The Expression Pattern of Adhesion G Protein-Coupled Receptor F5 Is Related to Cell Adhesion and Metastatic Pathways in Colorectal Cancer—Comprehensive Study Based on In Silico Analysis

Adhesion G protein-coupled receptor F5 (ADGRF5) is involved inthe neoplastic transformation of some cancer types. However, the significance of ADGRF5 expression signature and the impact of signaling pathways mediated by ADGRF5 during neoplastic transformation of the colon and colorectal cancer (CRC) progression has been poorly examined. Using Gene Expression Omnibus and The Cancer Genome Atlas datasets, we showed that ADGRF5 is overexpressed in the colons of patients with CRC. In line, combined analysis of ADGRF5 expression with clinical characterization revealed an increased expression of ADGRF5 in patients with more advanced stages of CRC compared to patients with early stages of CRC. The Spearman correlation analysis documented numerous genes positively and negatively correlated with the expression pattern of ADGRF5 in the colon of patients with CRC. In the colon of CRC patients, the expression signature of ADGRF5 was associated with genes participating in phosphatidylinositol 3-kinase/Akt, focal adhesion, cell adhesion molecules, and ribosome signaling pathways. Of note, ADGRF5 expression correlated with the levels of tumor-infiltrating immune cells in the colon of CRC patients. Moreover, we found that CRC patients with high expression of ADGRF5 had a significantly lower probability of overall survival and disease-free survival. In conclusion, our results support the prognostic value of ADGRF5 and its potent therapeutic implication in CRC.


Introduction
Among several cancer types, colorectal cancer (CRC) is one of the most diagnosed cancer types in men and women worldwide. Most CRCs are sporadic, and an accumulation of genetic and epigenetic alterations is observed. Genomic alterations are responsible for oncogenes activation and/or tumor suppressor genes inactivation leading to the neoplastic transformation of the colon [1,2]. Several processes, such as chromosomal and microsatellite instability, as well as aberrant CpG methylation, are responsible for CRC development and progression. On the contrary, numerous signaling pathways such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K), transforming growth factor-β (TGF-β),

Correlation Analysis
Positive and negative correlations between the expression of ADGRF5 and the other genes were estimated using the co-expression tool in cBioPortal for the Cancer Genomic and TCGA, Firehose Legacy dataset. Genes correlated positively with ADGRF5 expression and characterized by Spearman value higher or equal to 0.50 were employed in further analysis. The top 1000 genes negatively correlated with the expression of ADGRF5 were used in subsequent analysis. To note, genes negatively correlated with the expression of ADGRF5 were characterized by an R-value of the correlation co-efficiency significantly lower than 0.50, and for this reason, the top 1000 genes negatively correlated with the expression of ADGRF5 in the colon of CRC patients was obtained.

Pathway Enrichment Analysis
Pathway enrichment analyses were performed using selected genes correlated with the expression of ADGRF5, which were identified in the TCGA, Firehose Legacy dataset. Positively and negatively correlated genes with ADGRF5 expression patterns were classified into signaling pathways using Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional annotation analysis [26,27]. Gene lists included positively and negatively correlated genes with ADGRF5 expression and were uploaded separately using the official gene symbol with limited species to Homo sapiens in the background. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was used to evaluate signaling pathways based on the most correlated genes with the expression of ADGRF5 documented in TCGA, Firehose Legacy dataset.

Gene Expression Profiling
Gene expression data for 55 genes that positively correlated and 86 genes negatively correlated with the expression of ADGRF5 were ranked according to ADGRF5 expression signature in the colon of CRC patients derived from TCGA, Firehose Legacy dataset. Hierarchical clustering was performed in Cluster 3.0 using average linkage. Java TreeView 3.0 were used for the visualization of genes correlated with the expression of ADGRF5 belonging to the most significant signaling pathways.

Survival Analysis
The association of ADGRF5 expression signature with the probability of overall survival (OS) and disease-free survival (DFS) was assessed by examining the difference in survival between patients with high and low ADGRF5 expressions. The high expressions are above the upper tertile, and the low expressions are below the lower tertile. The Kaplan-Meier method was used to estimate OS and DFS probability, and the Log-rank test was used to compare the OS or DFS probabilities between the patient groups.

