Bioinformatics identification of adenosylhomocysteinase (AHCY) as a regulator of ferroptosis in nasopharyngeal carcinoma cells via the Hippo-Yes-associated protein (Hippo-YAP) pathway

Background: Nasopharyngeal carcinoma (NPC) is a widely prevalent malignant tumor with a marked tendency toward metastasis and recurrence. Ferroptosis-related genes (FRGs) are critically involved in the pathogenesis of NPC. This study aims to employ bioinformatics analysis methods to identify key genes influencing the malignant progression of NPC and to investigate the regulatory mechanisms of these genes. Methods: Bulk RNA sequencing datasets (GSE53819, GSE61218, GSE64634, GSE12452, and GSE102349) and a single-cell RNA sequencing dataset (GSE150825) were downloaded from the Gene Expression Omnibus. Integrated bioinformatics analyses—including differential expression analysis, weighted gene co-expression network analysis, machine learning, and survival analysis—were conducted to identify key FRGs associated with NPC. Intracellular expression levels of adenosylhomocysteinase (AHCY), acyl-CoA synthetase long chain family member 4, glutathione peroxidase 4, macrophage stimulating 1, and Yes1 associated transcriptional regulator were detected through western blot. Cell viability was assessed using the cell counting kit-8; cell death was determined by flow cytometry; and cell migration and invasion were evaluated using wound-healing and Transwell assays. Intracellular reactive oxygen species levels were determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, and malondialdehyde and glutathione levels were detected using their respective detection kits. Results: Four key FRGs—isocitrate dehydrogenase 1, AHCY, endothelial PAS domain-containing protein 1 (EPAS1), and ARHGEF26 antisense RNA 1—were identified. Survival analysis of publicly available cohorts highlighted AHCY and EPAS1 as potential biomarkers for survival in patients with NPC. We selected AHCY for further in vitro mechanistic analysis and found it to be upregulated in NPC cell lines (NPC/HK1 and c666-1) relative to the nasopharyngeal epithelial cell line NP69. Functionally, AHCY knockdown in c666-1 cells inhibited cell migration, viability, and invasion, as well as suppressing the Hippo-Yes-associated protein (YAP) pathway, while promoting ferroptosis. Conversely, AHCY overexpression in NPC/HK1 cells enhanced cell migration, viability, and invasion, as well as promoting the Hippo-YAP pathway, while inhibiting ferroptosis. Treatment of AHCY-knockdown c666-1 cells with the ferroptosis inhibitor ferrostatin-1 enhanced cell viability, migration, and invasion, while treatment with the Hippo-YAP pathway agonist PY-60 promoted cell viability, migration, and invasion, while inhibiting ferroptosis. Conclusions: Our in vitro findings indicate that AHCY suppresses ferroptosis, partly via the Hippo-YAP pathway, thereby promoting the invasion and migration of NPC cells. Further in vivo and clinical studies are warranted to validate these findings.