Prognostic and immune roles of synaptotagmin-4 in gastric cancer and brain lower-grade glioma


 Background Synaptotagmins (SYTs) are a family of proteins whose primary function is serving as a calcium sensor in vesicle transport and exocytosis, playing an important role in the function of immune cells. There is also a close relationship between immune cells and tumours. SYT4 is one molecule involved in this relationship, but the relationship between SYT4 and cancer remains unclear. Therefore, we hypothesize that SYT4 can affect the prognosis of cancer, and may be related to immune cells. Methods The following databases were used to study the immunological and prognostic role of SYT4 in cancers: Oncomine, Kaplan-Meier plotter, The Human Protein Atlas, CCLE, GEPIA2, TIMER, and CGGA. Results SYT4 expressions were lower in many cancers than in normal tissues. Specifically in gastric cancer and lower-grade gliomas, SYT4 played a protective and harmful role, respectively. Moreover, a difference between SYT4 expression and the levels of immune infiltration existed in stomach adenocarcinoma (STAD) and brain lower-grade glioma (LGG). In addition, we found that the relationship between markers of monocytes, M1 and M2 macrophages, tumour-associated macrophages (TAMs), Treg cells, B lymphocytes, dendritic cells (DCs) and SYT4 expression was opposite in STAD and LGG. Conclusions The effect of SYT4 on the prognosis of patients with STAD and LGG was opposite. And SYT4 has different effects on immune infiltration in these two tumours. Therefore, SYT4 might be a potential prognostic and tumour immune-related biomarker in STAD and LGG.

The expression levels of SYT4 in cancers First, we used the Oncomine database to explore the mRNA expression levels of SYT4 in different tumours ( Figure 1A). The results suggested that the mRNA expression of SYT4 was higher in the lung tumour tissues than in normal lung tissues. In contrast, the mRNA expression of SYT4 was lower in the brain and nervous system cancer, colorectal, oesophageal, gastric, and prostate cancer, as well as in sarcoma tissues, than in the respective normal tissues. Supplemental Table 1 explicitly presents the mRNA expression levels of SYT4 in tumours according to different studies.
Subsequently, we used the TIMER database to explore the expression levels of SYT4 in different cancers ( Figure 1B). We found a signi cant difference in the expression levels of SYT4 between cancer tissues and normal tissues in the following cancers: bladder To verify the above results at the tissue and cell levels, we analyzed SYT4 expression in HPA and CCLE. In the immunohistochemical staining data, we found that the expression level of SYT4 was not high in cancer tissues. Furthermore, compared to the levels in cell lines of neuroblastoma, small cell lung cancer and prostate cancer, the levels of SYT4 in other cancer cell lines were lower (Supplemental Figure 1A-P).

Prognostic signi cance of SYT4 in different cancers
We used gene chip data derived from the Kaplan-Meier plotter database to explore the association between SYT4 expression and the survival of breast, lung, gastric and ovarian cancer patients. The results are shown in Figure  After the exploration of SYT4 in the Kaplan-Meier plotter database, we analysed the impact of SYT4 on prognosis of different cancers by analysing RNA sequencing data from the TCGA database through GEPIA2 (Supplemental Figure 2). The results that the impacts of SYT4 on both the OS and disease-free survival (DFS) of LGG and STAD were consistent. High expression levels of SYT4 were related to a poor prognosis in patients with STAD (OS: HR=1.60, log-rank P = 0.006; DFS: HR=1.80, log-rank P = 0.0041). In contrast, high expression levels of SYT4 were correlated with a good prognosis in LGG patients (OS: HR=0.53, log-rank P = 0.00047; DFS: HR=0.58, log-rank P = 6e-04).
To further explore the correlation between the expression levels of SYT4 and the survival of LGG patients, we veri ed it in the CGGA database (Supplemental Figure 3). In the mRNAseq_325 dataset, the result suggested a signi cant correlation between the expression level of SYT4 and survival in all primary glioma patients, including patients with WHO grade II and III tumours, but there was no correlation between SYT4 and prognosis in patients with grade IV tumours. Similarly, in the mRNAseq_693 dataset, the result indicated a correlation between the expression level of SYT4 and the survival of all primary glioma patients, including patients with WHO grade III tumours, but there was no correlation between SYT4 and prognosis in patients with grade II and IV tumours. The above results partially verify our hypothesis, suggesting the prognostic value of SYT4 in LGG and STAD. SYT4 had a bene cial effect on the prognosis of LGG patients. In contrast, SYT4 had a detrimental effect on the survival of STAD patients.
