Research ArticleExperimental Studies
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Differential Proteomic Analysis of DEN-induced Hepatocellular Carcinoma in Male and Female Balb/c Mice Reveals Novel Sex-Specific Markers

SALMMA SALAMAH SALIHAH, MUHAMMAD TAHIR, BAREERA BIBI, RABIA SULTAN, MARTIN R. LARSEN, MUNAZZA RAZA MIRZA, SANA MAHMOOD, MUHAMMAD RIZWAN ALAM, JAMILA IQBAL, WILLIAM C. CHO and ASMA GUL
Cancer Genomics & Proteomics November 2025, 22 (6) 912-928; DOI: https://doi.org/10.21873/cgp.20540

Abstract

Background/Aim: 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.

Materials and 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 the 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 146 differentially expressed proteins (DEPs) in female and male mice, respectively. We identified chemical carcinogenesis, oxidative phosphorylation, actin binding and mitochondrial respiration 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 fatty acid biosynthesis, metabolism, degradation, and cytochrome P450 clusters. In contrast, in male mice, pathways were enriched in tryptophan metabolism, apoptotic execution phase and glutathione metabolism. Further, we identified the 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 up-regulation of previously unimplicated, NDUFA8 and ATP5H proteins which were associated with poor patient survival. On the other hand, in DEN-treated male mice up-regulation of RPS3 was associated with poor survival.

Conclusion: In conclusion, our research provides sex-specific proteomic signatures in DEN-induced HCC. The identification of proteins associated with ribosomal subunits in males, and two previously unimplicated mitochondrial complex I proteins in females as prognostic markers suggests novel therapeutic targets that may inform sex-tailored treatment strategies for HCC.

Keywords:

Introduction

Hepatocellular carcinoma (HCC) remains one of the most prevalent malignancies worldwide (1), posing a great burden on global health (2). Notably, significant sex-based differences exist in susceptibility and survival outcomes: males are more prone to hepatocarcinogenesis (3), while females often demonstrate better prognoses (4). These differences suggest underlying biological and molecular mechanisms that remain poorly understood, especially at the proteomic level. Investigating these mechanisms could unlock new opportunities for precision medicine and sex-specific treatment approaches.

Animal models are central to preclinical research in HCC, offering crucial insights into the molecular and pathophysiological landscape of the disease. While gene-editing and in situ transplantation models allow focused mechanistic studies, they often fall short in capturing the systemic tumor-immune dynamics seen in human disease (5). In contrast, the diethylnitrosamine (DEN)-induced mouse model closely mirrors the transcriptomic and proteomic characteristics of aggressive human HCC, particularly the poor-prognosis S-III subtype (6). It replicates key features such as low β-catenin expression, high proliferation, chromosomal instability, and altered apoptotic signaling (7). This makes the DEN model highly translational and suitable for investigating complex interactions in hepatocarcinogenesis, including potential sex-based differences. Two relevant studies have advanced this space. Zhang et al. (8) profiled the DEN and CCl4-induced male mouse model and aligned it with human HCC subtypes, identifying prognostic biomarkers. McGill and Jaeschke (9) explored sex-based proteomic differences in Ras-oncogene–induced HCC, finding that male hepatocytes were more vulnerable to oncogenic stress.

Despite these advances, critical gaps remain in understanding sex-specific molecular mechanisms in HCC. Zhang et al.’s study focused exclusively on male mice, leaving the female proteomic landscape unexamined (8). While McGill and Jaeschke shed light on sex-related vulnerability using a Ras-driven model, their findings were model-specific and may not generalize to chemically induced HCC (9). As a result, there is a lack of comprehensive, comparative proteomic data on male and female responses in a highly translational model like DEN.

In this study, we conducted a comprehensive proteomic analysis using a diethylnitrosamine (DEN)-induced mouse model of hepatocellular carcinoma (HCC), directly comparing tumor profiles between male and female subjects. This model, known for its strong translational relevance to aggressive forms of human HCC, enabled us to investigate sex-specific molecular differences in tumor biology. Our analysis revealed several key findings: (1) distinct proteomic signatures unique to male and female HCC, (2) pathways specifically involved in male and female hepatocarcinogenesis, (3) male and female-specific proteins associated with poorer outcomes, and (4) candidate therapeutic targets for developing sex-tailored treatment strategies. These results enhance our understanding of the molecular underpinnings of HCC and provide a foundation for future precision oncology approaches that consider biological sex as a critical variable.

