Phosphoproteomics Uncovers Tumor Heterogeneity and Therapeutic Opportunities in Early-Stage Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly lethal cancer, however, its mechanisms of initiation, progression, and metastasis are not fully clarified. Although prior large-scale phosphoproteomic studies in broader HCC have mapped phosphorylation dynamics and identified subgroups consistent with proteomic subgroups, the heterogeneity of phosphoproteome in early-stage HCC, which may determine patient outcomes, remains unexplored. Here, we aimed to provide a phosphoproteomic characterization in early-stage HCC, addressing a critical gap in understanding the signaling pathways, kinases and tumor heterogeneity. By integrative interrogating the phosphoproteome and proteome in HCC, unlike previous studies focusing on mRNA and protein levels, we identified a marked reduction in phosphorylation regulation of EGFR-mediated canonical ERK/MAPK signaling, accompanied by elevation of atypical MAPK signaling in HCC. Classification based on the heterogeneity of the phosphoproteome in tumors identified three distinct phosphoproteomic subtypes, each characterized by unique phosphorylation signatures, clinical characteristics, cellular signaling pathways, and kinase activities. The most malignant phosphoproteomic subtype is characterized by epithelial mesenchymal transition (EMT) and hyperphosphorylation of SRSF3. Further validation shows that SRSF3 hyperphosphorylation, driven by the activation of SRPK1/CLKs and potential suppression of PP1 phosphatase, promotes metastasis with EMT, and cell proliferation. Furthermore, inhibitors targeting SRPK1 or CLKs effectively suppressed hyperphosphorylation of SRSF3-mediated migration and invasion in HCC cells. Our study uncovers the dysregulated phosphorylation landscapes, offers insights into HCC heterogeneity and metastatic potential at an early stage, and identifies phosphorylation-dependent regulatory mechanisms and key kinases with therapeutic relevance.