DOCK4 Correlated with Immune Inltration is a Valuable Prognostic Biomarker in Stomach Adenocarcinoma.

Background:Dedicator for cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1, the mechanisms involved in immune inltration in STAD (stomachadenocarcinoma) remain unclear. Methods: The UALCAN database was used to analyze the expression of the DOCKfamily, and the Kaplan-Meier plotter and GEPIA databases were used to assess the prognostic value of the DOCK family in STAD. Furthermore,the correlation between DOCK4 expression withtumor immune inltration and the expression of related immune marker genes in STAD was explored using TIMER and GEPIA websites. Subsequently, the relationship between DOCK4 expression and clinical characteristics was veried using the UALCAN database. Finally, the mutation of DOCK4 was analyzed via TIMER2.0 and cBioPortal databases. And the protein-protein interaction(PPI) networks of DOCK4 were constructed using GeneMANIA and STRING websites. Results: DOCK4 was nally screened out, and its expression in tumors was signicantly evaluated relative to paracancerous tissues and had a negative impact on the prognosis of patients with STAD.DOCK4 was found signicantly related to tumor immune inltration in STAD. Conclusions:In summary, is a potential regulator of the recruitment and regulation of immune inltrating cells, thus becoming valuable in STAD. DOCK4 and explored the vital correlation of DOCK4 with the tumor microenvironment of STAD. These results revealed the prognostic value of DOCK4 in stomach adenocarcinoma and provided new evidence for the interaction of DOCK4 with tumor immunity.

sequence and domain, are divided into four subgroups: DOCK-A, DOCK-B, DOCK-C, and DOCK-D [17]. DOCK family proteins are the most researched in cell adhesion and migration. In addition to cell adhesion and migration, interactions between the immune system and DOCK family members have also been reported. DOCK2 and DOCK8 are widely expressed in immune cells, and as activators of Rac and Cdc42 they are necessary for mediating cytotoxic function and the cell-cell adhesion of immune cells, respectively [16,18,19]. There are only a few studies on gastric cancer. DOCK1 might be the target of mycophenolic acid (MPA) in modulating gastric cancer cell migration [20]. DOCK5 could also be a chemosensitivity-related gene in gastric cancer [21]. DOCK6 plays a pivotal role in regulating cancer stem cells of gastric cancer [22] and promotes GC metastasis through the Rac1/Cdc42 axis [23]. DOCK4 belongs to the DOCK-B subgroup. It is an atypical guanine nucleotide exchange factor for Rac1, whose encoding-gene is located on chromosome 7 [24]. A few studies have demonstrated a carcinogenic role in multiple tumors. Overexpressed DOCK4 promotes proliferation, migration and invasion of liver cancer [25]. lncRNA AC073284.4 suppresses cell invasion, metastasis and EMT of breast cancer via the miR-18b-5p/DOCK4 axis [26].DOCK4 regulates the migration of breast cancer cells by combining with SH3YL1 to activate Rho GTPase Rac1 [27]. Furthermore, DOCK4 promotes lung adenocarcinoma cell extravasation and metastasis through the TGF-β/Smad pathway [28]. DOCK4 ,as a prognostic biomarker in ovarian cancer, has been shown to be associated with immune in ltration [29]. However, the correlation between the expression and prognosis of DOCK4 and stomach adenocarcinoma remains unknown.
In the present study, we comprehensively analyzed the expression and clinical prognosis of the DOCK family in STAD. Then, we screened out DOCK4 and explored the vital correlation of DOCK4 with the tumor microenvironment of STAD. These results revealed the prognostic value of DOCK4 in stomach adenocarcinoma and provided new evidence for the interaction of DOCK4 with tumor immunity. Methods UALCAN database analysis UALCAN (http://ualcan.path.uab.edu.) aims to analyze the relative expression of genes as well as the correlation with diverse clinicopathologic features from the data of TCGA, MET500 and CPTAC [30]. This database was used to assess the expression level of the DOCK family in tumor tissues of STAD and paracancerous tissues, as well as the relationship between DOCK4 expression and clinical characteristics.

