Identification of potential targets of triptolide in regulating the tumor microenvironment of stomach adenocarcinoma patients using bioinformatics

Abstract

This study aimed to identify potential pharmacological targets of triptolide regulating the tumor microenvironment (TME) of stomach adenocarcinoma (STAD) patients. A total of 343 STAD cases from The Cancer Genome Atlas (TCGA) were assigned into high- or low-score groups applying Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE). Hub genes were identified from differentially expressed genes (DEGs) shared by stromal- and immune-related components in the TME of STAD patients using R software. Cox regression analysis was used to identify genes significantly correlated with STAD patient survival. Triptolide target genes were predicted from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Top 30 genes filtered by Cytohubba from 734 DEGs were screened as hub genes. Forty-two genes were found to be at high risk for STAD prognosis. Thirty-four targets of triptolide were predicted using the TCMSP database. Importantly, C-X-C chemokine receptor type 4 (CXCR4) was identified as a potential target of triptolide associated with the TME in STAD. Analysis of survival highlighted the association between CXCR4 upregulation with STAD progression and poor prognosis. Gene Set Enrichment Analysis (GSEA) confirmed that genes in the CXCR4- upregulated group had significant enrichment in immune-linked pathways. Additionally, triptolide targets were found to be significantly enriched in CXCR4-related chemokine and cancer-related p53 signaling pathways. Molecular docking demonstrated a high affinity between triptolide and CXCR4. In conclusion, CXCR4 may be a therapeutic target of triptolide in the treatment of STAD patients by modulating the TME.

Keywords: The cancer genome atlas (TCGA); bioinformatics; estimation of stromal and immune cells in malignant tumors using expression data (estimate); stomach adenocarcinoma; triptolide; tumor microenvironment.

Graphical abstract

Figure 1.

Box plots of correlation between ESTIMATE scores with clinicopathological staging characteristics. (2a, 2b, 2 c) StromalScore, ImmuneScore and ESTIMATEScore of STAD patients in each stage. (2d, 2e, 2 f) StromalScore, ImmuneScore and ESTIMATEScore of STAD patients in each grade. (2 g, 2 h, 2i) StromalScore, ImmuneScore and ESTIMATEScore of STAD patients in each tumor classification. (2 j, 2k, 2 l) StromalScore, ImmuneScore and ESTIMATEScore of STAD cases with or without lymph node metastasis. (2 m, 2 n, 2o) StromalScore, ImmuneScore and ESTIMATEScore of STAD cases with or without distant metastasis. P-values based on the Wilcoxon signed-rank test; p < 0.05 was statistically significant

Figure 2.

Heatmaps, Venn diagrams, and enrichment analysis of the DEGs. (2a) Heatmapped DEGs within the stromal high-scoring group versus low-scoring group (|logFC| >1; FDR < 0.05 based on Wilcoxon test); red represents higher expression and blue represents lower expression. (2b) Heatmapped DEGs in immune high-scoring group versus low-scoring group (|logFC| >1; FDR < 0.05 based on Wilcoxon test); red represents higher expression and blue indicates lower expression. (2 c) Venn diagram of 624 commonly upregulated genes (stromal/immune-linked scorings). (2d) Venn diagram of 110 commonly downregulated genes (stromal/immune-linked scorings). (2e) Ten highest-ranking GO terms for BP, CC and MF enriched using DEGs shared by stromal and immune scores (q value < 0.05). (2 f) Top 30 KEGG terms enriched by DEGs and ranked by q values from highest to lowest (q value < 0.05)

Figure 3.

CXCR4, a hub gene from the PPI network, correlated with STAD prognosis and was identified as a target gene of triptolide. (3a) PPI network (STRING) based on DEGs (combined score = 0.95). (3b) Interaction network constructed using Cytoscape v3.7.2 based on DEGs with a minimum required interaction score > 0.95; green and red illustrate downregulated and upregulated genes, respectively. (3 c) Sub-network of 30 hub genes filtered by Cytohubba MCC method; node score increases from light to dark color. (3d) Forest plot of Cox regression analysis with DEGs; genes with p < 0.05 and HR > 1 were identified as high-risk factors for STAD prognosis. (3e) Venn diagram of 30 hub genes from the PPI network, 42 prognosis-related genes based on Cox regression analysis, and 34 target genes of triptolide

Figure 4.

Comparison between CXCR4 expression with prognosis/clinicopathological features in STAD patients. (4a, 4b) Difference analysis and paired difference analysis showed significantly higher CXCR4 expression levels in STAD tissues versus normal tissues (p < 0.05, Wilcoxon test). (4 c) Patients were categorized into CXCR4 high-/low-expressing cohorts; survival curve showed that CXCR4 was linked to poor prognosis of STAD patients (p = 0.003). (4d) CXCR4 expression levels were significantly higher in T2/3/4 versus T1 and in T4 versus T2 (p < 0.05). (4e) No correlation detected between CXCR4 expression and N classification (p > 0.05). (4 f) CXCR4 showed no difference between patients with and without metastasis (p = 0.059)

Figure 5.

GSEA of top ten KEGG pathways with the highest FDR q values for samples with high CXCR4 expression. Individual lines represent a specific KEGG pathway and associated color; left-hand-side genes were positively associated with CXCR4, and genes on the right negatively correlated with CXCR4

Figure 6.

Enrichment analysis of triptolide target genes and molecular docking between triptolide and CXCR4. (6a) Ten highest-ranking GO terms for BP, CC, and MF enriched by triptolide targets (q value < 0.05). (6b) Top 15 KEGG pathways enriched by triptolide targets and ranked by q values from the highest to the lowest. (6 c) The optimal mode of molecular interaction between triptolide and CXCR4 is shown in its three-dimensional model and the binding point is located in the center of the active binding pocket. (6d) Hydrophobicity of molecular interaction between triptolide and CXCR4. (6e) Hydrogen bonds are formed with Tyr121, Arg188, and Tyr255