CUB domain-containing protein 1 signaling dysregulates gemcitabine metabolism contributing to therapeutic resistance in T24 cells.

Gemcitabine is commonly used in the standard first-line treatment of urothelial carcinoma (UC); however, the emergence of drug resistance significantly limits its clinical benefit. The present study aims to investigate the role of CUB domain-containing protein 1 (CDCP1) in mediating resistance to gemcitabine in UC cells. Gemcitabine-resistant T24 (T24-GR) cells exhibited downregulation of human equilibrative nucleoside transporter 1 and upregulation of cytidine deaminase, key regulators of gemcitabine metabolism, as well as increased CDCP1 expression. Notably, silencing CDCP1 reversed these resistance-associated expression patterns. Mechanistically, T24-GR cells displayed elevated expression of CDCP1 and increased phosphorylation of c-Src and PKCδ, indicating activation of downstream survival signaling. Overexpression of CDCP1 in T24-CD cells activated similar pathways and modulated regulators of gemcitabine metabolism. In contrast, CRISPR/Cas9-mediated knockout of CDCP1 in T24-CDKO cells suppressed c-Src/PKCδ signaling and increased sensitivity to gemcitabine-induced cytotoxicity. Using flow cytometry, we observed that treatment with gemcitabine induced apoptosis in parental T24 cells, as indicated by an increase in the sub-G1 population. In contrast, T24-GR and T24-CD cells showed minimal sub-G1 accumulation, suggesting resistance to gemcitabine-induced apoptosis. Western blot analysis revealed decreased levels of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in T24-GR and T24-CD cells following gemcitabine exposure, whereas these markers were upregulated in parental T24 and T24-CDKO cells. Furthermore, the knockdown of CDCP1 and the utilization of c-Src/PKCδ signaling inhibitors in T24-GR cells led to the restoration of sensitivity to gemcitabine. By suppressing apoptosis and altering drug metabolism pathways, highlighting CDCP1 as a potential therapeutic target for overcoming gemcitabine resistance in UC.