Kaplan-meier Plotter Database Analysis
The Kaplan-Meier plotter (http://kmplot.com/analysis/) was used to evaluate the prognostic impact of 54,675 genes in 10,461 cancer samples from 21 cancer types [31]. This database was rst used to explore the in uence of DOCK family expression on the OS of patients with STAD, and to analyze the impact of clinicopathological factors and immune cells on the OS of STAD patients.
Timer Database Analysis TIMER (https://cistrome.shinyapps.io/timer/) comprehensively assesses the correlation between tumorin ltrating immune cells (TIICs) and important tumoral genomic changes using six functional modules [32]. In our study, the correlation between the expression of DOCK3 and DOCK4 with the degree of immune in ltration of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells was analyzed using gene modules. Next, the tumor in ltration levels in different somatic copy number alterations were compared using the SCNA module. The relationship between DOCK4 expression and immune marker genes was explored using a correlation module. In addition, the mutation of DOCK4 in 34 cancer types was analyzed using gene mutation module in the TIMER2.0.

Gene Correlation Analysis In Gepia
The online database Gene Expression Pro ling Interactive Analysis (GEPIA) (http://gepia.cancerpku.cn/detail.php) provides RNA sequencing expression data to analyze the expression differences between tumor and control samples in multiple cancer types [33]. This database was used to assess the OS based on the expression of DOCK4, and the link between this gene and the expression of particular immune-related marker genes was veri ed.

Cbioportal Database Analysis
The cBioPortal (http://cbioportal.org) is a website for analyzing multidimensional cancer genomics data from cancer tissues and cells to providing the genetic, epigenetic, gene expression, and proteomic information for users [34]. The database was used to analyze the alterations of DOCK4 in stomach adenocarcinoma samples.

String Database Analysis
The STRING database ( https://string-db.org/) aims to collect and integrate the information of protein-protein interaction to constructing a comprehensive and objective global network for users [35]. The database was used to construct the PPI network of DOCK4. The Cytoscape software was used to visualize the results from the STRING database.

Genemania Database Analysis
The GeneMANIA (http://genemania.org) is an open website that can analyze correlated genes and predict gene function and the interactions of them [36]. The database was used to analyze the correlated genes of DOCK4 and construct the PPI network.

Statistical analysis
The hazard ratio (HR) and P-value of survival curves of OS were computed using the log rank test in the GEPIA and Kaplan-Meier Plotter databases. Spearman correlation was used to analyze the relationship between TIMER and GEPIA. The signi cance threshold was set at P < 0.05.

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Assessment of the expression level of DOCK family in STAD.
The Prognostic Value Of Dock Family In Stad Next, genes that showed no statistical difference in expression was excluded, including DOCK9 and DOCK11.
The impact of remaining DOCK family expression on OS in STAD was determined using the Kaplan-Meier plotter database. As shown in gure 2, overexpression of DOCK3 (Fig. 2C, HR=1.95, log rank P= 4.5e−05) and DOCK4 (Fig. 2D, HR=1.57, log rank P=0.016) were associated with poor prognosis in patients with STAD. DOCK1( Fig [39]. Furthermore, we analyzed the association between copy number variations of DOCK3 and DOCK4 with the above six immune in ltrates (Fig. 4). The results revealed that the levels of B cells(p<0.01), CD8+ T cells(p<0.001), CD4+ T cells(p<0.001), macrophage cells(p<0.001), neutrophil cells(p<0.001) and dendritic cells(p<0.001) were relatively low, as indicated by the deletion of these genes. Above all, these results showed that DOCK4 plays a vital role in immune in ltration.

Correlation Analysis Between Dock4 With Immune Marker Expression
To further investigate the underlying link between DOCK4 and immune in ltration, we next analyzed the correlation between DOCK4 expression and immune marker genes of numerous immune cells, including CD8+ T cells, T cells (general), B cells, monocytes, tumor-associated macrophages (TAMs), M1 and M2 macrophages, neutrophils, natural killer cells and dendritic cells (DCs) in STAD using TIMER and GEPIA databases. We additionally analyzed T cells with different subsets, such as T helper 1 cells (Th1), T helper 2 cells (Th2), T

