residues within the hinge region. Mutations that prevented SUMO modification impaired ERalpha-induced transcription without influencing ERalpha cellular localization. In addition to identifying protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIAS3 as E3 ligases for ERalpha, we also found that PIAS1 and PIAS3, as well as Ubc9, modulated ERalpha-dependent transcription independently from their SUMO-1 conjugation activity. These findings identify sumoylation as a new mechanism modulating ERalpha-dependent cellular response and provide a link between the SUMO and estrogen pathways.

Protein arginine methylation/demethylation and cancer.

Protein arginine methylation is a common post-translational modification involved in numerous cellular processes including transcription, DNA repair, mRNA splicing and signal transduction. Currently, there are nine known members of the protein arginine methyltransferase (PRMT) family, but only one arginine demethylase has been identified, namely the Jumonji domain-containing 6 (JMJD6). Although its demethylase activity was initially challenged, its dual activity as an arginine demethylase and a lysine hydroxylase is now recognized. Interestingly, a growing number of substrates for arginine methylation and demethylation play key roles in tumorigenesis. Though alterations in the sequence of these enzymes have not been identified in cancer, their overexpression is associated with various cancers, suggesting that they could constitute targets for therapeutic strategies. In this review, we present the recent knowledge of the involvement of PRMTs and JMJD6 in tumorigenesis.

Transcription activation of FLRG and follistatin by activin A, through Smad proteins, participates in a negative feedback loop to modulate activin A function.

Signaling of TGFbeta family members such as activin is tightly regulated by soluble binding proteins. Follistatin binds to activin A with high affinity, and prevents activin binding to its own receptors, thereby blocking its signaling. We previously identified FLRG gene from a B-cell leukemia carrying a t(11;19)(q13;p13) translocation. We and others have already shown that FLRG, which is highly homologous to follistatin, may be involved in the regulation of the activin function through its binding to activin. In this study, we found that, like follistatin, FLRG protein inhibited activin A signaling as demonstrated by the use of a transcriptional reporter assay, and blocked the activin A-induced growth inhibition of HepG2 cells. We have recently shown that the TGFbeta-induced expression of FLRG occurs at a transcriptional level through the action of Smad proteins. Here we show that activin A increases FLRG and follistatin at both the mRNA and protein levels. We found that Smad proteins are involved in the activin A-induced transcription activation of FLRG and follistatin. Finally we demonstrate that FLRG protein regulates its own activin-induced expression. In conclusion, activin A induces FLRG and follistatin expression. This observation, in conjunction with the antagonistic effect of FLRG and follistatin on activin signaling, indicates that these two proteins participate in a negative feedback loop which regulates the activin function.

Role of JMJD6 in Breast Tumourigenesis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Novel roles of the CCR4-NOT complex.

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.

JMJD6 regulates ERα methylation on arginine.

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.

Proximity ligation assay to detect and localize the interactions of ERα with PI3-K and Src in breast cancer cells and tumor samples.

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.