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.