Description
Gene markers STAD

Correlation Between Dock4 Expression And Clinical Characteristics Of Stad
Based on the prognostic value of DOCK4 in STAD, we further analyzed the relationship between DOCK4 expression and clinical characteristics in the UALCAN database. It is known that DOCK4 expression is higher in tumor tissues than in paracancerous tissues. The results showed that DOCK4 expression was elevated in cancer stage 3 relative to stage 2(p<0.01) and stage 1(p<0.05) (Fig. 6A). DOCK4 expression was lower in the 81-100 years age group than 61-80 years age group (Fig. 6D, p<0.05). Based on tumor grade, the expression of this gene in grade 3 was excessive to grade 2, as well as grade 3 to grade 1 (Fig. 6E, p<0.001). For TP53 mutation status, the DOCK4 expression of the TP-53 non-mutated subtype was higher than that of the TP53-mutated subtype (Fig. 6G, p<0.05). There was no statistical signi cance in the subgroups of race, gender, H. pylori infection status and nodal metastasis status of STAD patients. Next, in the strati ed prognostic analysis of DOCK4 expression in STAD ,the upregulated DOCK4 had a negative impact on OS in clinical stage 3 (OS,P-VALUE=0.025) and clinical stage 4 (OS,P-VALUE=0.036) using the Kaplan-Meier database (Table 3).Furthermore, DOCK4 also played an unfavorable role in the following clinicopathological characteristics: gender of female (OS, P-value =0.00035), race of white (OS, P =0.01) and mutation burden (OS, P =0.046)( Table 3).There was no statistically signi cant difference between the subgroups of the other clinicopathological characteristics(p>0.05)( Table 3).These results suggest that DOCK4 expression potentially in uences the prognosis of STAD patients with advanced clinical stages.

Ppi Networks Construction Analysis Of Dock4
The PPI networks of DOCK4 was constructed using the GeneMANIA website. The results revealed a list of correlated genes for DOCK4. The correlated genes were enriched in the pathways as follows: Rho GTPase binding, regulation of smooth muscle cell migration, smooth muscle cell migration, positive regulation of smooth muscle cell migration, regulation of vascular associated smooth muscle cell migration, vascular associated smooth muscle cell migration and muscle cell migration (Fig. 8A). In addition, the data of PPI network of DOCK4 from STRING website was visualized in the Cytoscape software (Fig. 8B). DOCK4 was interacted with ribosomal proteins such as RPL6, RPL24 and RPL18, and ribosomal protein subunit proteins such as RPS27A, RPS25, RPS29 and RPS15.