Materials and Methods

Mouse model and tissue collection. Balb/c mice were obtained from the National Institute of Health, Islamabad, Pakistan. The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of International Islamic University, Islamabad (April 02, 2021) for studies involving mice and carried out in accordance with institutional animal care guidelines. A total of 21 mice (11 females and 10 males) were housed under standard laboratory conditions with a 12-h light/dark cycle and a controlled temperature of 22-25°C. Animals had free access to a standard diet and water throughout the study. The mice were randomly divided into control and treatment groups, with each group consisting of 5 males and 6 females. At four weeks of age, mice in the treatment group received a single intraperitoneal injection of diethylnitrosamine (DEN) (100 mg/kg body weight, Sigma-Aldrich, St. Louis, MO, USA #N0756), while control animals were injected with an equivalent volume of normal saline. Both groups were monitored for 32 weeks.

At the end of the experimental period, surviving animals were terminally anesthetized with an overdose of sodium pentobarbital (Sigma-Aldrich, #P3761). Liver tissues were collected from all animals and rinsed in phosphate-buffered saline (PBS) (Sigma-Aldrich, #P4739). Each liver was examined macroscopically for visible nodules or lesions, then sectioned for further analysis. One portion was fixed in 4% formaldehyde (Sigma-Aldrich, #252549) for histological and immunohistochemical evaluation, while the remaining tissue was stored for subsequent proteomic analyses.

H&E staining & immunohistostaining. Formaldehyde-fixed tissues were embedded in paraffin and sectioned into 5μm slices. These sections were mounted onto glass slides and deparaffinized using three changes of xylene. Rehydration was performed through a graded ethanol series (100%, 95%, and 70%), after which the slides were stained with Mayer’s hematoxylin (Sigma-Aldrich, #MHS32) for 5 min and counterstained with eosin (Sigma-Aldrich, #15086-94-9). Sections were then dehydrated through an ascending ethanol series (70%, 95%, and 100%), followed by clearing in xylene. The stained slides were mounted using Permount (Sigma-Aldrich, #06522) and allowed to dry before microscopic evaluation.

For immunohistochemical analysis, antigen retrieval was performed using a microwave-based method in 0.01 M sodium citrate buffer (pH 6.0), as previously described by Maffini et al. (10). The following primary antibodies were used: Vimentin (#NCL-VIM-V9, Leica Biosystems, Nussloch, Baden-Württemberg, Germany; 1:1,000), Ki 67 (#PA0118, Leica Biosystems) and E-Cadherin (#PA0387, Leica Biosystems; 1:1,000-1:4,000). Antigen–antibody complexes were visualized using a streptavidin–peroxidase detection system with diaminobenzidine (DAB; Sigma-Aldrich) as the chromogen. Slides were counterstained with Harris’ hematoxylin (Sigma-Aldrich, #HHS32). Microscopic images were captured using an Olympus BX53 (Olympus Corporation, Tokyo, Japan) microscope equipped with a digital Olympus camera.

Sample preparation for proteome analysis. Liver sections of Balb/c reserved for proteome analysis were rinsed with phosphate-buffered saline (PBS) and immersed in freshly supplemented RIPA buffer (10 mM Tris-HCl, 140 mM NaCl, 0.1% SDS, 1% Triton X 100, and 0.01% Sodium deoxycholate) (R0278, Sigma Aldrich). The tissues were homogenized on ice for 20 min using a tissue homogenizer. After centrifugation (13,000 × g, 20 min, 4°C), supernatants were stored at −80°C. Protein estimation was performed through Bradford Assay.

Tryptic digestion. For the enzymatic digestion of protein standard (α-casein, β-casein, and ovalbumin) and protein samples, 0.1 mg/ml (200 ml) was aliquoted. To adjust the pH (~8) 1M NH4HCO3 (160 ml) was added to the aliquots. Protein samples were reduced and denatured by adding 45 mM dithiothreitol (DTT) and 40 mM nOGP. Afterwards, the vials were incubated on a thermomixer (Eppendorf, Germany) for 30 min at 90°C and 800 rpm. Protein solutions were allowed to cool at room temperature followed by alkylation at room temperature, carried out by adding 100 mM of iodoacetamide (IAA) and the vials were incubated in the dark for 15 min. Deionized water was added followed by the addition of 2 mg trypsin. After adding trypsin samples were digested on a thermomixer (Eppendorf, Germany) for 16 h at 600 rpm and 37°C. The digestion process was stopped by adding 2% trifluoroacetic acid TFA (60 ml, pH ≤3). All digests were stored at −20°C.

