Integrative Phosphoproteomics Uncovers Tumor Heterogeneity and SRSF3 Phosphoryaltion-Driven Metastasis in Early-Stage Hepatocellular Carcinoma

Abstract Mechanisms driving progression and metastasis in early-stage hepatocellular carcinoma (HCC) remain poorly clarified. Although phosphoproteomics has been applied to broader HCC cohorts, the heterogeneity of phosphorylation signaling in early-stage HCC remains unexplored. Here, we conducted integrated phosphoproteomic and proteomic analysis on early-stage HCC tumors and paired non-tumor tissues. By comparing tumor and non-tumor tissues, we identified markedly reduced phosphorylation of EGFR-mediated canonical ERK/MAPK signaling, alongside elevated atypical MAPK signaling in HCC. To further explore the signaling heterogeneity within tumors, we applied nonnegative matrix factorization (NMF) and consensus-clustering, and identified three distinct phosphoproteomic subtypes with unique phosphorylation signatures and clinical characteristics. Interrogating the most malignant subtype, we found markedly high abundance of phosphorylation of mRNA splicing regulators, and an enrichment of epithelial-mesenchymal transition (EMT) pathways in the proteome, with phosphorylation of the splicing factor SRSF3 emerging as a critical feature. Mechanistically, we show that SRSF3 hyperphosphorylation, driven by SRPK1/CLKs and potential PP1 phosphatase suppression, promotes metastasis and proliferation. Critically, SRPK1/CLKs inhibitors effectively suppressed SRSF3 hyperphosphorylation-mediated migration and invasion of HCC cells. Our study uncovers a new dimension of signaling-driven tumor heterogeneity in early-stage HCC and identifies the SRPK1/CLKs-SRSF3 hyperphosphorylation axis as a key metastatic driver, suggesting a potential therapeutic target for early intervention in HCC. Competing Interest Statement The authors have declared no competing interest. Footnotes Added new validation results, including external cohort validation and experiment validation and updates.