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

Hepatocellular carcinoma (HCC) is the third deadliest cancer in the world, however, the mechanisms of genesis, development and recurrence of HCC are not fully clarified yet. Previous studies on phosphoproteome in large-scale of HCC merely investigated the specific regulation on protein phosphorylation levels and identified subgroups consistent with proteomic subgroups. Here, we systematically interrogated the phosphoproteome of early-stage HCC and uncovered the dysregulated signaling pathways and kinases. Unlike previous studies on mRNA and protein levels, we observed a significant decline in phosphorylation regulation of EGFR-mediated canonical ERK/MAPK signaling alongside elevation of atypical MAPK signaling. Classification based on the heterogeneity of the phosphoproteome in tumor identified three phosphoproteomic subtypes, each characterized by distinct phosphorylation signatures, clinical characteristics, cellular signaling pathways and kinase activities. Further validation revealed hyperphosphorylation of SRSF3, one signature in the most malignant subtype, promote proliferation and metastasis of HCC cells. The mechanism of which was further demonstrated to be coordinated by activation of SRPK1/CLKs and potential suppression of PP1 phosphatase via phosphorylation of PPP1R7. Furthermore, inhibitors targeting SRPK1 or CLKs effectively suppressed hyperphosphorylation of SRSF3 mediated migration and invasion in HCC. Our study uncover the dysregulated phosphorylation signatures, offer new insights into the tumor heterogeneity, and identify phosphorylation regulating mechanism and corresponding key kinases with therapeutic value in early-stage HCC, which also spotlight the importance of protein phosphorylation in cancer progression.