2907 - Targeting O-GlcNAcylation Rearrangement and Radiotherapy Sensitivity in NSCLC via the Axis of OGA/HCF1/ RAD18

X. Wang^1,2, J. Yuan³, Y. Ma⁴, Z. Wang⁵, Z. Zhang¹, D. Chen⁶, and J. Yu⁷; ¹Shandong Cancer Hospital and Institute, Jinan, Shandong, China, ²Shandong University, Jinan, Shandong, China, ³Shandong Cancer Hospital and Institute, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, China, ⁴Shandong university, Jinan, Shandong, China, ⁵Shandong Second Medical University, Jinan, Shandong, China, ⁶Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ⁷Shandong Cancer Hospital and Institute, Jinan, China

Purpose/Objective(s): Metabolic reprogramming is one of the major features of cancer. And increasing evidence suggests that intermediary metabolites drive carcinogenesis through chemical posttranslational modifications (PTMs) that alter protein expression and function. Radiotherapy is an important mainstay for the treatment of Non-small-cell lung cancer (NSCLC). Our preliminary investigations have demonstrated that glucose uptake in tumor cells can be augmented and the glycosylation modification profile can be remodeled after radiotherapy. Considering the important roles of glucose metabolic reprogramming in radio-resistance, we aspire to potentiate the radiosensitivity by modulating the glycosylation modification levels in neoplastic cells. Materials/

Methods: Flow Cytometry and immunohistochemistry(IHC)were utilized to examine the glucose uptake and glycosylation levels of tumor cells in clinical and animal specimens after radiation therapy; The shOGA cell line was established and Flow Cytometry was used to measure the apoptosis rate and cell cycle after radiation therapy. Immunofluorescence and western blot were employed to assess DNA damage. DNA damage reporting system was used to evaluate DNA repair levels. Liquid chromatography–mass spectrometry (LC-MS/MS) Analysis was employed to identify key proteins and glycosylation sites. The OGA-specific inhibitor Thiamet-G (TMG) was also used for subcutaneous tumorigenesis experiments in nude mice.
Results: Our research revealed that the capacity of glucose uptake exhibit increased after radiotherapy, while O-GlcNAc glycosylation levels paradoxically decrease in NSCLC specimens. It is noteworthy that the sole enzyme responsible for deglycosylation, O-GlcNAcase (OGA), exerts a significant impact on the outcomes of radiotherapy. The strategic targeting of OGA amplifies the radiosensitivity of tumor cells. LC-MS/MS Analysis disclosed that Transcription factor HCF1, when glycosylated at the T490 site, reduces its own nuclear entry. Proteomic analysis has underscored a marked reduction in the expression of the HR repair gene RAD18 subsequent to HCF1 targeting. Targeting OGA increases HCF1 glycosylation, further curtailing its nuclear entry and leading to decreased RAD18 expression,which leads to an escalation in DNA damage, an upsurge in apoptosis rates, and a decline in proliferation among tumor cells post-radiotherapy. NSCLC radiotherapy sensitivity is ultimately enhanced. The amalgamation of the OGA-specific inhibitor TMG with radiotherapy significantly curtails the growth of NSCLC tumors in nude mice.
Conclusion: Our study delineates the axis of the OGA/HCF1/RAD18 after radiotherapy. The glycosylation level of HCF1 could serve as a significant predictor for the efficacy of radiation therapy, while targeting OGA may provide a new means to sensitize clinical radiation therapy.