The molecular sub-type and the development and validation of a prognosis prediction model based on endocytosis-related genes for hepatocellular carcinoma

Abstract

Background: Despite the critical role of endocytosis-related genes in oncogenic processes, research exploring their potential for prognosticating hepatocellular carcinoma (HCC) remains limited. Establishing a connection between endocytosis and HCC is imperative. This study aimed to create a gene signature related to endocytosis to identify HCC subtypes and predict outcomes.

Methods: RNA sequencing and clinical data of 371 HCC patients were obtained from The Cancer Genome Atlas (TCGA)-HCC dataset. Subtypes of HCC were identified through endocytosis-associated genes through consistent clustering analysis, and prognosis was assessed using an endocytosis-associated HCC model. Construction and validation of a prognostic endocytosis-related risk scoring system were created for HCC.

Results: A univariate Cox regression analysis was performed using the TCGA-HCC dataset, resulting in the identification of 4,354 genes significantly associated with patient prognosis. Subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these genes identified several biologically relevant pathways, particularly those related to endocytosis, autophagy, and cell cycle regulation. Through the application of consensus clustering methods, patients with TCGA-HCC were stratified into two distinct subtypes based on a selection of 82 genes associated with endocytosis. Importantly, the overall survival rate for the high-risk subtype (C1) was significantly higher than that of the low-risk subtype (C2). KEGG analysis indicated that the upregulated genes in the high-risk C1 subtype were predominantly related to various pathways, including the p53 signaling pathway, proteoglycans in cancer, cell cycle regulation, interactions between the extracellular matrix and receptors, and cellular senescence. In contrast, in the comparison between the C1 and C2 HCC samples, the genes exhibiting downregulation were predominantly linked to metabolic pathways, including tyrosine metabolism and steroid hormone biosynthesis. Boxplots showed significant differences in immune cell populations, including CD4⁺ T lymphocytes, endothelial cells, natural killer cells, and macrophages. From a pool of 82 endocytosis-related genes, 14 genes were identified through least absolute shrinkage and selection operator and Cox regression, including CLTA, STAM, RAB10, DAB2, VPS45, AGAP3, ARPC4, VPS29, HSPA8, DNAJC6, PARD6B, ACTR3B, PSD4, and ARRB2. Based on these genetic markers, patients were stratified into low-risk and high-risk categories. The prognostic performance of the model was validated using receiver operating characteristic curve analysis, which produced area under the curve values of 0.807, 0.757, and 0.716 for 1-, 3-, and 5-year survival predictions, respectively. The model of endocytosis-related genes was validated by external International Cancer Genome Consortium (ICGC)-HCC datasets.

Conclusions: Genes linked to endocytosis strongly correlate with tumor classification in patients with HCC. The related expression profiles may be valuable for predicting HCC prognosis and informing diagnosis and treatment.

Keywords: Hepatocellular carcinoma (HCC); biomarkers; endocytosis; prognostic model.

Figure 1

Prognostic gene signature analysis and pathway enrichment in hepatocellular carcinoma. (A) Survival-associated genes in hepatocellular carcinoma. Number outside parentheses represents the point estimate of the hazard ratio and number in parentheses represents the 95% CI of the HR. (B) Functional pathway enrichment of prognostic genes in hepatocellular carcinoma. Data are presented as the number of survival genes enriched in the signaling pathway (statistical P value). CI, confidence interval; HR, hazard ratio.

Figure 2

Subtypes of hepatocellular carcinoma identified through endocytosis-associated genes. (A) Survival-associated genes in hepatocellular carcinoma. Purple indicates high clustering stability, white indicates poor clustering stability, and the higher the consensus score, the more stable it is. (B) A heatmap of endocytosis-related genes in hepatocellular carcinoma. (C) Kaplan-Meier survival curves of the two endocytosis-related clusters. C1, cluster 1; C2, cluster 2; CI, confidence interval; HR, hazard ratio.

Figure 3

Differentially expressed gene and enrichment analyses of the HCC subtypes. (A) The volcano plot of the DEGs between C1 and C2 (the red color indicates upregulated genes, and the blue color indicates downregulated genes). (B) Heatmap of the DEGs between C1 and C2. (C) KEGG pathway enrichment analysis of the upregulated DEGs. (D) KEGG pathway enrichment analysis of the downregulated DEGs. (E) GO biological process enrichment for upregulated DEGs. (F) GO biological process enrichment for downregulated DEGs. C1, cluster 1; C2, cluster 2; DEG, differentially expressed gene; GO, Gene Ontology; HCC, hepatocellular carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Comparison of the immune activity between the two endocytosis-related hepatocellular carcinoma groups. The enrichment scores of six distinct immune cell types in HCC were analyzed and compared between the two groups associated with endocytosis. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. C1, cluster 1; C2, cluster 2; EPIC, estimating the proportion of immune and cancer cells; HCC, hepatocellular carcinoma; NK, natural killer.

Figure 5

Construction of a prognostic model based on endocytosis-related genes. (A) LASSO regression of endocytosis-related genes. (B) Cross-validation of the LASSO regression. (C) Patients with HCC from TCGA were stratified into low- (blue) and high-risk (red) groups based on the expression levels of endocytosis-related genes. (D) A Kaplan-Meier survival analysis was conducted to compare the survival outcomes between the high- and low-risk groups. (E) ROC curve analysis was performed to determine the AUC values for the stratification of risk groups. AUC, area under the curve; CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas.

Figure 6

Validation of a prognostic model based on endocytosis-related genes. (A) Patients with HCC from ICGC were stratified into low- (blue) and high-risk (red) groups based on the expression levels of endocytosis-related genes. (B) A Kaplan-Meier survival analysis was conducted to compare the survival outcomes between the high- and low-risk groups. (C) ROC curve analysis was performed to determine the AUC values for the stratification of risk groups. AUC, area under the curve; CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; ROC, receiver operating characteristic; ICGC, International Cancer Genome Consortium.