Mis-splicing Drives Loss of Function of p53E224D Point Mutation

Background: Tp53 is the most commonly mutated gene in cancer. Canonical Tp53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of Tp53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive expression of canonical targets, but is detected in human cancer. Methods: We established a novel mouse model with a single base pair mutation (GAG>GAC, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li Fraumeni syndrome. Using mouse p53 E221D and the the analogous human p53E224D mutant, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo. Results: Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53 E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense mediated decay. Moreover, fibroblasts derived from p53 E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53 E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53 WT animals. Conclusions: Mouse p53 E221D and human p53 E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo. Overall design: Wild-Type and homozygous P53E221D/E221D mutant mouse embryonic fibroblasts were sequenced without perturbation in triplicate. NCI-H716 and HCT116 cells were similarly sequenced in triplicate.

背景：Tp53是癌症中最常见的突变基因。经典Tp53 DNA损伤应答通路已得到充分表征，传统观点认为其介导了Tp53的肿瘤抑制功能。然而，该主流观点受到了挑战：小鼠模型研究显示，p53介导的细胞凋亡与细胞周期阻滞并非其发挥肿瘤抑制作用的必需条件。本研究针对一类在人类癌症中被检出的p53突变体展开反向研究，该突变被预测可激活经典靶基因的表达。方法：我们构建了一种位于DNA结合结构域（DNA-Binding domain）的新型单碱基对突变（GAG>GAC，p53E221D）小鼠模型，该突变在筛选实验中表现出野生型功能，但却意外在李-佛美尼综合征（Li Fraumeni syndrome）患者的癌症样本中被检出。我们分别利用p53 E221D小鼠模型及其对应的人类p53E224D突变体，在体外与体内环境中评估了基因表达、转录激活活性以及肿瘤抑制功能。结果：从互补DNA（cDNA）转录的人类p53E224D可翻译为功能完全正常的p53蛋白。然而，从内源基因座转录的p53 E221D/E221D RNA存在异常剪接，进而引发无义介导的mRNA降解（nonsense mediated decay）。此外，源自p53 E221D/E221D小鼠的成纤维细胞无法检测到可检出的p53蛋白产物。与野生型p53动物相比，p53 E221D纯合突变小鼠的肿瘤发生率升高，预期寿命缩短。结论：小鼠p53 E221D与人类p53 E224D突变可引发剪接异常，并在体外与体内环境中造成具有生物学意义的p53功能丧失。总体实验设计：对未施加任何扰动的野生型与p53E221D/E221D纯合突变小鼠胚胎成纤维细胞进行三次重复测序。对NCI-H716与HCT116细胞系也进行了相同的三次重复测序。