Nucleolin shields PTBP1 from autophagic degradation to sustain gemcitabine resistance in bladder cancer.

Chemoresistance is a principal cause of treatment failure in bladder cancer (BLCA), yet the mechanisms that actively maintain resistance remain poorly defined. Gemcitabine (GEM) resistance is classically attributed to impaired drug uptake via human equilibrative nucleoside transporter 1 (hENT1) and activation of pro-survival cell signaling pathways. However, these mechanisms do not explain how resistance is stably sustained over time. Here we uncover an autophagy-dependent pathway that maintains GEM chemoresistance, in which nucleolin (NCL) shields the RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) from microtubule-associated protein 1 light chain 3 beta (LC3B)-mediated autophagic degradation. Consequently, NCL-mediated stabilization of PTBP1 maintains glycolytic reprogramming in GEM-resistant BLCA cells, whereas NCL additionally promotes anti-apoptotic signaling. Notably, PTBP1 is enriched and preserved as a functional and surface-accessible dependency through NCL-mediated evasion of autophagic degradation. To exploit this vulnerability, we developed a monovalent dual-target DNA aptamer, SWL-3, that undergoes preferential internalization through surface-accessible PTBP1 while also binding NCL, enabling selective recognition of xenograft tumors in vivo and clinical BLCA specimens in vitro. By bypassing classical hENT1-dependent drug uptake, site-specifically conjugated SWL-3G achieves efficient intracellular drug accumulation and selectively reduces the viability of GEM-resistant, PTBP1-enriched cells. Together, these findings reveal NCL-mediated protein shielding from autophagic degradation as a mechanism that sustains GEM resistance and establish these resistance-maintaining dependencies as actionable targets for precision therapy in refractory disease.