Enfortumab vedotin promotes PD-L1 expression in urothelial carcinoma via NF-κB and STAT3 pathways highlighting mechanisms of immune evasion and potential for combination therapy.

Background: PD-1/PD-L1 inhibitors have revolutionized urothelial carcinoma (UC) treatment; however, the effects of prior or concurrent therapies on PD-L1 regulation remain unclear. This study investigates whether enfortumab vedotin (EV), a nectin-4-targeting antibody-drug conjugate, modulates PD-L1 expression in UC cells, and explores the underlying molecular pathways.

Methods: UC cell lines RT4 (high nectin-4 expression) and T24 (low nectin-4 expression) were treated with EV (10 µg/ml) for 6, 12, or 24 h, followed by a 48-hour drug-free period. Protein and mRNA expression levels of PD-L1, NF-κB, and STAT3 were quantified using western blotting and qRT-PCR.

Results: EV treatment upregulated PD-L1, NF-κB, and STAT3 in a time-dependent manner, with a more pronounced effect observed in RT4 than in T24 cells. PD-L1 protein levels increased 0.761-fold (12 h) and 2.399-fold (24 h) in RT4, whereas T24 showed a decrease (0.517-fold at 12 h) or minimal change (0.006-fold at 24 h). NF-κB expression increased 64.42-fold (12 h) and 97.03-fold (24 h) in RT4, compared to 1.251-fold (12 h) and 1.210-fold (24 h) in T24. STAT3 levels rose 2.334-fold (12 h) and 2.844-fold (24 h) in RT4, whereas T24 showed increases of 1.620-fold (12 h) and 1.379-fold (24 h). At the mRNA level (6 h post-treatment), PD-L1, NF-κB, and STAT3 were upregulated by 1.228-, 1.332-, and 1.225-fold, respectively, in RT4 cells.

Conclusion: EV is associated with increased PD-L1 expression, along with upregulation of NF-κB and STAT3, suggesting a mechanistic link that may contribute to immune modulation in nectin-4-high bladder cancer cells. These findings highlight the need for combination strategies integrating EV with PD-1/PD-L1 inhibitors to optimize therapeutic outcomes in UC.