Mass spectrometry analysis. Peptide digests were analyzed by nano-LC-MS/MS system on a Q Exactive HF Orbitrap LC-MS/MS system (Thermo Fisher, Waltham, MA, USA) coupled to an EASY-LC 1000 system (Thermo Fisher Scientific). Long-fused silica capillary column (PicoFrit 18 cm, 75 μm inner diameter) was packed in-house with reversed-phase Repro-SilPur C18-AQ 3 μm resin and used to load 1 μg of purified peptides. Peptides were eluted from buffer A (0.1% formic acid) at a flow rate of 250 nl/min followed by elution through buffer B (95% acetonitrile in 0.1% formic acid). The gradient gradually increased in B from 5% to 28% in 40 min, 45% in 60 min, and finally to 100% in the end. The Q-Exactive HF was set up to operate in data-dependent acquisition mode with the following parameters: full scan automatic gain control (AGC) target 3×106 at 120,000 FWHM resolution, scan range 350-1600 m/z, Orbitrap full scan maximum injection time 100 ms, normalized collision energy 28, a dynamic exclusion time of the 30s, an isolation window of 1.2 m/z and top 20 MS2 scans per MS full scan.

The raw Orbitrap files were processed in MaxQuant (version 2.7.3.0) (11) using the Andromeda search engine (12) with default settings. A false discovery rate (FDR) of 1% was applied at both the peptide-spectrum match (PSM) and protein levels. Spectra were searched against the Homo sapiens reference proteome from the UniProt/Swiss-Prot database (release 2023_01) (13). The mass tolerance was set to 4.5 ppm for precursor ions and 20 ppm for fragment ions. Enzyme specificity was defined as trypsin/P (cleavage C-terminal to arginine and lysine residues, including cleavage at arginine/lysine–proline bonds), allowing up to two missed cleavages. Carbamidomethylation of cysteine was specified as a fixed modification, while oxidation of methionine and acetylation of protein N-termini were included as variable modifications. Protein quantification was performed using the MaxLFQ algorithm (14) integrated into MaxQuant. For protein identification, a minimum of one unique or razor peptide was required, while a minimum ratio count of two unique or razor peptides was applied for quantification.

Statistical data analysis. The statistical analysis began by importing protein groups from MaxQuant. Initial pre-processing was performed in Perseus v1.6.10.50 (Max Planck Institute of Biochemistry, Martinsried, Germany) for both male and female cohorts, where potential contaminants, reverse database hits, and proteins identified only by site were removed, followed by log2 transformation. The data was then filtered based on 70% valid values, followed by median imputation of missing values. Imputed data was then subjected to significance testing.

Principal component analysis (PCA) was performed to confirm clear separation of control and treated samples in male and female datasets. Significance testing of protein LFQ intensities was performed separately for male and female cohorts using two-sample t-test in Perseus v.1.6.10.50. Proteins with a p-value ≤0.05 and Abs (log FC)>=1 were considered significant. PCA plots, heatmaps and volcano plots were generated in R version 4.5.1using ggplot2.

Gene enrichment analysis. The biological significance of each module was determined through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG: https://www.kegg.jp/kegg/pathway.html), and Reactome (https://reactome.org/Pathway.html) enrichment analyses to determine specific processes, molecular functions, or cellular components represented within each module. Due to low sample size, we validated our male results with an external dataset by Zhang et al. (8). Overrepresentation analysis of GO, KEGG, and Reactome terms was performed separately for our dataset and for the dataset from Zhang et al. (8) using the R package clusterProfiler (https://bioconductor.org/packages/clusterProfiler/). Terms with an adjusted p-value ≤0.05 were considered statistically significant. For the male cohort, overlapping GO terms and pathways between our analysis and that of Zhang et al. (8) were identified. For visualization, in the female cohort, redundant GO terms were removed and only the top five enriched terms in each category were plotted, whereas for the male cohort all overlapping terms were plotted.