Immune Cell Infiltration Analysis
TIMER 2.0 was used to study the correlation between the expression of ADGRF5 and immune cell infiltration in patients with CRC [28][29][30]. Systematic analysis of tumorinfiltrating immune cells was limited to the TIMER algorithm and immune cell types with a p-value < 0.05.

Statistical Analysis
Descriptive statistics were used to summarize the associations of ADGRF5 expression with the clinical characteristics of patients with CRC. Median ± interquartile range and mean ± standard deviation (SD) of ADGRF5 expression were used to estimate the ADGRF5 expression in the GEO and TCGA datasets, respectively. A Non-parametric Mann-Whitney U test was used to detect the difference of each variable. The Spearman correlation coefficient was used to identify genes whose expression was associated with that of the ADGRF5 expression signature. All the analyses were performed using GraphPad Prism 5.0 and R software.

ADGRF5 Is Overexpressed in the Colon of Patients with Colorectal Cancer
The ADGRF5 expression pattern was estimated using GSE21510, GSE32323, and GSE117606 datasets obtained from GEO, and the expression of ADGRF5 was evaluated in each dataset independently. As shown in Figure 1A-C, a statistically significant higher copy number of the ADGRF5 gene in the colon of CRC patients compared to controls (p < 0.05 for GSE32323; p < 0.001 for GSE21510 and GSE117606) was documented. coefficient was used to identify genes whose expression was associated with that of ADGRF5 expression signature. All the analyses were performed using GraphPad Pri 5.0 and R software.

ADGRF5 Is Overexpressed in the Colon of Patients with Colorectal Cancer
The ADGRF5 expression pattern was estimated using GSE21510, GSE32323, a GSE117606 datasets obtained from GEO, and the expression of ADGRF5 was evaluated each dataset independently. As shown in Figure 1A-C, a statistically significant high copy number of the ADGRF5 gene in the colon of CRC patients compared to controls ( 0.05 for GSE32323; p < 0.001 for GSE21510 and GSE117606) was documented.

ADGRF5 Expression Is Related to Clinical Characterization of Patients with Colorectal Cancer
In order to further explore the significance of ADGRF5 expression signature in colon of CRC patients, we assembled a large collection of CRC patients using the Fireho Legacy dataset available at TCGA. The data from the dataset for ADGRF5 expression a corresponding clinical characterization of CRC patients were used. The results are show in Table 1. The expression of ADGRF5 tended to be higher in the colon of black or Afric American patients with CRC (p = 0.058) compared to white patients with CRC. We fou an increased expression of ADGRF5 in CRC patients with a mucinous type of colon ad nocarcinoma compared to colon adenocarcinoma (p < 0.01). ADGRF5 expression chang were documented only in CRC patients with tumors localized in the colon but not in rectum. Moreover, in the colon of patients with CRC with more advanced stages, clas fied as III and IV stage, the expression of ADGRF5 was higher than in patients with C stages corresponding to I and II stage (p < 0.05). Analysis of ADGRF5 expression in C patients according to tumor-node-metastasis (TNM) staging system revealed higher pression of ADGRF5 when the extent of the tumors was classified as a T3 and T4 compar to the tumors described as a T1 and T2 (p < 0.01). Significantly higher copy numbers of ADGRF5 gene (p < 0.05) were found in CRC patients with nearby lymph nodes affect by tumor cells (N1/2) in relation to CRC patients, which were characterized by a lack tumor cells in the nearby lymph nodes (N0). Moreover, we did not identify statistica significant differences in the expression of ADGRF5 according to gender, age, race, or h tological diagnosis for rectum in patients with CRC. Collectively, these results indic that ADGRF5 participates in the progression of CRC.

