GRAP2 is a prognostic biomarker and correlated with immune infiltration in lung adenocarcinoma

Abstract Background GRAP2 is an adaptor protein involved in leukocyte signal activation; however, the prognostic value of GRAP2 and its correlation with immune infiltration in lung adenocarcinoma (LUAD) are unclear. Methods Original data were downloaded from the TCGA database and Gene Expression Omnibus (GEO) database. GRAP2 expression was analyzed with the TCGA and TIMER databases. We evaluated the influence of GRAP2 on clinical prognosis using the Kaplan–Meier plotter, GEO, and GEPIA database. The TIMER and TISIDB databases were used to investigate correlations between GRAP2 expression and cancer immune characteristics. Finally, we confirmed the expression of GRAP2 in LUAD by immunohistochemistry staining. Results The transcription levels of GRAP2 were significantly lower in several human cancer types, including LUAD, than in adjacent normal tissues. Immunohistochemistry staining confirmed that LUAD tumor tissues had lower GRAP2 protein expression levels than adjacent normal tissues. GRAP2 downregulation was associated with poorer overall survival, pathologic stage, T stage, N stage, and primary therapy outcome in LUAD. Mechanistically, we found a hub gene set that included a total of 91 genes coexpressed with GRAP2, which were closely related to the immune response in LUAD. The expression levels of GRAP2 were positively correlated with the infiltration levels of multiple immune cells and the cumulative survival time of a few immune cells. GRAP2 expression was found to be positively correlated with that of multiple immune markers, chemokines, chemokine receptors, and MHC molecules in LUAD. Conclusions GRAP2 can be used as a biomarker for assessing prognosis and immune infiltration levels in LUAD.


| INTRODUC TI ON
Lung cancer is one of the malignant tumors with the highest incidence and worst prognosis in the world. 1 Lung adenocarcinoma (LUAD) is a common subtype of lung cancer. [2][3][4] Despite improvements in systemic treatments for patients, the 5-year survival rate of LUAD patients is 15%. 5 Therefore, it is crucial to explore useful prognostic biomarkers and therapeutic targets against LUAD for diagnosis, prevention, and treatment.
The tumor microenvironment (TME) includes the extracellular matrix, stromal cells, and tumor-infiltrating immune cells (TIICs) that shape cancer development. 6 Increasing evidence indicates that TIICs determine the success of immunotherapy and affect the prognosis of patients. 7 Currently, which factors drive immune infiltration in LUAD is unclear. Therefore, it is necessary to find new biomarkers to assess immune infiltration in LUAD.

GRAP2 (Gads/Mona/GrpL/Grf40) is an important adaptor protein
in protein-tyrosine kinase signal transduction in leukocytes. 8 Studies have shown that GRAP2 is highly expressed in lymphoid organs and T lymphocytes. GRAP2 forms signaling complexes with different signaling molecules to mediate the activation and signal transduction of T cells. 9 At present, research on GRAP2 mainly focuses on the immune system. 10,11 The role of GRAP2 in tumors and its relationship with immune infiltration are largely unknown. There is only one study on the role of GRAP2 in cancer, and the research indicates that GRAP2 directly interacts with the tyrosine kinase RET and inhibits RET-induced NF-κB activation in a dose-dependent manner in medullary thyroid cancer cells. 12 In recent years, an increasing number of platforms and open databases have enabled researchers to use multiple datasets for cancer bioinformatics analysis. 4,13 To better explore the role of GRAP2 in LUAD, we assessed the relationship between GRAP2 expression and the prognosis of patients with LUAD using TCGA, TIMER, and the Kaplan-Meier plotter database. In addition, we investigated the correlation between GRAP2 expression and immune infiltration using the TIMER and TISIDB databases. Our findings revealed that GRAP2 can be used as a biomarker for assessing prognosis and immune infiltration levels in LUAD.

| Correlation heatmap and protein-protein interaction (PPI) network analysis
The GeneMANIA database (http://www.genem ania.org) 17 was used to generate a correlation heatmap for the genes coexpressed with GRAP2. The STRING database (https://strin g-db.org) was used to obtain PPI data based on protein interactions and signaling pathways, and Cytoscape 3.7.2 was used to construct the network.

| GEPIA database analysis
GEPIA (http://gepia.cance r-pku.cn/index.html) 16 was used to screen genes that were positively or negatively correlated with GRAP2 by applying the "Similar Genes" module. The "Survival" module in GEPIA was used to explore genes associated with LUAD prognosis.

