Zhou Jie, Luo Junwen, Li Peiwei, Zhou Yongjia, Li Peichao, Wang Fang, Mallio Carlo Augusto, Rossi Giulio, Jalal Ahmed Hasnain, Filipovic Nenad, Tian Zhongxian, Zhao Xiaogang
Department of Thoracic Surgery, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.
Institute of Medical Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.
Transl Lung Cancer Res. 2022 May;11(5):802-816. doi: 10.21037/tlcr-22-312.
The mutation rate of the tumor protein P53 (TP53) has been reported to be greater than 50% in non-small cell lung cancer (NSCLC), and gain-of-function (GOF) mutations in unfolded P53 (TP53 and TP53) have been associated with poor prognosis. However, the best treatment for patients with NSCLC harboring unfolded mutant P53 (mutp53) remains unclear. Triptolide is a natural compound derived from that has shown a strong antitumor effect in a variety of cancers. Our study aimed to explore the GOF mutations in unfolded mutp53 (TP53 and TP53) and to clarify the molecular mechanisms by which triptolide regulates the degradation of unfolded mutp53 proteins in NSCLC.
Two unfolded proteins harboring TP53 and TP53 mutations were selected to explore their functions in NSCLC progression. NCI-H1299 cells (TP53-null) were transfected with wild-type TP53 (TP53), TP53, or TP53 genes and treated with triptolide or a vehicle. Wound healing and transwell assays were performed to measure cell migration and invasion . Lung metastasis models were constructed through tail vein injection of mutant cells into BALB/c nude mice to evaluate the effect of triptolide on metastasis . Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunoprecipitation, and dual-luciferase reporter assays were performed to explore the relevant molecular mechanisms.
Our study revealed that triptolide treatment reduced TP53 levels and that the TP53 mutation enhanced the invasion and migration of NCI-H1299 cells. Mechanistically, triptolide promoted TP53 and TP53 protein proteasomal degradation mediated through the E3 ligase murine double minute 2 (MDM2) by directly interacting with heat shock protein 70 (HSP70). Moreover, by upregulating HSP70 transcription, triptolide contributed to the protein degradation of the GOF mutp53.
Our study reports, for the first time, the mechanism underlying triptolide-regulated protein degradation of TP53 or TP53, which offers new insight into developing a better therapeutic strategy for patients with NSCLC who express the unfolded mutp53 GOF protein.
据报道，非小细胞肺癌（NSCLC）中肿瘤蛋白P53（TP53）的突变率超过50%，未折叠型P53（TP53和TP53）的功能获得性（GOF）突变与预后不良相关。然而，对于携带未折叠型突变P53（mutp53）的NSCLC患者的最佳治疗方法仍不清楚。雷公藤内酯醇是一种从[来源未提及]中提取的天然化合物，已在多种癌症中显示出强大的抗肿瘤作用。我们的研究旨在探索未折叠型mutp53（TP53和TP53）中的GOF突变，并阐明雷公藤内酯醇调节NSCLC中未折叠型mutp53蛋白降解的分子机制。
选择两种携带TP53和TP53突变的未折叠蛋白，以探索它们在NSCLC进展中的功能。将野生型TP53（TP53）、TP53或TP53基因转染至NCI-H1299细胞（TP53基因缺失），并用雷公藤内酯醇或溶剂处理。进行伤口愈合和Transwell实验以测量细胞迁移和侵袭能力。通过尾静脉注射突变细胞至BALB/c裸鼠构建肺转移模型，以评估雷公藤内酯醇对转移的影响。进行蛋白质印迹法、定量实时聚合酶链反应（qRT-PCR）、免疫沉淀和双荧光素酶报告基因实验，以探索相关分子机制。
我们的研究表明，雷公藤内酯醇处理降低了TP53水平，并且TP53突变增强了NCI-H1299细胞的侵袭和迁移能力。机制上，雷公藤内酯醇通过直接与热休克蛋白70（HSP70）相互作用，促进了由E3泛素连接酶小鼠双微体2（MDM2）介导的TP53和TP53蛋白的蛋白酶体降解。此外，通过上调HSP70转录，雷公藤内酯醇促进了GOF mutp53的蛋白降解。
我们的研究首次报道了雷公藤内酯醇调节TP53或TP53蛋白降解的机制，这为为表达未折叠型mutp53 GOF蛋白的NSCLC患者制定更好的治疗策略提供了新的见解。