ADGRF5 Expression Is Related to Clinical Characterization of Patients with Colorectal Cancer
In order to further explore the significance of ADGRF5 expression signature in the colon of CRC patients, we assembled a large collection of CRC patients using the Firehose Legacy dataset available at TCGA. The data from the dataset for ADGRF5 expression and corresponding clinical characterization of CRC patients were used. The results are shown in Table 1. The expression of ADGRF5 tended to be higher in the colon of black or African American patients with CRC (p = 0.058) compared to white patients with CRC. We found an increased expression of ADGRF5 in CRC patients with a mucinous type of colon adenocarcinoma compared to colon adenocarcinoma (p < 0.01). ADGRF5 expression changes were documented only in CRC patients with tumors localized in the colon but not in the rectum. Moreover, in the colon of patients with CRC with more advanced stages, classified as III and IV stage, the expression of ADGRF5 was higher than in patients with CRC stages corresponding to I and II stage (p < 0.05). Analysis of ADGRF5 expression in CRC patients according to tumor-node-metastasis (TNM) staging system revealed higher expression of ADGRF5 when the extent of the tumors was classified as a T3 and T4 compared to the tumors described as a T1 and T2 (p < 0.01). Significantly higher copy numbers of the ADGRF5 gene (p < 0.05) were found in CRC patients with nearby lymph nodes affected by tumor cells (N1/2) in relation to CRC patients, which were characterized by a lack of tumor cells in the nearby lymph nodes (N0). Moreover, we did not identify statistically significant differences in the expression of ADGRF5 according to gender, age, race, or histological diagnosis for rectum in patients with CRC. Collectively, these results indicate that ADGRF5 participates in the progression of CRC. Values of ADGRF5 expression are presented as a means of z-scores ± SD; ns-not significant, * p < 0.05, ** p < 0.01.

ADGRF5 Expression Is Positively Correlated with Genes Participating in Cell Adhesion and Metastatic Pathways in Colorectal Cancer
We next performed correlation analysis using the above-mentioned dataset provided by TCGA, and we identified numerous genes positively correlated with the expression of ADGRF5 in the colon of CRC patients. We found 500 genes positively correlated with the expression of ADGRF5 and characterized by Spearman R-value higher or equal to 0.50 in the TCGA, Firehose Legacy dataset.
In the cell adhesion molecules signaling pathway, we identified numerous genes positively correlated with ADGRF5 expression signature such as CD34 (CD34) and CD86 (CD86) molecules, cadherin (CDH) 2 and 5, endothelial cell adhesion molecule (ESAM), ITGA4, ITGAV, ITGB1, junctional adhesion molecule (JAM) 2 and 3, neural cell adhesion molecule 2 (NCAM2), neuroligin 4 X-linked (NLGN4X), programmed cell death 1 ligand 2 (PDCD1LG2), platelet and endothelial cell adhesion molecule 1 (PECAM1), protein tyrosine phosphatase receptor (PTPR) type C and M, syndecan 2 (SDC2), selectin (SEL) E and L, vascular cell adhesion molecule 1 (VCAM1) and versican (VCAN) ( Table 4). Subsequently, we next performed expression profiling of 55 genes participating in PI3K/Akt, focal adhesion, and cell adhesion molecules signaling pathways according to ADGRF5 expression ranked from low to high expression of ADGRF5. As shown in Figure 3, the expression pattern of genes representing the above-mentioned signaling pathways was similar to the expression profile of ADGRF5 in the colon of CRC patients (Figure 3). Subsequently, we next performed expression profiling of 55 genes participating in PI3K/Akt, focal adhesion, and cell adhesion molecules signaling pathways according to ADGRF5 expression ranked from low to high expression of ADGRF5. As shown in Figure  3, the expression pattern of genes representing the above-mentioned signaling pathways was similar to the expression profile of ADGRF5 in the colon of CRC patients (Figure 3).

ADGRF5 Expression Is Negatively Correlated with Genes Participate in Ribosome Pathway in Colorectal Cancer
DAVID functional annotation analysis and KEGG pathway analysis revealed that the top 1000 genes negatively correlated with the expression of ADGRF5 are involved in various signaling pathways, including ribosome (hsa03010), which was the most significant