PPI and hub proteins. The protein–protein interaction (PPI) network was constructed using STRING database (version 12.0; https://string-db.org/) for differentially expressed proteins (DEPs) identified in the female cohort, as well as for overlapping DEPs obtained from the enrichment analysis of the male cohort using STRING DB (version 12.0) (https://string-db.org/). The PPI networks were visualized in Cytoscape (version 3.10.3). Functional modules were identified through MCODE plugin, and the top 3 modules were selected based on the MCODE score in Cytoscape (15). The analysis was carried out using default parameters (k-score=2, cut-off degree=2, max depth size=100 and node score cutoff=0.2). The Maximal Clique Centrality (MCC) algorithm was used to select hub genes from the PPI network using the CytoHubba plugin of Cytoscape.

Survival analysis. The clinical value of differentially expressed proteins was estimated by converting 20 hub proteins to their human homologs using the “biomaRt” R package (https://bioconductor.org/packages/biomaRt/). Protein expression of human homologous proteins corresponding to DEPs was matched with overall survival using the UALCAN tool (https://ualcan.path.uab.edu/).

Results

DEN-induced mouse model for male and female HCC. DEN-induced HCC mouse model for male and female Balb/c mice was established following one of the methods described by Peng et al. (15) with some modifications. All six female mice in the treatment group survived until 8 months whereas only 2 male mice survived in each group till the end. Subsequent analysis was performed with the surviving individuals of the groups. Normal and tumor tissues with internal and external controls were collected from control and treated mice of both sexes separately for the histological and proteomic analysis (Figure 1A).

Figure 1.
Figure 1.

Schematic diagram of the study, H&E analysis and immunohistochemical staining of tissue sections from treatment and control groups. (A) Schematic diagram of the development of DEN-induced HCC in Balb/c mice. (B, C) H&E staining [magnification 20× (scale bar 50mm), 40× (scale bar 10mm)] of liver sections obtained from treated and control groups of male and female mice, respectively. Half circular arrows in the treatment group indicate enlarged nuclei; arrows indicate immune infiltration and arrow heads show disorganized morphology. Percentage increase in the nuclear area is represented on the graph (n=3 for both); (D, E) E-Cadherin staining showing loss of E-Cadherin in the treatment groups of male and female mice (n=2 and n=3, respectively); (F, G) Ki67 staining showing gain of Ki67 in treated samples of male and female liver sections (n=3 for both); (H, I) Vimentin staining showing increase in the vimentin expression in treated and control section of male and female mouse livers (n=3 and n=2, respectively).

In the DEN-induced mouse model multiple tumors developed in the liver. We compared the morphology of control and tumor tissue using H&E staining. Control mice showed neatly arranged hepatocytes whereas tumor tissue showed disorganized morphology, large nuclei, and immune infiltration suggesting DEN-induced carcinogenesis in the mouse liver. The percentage area of the nuclei increased from 15% to 38% and from 19% to 31% in male and female groups, respectively (Figure 1B, C).

We also performed immunohistochemical analysis of tissue samples using tumor specific markers, E-Cadherin, vimentin and Ki67. The positive area stained with E-Cadherin decreased from 58% to 16% and 43% to 13% in treated samples of male and female mice respectively, suggesting loss of cellular adhesion (Figure 1D, E). The percentage area for the proliferation marker Ki67 was increased from 15% to 60%, and 8% to 33%, in tumor samples of male and female samples, respectively (Figure 1F, G). The increase in expression of Ki67 shows increased cellular proliferation in the treated samples. The positive area stained with vimentin was significantly increased from 16% to 54%, and 28% to 48%, in tumor tissue compared to normal tissues in male and female mice, respectively (Figure 1H, I). These results indicated that the DEN-induced mouse model was successfully generated with characteristic proliferative and adhesive features of the tumor.

Statistical data analysis. In the present study, protein samples isolated from 11 female and 4 male mice were analyzed through label-free LC-MS which led to the identification of a total of 2,897 proteins in each sample. After the filtration of contaminants, only identified by site and reverse hits, 2,803 proteins were selected. The data was log2-transformed and subsequently filtered within each group, reducing the number of proteins to 1,232. PCA revealed the spread of the data. Control and treated groups were clearly separated on the PCA plots for both male and female mice. PC1 contributed 29.4% and 52.7% whereas, PC2 contributed 19.9% and 37.9% for female and male mice respectively (Figure 2A).

