The role of ADAR1-mediated A-to-I RNA editing and non-editing functions in radioresistance of esophageal squamous cell carcinoma.

Abstract

e16099 Background: Radiotherapy (RT) is a critical treatment for esophageal squamous cell carcinoma (ESCC), but radioresistance poses a significant challenge. Previous research has indicated that Adar1-null tumors exhibit enhanced radiotherapy response in melanoma, suggesting that ADAR1 loss could improve RT efficacy. This study aims to investigate the role of ADAR1, including both its A-to-I RNA editing and non-editing functions, in the regulation of radioresistance in ESCC. Methods: ADAR1 expression was quantified in six ESCC tissue samples and adjacent normal tissues using qRT-PCR. A radiotherapy-resistant KYSE410 cell line was developed through serial irradiation. ADAR1 was downregulated in KYSE410 and TE-1 cell lines using siRNA, and recombinant plasmids were used to overexpress both the wild-type and edited forms of C16orf46. Cell proliferation and radiotherapy sensitivity were evaluated using the MTT assay and clonogenic assay. Pyroptosis was assessed using Annexin V staining, LDH release, and measurements of interleukin 1β (IL-1β) and interleukin 18 (IL-18). Transcriptome sequencing was performed on ADAR1-silenced cells to analyze changes in signaling pathways and RNA editing sites. Western blot experiments was used to detect alterations in the pyroptosis pathway and NF-κB signaling pathway. Results: ADAR1 expression was significantly higher in cancerous tissues compared to adjacent normal tissues (P < 0.05). Downregulation of ADAR1 significantly inhibited the proliferation of esophageal tumor cells, increased sensitivity to radiotherapy, and promoted radiotherapy-induced pyroptosis. Transcriptome analysis revealed that ADAR1 silencing activates the NF-κB signaling pathway. Inhibition of NF-κB with BAY 11-7082 reduced cell death following RT in ADAR1-knockdown KYSE410 cells, indicating a key role for NF-κB in pyroptosis triggered by ADAR1 loss. Western blot analysis demonstrated that ADAR1 activates cIAP2 in an RNA editing-independent manner, thereby regulating the NF-κB/GSDME pathway. A > G RNA editing events, particularly in the C16orf46 transcript, were identified in ADAR1-silenced cells, resulting in a Y335H amino acid change in the C16orf46 protein. The edited form of C16orf46 (edit-C16orf46) was found to enhance esophageal tumor cell proliferation and radiotherapy resistance. Conclusions: Our study underscores the potential of ADAR1 as a therapeutic target for overcoming radioresistance in ESCC through the modulation of pyroptosis, offering a new perspective for the development of targeted therapies.