Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities.
Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities.
复制标题
DOI：
10.18632/oncotarget.6634
复制
发表时间：
2016-02-02
期刊：
影响因子：
--
通讯作者：
Iwakuma T
中科院分区：
文献类型：
--
作者：
Iyer SV;Parrales A;Begani P;Narkar A;Adhikari AS;Martinez LA;Iwakuma T
许多P53热点突变体不仅失去转录活性，还表现出显性负性(DN)和致癌功能增强(GOF)活性。越来越多的证据表明，在癌细胞中敲除突变的p53会降低其侵袭性，这表明癌细胞的生存和增殖至少部分依赖于突变p53的存在。然而，这些p53 siRNAs可以下调野生型p53(Wtp53)和突变p53的表达，从而限制了它们的治疗应用。为了特异性地去除突变的p53，我们开发了针对p53热点突变的等位基因特异性siRNAs，并验证了它们在wtp53缺失或存在的情况下的生物学效应。首先，突变的p53特异性siRNAs选择性地降低了匹配的p53突变体的蛋白质水平，而wtp53水平的降低幅度最小。第二，在单独表达突变p53的癌细胞(P53mut)中，突变p53的下调导致细胞增殖和迁移显著减少。第三，在同时表达wtp53和突变p53的癌细胞中导入突变p53特异性siRNAs也减少了细胞的增殖和迁移，增加了p53下游靶基因的转录，当MDM2抑制剂Nutlin-3a或化疗药物阿霉素处理细胞时，这种作用变得更加明显。这些结果表明，在同时表达wtP53和突变P53的细胞中，通过其特定的siRNA去除突变P53，恢复了内源性WtP53的活性。这是首次在同时表达wtp53和mutp53的癌细胞中通过突变p53特异性siRNAs沉默突变p53的生物学效应和治疗潜力的研究，从而为靶向癌症治疗提供了一种新的策略。
Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.