Abstract

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a complex tumor ecosystem that contributes to its progression. Deubiquitinases (DUBs) are vital regulators in cancer. However, the overall activity of DUBs and their role in driving PDAC progression within immune microenvironment remain largely unknown. Methods We employed an integrative multi-omics strategy combining machine learning (ML) on bulk transcriptomic data, single-cell RNA sequencing and spatial transcriptomic profiling. We applied Coxnet and Fuzzy SVM for prognostic modeling, inferCNV for malignant cell identification, SCENIC for transcription factor regulon analysis, LIANA + for inferring inter−cellular communication networks and cell2location for spatial deconvolution. USP54 expression was detected by real-time quantitative PCR, western blotting and immunohistochemistry. USP54 function was validated through in vitro and in vivo assays. Results ML-based pathway analysis revealed post−translational modification as a major prognostic category, within which elevated DUBs activity emerged as an independent adverse prognostic factor. At the single−cell level, USP54 was upregulated along the trajectory of malignant ductal cells and correlated with an inflamed tumor microenvironment. Cell−cell communication analysis predicted signaling from monocytes/macrophages to tumor cells via the THBS1−integrin ligand−receptor pair. This immune−derived signaling potentially converged on KLF5−positive tumor cells, with KLF5 identified as a putative transcriptional activator of USP54. Spatial transcriptomics validated the co−localization of USP54 expression, elevated DUB activity, and KRAS signaling within specific tumor niches adjacent to THBS1−enriched immune regions. High USP54 expression was frequently observed in PDAC tissues and associated with poor patient survival. More importantly, in both BxPC-3 and PANC-1 cell lines, USP54 knockdown suppressed cell proliferation and met