RESUMO
The cell cycle regulator, SKP2, is overexpressed in various cancers and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. The highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range, 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site. This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We show by chromatin immunoprecipitation that SKP2 activation through the transcriptional start site in MYCN-amplified cells is associated with the low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 through this regulatory mechanism can be reestablished in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment-both leading to p53-p21 activation-or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter.
Assuntos
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas Quinases Associadas a Fase S/genética , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Fator de Transcrição E2F1/metabolismo , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Oncogênicas/biossíntese , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Quinases Associadas a Fase S/biossíntese , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
We analysed the expression of BIRC5 and BIRC5-2B in primary neuroblastoma (NB) tumors and NB model systems. In tumors, overexpression of BIRC5 correlated closely with its isoform BIRC5-2B. Expression of both transcripts was stage-dependent, associated with poor prognosis and with the expression of the transcription factor E2F1. In cell culture, we identified BIRC5 as a direct transcriptional target of activating E2Fs, primarily when p21(Cip1) and p27(Kip1), two other E2F1 targets, are strongly suppressed. Deregulated MYCN indirectly induces BIRC5 through suppression of CDKN1A/p21(Cip1) and induction of Skp2, which in turn favors the degradation of p27(Kip1). In addition, increased BIRC5 protein stability via phosphorylation is mediated by expression of E2F targets such as CDC2. In line with this, selective knock down of CDC2 inhibited BIRC5 abundance and suppressed its anti-apoptotic activities. We conclude that BIRC5 is induced via a functional cooperation between MYCN and E2F1.
