Effect of Sortilin1 on promoting angiogenesis and systemic metastasis in hepatocellular carcinoma via the Notch signaling pathway and CD133

Abstract

Hepatocellular carcinoma (HCC) is known to be lethal disease. However, its prognosis remains poor, primarily because the precise oncogenic mechanisms underlying HCC progression remain elusive, thus hampering effective treatment. Here, we aimed to identify the potential oncogenes in HCC and elucidate the underlying mechanisms of their action. To identify potential candidate genes, an integrative analysis of eight publicly available genomic datasets was performed, and the functional implications of the identified genes were assessed in vitro and in vivo. Sortilin 1 (SORT1) was identified as a potential candidate oncogene in HCC, and its overexpression in HCC cells was confirmed by analyzing spatial transcriptomic and single-cell data. Silencing SORT1 in Huh-7 and Hep3B cells significantly reduced HCC progression in vitro and in vivo. Functional analyses of oncogenic pathways revealed that SORT1 expression regulated the Notch signaling pathway activation and CD133 expression. Furthermore, analysis of epigenetic regulation of the candidate gene and its clinical implications using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) and our HCC cohort (AJOU_HCC cohort) data demonstrated an inverse correlation between the methylation status of the SORT1 promoter region, specifically at the cg16988986 site, and SORT1 mRNA expression, indicating the epigenetic regulation of SORT1 in HCC. In addition, the distinct methylation status of cg16988986 was significantly associated with patient survival. In conclusion, SORT1 plays a pivotal role in HCC by activating the Notch signaling pathway and increasing CD133 expression. These findings suggest SORT1 as a promising therapeutic target for HCC.

Fig. 1. Analysis of hepatocellular carcinoma (HCC) driver gene signatures across multiple datasets.

A Flow diagram detailing the methodology used to identify HCC driver gene signatures. B Venn diagram illustrating the overlap of significantly differentially expressed genes across the three datasets. C Serial pattern analysis of each dataset. The upper panels show line plots of gene expression patterns in different sample groups (NL, CH, LC, DN, eHCC, and aHCC) on the X-axis. The bottom panels display heat maps of the gene expression levels. NL, normal liver; CH, chronic hepatitis; LC, liver cirrhosis; DN, dysplastic nodules; eHCC, early HCC; aHCC, advanced HCC. D Venn diagram showing the overlap of common genes across the five RNA-seq datasets (TCGA, ICGC, GSE77314, GSE114564, and GSE124535). E Heatmap of the GSE114564 dataset showing gene expression patterns. Different genes are listed on the right side of the heatmap. F Box plots showing SORT1 expression at various stages of liver disease and cancer progression across the four datasets. The Y-axis represents SORT1 expression, whereas the X-axis represents different sample groups. G Paired plots showing the expression of SORT1 in HCC tumor (T) vs. non-tumoral (NT) tissues across four datasets: The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC), ICGC LIRI, GSE37991, and GSE77314. Each line connects the paired NT and T samples with a significant SORT1 upregulation in the tumor samples. H Kaplan–Meier survival analysis curve displaying survival probability over time for two groups: low and high SORT1 expression. The high SORT1 group showcases a reduced survival probability compared to the low SORT1 group, indicating the potential role of SORT1 as a prognostic marker. The hazard ratio (HR) of 1.44 (95% confidence interval (CI): 1.02–2.03) with a log-rank p-value of 0.039 suggests a significant association between high SORT1 expression and poor prognosis. I Bar graph showing fold-changes in SORT1 expression across a cohort of patients with HCC (n = 86), highlighting its differential expression in HCC. J Western blot analysis and quantitative optical density of SORT1 in paired HCC and adjacent normal tissues obtained from various patients. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The analysis was performed using one-way ANOVA.

Fig. 2. Verification of SORT1 overexpression in hepatocellular carcinoma (HCC) cells using the GepLiver and the Mendeley Databases (DBs).

A Heatmap of SORT1 expression (z-score) in 24 datasets from the GepLiver DB. B SORT1 expression (log₂(TPM + 1)) across tissues with different phenotypes. C Top: Representative hematoxylin and eosin (H&E) images. Middle: Spatial transcriptomics (ST) spots indicated by non-malignant (yellow) and malignant (purple) hepatocytes from tumor tissues. Bottom: SORT1 expression in the spatial sections. P: patient, T: tumor tissue. D Proportion of SORT1⁺ cells (%) (top) and SORT1 expression (bottom) in all analyzed tumor tissues. P: patient, T: tumor tissue. E UMAP plot of the GepLiver DB, an integrated liver scRNA-seq dataset. Each cell is colored according to the dataset (left) and major cell type (right). F UMAP plot of scAtlasLC. Each cell is colored by major cell type. G SORT1 expression (left) and proportion of SORT1⁺ cells (%) in hepatocytes of the GepLiver DB across different phenotypes. H SORT1 expression (left) and proportion of SORT1⁺ cells (%) in hepatocytes of the Single-cell Atlas in Liver Cancer (scAtlasLC) across different phenotypes. Statistical significance is indicated as ns, non-significant; *p < 0.05; ****p < 0.0001. Analyses were performed using the Welch’s t-test and one-way ANOVA.

