Multi-omics analysis identifies CKLF as a promoter for HCC progression by regulating the AKT, ERK pathways, and infiltrating immune cells

Background Hepatocellular carcinoma (HCC) is a highly malignant disease with limited therapeutic options, highlighting an urgent need to identify novel molecular regulators and potential intervention points. Methods We normalized single-cell data using SCTransform and removed batch effects with Harmony. Cell-cell communication was predicted using CellChat, and spatial cell-type mapping was performed using RCTD algorithms. For functional characterization, we conducted in vitro experiments including CKLF silencing via siRNA, followed by proliferation assays (CCK-8),Immunohistochemistry, wound healing, and colony formation assays. To verify the involved pathways, we performed complementary studies using constitutively active Ras mutants. Results CKLF was found to be upregulated in HCC tissues, and high CKLF levels correlated significantly with advanced pathological stages, distinct molecular classes, and poor clinical outcomes. Single-cell and spatial analyses revealed that CKLF is highly expressed not only in the hepatocellular compartment but also in infiltrated immune cells with a cytotoxic signature (CD8+ T cells and M1 macrophages), suggesting an impact on both tumor cells and immunity. CKLF knockdown significantly reduced tumor proliferation, motility, and colony-forming ability in HCC models in vitro. These effects were at least partly rescued by persistent Ras pathway activation, confirming that CKLF acts upstream to regulate the AKT/ERK pathway. Conclusions These results demonstrate that CKLF is a critical node linking gene copy number changes, oncogenic signaling pathways, and immunophenotypic alterations in hepatocellular carcinoma. Thus, CKLF represents a promising biomarker and therapeutic target for providing individualized therapies to HCC patients.