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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.
Biochem Pharmacol ; 192: 114692, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34298004

RESUMO

Triple-negative metaplastic breast carcinoma (MBC) poses a significant treatment challenge due to lack of targeted therapies and chemotherapy resistance. We isolated a novel MBC cell line, BAS, which showed a molecular and phenotypic profile different from the only other metaplastic cell model, HS578T cells. To gain insight behind chemotherapeutic resistance, we generated doxorubicin (HS-DOX, BAS-DOX) and paclitaxel (HS-TX, BAS-TX) resistant derivatives of both cell lines. Drug sensitivity assays indicated a truly multidrug resistant (MDR) phenotype. Both BAS-DOX and BAS-TX showed up-regulation of FOXC1 and its experimental down-regulation re-sensitized cells to doxorubicin and paclitaxel. Experimental modulation of FOXC1 expression in MCF-7 and MDA-MB-231 cells corroborated its role in MDR. Genome-wide expression analyses identified gene expression signatures characterized by up-regulation of TGFB2, which encodes cytokine TGF-ß2, in both BAS-DOX and BAS-TX cells. Pharmacological inhibition of the TGF-ß pathway with galunisertib led to down-regulation of FOXC1 and increase in drug sensitivity in both BAS-DOX and BAS-TX cells. MicroRNA (miR) expression analyses identified high endogenous miR-495-3p levels in BAS cells that were downregulated in both BAS MDR cells. Transient expression of miR-495-3p mimic in BAS-DOX and BAS-TX cells caused downregulation of TGFB2 and FOXC1 and re-sensitized cells to doxorubicin and paclitaxel, whereas miR-495-3p inhibition in BAS cells led to increase in resistance to both drugs and up-regulation of TGFB2 and FOXC1. Together, these data suggest interplay between miR-495-3p, TGF-ß2 and FOXC1 regulating MDR in MBC and open the exploration of novel therapeutic strategies.


Assuntos
Neoplasias da Mama/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fatores de Transcrição Forkhead/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Células Tumorais Cultivadas
4.
DNA Repair (Amst) ; 93: 102919, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33087260

RESUMO

Mammalian cells possess multiple closely related SWI/SNF chromatin remodelling complexes. These complexes have been implicated in the cellular response to DNA double strand breaks (DSBs). Evidence suggests that SWI/SNF complexes contribute to successful repair via both the homologous recombination and non-homologous end joining pathways. In addition, repressing transcription near DSBs is dependent on SWI/SNF activity. Understanding these roles is important because SWI/SNF complexes are frequently dysregulated in cancer, and DNA DSB repair defects have the potential to be therapeutically exploited. In this graphical review, we summarise what is known about SWI/SNF contribution to DNA DSB responses in mammalian cells and provide an overview of the SWI/SNF-encoding gene alteration spectrum in human cancers.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo de DNA por Recombinação , Animais , DNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
5.
Mol Cancer Ther ; 17(6): 1156-1166, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29545334

RESUMO

Recent reports indicate that some cancer types are especially sensitive to transcription inhibition, suggesting that targeting the transcriptional machinery provides new approaches to cancer treatment. Cyclin-dependent kinase (CDK)7 is necessary for transcription, and acts by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (PolII) to enable transcription initiation. CDK7 additionally regulates the activities of a number of transcription factors, including estrogen receptor (ER)-α. Here we describe a new, orally bioavailable CDK7 inhibitor, ICEC0942. It selectively inhibits CDK7, with an IC50 of 40 nmol/L; IC50 values for CDK1, CDK2, CDK5, and CDK9 were 45-, 15-, 230-, and 30-fold higher. In vitro studies show that a wide range of cancer types are sensitive to CDK7 inhibition with GI50 values ranging between 0.2 and 0.3 µmol/L. In xenografts of both breast and colorectal cancers, the drug has substantial antitumor effects. In addition, combination therapy with tamoxifen showed complete growth arrest of ER-positive tumor xenografts. Our findings reveal that CDK7 inhibition provides a new approach, especially for ER-positive breast cancer and identify ICEC0942 as a prototype drug with potential utility as a single agent or in combination with hormone therapies for breast cancer. ICEC0942 may also be effective in other cancers that display characteristics of transcription factor addiction, such as acute leukaemia and small-cell lung cancer. Mol Cancer Ther; 17(6); 1156-66. ©2018 AACR.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase Ativadora de Quinase Dependente de Ciclina
6.
Nat Commun ; 8(1): 1865, 2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29192207

RESUMO

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.


Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/análogos & derivados , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio/genética , Linhagem Celular Tumoral , Estradiol/uso terapêutico , Feminino , Fulvestranto , Humanos , Células MCF-7 , Mutação , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/uso terapêutico
7.
Clin Cancer Res ; 22(23): 5929-5938, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27301701

RESUMO

PURPOSE: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. RESULTS: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. CONCLUSIONS: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Ciclina H/genética , Quinases Ciclina-Dependentes/genética , Expressão Gênica/genética , Receptores de Estrogênio/genética , Adulto , Proteínas de Ciclo Celular , Feminino , Humanos , Pessoa de Meia-Idade , Fosforilação/genética , Prognóstico , Transdução de Sinais/genética , Fatores de Transcrição , Transcrição Gênica/genética , Quinase Ativadora de Quinase Dependente de Ciclina
8.
Cell Rep ; 13(1): 108-121, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26411678

RESUMO

Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.


Assuntos
Neoplasias da Mama/genética , Citidina Desaminase/genética , Citidina/metabolismo , Reparo do DNA por Junção de Extremidades , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , Desaminação , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Antígenos de Histocompatibilidade Menor , Prognóstico , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Transcrição Gênica , Fator Trefoil-1 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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