Fig. 3. Functional implications of SORT1 in hepatocellular carcinoma (HCC): impact on proliferation, colony formation, migration, apoptosis, and cell cycle dynamics.

A Proliferation curves of Huh-7 and Hep3B cells post-treatment with NC and siSORT1 monitored over 96 h. B Microscopic images showing the morphology of Huh-7 and Hep3B liver cancer cell lines after treatment with negative control (NC) or siSORT1. The bar graphs on the right quantify the cell numbers for each condition. C Colony formation assay for Huh-7 and Hep3B cells treated with NC or siSORT1. The number of colonies is quantified in the bar graphs. D Wound-healing assay showing the migratory potential of Huh-7 and Hep3B cells in response to SORT1 silencing at 0 and 48 h post-wound creation. Quantification of wound closure is shown in bar graphs on the right. E Cell cycle distribution of Huh-7 and Hep3B cells after post-siSORT1 transfection, as assessed via propidium iodide (PI) staining and flow cytometry. Quantitative data for cells in different phases are shown on the right. F Flow cytometry analysis of Annexin V/PI-stained Huh-7 and Hep3B cells transfected with siSORT1 or control siRNA (NC). The quantification of apoptotic cells is shown on the right side. G Expression profiles of cell cycle-related proteins (p-Wee1, Cyclin B1, Cyclin D1, Cyclin D3, Cdc2, and Cdc25A) in Huh-7 and Hep3B cells after siSORT1 transfection, as detected via western blotting. The densitometric analysis of protein bands is shown on the right. H Western blot analysis of apoptosis-related proteins, including cleaved PARP, cleaved caspase 3, and cleaved caspase 9, in Huh-7 and Hep3B cells transfected with siSORT1 or NC. The densitometric quantification of bands is shown on the right. I Fluorescence microscopy images of Huh-7 and Hep3B cells stained with Hoechst33342 and PI after SORT1 silencing. Apoptotic nuclei (yellow arrowheads) and damaged DNA (white arrowheads) are highlighted. The merged images provide a comprehensive view of the cell status with zoomed-in insets for clarity. Scale bars represent 100 µm. All experiments were repeated at least three times, and representative data are shown. Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. The analysis was performed using Welch’s t-test.

Fig. 4. In vivo attenuation of hepatocellular carcinoma progression via SORT1 silencing.

A Representative images showing tumor progression on day 15 in mice after SORT1 silencing, highlighting the reduced tumor size in the siSORT1 group compared to that in the NC group. B Body weight changes in mice over 15 d; NC vs. siSORT1 conditions. C Quantitative analysis of tumor growth over 15 d, highlighting a marked reduction in tumor size following SORT1 silencing (left). Comparative assessment of tumor weights between NC and siSORT1 mice (right). D Relative SORT1 mRNA expression in tumor tissues (left) and protein expression levels in NC versus siSORT1 conditions (right). E Histopathological examination of the tumors. Hematoxylin and eosin (H&E) staining showed the tumor architecture (leftmost). Subsequent panels display immunohistochemical staining for SORT1, Ki-67, and PCNA in both the NC and siSORT1 groups, with quantification of the stained areas on the right. F Visual representation of mouse liver on days 0 and 27 after siRNA treatment. G Gross anatomy of excised tumors from NC and siSORT1 mice. H Comparative display of metastatic nodules excised from the livers of the NC and siSORT1 groups (left). Quantitative analysis of the weights of these nodules indicated a decrease in metastatic potential upon SORT1 silencing (right). I Tumor histological assessment post-metastasis: H&E staining of metastatic nodules (left), followed by immunohistochemical analyses for SORT1, Ki-67, and PCNA. The right panel shows the quantification of the stained regions, confirming the reduced metastatic potential and cell proliferation upon SORT1 silencing. Scale bars represent 50 µm. All experiments were repeated at least three times, and representative data are shown. Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. The analysis was performed using Welch’s t-test.

Fig. 5. Functional impact of SORT1 on angiogenesis and systemic metastasis in HCC.

