RESUMO
High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, especially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase-anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuroblastoma cells.
Assuntos
Apoptose , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Ploidias , Fuso Acromático/genética , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hibridização in Situ Fluorescente , Lactente , Proteínas Mad2 , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Salivares Ricas em Prolina/genética , Proteínas Salivares Ricas em Prolina/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Catepsina D/fisiologia , Doxorrubicina/farmacologia , Neuroblastoma/tratamento farmacológico , Proteínas Reguladoras de Apoptose/fisiologia , Catepsina D/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Precursores Enzimáticos/metabolismo , Humanos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas de Ligação a RNA , Proteínas Ribossômicas/fisiologia , Transdução de SinaisRESUMO
Neuroblastoma (NB) is a solid childhood tumour that exhibits heterogeneous biological and clinical phenotypes. Multiple drug resistance marks a major complication especially in high-risk patients with advanced tumour stages and specific genetic aberrations, such as MYCN amplification and lp deletion. As an approach to further address the mechanisms of chemotherapeutic responsiveness of NB, we used a MYCN-inducible in vitro system and tested the susceptibility of NB cells to anti-tumour drugs currently included in NB treatment protocols dependent on MYCN expression. We observed cytotoxic effects using drug concentrations corresponding to blood plasma levels achieved in NB patients. The most potent drugs were microtubule inhibitors vindesin, paclitaxel and vincristin. Less effective were doxorubicine, arsenic trioxide, cisplatin, etoposide and carboplatin. Exposed to anti-tumour agents, NB cells with induced MYCN expression exhibited higher specific apoptosis than NB cells lacking MYCN expression. Anti-tumour drugs in MYCN-on cells accelerated G1-S phase transition, led to enhanced accumulation of cell populations in G2/M phase, and increased levels of apoptosis. In contrast, MYCN-off cell populations arrested in G1 and, to a smaller extent, in G2/M and exhibited delayed onset of apoptosis. In summary, apoptosis profiles and anti-proliferative potential of chemotherapeutic drugs, used at in vivo tolerable doses, are affected by MYCN overexpression and deregulated cell cycle in SH-EP(MYCN) cells.