Discussion
Stomach adenocarcinoma (STAD) is the fth most common malignant tumor and ranks third in cancer mortality worldwide [1]. In recent years, with the improvement of treatment methods of stomach adenocarcinoma including surgical treatment, neoadjuvant radiotherapy, chemotherapy and targeted therapy, the 5-year survival rate of patients with early diagnosis and treatment has increased, but the therapeutic effect in advanced/metastatic patients remains unsatisfactory [41].The possible development mechanism of stomach adenocarcinoma involves the malignancy of cancer cells and various immune cells in the tumor-related microenvironment [42]. The development of immune checkpoint inhibitors is bene cial for patients with advanced/metastatic stomach adenocarcinoma. For example, pembrolizumab prolongs the overall survival (OS) and reduces adverse effects of treatment in patients with advanced stomach adenocarcinoma [43]. The combination of nivolumab and ipilimumab is effective for chemotherapy-refractory gastroesophageal adenocarcinoma [14]. However, the incomplete validity of immune checkpoint inhibitors in GC patients has become a new challenge [15,44]. Thus, in order to optimize individualized treatment strategies, we aimed to explore new immunotherapeutic-related molecules for STAD. There are 20 Rho GTPases in the human genome, especially Rac1 and Cdc42, which are reported more frequently and have an important in uence on cell proliferation, differentiation, motility, adhesion, survival and secretion [45]. As Rho GTPase activators, the DOCK family was rst identi ed as the exchange factor of Rac(1/2/3) and Cdc42 more than ten years ago  [25][26][27]. However, no study has reported the interaction between DOCK4 and the tumor microenvironment of STAD. In this study, we found that high DOCK4 expression was a poor prognostic factor for STAD. DOCK4 expression was signi cantly related to the expression of different immune-related markers, highlighting its possible role in STAD immunity, making it a valuable biomarker for this cancer.
We rst found that the expression of most DOCK family members was higher than that of control samples using the UALCAN database (Fig. 1). Based on their prognostic value in the Kaplan-Meier Plotter and GEPIA databases, we selected DOCK3 and DOCK4, both of which had an unfavorable impact on the OS of STAD (Fig. 2). DOCK3 is speci cally expressed in neurons and participates in the development of Alzheimer's disease [52]. Furthermore, Twist1 promotes EMT through the NEDD9/DOCK3/Rac axis in head and neck squamous cell carcinoma [53].DOCK3 drives tumor cell adhesion, migration, and invasion by regulating Rac1 activity in lung cancer [54].Besides metastasis, the relationship between immune in ltration and the two genes in STAD was explored via TIMER and GEPIA websites. There was only a weak relationship between DOCK3 and B cells, CD8+ T cells, CD4+ T cells and macrophages (Fig. 3A). However, DOCK4 was positively correlated with CD8+ T cells, CD4+ T cells, macrophage cells, neutrophil cells, and dendritic cells, while it was negatively correlated with tumor purity (Fig. 3B). In addition to copy number variations, deletion of DOCK4 was signi cantly associated with lower levels of six immune cells (Fig. 4). Thus, DOCK4 is closely related to the tumor microenvironment of STAD.
Although the structure of DOCK4 has high homology with DOCK3, it can also be detected as a secreted protein in peripheral blood [29]. The function of chemokines in the migration of lymphocytes depends on the activation of DOCK2 and the mutations in DOCK8 also cause functional defects of the immune system, which proves that DOCK2 and DOCK8 are necessary regulators of the immune system [55,56].Therefore, this suggests that DOCK4 might have a particular correlation with immune in ltration, similar to DOCK2 and DOCK8. Thus, we further veri ed the link between the expression of immune-related gene markers and this gene. There was a signi cant correlation between DOCK4 and diverse marker genes of immune in ltrating cells, including monocytes, TAMs, M2 macrophages, DCs, Tregs, and exhausted T cells in the TIMER database. M1 (anti-tumor) and M2 (pro-tumor) phenotypes represent different functional states [57]. DOCK4 was weakly associated with M1 macrophage markers (IRF5, COX2) and strongly associated with M2 macrophage markers (CD163, VSIG4, MS4A4A) ( Table   1). These results revealed that DOCK4 participates in macrophage polarization. The high level of immune in ltration of Treg cells is related to the poor prognosis of cancer patients, and FOXP3 participates in the regulation of differentiation of regulatory T cells, thus suppressing the anti-tumor immune response[58]. PD-1, CTLA4, Tim-3, and LAG3 are vital markers of exhausted T cells, which represent a poor response to antigenmediated TCR stimulation [59]. There was a signi cant correlation between DOCK4 expression and Treg cell markers (FOXP3, TGFβ, CCR8, STAT5B) and T cell exhaustion markers (PD-1, CTLA4, TIM-3, LAG3) ( Table 1).
These results provided evidence that elevated DOCK4 levels suppressed the anti-tumor immune response of clinical patients with STAD. NRP1(DC marker) was strongly related to DOCK4, which was determined to be a prognostic factor for STAD [60].Furthermore, the T helper cells in ltration is conducive to prognostic prediction [61]. And the signi cant link between DOCK4 expression and several markers of T helper cells (Th1, Th2, Tfh, Th17) increased the accuracy of survival prognosis prediction of STAD patients (Table 1). Based on the homologous data from TCGA, we analyzed the above markers in the GEPIA database and obtained similar results (Fig. 5). Furthermore, we revealed that DOCK4 expression had an impact on OS in different stages of immune in ltration ( Table 2 Finally, we analyzed the correlation between DOCK4 levels and the clinical characteristics of STAD (Fig. 6).
Combining . Thus, it provides further evidence for the correlation between DOCK4 and p53 in STAD. We also observed that DOCK4 expression was higher in advanced subtypes of cancer stages and grades. Both the TGF-β/Smad pathway, which is an important signaling pathway of the metastatic phenotype and the transcription factor c-MAF, which is the key factor of bone metastasis, are related to DOCK4 expression [28,69]. And the correlated genes of DOCK4 are enriched in regulating cell migration pathways (Fig. 8A). This might be the potential mechanism by which DOCK4 participates in metastasis in STAD, but it was necessary for experimental veri cation. Reducing the high mortality of advanced/metastatic patients with STAD is a complicated problem [70]. However, DOCK4 expression had an impact on the prognosis of OS in STAD patients with advanced clinical stages (Table 3). Systemic chemotherapy is an effective method for treating advanced gastric cancers [71]. DOCK4 is associated with immune in ltration as a platinumchemosensitive gene in ovarian cancer [29]. In addition, DOCK4 plays an unfavorable prognosis by regulating the immune escape of the tumor microenvironment in STAD, which potentially as a target of platinum chemosensitivity, provided new treatment strategies for advanced/metastatic patients.

Conclusions
Our study revealed the crucial role of DOCK4 in tumorigenesis and the development of STAD. Above all, as a new prognostic factor of STAD, DOCK4 plays a vital role in immune in ltration and is a valuable molecule for

Availability of data and materials
All data supporting the ndings of this study are available within the article. These data come from public databases.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was nancially supported by the Natural Science Foundation of Tianjin City, China (Grant no. 17JCYBJC25600).
Authors' contributions LZ and LY designed and performed the research, analyzed data, and wrote the manuscript; YJX, DQP and DY participated in data preparation, analysis, and gure preparation. All authors have read and approved the