Pan-cancer analysis of ITGA5 expression levels
To determine ITGA5 mRNA expression levels in both normal and tumor tissues, Oncomine was interrogated to analyze ITGA5 expression in various cancer types. The expression levels of ITAG5 were greater in colorectal, esophageal, gastric and pancreatic cancers relative to normal tissues (Figure 1A). In addition, the transcriptional levels for ITGA5 were analyzed using RNA-seq data for multiple malignancies in the TCGA with the use of TIMER. Results revealed significant differences in ITGA5 expression levels when comparing normal and tumor tissues. This was significantly higher in, of which was liver hepatocellular carcinoma (LIHC) and stomach adenocarcinoma (STAD) compared to normal tissue levels (Figure 1B).
Correlation between ITGA5 expression levels and patient prognosis
The PrognoScan database was used to investigates the relationship between ITGA5 mRNA levels and the survival of cancer patients using high-throughput analysis and detailed clinical prognosis data. ITGA5 expression was found to significantly impact prognosis in colorectal and ovarian cancers. Specifically, the cohorts (GSE17536) (29) included 177 samples at different stages of colorectal cancer and showed a remarkable association between high ITGA5 expression levels and poor prognosis (OS HR = 1.66, 95% CI = 1.16 to 2.37, Cox P = 0.005; DFS HR = 2.97, 95% CI = 1.70 to 5.21, Cox P = 0.0004) (Figures 2A–B). Similarly, the cohorts (GSE26712) (30) showed that high ITGA5 expression levels were correlated with poor prognosis (OS HR = 1.58, 95% CI = 1.06 to 2.36, Cox P = 0.025; DFS HR = 1.54, 95% CI = 1.06 to 2.24, Cox P = 0.025) in ovarian cancers (Figures 2C–D). Subsequently, clinical data from TCGA were used to explore the prognostic value of ITGA5 expression using GEPIA2. High expression of ITGA5 was marginally correlated with poor prognosis in pancreatic cancer (OS HR=1.6, P = 0.12; DFS HR = 2.1, P = 0.015) and esophageal cancer (OS HR=1.4, P = 0.37; DFS HR = 1.2, P = 0.52) (Figure 2E-H).
To further identify the prognostic potential of ITGA5 in various cancers, the Kaplan-Meier plotter database was used to determine the prognostic value of ITGA5. Significant differences were observed between ITGA5 expression and prognosis in gastric cancer (OS HR=2.4, 95% CI = 1.96 to 2.97, P < 1e-16; DFS HR = 2.69, 95% CI = 2.04 to 3.54, P = 2.2e-13) and live cancer (OS HR=1.73, 95% CI = 1.22 to 2.44, P = 0.0017; DFS HR = 1.23, 95% CI = 0.88 to 1.72, P = 0.23) patients (Figure 2I-L). Interestingly, high expression of ITGA5 was also associated with poor prognosis in breast cancer (OS HR=1.21, 95% CI = 0.96 to 1.52, P = 0.11; DFS HR = 1.28, 95% CI = 1.14 to 1.44, P = 4.1e-05) and lung cancer (OS HR=1.6, 95% CI = 1.4 to 1.81, P = 5.9e-13; DFS HR = 1.75, 95% CI = 1.44 to 2.12, P = 1.2e-08) patients (Figure 2M-P).
Relationship between ITGA5 expression and immune infiltration levels in gastrointestinal cancers
Recent work has shown that gastrointestinal tumors exhibit extensive immune infiltration characteristics (31-33). Therefore, to better understand the role of ITGA5 in GI tumors, the relationship between ITGA5 expression and immune infiltration in GI tumors was investigated using the TIMER database. ITGA5 expression was found to be significantly correlated with tumor purity in colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), STAD and rectum adenocarcinoma (READ) (P < 0.05), CD8+ cell in COAD, LIHC and pancreatic adenocarcinoma (PAAD), CD4+T cells in COAD, PAAD, LIHC, READ and STAD (P < 0.05) (Figure 3A-F). Furthermore, ITGA5 expression was significantly associated with macrophages and dendritic cells in COAD, ESCA, READ, LIHC, PAAD and STAD (P < 0.05) (Figure 3A-F). ITGA5 expression was also significantly associated with neutrophils in COAD, LIHC, READ, PAAD and STAD (P < 0.05) (Figure 3A-F).
Specifically, ITGA5 expression levels showed significantly positive correlations with infiltrating levels of CD8+T cells (r =0.248, P = 4.18e-07), CD4+ T cells (r = 0.423, P = 6.67e-19), macrophages (r = 0.545, P = 1.20e-32), neutrophils (r = 0.590, P = 4.92e-39) and dendritic cells (r = 0.556, P = 5.48e-34) in COAD (Figure 3A). Similarly, positive correlations were identified with infiltrating levels of CD4+ T cells (r = 0.426, P =1.52e-17), macrophages (r = 0.447, P = 1.42e-19), neutrophils (r = 0.189, P = 2.47e-04) and dendritic cells (r = 0.28, P = 4.19e-08) in STAD (Figure 3F). These results revealed that ITGA5 expression is closely associated with immune infiltration in GI tumors.
