RESUMEN
Mutations of succinate dehydrogenase subunit B (SDHB) play a crucial role in the pathogenesis of the most aggressive and metastatic pheochromocytomas (PHEOs) and paragangliomas (PGLs). Although a variety of missense mutations in the coding sequence of the SDHB gene have been found in PHEOs and PGLs, it has been unclear whether these mutations impair mRNA expression, protein stability, subcellular localization, or intrinsic protein function. RT-PCR and Western blot analysis of SDHB mRNA and protein expression from SDHB-related PHEOs and PGLs demonstrated intact mRNA expression but significantly reduced protein expression compared to non-SDHB PHEOs and PGLs. A pulse-chase assay of common SDHB missense mutations in transfected HeLa cell lines demonstrated that the loss of SDHB function was due to a reduction in mutant protein half-life, whereas colocalization of SDHB with mitochondria and immunoprecipitation with SDHA demonstrated intact subcellular localization and complex formation. The half-life of the SDHB protein increased after treatment with histone deacetylase inhibitors (HDACis), implicating the protein quality control machinery in the degradation of mutant SDHB protein. These findings provide the first direct mechanism of functional loss resulting from SDHB mutations and suggest that reducing protein degradation with HDACis may serve as a novel therapeutic paradigm for preventing the development of SDHB-related tumors.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Mutación Missense , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Clonación Molecular , Estabilidad de Enzimas , Células HeLa , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , Paraganglioma/metabolismo , Feocromocitoma/metabolismo , Unión Proteica , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Pheochromocytomas (PHEOs) and paragangliomas (PGLs) related to mutations in the mitochondrial succinate dehydrogenase (SDH) subunits A, B, C, and D, SDH complex assembly factor 2, and the von Hippel-Lindau (VHL) genes share a pseudohypoxic expression profile. However, genotype-specific differences in expression have been emerging. Development of effective new therapies for distinctive manifestations, e.g., a high rate of malignancy in SDHB- or predisposition to multifocal PGLs in SDHD patients, mandates improved stratification. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggest novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies.