Cryptotanshinone differentially induces cell death in ATP6V0D1-deficient pancreatic cancer cells.

Aim: Dysregulation of tumor-suppressive pathways can lead to constitutive activation of multiple oncogenic signaling cascades. Such overactivation makes cancer cells highly dependent on these pathways, creating potential therapeutic vulnerabilities. Based on our previous findings and current data, genetic knockout of ATPase H(+) transporting V0 subunit D1 (ATP6V0D1) - a key mediator of alkaliptosis - induces hyperactivation of oncogenic pathways, including signal transducer and activator of transcription 3 (STAT3)-mediated lysosomal pH regulation and AKT serine/threonine kinase (AKT) signaling. It also alters cellular responses to cryptotanshinone therapy. This study aimed to investigate how ATP6V0D1 deficiency reshapes oncogenic signaling networks and cellular heterogeneity in pancreatic ductal adenocarcinoma (PDAC), while evaluating therapeutic strategies that exploit alkaliptosis-related vulnerabilities. Methods: ATP6V0D1-deficient SW1990 and MIAPaCa2 cells were generated via gene knockdown. Cell viability and death following various treatments were assessed using CCK-8 and propidium iodide assays. Transcriptomic analysis was conducted to identify feedback signaling pathways, while Western blotting was used to measure expression of signaling proteins. Macropinocytosis was evaluated by TRITC-dextran uptake. Additionally, The Cancer Dependency Map (DepMap) database was analyzed to explore background differences between SW1990 and MIAPaCa2 cells. Results: ATP6V0D1 deletion led to overactivation of STAT3-mediated lysosomal pH regulation and AKT signaling; inhibition of these pathways restored alkaliptosis. Notably, cryptotanshinone selectively induced cell death in ATP6V0D1-deficient MIAPaCa2 cells but not SW1990 cells. Resistance in SW1990 cells was mediated by FGFR2 upregulation, which was reversed upon FGFR2 inhibition. Conclusion: ATP6V0D1 deficiency drives PDAC progression via dual mechanisms: compensatory oncogenic signaling (STAT3/AKT) and FGFR2-mediated cellular heterogeneity. While targeting these pathways may offer therapeutic potential, tumor heterogeneity remains a major clinical challenge.