The expression of SYT4 has an impact on the survival of gastric patients with lymphatic metastasis To explore the mechanism by which the expression level of SYT4 affected the survival of gastric cancer patients, we used the Kaplan-Meier plotter database to explore the association between the expression levels of SYT4 and clinical factors of gastric cancer patients ( SYT4 expression is related to the level of immune in ltration in gastric cancer and lower-grade brain glioma Among the various factors affecting the survival and lymph node metastasis of cancer patients, lymphocyte in ltration is a signi cant independent predictor [25]. Therefore, we analysed the association between the expression levels of SYT4 and the levels of immune in ltration in 39 types of cancer in the TIMER database (Supplemental Figure 4). According to the results, we found that tumour purity Correlations between the expression levels of SYT4 and markers of immune cells To further explore the potential mechanisms of interaction between SYT4 and various immune in ltrating cells, such as CD8+ T cells, T cells (general), B cells, monocytes, TAMs, M1 and M2 macrophages, neutrophils, natural killer cells, and dendritic cells, in STAD and LGG, we analysed the correlations between the expression levels of SYT4 and immune markers of multiple immune cells in STAD and LGG based on the TIMER and GEPIA databases, In addition, we also performed correlation analysis with the immune markers of the following types of functional T cells: T helper cells, follicular helper T cells, regulatory T cells, and exhausted T cells. The correlation coe cient was adjusted based on tumour purity[26] ( Table 2). According to the correlation analysis between the expression levels of 56 immune cell markers and the expression levels of SYT4, we found that the purity-adjusted correlation coe cients of 35 markers were statistically signi cant in STAD patients, and they were all positive. The purity-adjusted correlation coe cients of 46 markers were statistically signi cant in LGG patients, however, most of them were negatively. Only ten markers had statistically signi cant purity-adjusted correlations with the expression levels of SYT4 in BRCA patients. In addition, we also found signi cant correlations between the expression levels of markers of monocytes, TAMs and M2 macrophages and the expression levels of SYT4 in patients with STAD and LGG but not in BRCA (Table 2, Figure 4). In detail, these markers, such as CD115, CCL2, IL10, VSIG4, and MS4A4A, had signi cantly positive correlations with SYT4 expression levels in STAD (P <0.0001, Figure 4A-D). For LGG, these markers, such as CD163 of M2 macrophages, NOS2, IRF5 and COX2 of M1 macrophages, and CD86 of monocytes, also showed a signi cant correlation with SYT4 expression levels but the markers that were correlated with SYT4 expression in STAD did not show a correlation in LGG (P <0.0001, Figure 4I-L). However, the expression of SYT4 in BRCA did not show signi cant correlations with the above markers ( Figure   4E-H). Then, to verify the results, we analysed the correlation between monocytes, TAMs, and M1 and M2 macrophages immune markers and the expression levels of SYT4 in STAD, LGG, and BRCA with the GEPIA2 database. The results were similar to those achieved in the TIMER analysis (Table 3). Moreover, it is worth noting that the correlations between the expression level of SYT4 and the levels of immune markers were positive and negative in STAD and LGG, respectively. Hence, we concluded that SYT4 likely interactes with various immune cells in STAD and LGG in the opposite ways Discussion Calcium-mediated exocytosis is an integral part of the human immune system. Exocytosis of mast cells, natural killer cells, and cytotoxic lymphocytes protects humans from pathogen invasion, infected cells and malignant cells [27]. SYTs, a large class of membrane transporters, are the primary calcium sensors during exocytosis. They have signi cant regulatory effects in nerves, the endocrine system, and the immune system [28]. Baram D et al. con rmed that SYT1 expression was observed in mouse bone marrowderived mast cells (BMMCs) and rat abdominal mast cells (RPM-Cs) by immunoblotting, and played a decisive regulatory role in mast cell exocytosis. SYT2 plays a negative supervisory role in lysosomal exocytosis of mast cells [29]. Lindmark IM et al. con rmed that SYT2 was expressed in human neutrophils (PMNs), while SYT1, SYT3, and SYT4 were not, and SYT2 was involved in PMN phagocytosis and exocytosis [30]. These studies re ect the effects of the SYT family members on immune cells and the diversity of their functions. In terms of the relationship between SYTs and tumours, one study found that SYT13 was helpful for presicting the occurrence and development of colorectal cancer [10]. SYT7 was bene cial for monitoring the development of osteosarcoma [11]. A recent study found that SYT4 regulates Ca2+ in ux through TRPM1, leading to melanin production and axon elongation in alpaca melanocytes, which suggests that the growth and metastasis of melanoma are controlled by the suppression of SYT4 expression in melanoma cells [12]. Therefore, we hypothesized that SYT4 can affect the prognosis of cancer, and its mechanism may be related to tumour immunity.