ADGRF5 Expression Is Negatively Correlated with Genes Participate in Ribosome Pathway in Colorectal Cancer
DAVID functional annotation analysis and KEGG pathway analysis revealed that the top 1000 genes negatively correlated with the expression of ADGRF5 are involved in various signaling pathways, including ribosome (hsa03010), which was the most significant signaling pathway (adjusted p < 0.00001) associated with the expression signature of ADGRF5 in CRC patients. The symbol and description of genes enriched in ribosome signaling pathways and the R-value of the correlation co-efficiency are presented in Table 5. In the ribosome signaling pathway, we identified negatively correlated genes such as FAU ubiquitin-like and ribosomal protein S30 fusion (FAU), mitochondrial ribosomal protein L (MRPL) 1,2,3,4,11,12,14,15,16,17,20,21,22,23,24,27,32,34    Next, we ranked the expression of 86 genes in the ribosome signaling pathway according to the expression pattern of ADGRF5 in the colon of patients with CRC. As shown in Figure 4, lower expression of ADGRF5 was associated with higher expression of genes participating in the ribosome signaling pathway, and conversely, higher expression of ADGRF5 was associated with lower expression of genes regulating ribosome. Nevertheless, with weak correlation co-efficiency for genes related to ribosome signaling pathway, the expression pattern of genes negatively correlated with the expression of ADGRF5 is not so pronounced as in the case of genes positively correlated with ADGRF5 expression signature.
in Figure 4, lower expression of ADGRF5 was associated with higher expression of genes participating in the ribosome signaling pathway, and conversely, higher expression of ADGRF5 was associated with lower expression of genes regulating ribosome. Nevertheless, with weak correlation co-efficiency for genes related to ribosome signaling pathway, the expression pattern of genes negatively correlated with the expression of ADGRF5 is not so pronounced as in the case of genes positively correlated with ADGRF5 expression signature.

Increased Expression Pattern of ADGRF5 in the Colon of Patients with CRC Is Associated with Poor Probability of Overall Survival and Disease-Free Survival
We investigated the association of ADGRF5 expression signature with CRC patient survival using data obtained from the TCGA, Firehose Legacy dataset. We found that the expression of ADGRF5 in the colon of CRC patients was associated with the probability of both OS and DFS. As shown in Figure 5A,B, higher expression of ADGRF5 was accompanied by a worse probability of OS and DFS in CRC patients, which was confirmed by HR = 1.51 (p < 0.01) and HR = 1.49 (p < 0.01) for CRC patients, respectively. To note, CRC patients with higher expression of ADGRF5 were characterized by a median survival time of 34 months for OS and 32 months for DFS when compared to CRC patients with lower expression of ADGRF5 whose median survival time was evaluated as 22 months for both OS and DFS.

with Poor Probability of Overall Survival and Disease-Free Survival
We investigated the association of ADGRF5 expression signature with CRC patient survival using data obtained from the TCGA, Firehose Legacy dataset. We found that the expression of ADGRF5 in the colon of CRC patients was associated with the probability of both OS and DFS. As shown in Figure 5A,B, higher expression of ADGRF5 was accompanied by a worse probability of OS and DFS in CRC patients, which was confirmed by HR = 1.51 (p < 0.01) and HR = 1.49 (p < 0.01) for CRC patients, respectively. To note, CRC patients with higher expression of ADGRF5 were characterized by a median survival time of 34 months for OS and 32 months for DFS when compared to CRC patients with lower expression of ADGRF5 whose median survival time was evaluated as 22 months for both OS and DFS.

Expression Pattern of ADGRF5 Is Correlated with the Levels of Tumor-Infiltrating Immune Cells in the Colon of Patients with CRC
Finally, we evaluated the association between ADGRF5 expression and the level of immune cell infiltration in the colon of CRC patients using TIMER 2.0. Among distinct types of immune cells, the levels of dendritic cells (R = 0.62), macrophages (R = 0.56), neutrophils (R = 0.54), and CD4 + (R = 0.39), as well as CD8 + (R = 0.23) T cells infiltration were positively correlated (p < 0.05) with the expression of ADGRF5 in the colon of patients with adenocarcinoma ( Figure 6A). On the other hand, the level of B cells infiltration was negatively correlated (R = −0.12, p < 0.05) with the expression of ADGRF5 in the colon of patients with adenocarcinoma ( Figure 6A). As shown in Figure 6B, the levels of dendritic cells (R = 0.61), macrophages (R = 0.56), neutrophils (R = 0.55), CD8 + (R = 0.32), and CD4 + (R = 0.31) T cells, as well as B cells (R = 0.10) infiltration, were positively correlated (p < 0.05) with the expression of ADGRF5 in the rectum of patients with adenocarcinoma.