Figure 2.
Figure 2.

Proteomic analysis of DEN-induced HCC mouse models. (A) PCA shows separation between the treatment (red) and control (blue) groups of female mice (left) and male cohort (right). (B) Heatmap of DEP in liver tissue of treatment (red) and control groups (blue) in both sexes. (C) Volcano plots of differentially expressed proteins in male and female mice. Blue dots represent down-regulated proteins, red dots represent up-regulated proteins, and grey dots represent proteins with insignificant differences (p-value ≤0.05, |log2FC| ≥1). All plots were created with ggplot2.

Significance of the expressed protein based on the difference between the LFQ intensities of treatment and control groups in both the male and female cohort was calculated using two-sample t-test. In female mice, 170 proteins were significantly differentially expressed, among which 62 proteins were down-regulated, and 108 proteins were up-regulated. In male mice, 86 proteins were found to be significantly differentially expressed, among which 40 proteins were up-regulated whereas 46 proteins were down-regulated. Heatmaps of the DEPs were plotted based on Euclidean distance in both male and female groups (Figure 2B). In addition, volcano plots for both groups with a cutoff (p-value ≤0.05, |log2FC| ≥1) are shown in Figure 2C.

Jiang et al. (16) utilized the proteome to analyze paired tumor and non-tumor tissues from early-stage HCC patients. Their findings divided the cohort into three subtypes: S-I with hepatocyte-like traits, S-II with proliferative traits, and S-III showcasing proliferation, aggressiveness, and heightened immune infiltration, along with the poorest prognosis. The differentially expressed proteins in the female and male cohort showed greater than 50% similarity with the expression pattern in human S-III and S-II subtypes respectively. In the female cohort, metabolic pathways were more consistent with the human data. To determine the value of the DEN-induced HCC mouse model for clinical studies, we used heatmaps to demonstrate the expression of human homologs of these proteins in tumor and non-tumor tissues of the three subtypes of HCC (Figure 3).

Figure 3.
Figure 3.

Heatmap of human homologs of mouse DEPs in non-tumor (N) and tumor tissues (T) of three proteomic subtypes of Jiang et al.’s HCC cohort, S-I (n=36), S-II (n=31) and S-III (n=31). (A) Human subtype specific expression of up-regulated DEPs in the female cohort. (B) Human subtype specific expression of down-regulated DEPs in the female cohort. (C) Human subtype specific expression of up-regulated DEPs in the male cohort. (D) Human subtype specific expression of down-regulated DEPs in the male cohort. R software, package ggplot2 (version 4.5.1) was used to create heatmaps.

Oxidative phosphorylation and fatty acid metabolism in females. Functional enrichment analysis, Gene Ontology (GO), Reactome Analysis, and KEGG pathway analysis of all DEPs was performed to study the biological relevance of significantly altered proteins. GO analysis revealed significant enrichment in metabolic processes associated with fatty acid, sulfur compound, acyl-CoA, ATP and carboxylic acid catabolism. Moreover, DEPs related to mitochondrial structure and respiration such as respiratory chain complex, mitochondrial respirasome, mitochondrial protein–containing complex, electron transfer activity and proton motive force–driven mitochondrial ATP synthesis were also enriched (Figure 4A). GO term analysis of all DEPs showed enrichment in cortical cytoskeleton and actin filament binding processes.

Figure 4.
Figure 4.
Figure 4.

Overrepresentation analysis of the female and male cohort (overlap with Zhang et al. (8)). Red bars represent up-regulated processes and pathways, whereas blue bars represent down-regulated processes and pathways. (A) The top significant terms of biological processes, cellular composition, and molecular function from GO analysis of the female cohort, Benjamini–Hochberg adjusted p-value < 0.05. (B) The top significant GO terms of the male cohort showing overlapping processes between our study and the study of Zhang et al. (C) The top pathways of the female cohort from KEGG and Reactome pathway analysis. (D) Top KEGG and Reactome pathways from the male cohort showing overlap between our study and the study of Zhang et al.

