SRS85 Patey Prize Entrant - hENT1 inhibition as a strategy to overcome fluoropyrimidine resistance in colorectal cancer

Abstract Background Colorectal cancer (CRC) outcomes remain limited through resistance to fluoropyrimidine-based therapy. Human equilibrative nucleoside transporter 1 (hENT1) regulates nucleoside analogue uptake and may drive chemoresistance through altered expression. To date, its role in CRC remains poorly defined. Methods Paired tumour and normal tissues from a clinical CRC cohort were analysed for hENT1 expression and correlation with outcome. Results were then validated using public transcriptomic datasets. CRC cell lines were profiled at transcriptomic and proteomic levels, and selective lentiviral knockdown models were established and validated. Functional assays assessed proliferation, cell adhesion and barrier function, and sensitivity to 5-FU and capecitabine, with and without pharmacological hENT1 inhibition. Results High hENT1 expression correlated with adverse survival outcomes, including reduced disease-free and metastasis-free survival, findings reproduced in external datasets. In vitro, validated hENT1 knockdown reduced proliferative capacity and demonstrated impaired adhesion and spreading, consistent with cellular stress. Functionally, hENT1 knockdown increased sensitivity to both 5-FU and capecitabine. Pharmacological inhibition with NBMPR further enhanced cytotoxicity in both wildtype and knockdown cells, confirming that hENT1 activity directly modulates fluoropyrimidine response. Conclusions hENT1 functions as both a prognostic and predictive biomarker in CRC. Functional data demonstrate that knockdown reduces proliferation and adhesion while sensitising cells to fluoropyrimidines, with pharmacological inhibition producing the greatest enhancement of efficacy. These findings highlight hENT1's potential as a prognostic biomarker and a translational therapeutic target in CRC.