Expression Pattern of ADGRF5 Is Correlated with the Levels of Tumor-Infiltrating Immune Cells in the Colon of Patients with CRC
Finally, we evaluated the association between ADGRF5 expression and the level of immune cell infiltration in the colon of CRC patients using TIMER 2.0. Among distinct types of immune cells, the levels of dendritic cells (R = 0.62), macrophages (R = 0.56), neutrophils (R = 0.54), and CD4 + (R = 0.39), as well as CD8 + (R = 0.23) T cells infiltration were positively correlated (p < 0.05) with the expression of ADGRF5 in the colon of patients with adenocarcinoma ( Figure 6A). On the other hand, the level of B cells infiltration was negatively correlated (R = −0.12, p < 0.05) with the expression of ADGRF5 in the colon of patients with adenocarcinoma ( Figure 6A). As shown in Figure 6B

Discussion
According to The Human Protein Atlas, ADGRF5 is widely expressed in many types of human tissues, which suggests that signaling pathways mediated by ADGRF5 may be involved in the regulation of both physiological and pathophysiological conditions. Accumulating evidence indicates that ADGRF5 is a crucial member of adhesion GPCRs that mediate numerous processes such as proliferation, apoptosis, and migration, as well as

Discussion
According to The Human Protein Atlas, ADGRF5 is widely expressed in many types of human tissues, which suggests that signaling pathways mediated by ADGRF5 may be involved in the regulation of both physiological and pathophysiological conditions. Accumulating evidence indicates that ADGRF5 is a crucial member of adhesion GPCRs that mediate numerous processes such as proliferation, apoptosis, and migration, as well as invasion in the progression of some cancer types [17][18][19][20][21][22][23]. In fact, Tang et al. identified that ADGRF5 acts as a critical regulator of breast cancer metastasis. An increased ADGRF5 expression is associated with breast cancer progression and recurrence and poor prognosis of patients with breast cancer [18]. It should also be noted that the significance of ADGRF5 in breast cancer was evaluated in vitro and in vivo, and both approaches confirmed that ADGRF5 depletion in breast cancer cells was related to the reduced ability of breast cancer cells to migrate and invade [18].
Here, by employing three independent GEO datasets, we demonstrated that ADGRF5 is overexpressed in the colon of patients with CRC when compared to controls. In addition, the analysis of the ADGRF5 expression signature in the TCGA, Firehose Legacy dataset revealed a stage-dependent increase of ADGRF5 expression in CRC patients, and ADGRF5 expression patterns were associated with the progression of CRC. Yang et al. linked the deregulation of ADGRF5 expression to the differentiation stage and distant metastasis in CRC [19]. In our study, we demonstrated that the ADGRF5 expression pattern is associated not only with the CRC stage but also with lymph node metastasis, suggesting that ADGRF5 may be involved in the regulation of epithelial-mesenchymal transition of CRC cells. In addition, we found an increased expression of ADGRF5 in patients with a mucinous type of colon adenocarcinoma compared to colon adenocarcinoma. Colon mucinous adenocarcinoma is manifested by the presence of abundant mucous secretion comprising at least 50% of the tumor volume [31]. Mucin 2 and 5AC are two major types of mucins that are involved in the development of colon mucinous adenocarcinoma. However, the mechanisms by which both mucins regulate CRC progression are poorly understood. Clinically, colon mucinous adenocarcinoma is associated with worse clinical characteristics and worse prognosis compared to patients with colon adenocarcinoma [32,33]. Collectively, our findings suggest that CRC progression co-exists with ADGRF5 overexpression in the colon of CRC patients, suggesting the roles of ADGRF5 in the development and progression of CRC.