| Linked omics database analysis
The Linked Omics database (http://www.linke domics.org) 18 contains 32 tumor types from the TCGA database and multiomics data and clinical data of a total of 11,158 patients. In this study, the gene ontology biological process (GO_BP) and KEGG pathway analyses were performed using the Linked Omics database.

| Timer database analysis
In this study, the TIMER database (http://www.cistr ome.shiny apps. io) 19 was used to examine gene expression in normal tissues and tumor tissues from a variety of tumor types. Based on the gene expression profile of LUAD samples in TCGA data, the abundance of TIICs was also analyzed using the TIMER database.

| TISIDB database analysis
The TISIDB database (http://cis.hku.hk/TISIDB) 20 is a highly cited portal that allows researchers to interactively explore tumor and immune system interactions. In this study, the "Immunomodulator" module of the TISIDB database was utilized to explore the correlation between GRAP2 expression and MHC molecules. To investigate the relationship between GRAP2 and chemokine/chemokine receptor expression, we examined the chemokine/chemokine receptor expression level in TIICs using the "chemokine" module.

| Immunohistochemistry (IHC)
Seventy-six paraffin-embedded LUAD tissues and para-carcinoma tissues were used for IHC staining. Tissues were incubated with anti-

| Statistical analysis
The original data were calculated using GraphPad Prism software (version 8.0). Gene expression correlations were evaluated via Spearman's correlation, and statistical significance was determined.
Univariate and multivariate Cox analyses were used to screen for potential prognostic factors. A Mann-Whitney test and an independent t test were used to analyze differences between two groups of data. Differences among multiple groups were calculated using oneway ANOVA with a post hoc Tukey test and chi-square test. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

| Decreased GRAP2 expression in LUAD
The TIMER database was used to assess the transcriptional level of   Table 1 summarizes the correlation between GRAP2 expression and clinical characteristics in LUAD. The results showed that low expression of GRAP2 was associated with late T stage, N stage, pathologic stage, and worse primary therapy outcome ( Figure 1D-G and Table 1). The protein expression level of GRAP2 was further investigated by immunohistochemical staining, the clinicopathological features of the LUAD cases are presented in Supplementary Table S1, and we found that the GRAP2 protein level was obviously decreased in LUAD tissues compared with adjacent normal tissues ( Figure 2A,B).
Finally, we investigated GRAP2 expression in LUAD cell lines, and the data showed that GRAP2 mRNA levels were significantly downregulated in three LUAD cell lines (A549, H1975, and H1299) compared with levels in a normal lung epithelial cell line (BEAS-2B) ( Figure 2C).

| Low GRAP2 expression is an independent prognostic factor for overall survival in LUAD
We investigated whether GRAP2 expression is correlated with prognosis in patients with LUAD. We divided the LUAD patients from the TCGA data- we used an independent external GEO dataset, GSE37745, to verify our results, and the results were consistent with the above findings. LUAD patients with higher GRAP2 expression showed higher OS than patients with lower GRAP2 expression ( Figure S2). These data indicate that GRAP2 is a tumor suppressor gene and can be used as an independent prognostic factor for OS in LUAD.

| GRAP2 is associated with the immune response in LUAD
To examine the biological function of GRAP2 in LUAD, we used the GEPIA database to detect the coexpression pattern of GRAP2  (Table S2). These data suggest that GRAP2 is closely related to the regulation of immune cell function.
We also performed GO and KEGG annotation analyses in LUSC.
The data showed that enriched pathways in the genes coexpressed with GRAP2 (top 600) were closely associated with the immune response in LUSC, but there were few overlapping enrichment items between LUAD and LUSC ( Figure S3).
To gain further insight into the underlying mechanisms by which GRAP2 regulates LUAD prognosis, the survival-related and downregulated genes in LUAD were screened using the GEPIA database.
We crossed the 600 genes that were coexpressed with GRAP2 with 731 survival-related and downregulated genes in LUAD, and we identified a gene set containing 91 genes at the intersection ( Figure 5C). We performed functional enrichment in these 91 genes using GO and KEGG annotations, and several biological processes appeared to be particularly enriched, including the external side of the plasma membrane, specific granule membrane, MHC protein complex, T-cell activation, and lymphocyte differentiation ( Figure 5D).
We further used protein-protein interactions (PPIs) and correlation analysis to explore the interactions between these 91 proteins.
We found a higher enriched protein-protein interaction network among these proteins than random proteins ( Figure 6A). Furthermore, most of the proteins in the network had a strong positive correlation with each other ( Figure 6B). Therefore, these established genes coexpressed with GRAP2 are particularly related to the immune response, which may be the molecular mechanism by which GRAP2 affects the prognosis of patients with LUAD. Moreover, these coexpressed genes can potentially be used in a multigene biomarker panel to predict survival in LUAD.

