ATP6V1D drives hepatocellular carcinoma stemness and progression via both lysosome acidification-dependent and -independent mechanisms.

Metabolic reprogramming is pivotal in cancer stem cell (CSC) self-renewal. However, the intricate regulatory mechanisms governing the crosstalk between metabolic reprogramming and liver CSCs remain elusive. Here, using a metabolic CRISPR-Cas9 knockout screen, we identify ATP6V1D, a subunit of the vacuolar-type H⁺-translocating ATPase (V-ATPase), as a key metabolic regulator of hepatocellular carcinoma (HCC) stemness. Elevated ATP6V1D expression correlates with poor clinical outcomes in HCC patients. ATP6V1D knockdown inhibits HCC stemness and malignant progression both in vitro and in vivo. Mechanistically, ATP6V1D enhances HCC stemness and progression by maintaining macroautophagic/autophagic flux. Specifically, ATP6V1D not only promotes lysosomal acidification, but also enhances the interaction between CHMP4B and IST1 to foster ESCRT-III complex assembly, thereby facilitating autophagosome-lysosome fusion to maintain autophagic flux. Moreover, silencing CHMP4B or IST1 attenuates HCC stemness and progression. Notably, low-dose bafilomycin A₁ targeting the V-ATPase complex shows promise as a potential therapeutic strategy for HCC. In conclusion, our study highlights the critical role of ATP6V1D in driving HCC stemness and progression via the autophagy-lysosomal pathway, providing novel therapeutic targets and approaches for HCC treatment.