RESUMO
Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the expansion of multiple myeloma cells (MMCs) in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the most downregulated genes. RecQ helicases are DNA unwinding enzymes involved in the maintenance of chromosome stability. Here we show that RECQ1 is significantly overexpressed in MMCs compared to normal plasma cells and that increased RECQ1 expression is associated with poor prognosis in three independent cohorts of patients. Interestingly, RECQ1 knockdown inhibits cells growth and induces apoptosis in MMCs. Moreover, RECQ1 depletion promotes the development of DNA double-strand breaks, as evidenced by the formation of 53BP1 foci and the phosphorylation of ataxia-telangiectasia mutated (ATM) and histone variant H2A.X (H2AX). In contrast, RECQ1 overexpression protects MMCs from melphalan and bortezomib cytotoxicity. RECQ1 interacts with PARP1 in MMCs exposed to treatment and RECQ1 depletion sensitizes MMCs to poly(ADP-ribose) polymerase (PARP) inhibitor. DNMT inhibitor treatment results in RECQ1 downregulation through miR-203 deregulation in MMC. Altogether, these data suggest that association of DNA damaging agents and/or PARP inhibitors with DNMT inhibitors may represent a therapeutic approach in patients with high RECQ1 expression associated with a poor prognosis.
Assuntos
DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Mieloma Múltiplo/enzimologia , Proteínas de Neoplasias/fisiologia , RecQ Helicases/fisiologia , Bortezomib/farmacologia , Ciclo Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Metilação de DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , DNA-Citosina Metilases/antagonistas & inibidores , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Melfalan/farmacologia , MicroRNAs/genética , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RecQ Helicases/antagonistas & inibidores , RecQ Helicases/genética , Células Tumorais CultivadasRESUMO
Multiple tumorigenic pathways converge on the activating protein-1 (AP-1) family of dimeric transcription complexes by affecting transcription, mRNA decay, posttranslational modifications, as well as stability of its JUN and FOS components. Several mechanisms have been implicated in the phosphorylation- and ubiquitylation-dependent control of c-Jun protein stability. Although its dimer composition has a major role in the regulation of AP-1, little is known about the influence of heterodimerization partners on the half-life of c-Jun. The FOS family member Fra-1 is overexpressed in various tumors and cancer cell lines wherein it controls motility, invasiveness, cell survival and cell division. Oncogene-induced accumulation of Fra-1 results from both increased transcription and phosphorylation-dependent stabilization of the protein. In this report, we describe a novel role of Fra-1 as a posttranslational regulator of c-Jun. By using both constitutively and inducible transformed rat thyroid cell lines, we found that c-Jun is stabilized in response to RAS oncoprotein expression. This stabilization requires the activity of the extracellular signal-related kinase (ERK) pathway, along with c-Jun heterodimerization with Fra-1. In particular, heterodimerization with Fra-1 inhibits c-Jun breakdown by a mechanism dependent on the phosphorylation of the Fra-1 C-terminal domain that positively controls the stability of the protein in response to ERK signaling. Therefore, Fra-1 modulates AP-1 dimer composition by promoting the accumulation of c-Jun in response to oncogenic RAS signaling.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Linhagem Celular Transformada , Dimerização , Fosforilação , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , Ratos , Glândula Tireoide/enzimologia , Glândula Tireoide/metabolismo , Fator de Transcrição AP-1/metabolismo , Regulação para Cima , Proteínas ras/biossíntese , Proteínas ras/genéticaRESUMO
cAMP response element binding protein-2 (CREB-2) is a basic leucine zipper (bZIP) factor that was originally described as a repressor of CRE-dependent transcription but that can also act as a transcriptional activator. Moreover, CREB-2 is able to function in association with the viral Tax protein as an activator of the human T-cell leukemia virus type I (HTLV-I) promoter. Here we show that CREB-2 is able to interact with C/EBP-homologous protein (CHOP), a bZIP transcription factor known to inhibit CAAT/enhancer-dependent transcription. Cotransfection of CHOP with CREB-2 results in decreased activation driven by the cellular CRE motif or the HTLV-I proximal Tax-responsive element, confirming that CREB-2 and CHOP can interact with each other in vivo.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Zíper de Leucina , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , Fator de Transcrição CHOP , Fatores de Transcrição/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.