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

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. 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. 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. 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.