NFYA transcriptionally activates GPX4 inhibiting ferroptosis in colorectal cancer.

[BACKGROUND] Nuclear transcription factor Y subunit α (NFYA) has been reported to play functional roles in a range of malignancies, while its precise mechanistic function in the context of colorectal cancerer (CRC) progression has yet to be established.

[METHODS] TIMER2, UALCAN, and Kaplan-Meier Plotter databases were leveraged to evaluate the expression of NFYA and to gauge its prognostic relevance in CRC. Determine NFYA and glutathione peroxidase 4 (GPX4) expression in cell lines (NCM460, DLD1 and HCT116) and clinical samples using Quantitative Real-time PCR (qPCR) and western immunoblotting assay. Assessment of cell viability, proliferation, and migration involved MTT, EdU staining assay, colony formation, and flow cytometry. Malondialdehyde (MDA) content and lipid reactive oxygen species (ROS) levels were assessed using specialized kits. Mitochondrial ultrastructural changes were observed using transmission electron microscopy (TEM). The transcriptional regulation of GPX4 by NFYA was investigated through Luciferase reporter assays and Chromatin Immunoprecipitation Assay (ChIP) experiments.

[RESULTS] Higher levels of NFYA were expressed in CRC cells and clinical samples, and the upregulation of this target was associated with a worse prognostic outcome for patients with CRC. In vitro,the inhibition of NFYA expression impaired CRC cell proliferative and migratory activity. NFYA was found to function in part by binding to the GPX4 promoter and activating transcription, leading to an increase in GPX4 levels and consequent reductions in lipid peroxide levels and ferroptosis. NFYA binds to the promoter region of GPX4, regulating the transcription of GPX4. In the recovery experiment, inhibition of NFYA-induced ferroptosis in cells was reversed by GPX4. Correspondingly, the ferroptosis caused by GPX4 suppression can be reversed by NFYA.

[CONCLUSION] NFYA is thus a promising target for therapies aimed at treating CRC. NFYA is a key regulator of ferroptosis resistance in CRC while also providing direct mechanistic insights into the oncogenic function that this protein plays in these tumor cells. NFYA/GPX4 may be one of the underlying reasons for malignant growth of CRC cells.