RESUMEN
Myeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic Bcl-2 family protein that plays essential roles in multiple myeloma (MM) survival and drug resistance. In MM, it has been demonstrated that proteasome inhibition can trigger the accumulation of Mcl-1, which has been shown to confer MM cell resistance to bortezomib-induced lethality. However, the mechanisms involved in this unwanted Mcl-1 accumulation are still unclear. The aim of the present study was to determine whether the unwanted Mcl-1 accumulation could be induced by the unfolded protein response (UPR) and to elucidate the role of the endoplasmic reticulum stress response in regulating Mcl-1 expression. Using quantitative RT-PCR and Western blot, we found that the translation of activating transcription factor-4 (ATF4), an important effector of the UPR, was also greatly enhanced by proteasome inhibition. ChIP analysis further revealed that bortezomib stimulated binding of ATF4 to a regulatory site (at position -332 to -324) at the promoter of the Mcl-1 gene. Knocking down ATF4 was paralleled by down-regulation of Mcl-1 induction by bortezomib and significantly increased bortezomib-induced apoptosis. These data identify the UPR and, more specifically, its ATF4 branch as an important mechanism mediating up-regulation of Mcl-1 by proteasome inhibition.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 4/genética , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Proliferación Celular , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Pirazinas/farmacología , Empalme del ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Recently, we showed that hypoxia is a critical microenvironmental factor in multiple myeloma, and that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells and improves multiple disease parameters in vivo. To explore approaches for sensitizing multiple myeloma cells to TH-302, we evaluated in this study the antitumor effect of TH-302 in combination with the clinically used proteasome inhibitor bortezomib. First, we show that TH-302 and bortezomib synergistically induce apoptosis in multiple myeloma cell lines in vitro. Second, we confirm that this synergism is related to the activation of caspase cascades and is mediated by changes of Bcl-2 family proteins. The combination treatment induces enhanced cleavage of caspase-3/8/9 and PARP, and therefore triggers apoptosis and enhances the cleavage of proapoptotic BH3-only protein BAD and BID as well as the antiapoptotic protein Mcl-1. In particular, TH-302 can abrogate the accumulation of antiapoptotic Mcl-1 induced by bortezomib, and decreases the expression of the prosurvival proteins Bcl-2 and Bcl-xL. Furthermore, we found that the induction of the proapoptotic BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA is associated with this synergism. In response to the genotoxic and endoplasmic reticulum stresses by TH-302 and bortezomib, the expression of PUMA and NOXA were upregulated in p53-dependent and -independent manners. Finally, in the murine 5T33MMvv model, we showed that the combination of TH-302 and bortezomib can improve multiple disease parameters and significantly prolong the survival of diseased mice. In conclusion, our studies provide a rationale for clinical evaluation of the combination of TH-302 and bortezomib in patients with multiple myeloma.