Little is known about the possible mechanisms responsible for the induction of AD-GRF5 expression in cancer. Among several post-transcriptional mechanisms, RNA interference seems to be involved in the regulation of ADGRF5 gene expression in CRC. Studies by Wang et al. demonstrated that ADGRF5 is a direct target for miR-511-5p [21]. Functionally, miR-511-5p mediates neoplastic transformation and progression of lung squamous cell carcinoma and CRC, as well as playing a role in the regulation of immune response by monocytes [21,34,35]. In CRC, miR-511-5p acts as a tumor suppressor microRNA, and the expression of miR-511-5p is reduced in both CRC tissues and cell lines compared to the controls. Moreover, miR-511-5p expression is inversely correlated with the differentiation of CRC and TNM stages of CRC patients. Furthermore, survival analysis revealed that CRC patients with low expression of miR-511-5p are characterized by a poor probability of OS and DFS [21]. It was estimated that the 3 UTR region of the ADGRF5 mRNA sequence contains the binding site for miR-511-5p and ADGRF5-miR-511-5p interaction seems to be responsible for modulation of proliferation, apoptosis, and invasion of CRC cells. In vitro studies on HCT116 and LOVO cells conducted by Yang et al. also noted that silencing of the ADGRF5 gene was accompanied by the reduced ability of CRC cells to proliferate and invade [19]. Above mentioned processes seem to be mediated by the regulation of both Akt and extracellular signal-regulated kinase (ERK) activity. In fact, in vitro analysis using CRC cells and siRNA against ADGRF5 documented that the silencing of ADGRF5 led to a reduction of CDH2, Snail family zinc finger transcriptional factor, phospho-Akt, and phospho-ERK 1/2 protein level suggesting that its role in the progression of CRC [19].
The main hallmark of cancer progression is epithelial-mesenchymal transmission underlying cancer cells' migratory and invasive capabilities. Accumulating evidence indicates that numerous signaling pathways participate in epithelial-mesenchymal transmissions, such as TGF-β, receptor tyrosine kinase, extracellular matrix, or PI3K/Akt, among others [36]. Our study documented a strong positive correlation between ADGRF5 expression pattern and expression of a number of genes involved in PI3K/Akt, focal adhesion, and cell adhesion molecules signaling pathways. Then PI3K/Akt signaling pathway is a major effector of epithelial-mesenchymal transmission and immunosuppression, and according to numerous reports, the action of the PI3K/Akt signaling pathway in cancers is multidirectional. In CRC, the hyperactivated PI3K/Akt signaling pathway positively regulates transcription mediated by NF-κB and β-catenin, which in turn are responsible for the pro-tumorigenic and invasive capacity of cancer cells [4,37,38]. It has to be highlighted that ADGRF5 may mediate the pro-tumorigenic action of the PI3K/Akt pathway through G protein activity regulation. The previous findings by Tang et al. documented that ADGRF5 affects signaling pathways responsible for breast cancer progression coupling to Gαq [18]. On the contrary, the PI3K/Akt signaling pathway plays a role in the negative regulation of the CDH1 gene encoding E-cadherin, which is a crucial regulator of cell adhesion and tight junctions and serves as a marker of epithelial phenotype cells [39,40]. In fact, mesenchymaltype cancer cells have low expression of epithelial markers like CDH1 and high expression of mesenchymal markers such as CDH2 gene encoding N-cadherin, fibronectin, and vimentin, among others. Additionally, invasive cancer cells are characterized by a lack of polarization and decreased cell adhesion, as well as disturbed cell-cell and cell-matrix interactions [39]. Cadherins, caveolins, collagens, integrins, junctional adhesion molecules, laminins, and selectins all maintain proper cell function and physiological morphology of cells. Observational studies documented aberrant expression of CAV1, COL1A2, COL3A1, COL5A2, COL6A3, COL11A1, ITGA1, ITGA5, ITGB1, LAMA4, JAM2, JAM3, and VCAM1 in CRC, among others [41][42][43][44][45][46][47][48][49][50][51][52][53]. Numerous of the above-mentioned genes represent focal adhesion regulators and extracellular matrix/receptor interaction signaling molecules. The expression of these genes was positively correlated with the expression of ADGRF5 signature in the colon of patients with CRC in our studies (refer to Tables 2-4). Our results suggest that ADGRF5 may participate in cell junction machinery and may be involved in epithelial-mesenchymal transmission during the development of CRC. Nevertheless, further studies employing in vitro and in vivo approaches are required to dissect the mechanism by which ADGRF5 contributes to the change of epithelial to mesenchymal phenotype of cells during CRC progression.
