Comprehensive phosphoproteomic profiling reveals sex-specific regulatory mechanisms in HrasG12V-driven hepatocarcinogenesis.

OBJECTIVE: To identify common and sex-specific phosphorylation dynamics during hepatocarcinogenesis in HrasG12V transgenic mice (Hras-Tg). METHODS: We constructed a phosphoproteomic database using male/female (M/F) tumor (T), precancerous (P), and wild-type liver tissues (W) from Hras-Tg, validated via parallel reaction monitoring (PRM). Comparative analysis and hierarchical clustering were employed to delineate shared and sex-stratified phosphorylation signatures in hepatocellular carcinoma (HCC) development. RESULTS: PRM-validated phosphoproteomic profiling quantified 5,410 phosphorylation sites across 2,427 proteins. Analysis of common features revealed nuclear-enriched phosphoprotein accumulation in tumors versus precancerous/wild-type tissues (T vs. P/W). KEGG pathway analysis identified consistently dysregulated pathways including MAPK signaling, focal adhesion, and protein digestion/absorption. Protein-protein interaction (PPI) network analysis of shared phosphoproteins pinpointed key regulators (Alb, Hspa5, Psn), with high connectivity, suggesting potential regulatory roles in the network. Sex-specific analyses demonstrated distinct phosphorylation patterns: males exhibited extensive membrane protein phosphorylation alterations, while females showed predominant cytoplasmic modifications. KEGG pathway mapping revealed male-biased dysregulation in Ras signaling, mTOR pathways, and actin cytoskeleton regulation. Functional annotation indicated greater complexity of phosphorylated proteins in males. Notably, phosphorylation events occurring on proteins with annotated kinase or phosphatase activity were more prevalent in males, suggesting enhanced phosphorylation-mediated signaling dynamics. SIGNIFICANCE: This study establishes the first sexual dimorphism-aware phosphoproteomic resource for Ras-driven hepatocarcinogenesis, systematically characterizing conserved and sex-divergent phosphorylation networks. The findings provide preliminary molecular insights into gender disparities in HCC progression and may guide future therapeutic exploration.