Differential Proteomic Analysis of DEN-Induced Hepatocellular Carcinoma in Male and Female Balb/c Mice Reveals Novel Gender Specific Markers

Abstract Background Hepatocellular carcinoma (HCC) is one of the leading causes of hepatic malignancy with a higher prevalence in males compared to females, however, the distinct underlying mechanisms contributing to this disparity remain poorly understood. Methods In this study, we aimed to investigate comparative proteome profiling of a diethylnitrosamine (DEN) induced HCC model in male and female Balb/c mice. We extracted proteins from liver tissue of DEN treated male and female mice and their corresponding controls and subjected them to mass spectrometry and subsequent bioinformatics analyses. Results We identified 170 and 233 differentially expressed proteins (DEPs) in female and male mice, respectively. We identified chemical carcinogenesis and cortical cytoskeleton as the shared pathways between the two groups. In addition, we identified distinct signaling pathways in DEN-treated male and female mice. Female mice showed enrichment in oxidative phosphorylation, fatty acid biosynthesis, metabolism and degradation and cytochrome P450 clusters. In contrast, in male mice, these pathways were enriched in cholesterol metabolism, coagulation and mRNA processing. Further, we identified top ten genes ranked by highest maximal clique centrality, by protein-protein interaction analysis of differentially expressed proteins (DEPs) in both sexes. Of these hub genes, female mice showed upregulation of NDUFA8 and ATP5H and were associated with poor patient survival. On the other hand, In DEN-treated male mice upregulation of FGG, FGA, HPX and SERPINC1 were associated with poor survival. Conclusion In conclusion, our research provides gender-specific proteomic signatures in DEN-induced HCC. The identification of proteins associated cholesterol metabolism and coagulation in males, and mitochondrial complex I proteins in females as prognostic markers suggests novel therapeutic targets that may inform gender-tailored treatment strategies for HCC. Simple Summary Hepatocellular carcinoma (HCC) is a common and deadly liver cancer that affects men more than women. To understand the biological reasons behind this difference, we developed a liver cancer model in male and female mice using a chemical called diethylnitrosamine (DEN). We then studied the proteins involved in tumor development using advanced techniques like mass spectrometry and bioinformatics. We found that different sets of proteins and biological pathways were active in males and females. In males, ribosomal and RNA-binding proteins were linked to worse survival, while in females, mitochondrial proteins were more important. These findings suggest that men and women may need different strategies for diagnosing and treating HCC, and they offer new gender-specific targets for future therapies.