Abreu Renata B V, Gomes Thiago T, Nepomuceno Thales C, Li Xueli, Fuchshuber-Moraes Mateus, De Gregoriis Giuliana, Suarez-Kurtz Guilherme, Monteiro Alvaro N A, Carvalho Marcelo A
Divisão de Pesquisa Clínica, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.
Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, United States.
Front Pharmacol. 2022 Jun 28;13:935995. doi: 10.3389/fphar.2022.935995. eCollection 2022.
BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene .
BRCA1是一种主要的肿瘤抑制因子，通过同源重组（HR）在DNA双链断裂的精确修复中发挥作用。其无义突变会导致无活性的截短蛋白产物，并与乳腺癌和卵巢癌的高风险相关。这些突变产生过早终止密码子（PTC）。不同研究表明，氨基糖苷类药物可通过促进终止密码子通读并恢复全长（FL）蛋白表达来诱导PTC抑制。这些化合物已在囊性纤维化和杜氏肌营养不良等遗传疾病的临床试验中进行了研究，结果令人鼓舞。在此，我们展示了概念验证数据，表明氨基糖苷类药物G418可诱导PTC通读并恢复FL蛋白合成及功能。我们首先证明，G418处理可在携带R1751X突变的人乳腺肿瘤细胞系HCC1395中恢复FL蛋白合成。用G418处理的HCC1395细胞也恢复了HR DNA修复并恢复了细胞周期检查点激活。在绿色荧光蛋白（GFP）C末端BRCA1构建体模型中评估了一组编码不同PTC的天然无义变体，PTC通读水平因终止密码子上下文而异。由于PTC通读可能产生携带致病性错义突变的FL蛋白，因此通过经过验证的转录激活试验对代表通读后最可能获得的氨基酸取代的变体进行了功能评估。总体而言，这是第一项评估具有临床相关性的PTC变体在乳腺癌和卵巢癌易感基因中通读情况的研究。