A Pathway analysis showing the most significant pathways related to SORT1 from the MSigDB Hallmark 2020 and Panther 2016 databases sorted by combined score. The p value is represented by color intensity. B Tube-formation assay in NC or siSORT1 transfected HUVECs and Hep3B cells. Images are shown at 4× and 10× magnifications. A quantitative analysis of the total tube length is shown on the right. C Western blot analysis of various epithelial-to-mesenchymal transition (EMT) markers in NC- or siSORT1-treated Huh-7 and Hep3B cells. Densitometric quantification is shown on the right-hand side. D Scatter plots illustrating the correlation between SORT1 and CDH2 (N-cadherin) and SORT1 and FN1 (fibronectin) based on TCGA LIHC data. The correlation coefficients (r) and p-value are provided. E Immunohistochemical staining of CD31, VEGF, ZO-1, and vimentin in tumors from mice with subcutaneous and orthotopic xenograft injections of either NC or siSORT1-transfected cells. F Body weight of mice subjected to either NC or siSORT1 treatment over a specified period. G Representative images of lung tumor nodules in mice on days 23, 35, 55, and 65 post-injection with either NC or siSORT1-transfected cells. Yellow arrows indicate precancerous blood dots and red arrows indicate tumors. H Quantitative analysis of the number of nodules formed in the lungs of mice treated with NC or siSORT1. Scale bars represent 50 µm. All experiments were repeated at least thrice, and representative data are shown. Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. The analysis was performed using Welch’s t-test.

Fig. 6. Correlation of SORT1 expression with CD133 and the Notch Signaling pathway in HCC.

A GSEA hallmark pathways analysis identifying the Notch signaling pathway as one of the top-ranked pathways in SORT1^high samples. B GO enrichment analysis confirming the enrichment of Notch signaling-related gene sets in SORT1^high samples. C Wilcoxon rank-sum test results showing ssGSEA scores of each Notch signaling pathway in samples stratified by SORT1 expression (top: scRNA-seq; bottom: bulk RNA-seq data). D Scatter plots representing the positive correlation between SORT1 and PROM1 (CD133), PROM1 and NOTCH1, and SORT1 and NOTCH1 expression in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) and ICGC LIRI datasets. Each dot represents a single sample. Pearson’s correlation coefficients (r) and p values are indicated for each plot. E Heatmap illustrating the correlation between SORT1 and selected genes involved in the Notch signaling pathway derived from the KEGG, Hallmark, and BIOCARTA datasets. Genes were chosen based on their established role in the Notch pathway and their significant positive correlation with SORT1 in TCGA LIHC dataset. The color intensities of the circles are proportional to the correlation coefficients. F Western blot analyses of Huh-7 and Hep3B cells treated with NC or siSORT1 showing the expression levels of Notch1, cleaved Notch1, and other pivotal Notch signaling molecules, along with CD133, in HCC cells. Statistical significance is indicated by **p < 0.01, ***p < 0.001. The analysis was performed using Welch’s t-test.

Fig. 7. Comprehensive analysis of SORT1 expression, genetic alteration, and clinical relevance in patients with hepatocellular carcinoma (HCC).

A SORT1 mRNA expression level based on gene copy number status from TCGA LIHC cohort (n = 371, left). Scatter plot illustrating the correlation between SORT1 mRNA expression and copy number (GISTIC) in TCGA LIHC cohort (n = 364, right). B Scatter plot illustrating the correlation between SORT1 mRNA expression and methylation in TCGA LIHC cohort (n = 371, left). Density plot displaying the methylation beta values for the (NT) and tumor (T) groups from the same cohort (right). C Genome-wide association between SORT1 copy number variations and differential mRNA expression in HCC tumor tissues. The red line indicates the 5′ promoter region corresponding to SORT1. D Dot plots displaying differential methylation levels for four significant CpG sites related to SORT1 between non-tumoral (NT) and tumoral (T) liver tissues, with significance values presented for each site. E Kaplan–Meier survival curves comparing overall survival (OS) and disease-free survival (DFS) between HCC patients with high and low SORT1 expression from The Cancer Genome Atlas (TCGA) database, accompanied by hazard ratio (HR) and log-rank p values. F Methylation levels at multiple CpG sites of SORT1 in non-tumoral (NT) and tumoral (T) liver tissues, assessed in both the test (left) and validation cohorts (middle) from Ajou University Hospital. Violin plot of SORT1 methylation in non-tumor (NT) and tumor (T) liver tissues in the total cohort from Ajou University Hospital (right). G Receiver operating characteristic (ROC) curve assessing the diagnostic performance of SORT1 methylation in differentiating T from NT tissues. H Violin plot of SORT1 methylation based on the vascular invasion status (left) and HCC stage (right). I Scatter plot depicting the inverse correlation between SORT1 expression and its methylation level in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) and AJOU_HCC cohorts. Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. The analysis was performed using Student’s t-test and Welch’s t-test.