Triptolide promotes degradation of the unfolded gain-of-function Tp53R175H/Y220C mutant protein by initiating heat shock protein 70 transcription in non-small cell lung cancer

BACKGROUND: 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(R175H) and TP53(Y220C)) 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 Tripterygium wilfordii that has shown a strong antitumor effect in a variety of cancers. Our study aimed to explore the GOF mutations in unfolded mutp53 (TP53(R175H) and TP53(Y220C)) and to clarify the molecular mechanisms by which triptolide regulates the degradation of unfolded mutp53 proteins in NSCLC. METHODS: Two unfolded proteins harboring TP53(R175H) and TP53(Y220C) mutations were selected to explore their functions in NSCLC progression. NCI-H1299 cells (TP53-null) were transfected with wild-type TP53 (TP53(WT)), TP53(R175H), or TP53(Y220C) genes and treated with triptolide or a vehicle. Wound healing and transwell assays were performed to measure cell migration and invasion in vitro. 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 in vivo. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunoprecipitation, and dual-luciferase reporter assays were performed to explore the relevant molecular mechanisms. RESULTS: Our study revealed that triptolide treatment reduced TP53(R175H) levels and that the TP53(Y220C) mutation enhanced the invasion and migration of NCI-H1299 cells. Mechanistically, triptolide promoted TP53(R175H) and TP53(Y220C) 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. CONCLUSIONS: Our study reports, for the first time, the mechanism underlying triptolide-regulated protein degradation of TP53(R175H) or TP53(Y220C), which offers new insight into developing a better therapeutic strategy for patients with NSCLC who express the unfolded mutp53 GOF protein.