Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Neuro Oncol. 2022 Oct 3;24(10):1712-1725. doi: 10.1093/neuonc/noac105.
Epidermal growth factor receptor (EGFR) amplification and TP53 mutation are the two most common genetic alterations in glioblastoma multiforme (GBM). A comprehensive analysis of the TCGA GBM database revealed a subgroup with near mutual exclusivity of EGFR amplification and TP53 mutations indicative of a role of EGFR in regulating wild-type-p53 (wt-p53) function. The relationship between EGFR amplification and wt-p53 function remains undefined and this study describes the biological significance of this interaction in GBM.
Mass spectrometry was used to identify EGFR-dependent p53-interacting proteins. The p53 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interaction was detected by co-immunoprecipitation. We used CRISPR-Cas9 gene editing to knockout EGFR and DNA-PKcs and the Edit-R CRIPSR-Cas9 system for conditional knockout of EGFR. ROS activity was measured with a CM-H2DCFDA probe, and real-time PCR was used to quantify expression of p53 target genes.
Using glioma sphere-forming cells (GSCs), we identified, DNA-PKcs as a p53 interacting protein that functionally inhibits p53 activity. We demonstrate that EGFR knockdown increased wt-p53 transcriptional activity, which was associated with decreased binding between p53 and DNA-PKcs. We further show that inhibition of DNA-PKcs either by siRNA or an inhibitor (nedisertib) increased wt-p53 transcriptional activity, which was not enhanced further by EGFR knockdown, indicating that EGFR suppressed wt-p53 activity through DNA-PKcs binding with p53. Finally, using conditional EGFR-knockout GSCs, we show that depleting EGFR increased animal survival in mice transplanted with wt-p53 GSCs.
This study demonstrates that EGFR signaling inhibits wt-p53 function in GBM by promoting an interaction between p53 and DNA-PKcs.
表皮生长因子受体（EGFR）扩增和 TP53 突变是胶质母细胞瘤（GBM）中最常见的两种遗传改变。对 TCGA GBM 数据库的综合分析显示，存在一个亚组，其 EGFR 扩增和 TP53 突变近乎相互排斥，表明 EGFR 在调节野生型-p53（wt-p53）功能方面发挥作用。EGFR 扩增与 wt-p53 功能之间的关系尚未确定，本研究描述了这种相互作用在 GBM 中的生物学意义。
使用质谱法鉴定 EGFR 依赖性 p53 相互作用蛋白。通过共免疫沉淀检测 p53 和 DNA 依赖性蛋白激酶催化亚基（DNA-PKcs）的相互作用。我们使用 CRISPR-Cas9 基因编辑敲除 EGFR 和 DNA-PKcs，以及 Edit-R CRISPR-Cas9 系统条件敲除 EGFR。使用 CM-H2DCFDA 探针测量 ROS 活性，并使用实时 PCR 定量 p53 靶基因的表达。
使用神经胶质瘤球体形成细胞（GSCs），我们鉴定出 DNA-PKcs 是一种与 p53 相互作用的蛋白，可抑制 p53 活性。我们证明，EGFR 敲低增加了 wt-p53 的转录活性，这与 p53 与 DNA-PKcs 之间的结合减少有关。我们进一步表明，通过 siRNA 或抑制剂（nedisertib）抑制 DNA-PKcs 增加了 wt-p53 的转录活性，而 EGFR 敲低并未进一步增强其活性，表明 EGFR 通过与 p53 结合抑制 wt-p53 活性。最后，使用条件性 EGFR 敲除 GSCs，我们表明，耗尽 EGFR 增加了移植 wt-p53 GSCs 的小鼠的动物存活率。
本研究表明，EGFR 信号通过促进 p53 与 DNA-PKcs 之间的相互作用抑制 GBM 中的 wt-p53 功能。
