3.1 Decreased GRAP2 expression in LUAD
In order to explore the expression level of GRAP2 in tumor tissues and normal tissues, we used the TIMER database to analyze the expression level of GRAP2 mRNA in different tumors. The results showed that, compared with normal tissues, GRAP2 expression levels in bladder urothelial carcinoma, breast invasive carcinoma, colon adenocarcinoma, LUAD, lung squamous cell carcinoma (LUSC), prostate adenocarcinoma, rectum adenocarcinoma and other tumor tissues were significantly lower than normal tissues. However, higher expression was observed in tumors such as esophageal carcinoma and kidney renal clear cell carcinoma (Figure 2A).
We further used the TCGA database to evaluate the mRNA expression level of GRAP2 in tumor tissues compared with adjacent normal tissues in LUAD. The results showed that the expression level of GRAP2 was significantly lower than that in unmatched adjacent normal tissues (P<0.01) (Figure 2B). These results were verified in matched tumor tissues and adjacent normal tissues (Figure 2C). We also found that the mRNA expression level of GRAP2 was significantly lower than that in adjacent normal tissues in LUSC (Supplementary Figure 1A and 1B).
To investigate the correlation between GRAP2 expression and clinical characteristics in LUAD patients, we analyzed the mRNA expression levels of GRAP2 in different clinical categories in the TCGA database. Table 1 summarizes the correlation between GRAP2 expression and clinical characteristics in LUAD. The results show that the low expression of GRAP2 is associated with late T stage, N stage, Pathologic stage, and worse Primary therapy outcome (Figure 2D-2G and Table 1). The protein expression level of GRAP2 was further investigated by IHC staining, and we found that the GRAP2 protein level was obviously decreased in LUAD tissues compared with adjacent normal tissues (Figures 2A and 2B). Moreover, we found that GRAP2 mRNA expression was significantly down-regulated in three LUAD cell lines (A549, H1975 and H1299) compared to that in a nonmalignant lung epithelial cell line (BEAS -2B) (Figure 2C).
3.2 Low GRAP2 expression is an independent prognostic factor for overall survival in LUAD
We investigated whether GRAP2 expression correlates with prognosis in cancer patients. We divided the LUAD patients in the TCGA database into high (top 50% samples with the highest expression) and low (50% remaining samples) cohorts according to GRAP2 expression level for survival analysis. LUAD patients with higher expression of the GRAP2 expression exhibited good overall survival (OS) (HR=0.61, P=0.001) according to the Kaplan-Meier survival analysis (Figure 3A). However, the correlation between GRAP2 expression and the OS in LUSC was not significant (HR=0.95, P=0.727) (Supplementary Figure 1C). Therefore, we will only analyze the role of GRAP2 in LUAD in the follow-up. Subgroup analysis showed that high GRAP2 expression was significantly associated with longer OS in LUAD under the following features: T2 stage (HR = 0.66, P = 0.035), N0&N1 stage (HR = 0.67, P = 0.018), M0 stage (HR = 0.54, P = 0.001), Pathological stage III (HR = 0.55, P = 0.048), Primary therapy outcome, PD&SD (HR = 0.47, P = 0.008), Residual tumor R0 (HR = 0.54, P = 0.001), Smoker ( HR = 0.61, P = 0.004), Male patients (HR=0.63, P=0.034), Female patients (HR=0.59, P=0.012), Age >65 years (HR=0.57, P=0.007) and Age <=65 years (HR=0.62, P=0.003) (Figure 3B-3L). Cox analysis was also used to explore the correlation between GRAP2 expression and OS. Data showed that high GRAP2 expression was significantly associated with longer overall survival (Univariate Cox: hazard ratio HR = 602, 95% CI = 0.448–0.808, P <0.001; Multivariate Cox: hazard ratio HR = 602, 95% CI = 0.448–0.808, P <0.001) (Figure 4). Finally, we used an independent external GEO data set GSE37745 to verify our results, the result showed that the overall survival of LUAD patients with high expression of GRAP2 were significantly higher than those of patients with low expression (Supplementary Figure 2). These data indicate that GRAP2 is a tumor suppressor gene and can be used as an independent prognostic factor for OS in LUAD.
3.3 GRAP2 is associated with immune response in LUAD
To examine the biological function of GRAP2 in LUAD, we used the GEPIA database to detect the co-expression pattern of GRAP2 in LUAD. The first 25 genes are positively correlated with GRAP2 expression, and the last 25 genes are negatively correlated with GRAP2 expression in the heat map (Figure 5A).
We used the “Link Interpreter” module of the Linked Omics website to identify the GO functional enrichment and KEGG pathway in the co-expressed genes of GRAP2 (top 600), and found that these genes were enriched in immune response processes, such as Th17 cell differentiation, T cell activation, initial immune deficiency, cytokine receptor activation and so on (Figure 5B). We have also performed GO functional enrichment and KEGG pathway analysis in LUSC. The data showed that GRAP2 was closely associated with the immune response-related pathways in LUSC, but there are few overlapping enrichment items between LUAD and LUSC (Supplementary Figure 3).
