RHBDD1 induces cell cycle arrest in TP53-mutant NSCLC cells by promoting endoplasmic reticulum-associated degradation of p53 and DNA-PKcs.

Background: RHBDD1 is a tumor-promoting protein that enhances endoplasmic reticulum-associated degradation (ERAD). This study aimed to explore its effects on non-small cell lung cancer (NSCLC) cells.

Methods: The potential roles of RHBDD1 in NSCLC were predicted through bioinformatics analysis. The predicted regulation of p53/DNA-PKcs signaling by RHBDD1-associated ERAD was further validated in TP53 wild-type and mutant NSCLC cells.

Results: Bioinformatics analysis revealed that in the RHBDD1-highly expressed NSCLC samples, cell cycle was significantly downregulated, protein processing in endoplasmic reticulum (ER) was significantly upregulated, and p53 signaling pathway was inhibited. TP53 and PRKDC were identified as hub genes. Inhibition of cell cycle and activation of protein processing in ER were specifically enriched in TP53-mutant, RHBDD1-highly expressing samples, but not in TP53 wild-type ones. The protein levels of p53 and DNA-PKcs (encoded by the PRKDC gene) were more significantly altered by the DNA-PKcs inhibitor STL127705 and sh-RHBDD1 in NCI-H596 cells compared to A549 cells. In NCI-H596 cells transfected with pcDNA-RHBDD1, the reduced levels of DNA-PKcs and p53 were restored by co-treatment with p53 activators HBX41108 or Nutlin-3a. Cell cycle progression in NCI-H596 cells was significantly arrested at the S phase in both sh-RHBDD1 and pcDNA-RHBDD1 transfected groups. Both interventions led to decreased cell viability, which was reversed by co-treatment with the DNA-PKcs inhibitor STL127705 and further enhanced by Nutlin-3a. Increased levels of DNA-PKcs and p53, along with a modest reduction in ER stress markers, were observed in the sh-RHBDD1 group, whereas opposite trends were seen in the pcDNA-RHBDD1 group. Apoptosis was obviously inhibited in pcDNA-RHBDD1 transfected NCI-H596 cells, but not in transfected A549 cells. Co-localization of p53 with RHBDD1 and DNA-PKcs was prominently observed in TP53-mutant NSCLC cells, but not in wild-type cells.

Conclusion: RHBDD1 arrests the cell cycle in TP53-mutant NSCLC cells through ERAD-dependent downregulation of p53 and DNA-PKcs. However, cell survival may be enhanced due to the inhibition of DNA-PKcs-regulated apoptosis.