In the colon of patients with CRC, we identified a negative correlation between ADGRF5 expression and the expression of 86 genes of the ribosome signaling pathway. It should also be noted that the R-value describing the Spearman correlation between ribosomal genes was lower than for genes positively correlated with ADGRF5 expression signature, and weak to moderate association between ADGRF5 expression pattern and ribosomal genes expression was documented. Nevertheless, in CRC, alteration of various ribosomal genes is recognized, which indicates that ribosomal stress contributes to CRC development and progression. Indeed, ribosomal protein genes contribute to processes such as proliferation, apoptosis, hypoxia, glycolysis, and cell cycle regulation during neoplastic transformation of the colon, and their action seems to be associated, for instance, with the formation of ribosomal subunits or rRNA processing [54][55][56][57].
Finally, using Kaplan-Meier survival curves, we analyzed the clinical significance of ADGRF5 expression signature in patients with CRC and showed that CRC patients with increased expression of ADGRF5 in the colon had an inferior probability of OS and DFS compared to CRC patients with reduced ADGRF5 expression in the colon. The results from survival and pathway enrichment analyses indicate that ADGRF5 may modulate CRC progression in patients promoting pro-metastatic pathways. Additionally, our analyses document the link between ADGRF5 expression and the levels of tumor-infiltrating immune cells in the colon and rectum of patients with adenocarcinomas. The levels of immune cell infiltration and composition of immune cells in the tumor microenvironment are crucial for patients' survival and cancer prognosis. However, conflicting results regarding the tumor-infiltrating cells seem directly related to tumor microenvironment plasticity, which is continuously changing in response to signals from surrounding tissues. Nevertheless, accumulating evidence highlighted the crucial role of T cells, neutrophils, and macrophages, as well as dendritic cells, in the development and progression of CRC. Wang et al. found that the levels of T cells may predict the treatment efficiency and OS as well as progression-free survival in CRC patients [58]. On the contrary, fibroblasts, macrophages, and neutrophils may support tumor growth and the invasion ability of cancer cells. For instance, neutrophils are one of the major sources of matrix metallopeptidase 9, which promotes extracellular matrix remodeling and angiogenesis [59,60]. Furthermore, macrophages also affect the above-mentioned processes, as evidenced by their role in the regulation of proteins related to epithelial-mesenchymal transition [61]. In our study, dendritic cells showed the strongest correlation with the expression of ADGRF5 among all the tested types of immune cells. Strikingly, studies by Sandel et al. showed that tumor-infiltrating dendritic cells affected local tumor cell and immune cell interactions, and their levels were associated with prognosis in CRC patients [62]. The link between ADGRF5 and inflammation was pointed out in the study performed by Kubo et al. [63]. Our evidence from immune cell infiltration analysis in the colon of CRC patients supports previous findings about ADGRF5 function in immunity. Moreover, correlation analysis indicated that ADGRF5 expression is associated with genes related to immune response, such as TLR2, which plays a fundamental role in the activation of innate immune response, among others [64].
Nevertheless, the role of ADGRF5 in both immunomodulation and neoplastic transformation of the colon remains elusive. Thus, the results of our comprehensive in silico study provide a basis for further investigation of the roles of ADGRF5 in CRC and other tumors.

Conclusions
In this study, we identified that ADGRF5 is overexpressed in the colons of patients with CRC, and ADGRF5 expression patterns are associated with the disease stage. Moreover, patients with high expression of ADGRF5 are characterized by a shorter probability of overall and disease-free survival. Our results suggest that ADGRF5 may play a role in epithelial-mesenchymal transmission and may be responsible for the modulation of cell adhesion and metastatic pathways during the neoplastic transformation of the colon, and CRC progression. Further studies on identifying ADGRF5-mediated pathways are warranted to develop a better understanding of the roles of ADGRF5 in the development and progression of CRC.