Reactome pathway analysis further indicated the enrichment of mitochondrial respiratory pathways including respiratory electron transport, aerobic respiration and ATP synthesis by chemiosmotic coupling and other mitochondrial processes including heat production by uncoupling proteins (Figure 4B). Oxidative phosphorylation and fatty acid metabolism were found to be enriched in both GO and KEGG analysis. KEGG pathway analysis also represented enrichment of pathways of chemical carcinogenesis and fatty acid metabolism (Figure 4A, B).

Metabolism and mitochondrial pathways in male mice. In order to validate our results, separate enrichment analysis was carried out for our dataset and the dataset reported by Zhang et al. (8). The overlapping pathways were identified between the two independent datasets to detect the true signal that revealed common biological processes and pathways associated with DEN treatment.

GO term analysis indicated significant enrichment in biological processes such as glutathione metabolism, NADH metabolism, cellular ketone metabolism, and amino acid metabolism. Enriched cellular component included the mitochondrial protein containing complex, respiratory chain complex, inner mitochondrial membrane protein complex, respiratory chain complex and mitochondrial matrix. Enrichment in molecular function highlighted glutathione transferase activity, oxidoreductase activity and actin binding (Figure 4C).

Reactome analysis demonstrated over-representation of apoptotic execution phase (Figure 4D). KEGG pathway analysis further confirmed enrichment in glutathione and tryptophan metabolism and revealed enrichment in chemical carcinogenesis pathways and pantothenate and CoA biosynthesis (Figure 5D).

Figure 5.
Figure 5.

Protein-protein interactions in the DEPs of both sexes. (A) Pathway interaction in the top three modules of male and female DEPs. (B) Hub protein analysis of the top cluster.

PPI networks in male and female mice. For DEPs in male and female mice, the PPI network was constructed using Cytoscape version 3.10.3. The female PPI network consisted of 143 nodes with 605 edges while the male PPI network consisted of 50 nodes with 97 edges. The MCODE score of the 1st module in the female group was 14 (14 nodes and 91 edges), whereas the score of the second and the third group was 8.75 (9 nodes and 35 edges) and 8 (8 nodes and 28 edges) respectively (Figure 5A). For the male cohort, proteins involved in overlapping pathways were extracted from the enrichment analysis and used for the subsequent downstream analysis. The MCODE score of first, second and third module in the male group was 5.6 (6 nodes, 14 edges), 4 (4 nodes, 6 edges) and 3 (3 nodes, 3 edges), respectively. The ten highest ranked genes based on the MCC algorithm were selected as hub genes (Figure 5B).

Prognostic value of ribosomal proteins in male mice and mitochondrial proteins in female mice. To identify the relationship between the expression of hub proteins and overall survival time of patients and to evaluate the prognostic value of hub proteins, survival analysis of human homologs of all hub genes was carried out using the online tool UALCAN. The results indicated that three hub genes were significantly associated with overall patient survival in LIHC. Among the hub genes of DEN-treated female mice, only the up-regulation of NDUFA8 (p<0.017) and ATP5H (p<0.0066) was associated with poorer patient survival (Figure 6A, B). In DEN-treated male mice, up-regulation of RPS3 was associated with poor patient survival (Figure 6C).

Figure 6.
Figure 6.

Overall survival curves for three proteins with significant prognostic value. (A) NDUFA8, (B) ATP5H, (C) RPS3. Survival plots were generated using UALCAN, based on RNA-seq data from The Cancer Genome Atlas (TCGA).

Discussion

In the present study, differential proteomic analysis of DEN-induced male and female HCC mouse models provided insights into sex-specific proteomic signatures and associated molecular mechanisms. Two previously unimplicated proteins, NDUFA8 and ATP5H in the female cohort, were significantly associated with poor survival outcomes, whereas RPS3 in the male cohort was significantly associated with poor prognosis. The study provides a comprehensive proteomic landscape of mouse HCC that emphasizes the need of using sex-specific mouse models in HCC research. The study also provides the proteomic data from DEN-induced HCC in mice that closely resembles human HCC of poor prognosis, establishing the clinical relevance of the model.

