Upregulation of CDKN2B in 5-fluorouracil-resistant colorectal cancer cells: a pilot study of its dual role and potential clinical relevance

Abstract Background Resistance to 5-fluorouracil (5-FU) is almost inevitable during chemotherapy for advanced colorectal cancer (CRC). Early identification of resistant patients and overcoming resistance remain major clinical challenges. Objective This study aimed to investigate the role of CDKN2B in 5-FU resistance in CRC and to explore its potential as a predictive biomarker. Methods The 5-FU-resistant cell line HT-29/5FU-R was established by concentration-gradient induction. Differentially expressed genes were screened by transcriptome sequencing, and CDKN2B expression was verified by qPCR and Western blot. The expression, methylation status, prognostic value, and association with 5-FU response of CDKN2B were analyzed using the TCGA-COAD cohort (286 tumors and 41 normal tissues) and two GEO chemotherapy cohorts (GSE28702, n = 83; GSE72970, n = 124). CD8 + T-cell infiltration was evaluated using TIMER2.0. Results A total of 3,293 differentially expressed genes were identified (1,780 upregulated and 1,513 downregulated). CDKN2B was upregulated in resistant cells (fold change 2.31, p = 0.0006) and confirmed at the mRNA and protein levels. In TCGA-COAD, CDKN2B was significantly downregulated in tumor tissues compared with normal tissues ( p = 1.3×10⁻ 23 ), and its expression was not associated with overall survival ( p = 0.26). In GSE28702, non-responders showed higher CDKN2B expression, an association that reached significance in primary-tumor biopsies ( p = 0.048); this association was not replicated in the validation cohort GSE72970. CDKN2B expression was negatively correlated with promoter methylation (r = -0.245, p < 0.001) and modestly associated with CD8 + T-cell infiltration. Conclusion CDKN2B is upregulated in 5-FU-resistant CRC and is epigenetically regulated by promoter methylation. CDKN2B is not a prognostic marker, but it is a candidate predictive biomarker of fluoropyrimidine response that requires prospective validation. CDKN2B may help identify patients who are unlikely to benefit from 5-FU-based chemotherapy and guide the selection of later-line treatment, and it may also serve as a candidate target for overcoming 5-FU resistance.