| The expression level of GRAP2 is correlated with immune infiltration in LUAD
Since the hub genes were enriched in immune response-related pathways, the association between the immune score and GRAP2 expression was further explored. We divided LUAD cases into two cohorts according to the expression level of GRAP2 and estimated the immune score using the ESTIMATE database. The immune score was significantly higher in the cohort with high GRAP2 expression than in the cohort with low GRAP2 expression ( Figure 7A).
Studies have shown that the survival time of many cancer patients is determined by the number and activity status of TIICs. 6 Therefore, we used the TIMER database to explore the correlation between GRAP2 but not CD8 + T cells, CD4 + T cells, neutrophils, or macrophages ( Figure 7D). These data suggest that GRAP2 plays a specific role in immune infiltration in LUAD.

| GRAP2 is positively correlated with various immune markers
To further explore the relationship between GRAP2 and the immune response, the TIMER database was used to investigate correlations between GRAP2 expression and diverse immune markers in LUAD. These immune markers can be used to characterize immune

cells, including T cells (general), CD8+ T cells, Th1 cells, Th2 cells, follicular helper T cells, Th17 cells, Tregs, effector T cells, effector memory T cells, resident memory T cells, general memory T cells, exhausted T cells, B cells, monocytes, neutrophils, natural killer cells,
and dendritic cells. In clinical cancer biopsies, tumor purity is an important parameter reflecting the level of immune infiltration. 23 Our data showed that GRAP2 expression was positively correlated with most immune markers in multiple types of immune cells, and 58 of 59 immune markers presented a positive correlation with GRAP2 expression in LUAD (Table 2). These data further support the notion that GRAP2 expression is significantly correlated with immune infiltration.

| GRAP2 is positively correlated with chemokine/chemokine receptors and MHC molecules
Chemokines and chemokine receptors play important roles in the infiltration of immune cells into tumors. 24 To investigate the potential mechanism by which GRAP2 regulates immune infiltration in LUAD, the TISIDB database was used to examine correlations between the expression level of GRAP2 and those of chemokines/ chemokine receptors. A heatmap showed that GRAP2 expression was positively correlated with that of various chemokines and chemokine receptors in multiple tumor types ( Figure 8A,C). We also comprehensively explored the correlation between GRAP2 expression and chemokine/chemokine receptors using scatter plots. The data showed that GRAP2 expression was positively correlated with multiple chemokines, such as CCL4, CCL5, CCL18, CCL19, CXCL9, CXCL10, CXCL11, CXCL13, and XCL2 ( Figure 8B). The GRAP2 expression level was also positively associated with multiple chemokine receptors, such as CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, CXCR4, and CXCR6 ( Figure 8D).

| DISCUSS ION
GRAP2 is an adaptor protein in the GRB2 family. 8 subsets. Therefore, the precise role of GRAP2 in the TME needs further exploration.
Chemokines are secreted proteins with a low molecular weight that mainly mediate immune cell trafficking and lymphoid tissue development. 24 Immune cells are recruited into the TME by interactions between chemokines and paired chemokine receptors. 6  immune escape of tumor cells. 39,40 Here, our data indicate that GRAP2 expression is positively correlated with numerous MHC molecules.
These data strongly suggest that GRAP2 plays an important role in the presentation of tumor antigens in LUAD.
However, despite our systematic analysis of GRAP2, there are still some limitations in this study. First, in vivo/in vitro experiments are needed to demonstrate the effect of GRAP2 on tumor progression to improve the reliability of our results. Second, although we concluded that GRAP2 expression is closely related to immune infiltration in LUAD and patient prognosis, we do not have direct evidence that GRAP2 influences prognosis by regulating immune infiltration. These problems deserve further experimental verification in the future.
Third, this study is mainly based on public databases, and thus, the quality and uniformity of the data from different databases can affect the interpretation of the research results. However, the analysis of data from multiple databases produced similar results, which supports the conclusions of our study.

| CON CLUS ION
We conclude that GRAP2 is a tumor suppressor gene and can po-

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available from the corresponding author upon reasonable request.