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1.
Sci Rep ; 14(1): 6873, 2024 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519482

RESUMO

Three quarters of all breast cancers express the estrogen receptor (ER, ESR1 gene), which promotes tumor growth and constitutes a direct target for endocrine therapies. ESR1 mutations have been implicated in therapy resistance in metastatic breast cancer, in particular to aromatase inhibitors. ESR1 mutations promote constitutive ER activity and affect other signaling pathways, allowing cancer cells to proliferate by employing mechanisms within and without direct regulation by the ER. Although subjected to extensive genetic and transcriptomic analyses, understanding of protein alterations remains poorly investigated. Towards this, we employed an integrated mass spectrometry based proteomic approach to profile the protein and phosphoprotein differences in breast cancer cell lines expressing the frequent Y537N and Y537S ER mutations. Global proteome analysis revealed enrichment of mitotic and immune signaling pathways in ER mutant cells, while phosphoprotein analysis evidenced enriched activity of proliferation associated kinases, in particular CDKs and mTOR. Integration of protein expression and phosphorylation data revealed pathway-dependent discrepancies (motility vs proliferation) that were observed at varying degrees across mutant and wt ER cells. Additionally, protein expression and phosphorylation patterns, while under different regulation, still recapitulated the estrogen-independent phenotype of ER mutant cells. Our study is the first proteome-centric characterization of ESR1 mutant models, out of which we confirm estrogen independence of ER mutants and reveal the enrichment of immune signaling pathways at the proteomic level.


Assuntos
Neoplasias da Mama , Quinases Ciclina-Dependentes , Humanos , Feminino , Quinases Ciclina-Dependentes/genética , Proteoma/genética , Proteômica , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Neoplasias da Mama/patologia , Mutação , Estrogênios , Receptores de Estrogênio/genética , Fosfoproteínas/genética
2.
Oncogene ; 41(44): 4905-4915, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36198774

RESUMO

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.


Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Humanos , Feminino , Receptores de Estrogênio/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Prognóstico , Antagonistas de Estrogênios/uso terapêutico , Mutação , Estrogênios/farmacologia
3.
Cancers (Basel) ; 13(24)2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34944934

RESUMO

While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15-40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatment.

4.
Oncogene ; 40(6): 1077-1090, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33323971

RESUMO

The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.


Assuntos
Citidina Desaminase/genética , Antígenos de Histocompatibilidade Menor/genética , Neoplasias/genética , Fator de Transcrição RelA/genética , Proteína Supressora de Tumor p53/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sistemas CRISPR-Cas/genética , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética , Células HCT116 , Humanos , Células MCF-7 , Mutação/genética , NF-kappa B/genética , Neoplasias/patologia
5.
Cancer Metastasis Rev ; 39(3): 805-823, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32385714

RESUMO

Cyclin-dependent kinase 7 (CDK7), along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs progression through the cell cycle via T-loop phosphorylation of cell cycle CDKs. CAK is also a component of the general transcription factor, TFIIH. CDK7-mediated phosphorylation of RNA polymerase II (Pol II) at active gene promoters permits transcription. Cell cycle dysregulation is an established hallmark of cancer, and aberrant control of transcriptional processes, through diverse mechanisms, is also common in many cancers. Furthermore, CDK7 levels are elevated in a number of cancer types and are associated with clinical outcomes, suggestive of greater dependence on CDK7 activity, compared with normal tissues. These findings identify CDK7 as a cancer therapeutic target, and several recent publications report selective CDK7 inhibitors (CDK7i) with activity against diverse cancer types. Preclinical studies have shown that CDK7i cause cell cycle arrest, apoptosis and repression of transcription, particularly of super-enhancer-associated genes in cancer, and have demonstrated their potential for overcoming resistance to cancer treatments. Moreover, combinations of CDK7i with other targeted cancer therapies, including BET inhibitors, BCL2 inhibitors and hormone therapies, have shown efficacy in model systems. Four CDK7i, ICEC0942 (CT7001), SY-1365, SY-5609 and LY3405105, have now progressed to Phase I/II clinical trials. Here we describe the work that has led to the development of selective CDK7i, the current status of the most advanced clinical candidates, and discuss their potential importance as cancer therapeutics, both as monotherapies and in combination settings. ClinicalTrials.gov Identifiers: NCT03363893; NCT03134638; NCT04247126; NCT03770494.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Quinases Ciclina-Dependentes/metabolismo , Humanos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Quinase Ativadora de Quinase Dependente de Ciclina
6.
Oncogene ; 39(3): 651-663, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31530935