Correlation between ITGA5 expression levels and immune markers
Gastric cancer and colorectal cancer are the most common GI-related malignancies. Therefore, COAD and STAD were chosen to further investigate the relationship between ITGA5 expression and immune marker genes of diverse immune cells using TIMER databases. The immune cells analyzed included CD8+ T cells, general T cells, different functional T cells, B cells, monocytes, TAMs, M1 macrophages and M2 macrophages, neutrophils, NK cells and dendritic cells. After adjusting correlation by purity, ITGA5 expression was found to be significantly correlated with immune markers of most immune cells in STAD and COAD (Table 1 and Figure 4).
There is a great deal of evidence suggesting that an increased number of M2 macrophages is associated tumor growth, angiogenesis, invasion and metastasis (34, 35). M2 cells promote tumor and local immunosuppression (36). Th2 cells release IL4 and IL13 and induce M2 cell polarization (37, 38). Immune markers for monocytes, TAMs, M2 and Th2 phenotypes were found to be significantly and positively correlated with ITGA5 expression levels in COAD and STAD (P < 0.05; Figures 4A–H). These findings support the hypothesis that ITGA5 may be a critical factor for macrophage polarization and immune escape in COAD and STAD.
High ITGA5 expression affects the prognosis of gastric cancer patients exhibiting lymph node metastasis
To better understand the relationship between ITGA5 expression levels and clinicopathological features, further research was focused on gastric cancer. According to Kaplan-Meier plotter database analyses, overexpression of ITGA5 is strongly correlated with the deterioration of OS and progression-free survival (PFS) based on gender, differentiation and Lauren classification (P < 0.05). In addition, stage 1 through 4 cancer patients with high expression levels of ITGA5 showed worse OS (P < 0.05). Similarly, high expression levels of ITGA5 were associated with worse PFS in stage 2 through 4 gastric cancer patients (P < 0.05) but not in stage 1 gastric cancer patients (PFS). However, there was no strong correlation observed between ITGA5 expression levels and patient prognosis in stage N0 (OS HR = 1.92, P = 0.13; PFS HR = 1.95, P = 0.11), mixed Lauren classification (OS HR = 1.88, P = 0.22; PFS HR = 0.48, P = 0.16) or poor differentiation (OS HR = 0.75, P = 0.16; PFS HR = 1.39, P = 0.25) (Table 2). N category refers to lymph node involvement . Moreover, high ITGA5 levels express the highest HR value of N1 with OS and PFS among four N categories (Table 2). These results indicate that the expression levels of ITGA5 are related to lymph node metastasis in gastric cancer patients.
ITGA5 protein expression and M2 and Th2 immune marker genes in STAD
To confirm the results obtained from publicly available databases, IHC were performed on adjacent normal and STAD tumor tissues obtained from 40 gastric patients. Compared with adjacent normal tissues, ITGA5, CD163, STAT6 and GATA3 levels were greater in most tumor tissues (Figure 5A). In addition, these results revealed that the expression levels of CD163 (χ2 = 8.750, P = 0.003), STAT6 (χ2 = 8.174, P = 0.004) and GATA3 (χ2 = 5.079, P = 0.024) in the ITGA5 high-expression group were significantly higher than what was observed in the ITGA5 low-expression group (Table 3). In addition, we attempted to detect the proteins levels of ITGA5, CD163, STAT6 and GATA3 in patient tissues by western blot. As figure 5B shows, the expression levels in tumor tissues were indeed higher than that in adjacent normal tissues.
ITGA5 PPI network and functional enrichment
PPI network analysis revealed interactions between ITGA5 and specific genes. As shown by GeneMANIA, genes interacting with ITGA5 included SPP1, ITGB1, ITGB3, COL18A1, HOXD3, ANGPTL3, CD9, FBN1, VEGFD, ITGA2B, ITGA4, FN1, FLT4, TLN1, ACVRL1, FUBP1, THBS4, ACTN1, ANGPT2 and ITGA8 (Figure 6A).
To further predict enriched functional information of ITGA5 interacting genes, GO and KEGG pathway analyses were performed using Metascape. According to GO term analysis, genes interacting with ITGA5 in the PPI network are mainly related with cell-substrate adhesion, regulation of leukocyte migration, response to wounding and myeloid cell differentiation. KEGG pathway analysis showed these genes are enriched in focal adhesion, PID avb3 integrin pathway and PID integrin5 pathway. Based on these results, ITGA5 and its interacting proteins were shown to play an important role in the integrin mediated signaling pathway, leukocyte migration, cell-substrate adhesion and other essential biological processes (Figure 6B). In addition, Cytoscape was used to determine the relationship for the enriched terms and to build a network diagram (Figure 6C and 6D)