RESUMO
The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Mitose , Proteína SUMO-1/metabolismo , Sumoilação , Antibióticos Antineoplásicos/farmacologia , Antígenos CD , Sítios de Ligação , Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Epirubicina/farmacologia , Proteína Forkhead Box M1 , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HeLa , Humanos , Células MCF-7 , Nocodazol/farmacologia , Transporte Proteico , ProteóliseRESUMO
In this study, we investigated the regulation of FOXM1 expression by estrogen receptor alpha (ERalpha) and its role in hormonal therapy and endocrine resistance. FOXM1 protein and mRNA expression was regulated by ER-ligands, including estrogen, tamoxifen (OHT) and fulvestrant (ICI182780; ICI) in breast carcinoma cell lines. Depletion of ERalpha by RNA interference (RNAi) in MCF-7 cells downregulated FOXM1 expression. Reporter gene assays showed that ERalpha activates FOXM1 transcription through an estrogen-response element (ERE) located within the proximal promoter region. The direct binding of ERalpha to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation (ChIP) analysis. Our data also revealed that upon OHT treatment ERalpha recruits histone deacetylases to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity. Importantly, silencing of FOXM1 by RNAi abolished estrogen-induced MCF-7 cell proliferation and overcame acquired tamoxifen resistance. Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT. OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines. Furthermore, qRT-PCR analysis of breast cancer patient samples revealed that there was a strong and significant positive correlation between ERalpha and FOXM1 mRNA expression. Collectively, these results show FOXM1 to be a key mediator of the mitogenic functions of ERalpha and estrogen in breast cancer cells, and also suggest that the deregulation of FOXM1 may contribute to anti-estrogen insensitivity.
Assuntos
Neoplasias da Mama/genética , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/fisiologia , Estrogênios/farmacologia , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Resistencia a Medicamentos Antineoplásicos , Ensaio de Desvio de Mobilidade Eletroforética , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Fulvestranto , Humanos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The Ewing's sarcoma family of tumours (ESFT) are small round cell tumours characterized by the non-random EWS-ETS gene rearrangements. We have previously demonstrated that ESFT are highly sensitive to fenretinide-induced death, effected in part through a reactive oxygen species (ROS)-dependent pathway. Here, we demonstrate for the first time that the sensitivity of ESFT cells to fenretinide-induced cell death is decreased following downregulation of the oncogenic fusion protein EWS-Fli1; siRNA targeting EWS-Fli1 attenuated fenretinide-induced cell death in cell lines expressing EWS-Fli1, but not EWS-ERG. This decrease in cell death was independent of the level of ROS produced following exposure to fenretinide, but was effected through EWS-Fli1-dependent modulation of p38(MAPK) activity. Furthermore, inhibition of p38(MAPK) activity and knockdown of EWS-Fli1 reduced fenretinide-induced mitochondrial permeabilization, cytochrome c release, caspase and PARP cleavage, consistent with the hypothesis that p38(MAPK) is critical for activation of the death cascade by fenretinide in ESFT cells. These data demonstrate that expression of EWS-Fli1 enhances fenretinide-induced cell death in ESFT and that this is effected at least in part through modulation of p38(MAPK) activity.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fenretinida/farmacologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Regulação para Baixo , Eletroporação , Citometria de Fluxo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/genética , RNA Interferente Pequeno/farmacologia , Proteína EWS de Ligação a RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
FoxO transcription factors are an evolutionary conserved subfamily of the forkhead transcription factors, characterized by the forkhead DNA-binding domain. FoxO factors regulate a number of cellular processes involved in cell-fate decisions in a cell-type- and environment-specific manner, including metabolism, differentiation, apoptosis and proliferation. A key mechanism by which FoxO determines cell fate is through regulation of the cell cycle machinery, and as such the cellular consequence of FoxO deregulation is often manifested through perturbation of the cell cycle. Consequently, the deregulation of FoxO factors is implicated in the development of numerous proliferative diseases, in particular cancer.