While the study offers important insights, certain limitations should be acknowledged. Early mortality in male mice reduced statistical power for detecting subtle proteomic changes. To address this, we validated our data with proteomic data of HCC related processes and pathways from eight DEN and CCl4-treated male mice from Zhang et al. (8). Furthermore, our proteomic findings were internally consistent and validated through literature cross-referencing. However, due to resource limitations, we were unable to experimentally confirm previously unimplicated proteins via techniques such as Western blotting or immunohistochemistry. Nevertheless, the use of integrative and literature-supported validation strengthens the reliability of our observations and lays the foundation for future targeted experimental studies.

Our findings build on and extend prior work by Salim et al. (17) and Zhang et al. (8) which investigated transcriptomic and proteomic changes in DEN and CCl4-induced HCC mouse models. Salim et al. (17) identified enrichment of pathways such as steroid hormone biosynthesis, retinol metabolism, and cytochrome P450 via transcriptomic profiling. Zhang et al. (8) reported 4,383 quantified proteins and 432 differentially expressed proteins (DEPs) in male mice, with up-regulation of actin cytoskeleton components linked to tumor progression. While these studies provided foundational insights, Salim et al. (17) focused solely on transcriptomics, and Zhang et al. (8) limited their proteomic analysis to male mice. Our study complements and expands on these works by providing sex-specific proteomic data, capturing both overlapping and distinct biological pathways. Consistent with Zhang et al. (8), our study also showed up-regulation of cortical cytoskeleton in both the male and female cohort. Previous studies had suggested that DEN and CCl4 cause hepatic injury by stimulating hepatic stellate cells which lead to the genesis of actin cytoskeleton, which subsequently plays a role in cell-to-cell communication and increased expression of extracellular matrix (18, 19). The actin cytoskeleton is essential for numerous cellular functions, including maintaining intracellular stability and facilitating signal transduction. Actin and actin-binding proteins play key roles in all stages of cancer progression, such as epithelial to mesenchymal transition, migration, invasion, and angiogenesis (19-21).

In the female cohort, we observed significant up-regulation of oxidative phosphorylation and down-regulation of fatty acid metabolism and xenobiotic detoxification. We found enrichment in mitochondrial respiratory chain components, particularly Complex I subunits such as NDUFA2, NDUFA7, NDUFA8, NDUFS6, NDUFV3, and ATP5H. Two of these, NDUFA8 and ATP5H, were associated with poor survival in human HCC patients. NDUFA8, a critical Complex I subunit, regulates electron transport and ATP production and has been implicated in oncogenic proliferation in cervical cancers (22). Complex I, comprising over 40 subunits, initiates the mitochondrial electron transport chain and is the largest and first point of electron entry for NADH oxidation (23). While mitochondrial metabolism has been increasingly recognized in cancer biology, the role of Complex I subunits in hepatocellular carcinoma remains poorly characterized (24). The observed mitochondrial shift toward enhanced respiratory metabolism may reflect an adaptive oncogenic response to sustain energy production and redox balance in tumor cells (24, 25).

Concurrently, we observed the down-regulation of fatty acid metabolism and xenobiotic detoxification pathways, particularly those mediated by CYP450 enzymes and associated with chemical carcinogenesis through DNA adduct formation. This suppression may impair the liver’s intrinsic ability to metabolize and eliminate carcinogens, thereby allowing for ROS accumulation and prolonged genotoxic stress (26). Elevated oxidative phosphorylation, combined with reduced detoxification capacity, may promote ROS buildup-further supported by our observation of up-regulated ROS-associated signaling. This pro-oxidative environment can enhance DNA damage, impair lipid homeostasis, and promote tumorigenesis (27). Down-regulation of fatty acid β-oxidation, as reported in several HCC subtypes (28), likely contributes to lipid accumulation and metabolic stress, compounding the oncogenic effect. Collectively, our results suggest that female HCC may harbor a unique metabolic phenotype characterized by mitochondrial overactivation and impaired hepatic detoxification, which could represent a therapeutic vulnerability for sex-specific interventions targeting mitochondrial bioenergetics and ROS regulation.