To better understand the relevance and underlying mechanisms of GRAP2 expression on the prognosis of LUAD. The survival-related and down-regulated genes in LUAD were screened using GEPIA database. We crossed the 600 genes that co-expressed with GRAP2 with 731 survival-related and down-regulated genes in LUAD, and detected 91 genes at the intersection (Figure 5C). These 91 protein-coding genes may be the potential genetic biomarkers for LUAD patients. GO functional enrichment and KEGG pathway analysis were performed in these 91 genes, and the results showed that these genes were enriched in external side of plasma membrane, specific granule membrane, MHC protein complex, T cell activation and lymphocyte differentiation and so on (Figure 5D).
We further used protein-protein interaction (PPI) and correlation analysis to identify the interactions between these 91 proteins. We found that there is a stronger enrichment network between these proteins than random proteins, and these genes are particularly related to the immune response pathway (Figure 6A). Gene co-expression correlation analysis showed that most of the proteins in the network have a strong positive correlation with each other (Figure 6B). Therefore, these established genes co-expressed with GRAP2 particularly related to immune response, which may be the molecular mechanisms that GRAP2 affecting the prognosis, and can be used as multi-gene biomarkers to predict the survival in LUAD.
3.4 The levels of GRAP2 expression correlate with the immune infiltration level in LUAD
Since the signature was composed of immune-related genes, the association between the immune score and GRAP2 expression was further explored. We divided the cases into high-expression and low-expression cohorts according to the expression level of GRAP2 and estimated the immune score by the ESTIMATE database. The results showed that the immune score in GRAP2 high expression cohort was significantly higher than that in low expression cohort (Figure 7A).
Researches show that the survival time of several cancer patients is determined by the number and activity status of TIICs . Therefore, we used the TIMER database to analyze the correlation between GRAP2 expression and immune infiltration in LUAD. The results showed that numerous immune cells (except γδ T cells and Th2 cells) showed higher immune infiltration level in GRAP2 high expression cohort (Figure 7B). Furthermore, GRAP2 expression level was negatively correlated with tumor purity, however, it was positively correlated with infiltration levels of B cells (r=0.579, P<0.001), CD8+ T cells (r= 0.512, P<0.001), CD4+ T cells (r= 0.562, P <0.001), macrophages (r =0.252, PP<0.001), neutrophils (r=0.484, P<0.001) and dendritic cells (r =0.536, P<0.001) (Figure 7C). We also analyzed the correlation between GRAP2 expression level and immune cell survival in LUAD. The data showed that cohort of high GRAP2 expression had a higher cumulative survival time in B cells (P=0) and dendritic cells (P=0.048), but not in CD8+ T cells, CD4+ T cells, neutrophils and macrophages (Figure 7D). These data suggest that GRAP2 plays a specific role in immune infiltration in LUAD.
3.5 GRAP2 is positively correlated with various immune markers
To deepen our understanding of GRAP2 crosstalk with the immune response, TIMER database was used to investigate the correlations between GRAP2 expression and diverse immune signatures in LUAD. The genes listed were used to characterize immune cells, including T cell (general), CD8+ T cell, Th1, Th2, Follicular helper T cell, Th17, Treg, Effector T-cell, Effector memory T-cell, Resident memory T-cell, General memory T-cell, Exhausted T-cell, B cell, Monocyte, Neutrophils, Natural killer cell and Dendritic cells. Tumor purity is an important aspect affecting the dissection of immune infiltration in clinical cancer biopsies. After adjusting for tumor purity, GRAP2 expression was significantly associated with most immune markers in divergent types of immune cells, the GRAP2 expression level was significantly correlated with 58 of 59 immune markers in LUAD (Table 2). These data further support that GRAP2 expression is significantly correlated with immune infiltration.
3.6 GRAP2 is positively correlated with chemokines/ chemokines receptors and MHC molecules
Chemokines and chemokine receptors are essential for the immune cells towards the tumors . Therefore, we used the TISIDB database to analyze the correlation between the expression level of GRAP2 and immune cell related chemokines/ chemokine receptors in LUAD. The heat map results showed that GRAP2 expression was positively correlated with various chemokines and chemokine receptors in multiple tumors (Figure 8A, 8C). In order to further clarify the relationship between GRAP2 expression and immune cell migration, we comprehensively analyzed the correlation between GRAP2 expression and chemokine/chemokine receptors. The results show that GRAP2 expression was positively correlated with immune cell-related chemokines, such as CCL4, CCL5, CCL18, CCL19, CXCL9, CXCL10, CXCL11, CXCL13 and XCL2 (Figure 8B). GRAP2 expression was also positively associated with immune cell-related chemokine receptors, such as CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, CXCR4, and CXCR6 (Figure 8D).
A large number of studies have shown that downregulation of major histocompatibility complex class-I and -II (MHC-I and MHC-II) has been linked to immune suppression, metastatic progression and a poor prognosis in numerous tumors . Therefore, we used the TISIDB database to analyze the correlation between GRAP2 expression and MHC molecules. The heat map results showed that the MHC molecules in numerous tumors were significantly positively correlated with the expression of GRAP2 (Figure 9A). The expression level of GRAP2 was positively correlated with the expression of multiple MHC molecules, including B2M, HLA-B, HLA-C, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA- DOA1, HLA-DOA2, HLA-DRA, HLA-DRB1, HLA-E, HLA-F and TAP1 (Figure 9B-9Q). Therefore, it was confirmed that GRAP2 participating widely in modulating various immune molecules in LUAD to affect immune infiltration in TME.