Single-cell multi-omics reveals DUSP9 as a key regulator of cancer stemness and a potential therapeutic target in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) exhibits pronounced intratumoral heterogeneity largely driven by cancer stem cells (CSCs). However, the molecular regulators that connect CSC stemness to tumor progression remain incompletely understood. METHODS: We performed an integrative analysis combining bulk RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), and spatial transcriptomics to identify regulators potentially associated with CSC-related stemness in HCC. High-dimensional co-expression network analysis and regulatory network inference were applied to delineate stemness-associated modules, while cell–cell interaction analyses explored the tumor microenvironment. Functional validation was conducted through in vitro assays, and candidate compounds were identified using multi-platform drug screening followed by experimental testing. RESULTS: Dual-specificity phosphatase 9 (DUSP9) was identified as a potential regulator enriched in a malignant subpopulation characterized by elevated ERK activation, oxidative metabolism, and progenitor-like features. Co-expression and regulatory network analyses revealed a DUSP9⁺-specific module enriched for stemness- and invasion-related genes, with Forkhead Box Protein O3 (FOXO3) identified as a core downstream effector. Functional assays confirmed that DUSP9 enhances stemness through the ERK–FOXO3 axis. Spatial transcriptomics demonstrated that DUSP9⁺ cells localized within immunosuppressive niches enriched with cancer-associated fibroblasts (CAFs). Moreover, a candidate compound, AKOS000434153, inhibited HCC cell proliferation, migration, and sphere formation in vitro, accompanied by increased p-ERK and decreased p-FOXO3 levels. CONCLUSIONS: This study suggests that DUSP9 may serve as a potential regulator linking CSC-associated stemness with tumor progression in HCC. By integrating multi-omics datasets, our findings provide a comprehensive view of DUSP9-associated regulatory networks and highlight its promise as a biomarker for stemness-driven hepatocarcinogenesis. Further in vivo validation will be necessary to confirm these regulatory relationships.