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1.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076907

RESUMO

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.


Assuntos
Neoplasias da Mama , Proteína-Arginina N-Metiltransferases , Receptores de Progesterona , Neoplasias da Mama/metabolismo , Feminino , Humanos , Progesterona/metabolismo , Progestinas , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia
2.
Breast Cancer Res Treat ; 190(3): 389-401, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34561764

RESUMO

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.


Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Animais , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito , Humanos , Células MCF-7 , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética
3.
iScience ; 23(6): 101236, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32563156

RESUMO

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.

4.
Oncogene ; 38(21): 4015-4027, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30692633

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Genes src/genética , Humanos , Células MCF-7 , Metilação , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/fisiologia , Receptor IGF Tipo 1/metabolismo
5.
Int J Cancer ; 144(3): 595-606, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30289978

RESUMO

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.


Assuntos
Neoplasias da Mama/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Células MCF-7 , Pessoa de Meia-Idade , Fosforilação
6.
Nucleic Acids Res ; 45(14): 8508-8523, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28591869

RESUMO

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.


Assuntos
Processamento Alternativo , Citoplasma/genética , Perfilação da Expressão Gênica/métodos , Fatores de Transcrição/genética , Arginina/genética , Arginina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/genética , Citoplasma/metabolismo , Exorribonucleases , Células HEK293 , Humanos , Células MCF-7 , Metilação , Microscopia Confocal , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
7.
Oncotarget ; 7(41): 67532-67550, 2016 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-27556302

RESUMO

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.


Assuntos
Arginina/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Desmetilação , Humanos , Metilação
8.
PLoS One ; 10(5): e0126181, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25951181

RESUMO

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.


Assuntos
Neoplasias da Mama/patologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico
9.
Breast Cancer Res ; 17: 13, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25633049

RESUMO

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.


Assuntos
Inibidores da Aromatase/farmacologia , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Antineoplásicos/farmacologia , Antineoplásicos Hormonais , Inibidores da Aromatase/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Análise por Conglomerados , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Letrozol , Células MCF-7 , Recidiva Local de Neoplasia , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Triazóis/farmacologia , Células Tumorais Cultivadas , Regulação para Cima
10.
Methods Mol Biol ; 1204: 135-43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25182767

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Quinases da Família src/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Microscopia de Fluorescência/métodos , Mapas de Interação de Proteínas
11.
Wiley Interdiscip Rev RNA ; 5(6): 883-901, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25044499

RESUMO

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.


Assuntos
Eucariotos/fisiologia , Regulação da Expressão Gênica , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Eucariotos/genética , Complexos Multienzimáticos/metabolismo , Estresse Fisiológico
12.
Mol Oncol ; 8(8): 1441-57, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24973012

RESUMO

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.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Transativadores/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Imunoprecipitação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Transativadores/genética
13.
Int J Cancer ; 135(6): 1307-18, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24615515

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Metilação , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Transfecção , Quinases da Família src/metabolismo
14.
PLoS One ; 9(2): e87982, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24498420

RESUMO

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.


Assuntos
Arginina/química , Neoplasias da Mama/genética , Metilação de DNA , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Processamento de Proteína Pós-Traducional , Western Blotting , Neoplasias da Mama/metabolismo , Cromatografia de Afinidade , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imunoprecipitação , Histona Desmetilases com o Domínio Jumonji/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais Cultivadas
15.
Mol Endocrinol ; 28(2): 183-96, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24422630

RESUMO

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 3 (HDAC3), 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.


Assuntos
Cisteína Endopeptidases/fisiologia , Receptor alfa de Estrogênio/fisiologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias da Mama , Proliferação de Células , Estradiol/fisiologia , Feminino , Histona Desacetilases/metabolismo , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Transcrição Gênica
16.
Int J Cancer ; 133(7): 1589-602, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23553037

RESUMO

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.


Assuntos
Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Nitrilas/farmacologia , Triazóis/farmacologia , Anastrozol , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/biossíntese , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Recidiva Local de Neoplasia/metabolismo , Nitrilas/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Triazóis/uso terapêutico
17.
EMBO J ; 32(5): 688-700, 2013 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-23386060

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Interferons/farmacologia , Fator de Transcrição STAT1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Replicação Viral/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Exorribonucleases , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Imunoprecipitação , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
18.
EMBO Mol Med ; 4(11): 1200-13, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23065768

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Estrogênios/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Análise de Sobrevida
19.
Cancer Res ; 72(14): 3593-606, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22593193

RESUMO

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-ß-activated Smad signaling pathway was identified as a major driver of ZNF217-induced EMT. In addition, a TGF-ß autocrine loop sustained activation of the TGF-ß pathway in ZNF217-overexpressing mammary epithelial cells, most likely because of ZNF217-mediated direct upregulation of TGFB2 or TGFB3. Inhibition of the TGF-ß 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-ß signaling pathway may benefit the subset of patients whose tumors express high levels of ZNF217.


Assuntos
Biomarcadores Tumorais/análise , Transição Epitelial-Mesenquimal/genética , Metástase Neoplásica/genética , Transativadores/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/secundário , Metástase Linfática , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Prognóstico , RNA Mensageiro/análise , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transplante Heterólogo
20.
Endocr Rev ; 32(5): 597-622, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21680538

RESUMO

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.


Assuntos
Neoplasias da Mama , Processamento de Proteína Pós-Traducional , Receptores de Estrogênio , Acetilação , Animais , Antineoplásicos Hormonais , Neoplasias da Mama/química , Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/análise , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/fisiologia , Estrogênios/fisiologia , Feminino , Humanos , Metilação , Mutação , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologia , Transdução de Sinais , Ubiquitinação
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