Shikonin inhibits bladder cancer progression by targeting deoxyribonuclease 2 to suppress reticulon 3-dependent endoplasmic reticulum autophagy.

BACKGROUND: Bladder cancer (BCa) is a prevalent urinary malignancy with unmet clinical therapeutic needs. Shikonin, a natural anti-tumor compound, exerts anti-BCa activity, yet its direct molecular target and underlying mechanism remain undefined. PURPOSE: This study aimed to identify the direct target of shikonin in BCa and to elucidate the mechanism by which shikonin suppresses tumor progression. METHODS: The anti-BCa effects of shikonin were assessed in vitro and in vivo. Drug affinity responsive target stability (DARTS) combined with 4D quantitative proteomics identified shikonin's direct target, which was validated by molecular docking and cellular thermal shift assay (CETSA). Gain- and loss-of-function experiments and ER-phagy-related assays elucidated the underlying molecular mechanism. RESULTS: Shikonin inhibited BCa cell proliferation, migration, invasion and induced senescence in vitro, and suppressed tumor growth in vivo with no obvious toxicity. Deoxyribonuclease 2 (DNASE2) was identified as shikonin's direct target; shikonin promoted DNASE2 ubiquitination and degradation without altering its mRNA level. DNASE2 was highly expressed in BCa and correlated with poor prognosis, and its knockdown mimicked and enhanced shikonin's anti-BCa effects. Mechanistically, shikonin targeted DNASE2 to downregulate Reticulon 3 (RTN3), thereby inhibiting RTN3-dependent endoplasmic reticulum autophagy (ER-phagy) in BCa. CONCLUSION: This study first identifies DNASE2 as the direct target of shikonin in BCa, and reveals that shikonin exerts anti-BCa activity by promoting DNASE2 ubiquitination/degradation to inhibit the DNASE2/RTN3 axis-mediated ER-phagy. The DNASE2/RTN3 axis is a novel ER-phagy regulatory node in BCa, providing a potential therapeutic target for BCa treatment.