O-GlcNAcylation of PRDX1 and Radiation Resistance in Non-Small Cell Lung Cancer via Suppressing TRIM21 Mediated Ubiquitination

Abstract

Purpose/Objective(s)

The molecular mechanism underlying radiation resistance in non-small cell lung cancer (NSCLC) is not well understood. This study aimed to investigate the potential role of Peroxiredoxin-1 (PRDX1) O-GlcNAcylation and its specific regulatory mechanism in NSCLC radiotherapy resistance.

Materials/Methods

To establish radiotherapy-resistant NSCLC cell lines, multiple radiotherapy treatments were administered. The radiosensitivity of cells was evaluated using colony formation assay, western blot, and immunofluorescence. Metabolic changes in response to irradiation challenge were comprehensively analyzed through a metabolomics assay comparing radioresistant NSCLC cell lines (A549R or LLCR) with their parental cell lines (A549 or LLC). The correlation between PRDX1 and O-GlcNAcylation expression was investigated using a tissue microarray (TMA) of human NSCLC tissue (n = 90). Co-immunoprecipitation coupled with mass spectrometry (Co-IP-MS) was employed to identify proteins regulating PRDX1 expression. The functional role of PRDX1 O-GlyNAcylation in NSCLC radiotherapy resistance was validated through multicolor immunofluorescence staining in tissue samples from pre-radiotherapy biopsies (n = 79). Lastly, a short peptide was designed to target PRDX1 glycosylation both in vitro and in vivo.

Results

Metabolomic analyses revealed a shift in metabolic flux to the hexosamine biosynthesis pathway (HBP) with higher levels of UDP-GlcNAc in radioresistant NSCLC cells, a substrate for O-GlcNAcylation. Subsequently, O-GlcNAcylation and PRDX1 were found to be significantly up-regulated in human NSCLC tissues compared to adjacent benign tissues. O-GlcNAcylation of PRDX1, mediated by OGT, was identified as critical for radioresistance in NSCLC. Specifically, PRDX1 was O-GlcNAcylated at the conserved serine 2/3, and radiation-resistant NSCLC cells exhibited elevated levels of O-GlcNAcylation of PRDX1. This post-translational modification protected the protein stability of PRDX1, while TRIM21 was discovered as an E3 ubiquitin ligase promoting PRDX1 degradation, with its interaction affected by O-GlcNAcylation. Conversely, pharmacological reduction in the O-GlcNAcylation of PRDX1 (OSMI treatment) enhanced the radiotherapy sensitivity of NSCLC. Notably, multicolor immunofluorescence staining of tissue samples from pre-radiotherapy biopsies (n = 79) confirmed a positive correlation between PRDX1 O-GlyNAcylation levels and OGT expression, as well as radiotherapy tolerance, and a negative correlation with TRIM21 expression levels.

Conclusion

Our findings highlight the regulatory role of PRDX1 in NSCLC radiation resistance, emphasizing how O-GlcNAcylation stabilizes and shields PRDX1 from TRIM21-mediated ubiquitination. These results uncover a potential mechanism of NSCLC radiation resistance and propose a promising therapeutic approach for NSCLC treatment.