RESUMO

The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Seleção de Pacientes , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinase Ativadora de Quinase Dependente de Ciclina
7.
Breast Cancer Res Treat ; 150(2): 335-46, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25762479

RESUMO

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Receptores de Glucocorticoides/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/secundário , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Carga Tumoral
8.
Nucleic Acids Res ; 41(10): 5400-12, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23580553

RESUMO

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular , Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Animais , Neoplasias da Mama/metabolismo , Miosinas Cardíacas/metabolismo , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Adesões Focais/ultraestrutura , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Cadeias Leves de Miosina/metabolismo , Metástase Neoplásica , Fosforilação , Regiões Promotoras Genéticas , Pseudópodes/ultraestrutura , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Quinases Ativadas por p21/metabolismo
9.
J Med Chem ; 55(4): 1731-50, 2012 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-22280363

RESUMO

Psammaplin A (11c) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that 11c functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC(50) 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for 11c functioning as a DNMT inhibitor.


Assuntos
Antineoplásicos/farmacologia , Dissulfetos/farmacologia , Epigênese Genética , Inibidores de Histona Desacetilases/farmacologia , Pró-Fármacos/farmacologia , Tirosina/análogos & derivados , Acetilação , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Dimerização , Dissulfetos/síntese química , Dissulfetos/química , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histonas/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Modelos Moleculares , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Tirosina/síntese química , Tirosina/química , Tirosina/farmacologia
10.
Nucleic Acids Res ; 34(21): 6126-36, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17085477

RESUMO

The regulation of gene expression by estrogen receptor-alpha (ERalpha) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERalpha as bait in a yeast two-hybrid screen, we have identified a novel ERalpha-interacting protein, ZNF366, which is a potent corepressor of ERalpha activity. The interaction between ZNF366 and ERalpha has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERalpha corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células COS , Proteínas de Transporte/análise , Proteínas de Transporte/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteína 1 de Interação com Receptor Nuclear , Distribuição Tecidual , Dedos de Zinco
11.
Nucleic Acids Res ; 33(19): 6393-404, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16282588

RESUMO

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-alpha. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.


Assuntos
Timina DNA Glicosilase/química , Timina DNA Glicosilase/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Histona Acetiltransferases , Humanos , Dados de Sequência Molecular , Coativador 1 de Receptor Nuclear , Sequências Repetitivas de Aminoácidos , Transativadores/química , Fatores de Transcrição/química , Tirosina/análise
13.
J Biol Chem ; 278(40): 38586-92, 2003 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-12874288

RESUMO

Nuclear receptors (NR) classically regulate gene expression by stimulating transcription upon binding to their cognate ligands. It is now well established that NR-mediated transcriptional activation requires the recruitment of coregulator complexes, which facilitate recruitment of the basal transcription machinery through direct interactions with the basal transcription machinery and/or through chromatin remodeling. However, a number of recently described NR coactivators have been implicated in cross-talk with other nuclear processes including RNA splicing and DNA repair. T:G mismatch-specific thymine DNA glycosylase (TDG) is required for base excision repair of deaminated methylcytosine. Here we show that TDG is a coactivator for estrogen receptor alpha (ERalpha). We demonstrate that TDG interacts with ERalpha in vitro and in vivo and suggest a separate role for TDG to its established role in DNA repair. We show that this involves helix 12 of ERalpha. The region of interaction in TDG is mapped to a putative alpha-helical motif containing a motif distinct from but similar to the LXXLL motif that mediates interaction with NR. Together with recent reports linking TFIIH in regulating NR function, our findings provide new data to further support an important link between DNA repair proteins and nuclear receptor function.


Assuntos
Estrogênios/metabolismo , N-Glicosil Hidrolases/química , Receptores de Estrogênio/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Pareamento Incorreto de Bases , Células COS , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatina/metabolismo , DNA/metabolismo , DNA Glicosilases , Reparo do DNA , Receptor alfa de Estrogênio , Vetores Genéticos , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Ligantes , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , RNA/metabolismo , Splicing de RNA , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Timidina/química , Transcrição Gênica , Ativação Transcricional , Transfecção , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismo
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