In the male cohort of DEN-induced hepatocellular carcinoma (HCC), proteomic analysis revealed significant up-regulation of processes related to the metabolism, mitochondrial complexes, oxidoreductase and glutathione transferase activity, alongside chemical carcinogenesis and glutathione metabolism, as shown by KEGG pathway enrichment. Enrichment in these processes and pathways indicates active metabolic reprogramming in tumor tissues. Several hub proteins identified in this group, including UQCRB, COX4I1, COX5A, COX6B1, GSTP1, CYC1, IDH3A, ENO1, RPS3, SLC25A5 are associated with mitochondrial complexes, xenobiotic metabolism and DNA repair (29-35). Many of these proteins also demonstrated prognostic significance: high expression of RPS3 was associated with poorer overall survival (36).

In hepatocellular carcinoma, RPS3 is frequently up-regulated in tumor tissues compared to non-tumor counterparts and is significantly associated with poor overall survival, elevated alpha-fetoprotein levels, advanced tumor staging, and aggressive clinicopathological features, suggesting a pro-tumorigenic role in HCC progression (37). In contrast, SLC25A5, a mitochondrial ADP/ATP antiporter that maintains cellular energy balance and inhibits apoptosis is down-regulated in HCC, and its reduced expression has been linked to enhanced tumor growth and metastasis through suppression of fatty acid oxidation and promotion of epithelial-to-mesenchymal transition and cell-cycle progression (35). Although ENO1 is generally overexpressed in HCC and promotes proliferation, migration, invasion, epithelial–mesenchymal transition, and ferroptosis resistance (38, 39), down-regulation of ENO1 might attenuate these malignant phenotypes, underscoring the enzyme’s context-dependent oncogenic functions.

Conclusion

In conclusion, this study presents a comprehensive proteomic characterization of male and female DEN-induced HCC mouse models, revealing distinct sex-specific molecular signatures with implications for tumor progression and prognosis. While female HCC was marked by enhanced oxidative phosphorylation and suppressed detoxification pathways, male HCC exhibited up-regulation of ribosomal and mitochondrial proteins. Despite limitations such as early mortality in male mice and the translational gap between mouse and human models, the literature validation supports the biological relevance of our findings. Future studies should include functional validation of hub proteins and their impact on sex-informed therapeutic strategies. This work lays a foundation for more personalized approaches in liver cancer research and may inform future clinical trial designs.

Acknowledgements

We are grateful to the Pakistan Science Foundation for partially funding this research via PSF grant # 733. We are thankful to Molecular Oncology Research Lab for serving as a base camp for this project. We also appreciate Dr. Ikramullah for allowing us to use the animal house facility at the Center for Interdisciplinary Research in Basic Science (CIRBS), International Islamic University Islamabad. The study was supported by the VILLUM Center for Bio analytical Sciences at University of Southern Denmark and by PRO-MS, Danish National Mass Spectrometry Platform for Functional Proteomics.

Footnotes

  • Data Availability

    Data is provided within the manuscript. Raw data is available on the online repository PRIDE.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Authors’ Contributions

    Conceptualization, Salmma Salamah Salihah, Jamila Iqbal and Sana Mahmood; methodology, Salmma Salmah Salihah, Muhammad Tahir, Rabia Sultan and Bareera Bibi; software, Bareera Bibi and Rabia Sultan; validation, Bareera Bibi; resources, Asma Gul, Munazza Raza Mirza, Muhammad Rizwan Alam and Martin R. Larsen; writing – original draft preparation, Salmma Salamah Salihah; writing – review and editing, Muhammad Tahir, Jamila Iqbal and Muhammad Rizwan Alam; visualization, Bareera Bibi; supervision, Jamila Iqbal, Asma Gul; project administration, Asma Gul and Martin R. Larsen; funding acquisition, Asma Gul. All Authors have read and agreed to the published version of the manuscript.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received July 29, 2025.
  • Revision received August 27, 2025.
  • Accepted September 4, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Differential Proteomic Analysis of DEN-induced Hepatocellular Carcinoma in Male and Female Balb/c Mice Reveals Novel Sex-Specific Markers
SALMMA SALAMAH SALIHAH, MUHAMMAD TAHIR, BAREERA BIBI, RABIA SULTAN, MARTIN R. LARSEN, MUNAZZA RAZA MIRZA, SANA MAHMOOD, MUHAMMAD RIZWAN ALAM, JAMILA IQBAL, WILLIAM C. CHO, ASMA GUL
Cancer Genomics & Proteomics Nov 2025, 22 (6) 912-928; DOI: 10.21873/cgp.20540
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