STK31 enhances gallbladder cancer development by modulating DNA replication machinery.

Gallbladder cancer (GBC) is a highly aggressive and insidious malignancy with a poor prognosis, primarily due to the lack of specific therapeutic targets and early diagnostic biomarkers. Therefore, identifying novel oncogenes involved in GBC and exploring targeted therapeutic strategies against them are of particular importance. STK31 is a cancer/testis antigen, but its biological function and mechanistic role in GBC remain unclear. This study investigates how STK31 influences GBC. STK31 expression in GBC and gastrointestinal tumors were analyzed using collected clinical samples and TCGA database respectively. Its pro-tumorigenic functions were then confirmed by in vivo and in vitro assays, while downstream regulatory mechanisms were uncovered via multi-platform proteomic strategies. Molecular docking was employed to screen 1,008 ChEMBL small molecules post STK31 functional validation. Significant upregulation of STK31 expression was observed in both GBC and gastrointestinal malignancies through our comprehensive analysis. STK31 depletion decreased GBC cell proliferation, migration and induced G1/S phase arrest and cellular polyploidization. Gene Set Enrichment Analysis (GSEA) demonstrated that STK31 functionally correlates with core biological processes including DNA Replication, Cell Cycle and Extracellular Matrix (ECM) Receptor Interaction. STK31 directly binds to both c-Myc and FOXP1, thereby regulating MCM and ITG family proteins to exert its biological function. In addition, STK31 influences the phosphorylation of c-Myc through its kinase activity. Atovaquone identified through molecular docking screening, binds STK31 and reduces its expression and kinase activity, demonstrating anti-tumor effects against GBC both in vitro and in vivo. We demonstrate that STK31 facilitates GBC progression through c-Myc/FOXP1-mediated DNA replication dysregulation. Its inhibition triggers lethal polyploidization, with ATO identified as a potential STK31-targeting drug candidate.