RESUMO
Rhabdomyosarcoma (RMS), a neoplasm characterised by undifferentiated myoblasts, is the most common soft tissue tumour of childhood. Although aggressive treatment of RMS could provide long-term benefit, resistance to current therapies is an ongoing problem. We report here that insulin-like growth factor 2-binding protein 1 (IGF2BP1), an oncofetal protein, is expressed in RMS patient-derived cell lines and in primary tumours where it drives translation of the cellular inhibitor of apoptosis 1 (cIAP1), a key regulator of the nuclear factor-κB signalling pathway and of caspase-8-mediated cell death. We demonstrate that reducing the levels of cIAP1 in RMS, either by IGF2BP1 knockdown or by IAP antagonists, sensitises these cells to tumour necrosis factor-α-mediated cell death. Finally, we show that targeting cIAP1 by IAP antagonists delays RMS tumour growth and improve survival in mice. Our results identify IGF2BP1 as a critical translational regulator of cIAP1-mediated apoptotic resistance in RMS and advocate for the combined use of IAP antagonists and tumour necrosis factor-α as a therapeutic approach for this type of cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Inibidoras de Apoptose/genética , Proteínas de Ligação a RNA/metabolismo , Rabdomiossarcoma/metabolismo , Alcinos/farmacologia , Animais , Apoptose , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Biossíntese de Proteínas , Proteínas de Ligação a RNA/antagonistas & inibidores , Rabdomiossarcoma/tratamento farmacológico , Transdução de Sinais , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Expression of the cellular inhibitor of apoptosis protein 1 (cIAP1) is unexpectedly repressed at the level of translation under normal physiological conditions in many cell lines. We have previously shown that the 5' untranslated region of cIAP1 mRNA contains a stress-inducible internal ribosome entry site (IRES) that governs expression of cIAP1 protein. Although inactive in unstressed cells, the IRES supports cap-independent translation of cIAP1 in response to endoplasmic reticulum stress. To gain an insight into the mechanism of cIAP1 IRES function, we empirically derived the minimal free energy secondary structure of the cIAP1 IRES using enzymatic cleavage mapping. We subsequently used RNA affinity chromatography to identify several cellular proteins, including nuclear factor 45 (NF45) as cIAP1 IRES binding proteins. In this report we show that NF45 is a novel RNA binding protein that enhances IRES-dependent translation of endogenous cIAP1. Further, we show that NF45 is required for IRES-mediated induction of cIAP1 protein during the unfolded protein response. The data presented are consistent with a model in which translation of cIAP1 is governed, at least in part, by NF45, a novel cellular IRES trans-acting factor.
Assuntos
Apoptose/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Proteína do Fator Nuclear 45/metabolismo , Biossíntese de Proteínas/fisiologia , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia de Afinidade , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Ativação Enzimática/fisiologia , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteína do Fator Nuclear 45/genética , Estrutura Terciária de Proteína/fisiologia , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genéticaRESUMO
cIAP1 is an important member of the inhibitor of apoptosis family of proteins and is involved in the regulation of the NF-kappaB-signalling pathway downstream of the TNF receptor. We report here that UV irradiation leads to downregulation of cIAP1 expression because of enhanced cIAP1 mRNA destabilization. An AU-rich element located within the 3' untranslated region of cIAP1 mRNA is sufficient to mediate cIAP1 mRNA instability. Furthermore, we have identified hnRNP A1 as a cIAP1 3'UTR-binding protein. hnRNP A1 is a primarily nuclear protein, but accumulates in the cytoplasm after exposure of cells to UV irradiation. Indeed, we find that hnRNP A1 enhances the destabilization of cIAP1 mRNA during UV irradiation. Moreover, siRNA-mediated knockdown of hnRNP A1 restores cIAP1 levels and prevents UV irradiation-induced activation of the NF-kappaB signal transduction pathway, suggesting that hnRNP A1 is an essential post-transcriptional modulator of cIAP1 expression, and thus cIAP1 activity.