This study used the following databases to explore the associations between SYT4 and cancers: Oncomine, Kaplan-Meier plotter, GEPIA2, TIMER, HPA, CCLE and CGGA. Data from Oncomine showed that SYT4 was expressed at low levels in brain and nervous system cancer, colorectal cancer, oesophageal cancer, gastric cancer, prostate cancer, and sarcoma compared to normal tissues. Only one dataset showed higher expression levels of SYT4 in lung cancer than in the respective normal tissues ( Figure 1A). The results from TIMER showed that compared to those in normal adjacent tissues, the expression levels of SYT4 were signi cantly different in the following cancers: BLCA, BRCA, COAD, ESCA, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA, and UCEC ( Figure   1B). In addition, we veri ed the expression of SYT4 in the HAL and CCLE databases at the tissue and cell levels, and obtained similar results. Throughout these databases, we determined the consistency of SYT4 expression in cancers. In the analysis of patient prognosis, we employed the Kaplan-Meier plotter and GEPIA2 databases. The results suggested that high expression of SYT4 was related to a favourable prognosis of LGG patients. In contrast, high expression of SYT4 was correlated with an unfavourable prognosis in STAD. The CGGA database veri ed the above result, and similar results were obtained. In addition, immunohistochemical staining data from HPA also suggested that SYT4 was expressed at low levels in STAD and LGG tissues than in normal gastric and brain tissues, respectively (Supplemental Figure 1-Q). These ndings suggested that the relationships between the expression level of SYT4 and prognosis is different in different cancers,, indicating that SYT4 might be a potential prognostic biomarker, at least in STAD and LGG.
To continue to investigate how the expression level of SYT4 affects the prognosis of cancer patients, we conducted a strati ed analysis of the clinical characteristics of STAD patients and found that the expression of SYT4 had an impact on the survival of gastric patients with lymphatic metastasis (Table 1), which suggested that SYT4 might be related to the level of tumour immune in ltration. Therefore, we explored the associations between the expression level of SYT4 and the levels of immune in ltration in STAD and LGG with in TIMER database. Moreover, the associations were also analysed in BRCA as a control, because the prognosis of BRCA patients was not related to the expression of SYT4. First, there was no correlation between SYT4 expression and tumour purity in LGG, but a negative correlation with tumour purity was found in STAD. We speculated that the difference might be due to the different expression patterns of SYT4 in the TME. Genes that had a high expression in cells of the TME compared with tumour cells were negatively related to the level of tumour purity, while genes with a high expression in tumour cells had a positive correlation with tumour purity [26]. The expression of SYT4 had different associations with tumour purity under different circumstances, indicating that SYT4 might have different functions in various tumours. Second our ndings indicated that the expression of SYT4 was related to the in ltration levels of B cells, CD4+ T cells, macrophages, and DCs in STAD and LGG, but this correlation was not present in BRCA ( Figure  3). Interestingly, there was a positive correlation coe cient between the levels of immune cell in ltration and SYT4 expression levels in STAD, while a negative correlation coe cient existed in LGG. Although causality cannot be established through the current research, we did nd different correlations between the expression levels of SYT4 and the levels of immune cell in ltration and different correlations between the expression of SYT4 and prognosis in STAD and LGG.
In addition, the correlation between the expression of SYT4 and immune cell marker genes suggested that SYT4 had a role in regulating tumour immunity in LGG and STAD. We rst focused on monocytes, M1 and M2 macrophages, and TAMs ( Figure 4). Some studies found that TAMs aid tumour cell migrationin a mechanism that involves secretion of epidermal growth factor family ligands by the macrophages and secretion of colony-stimulating factor-1 by tumour cells [31] , and M2 macrophages were detrimental to the prognosis of cancer patients because they stimulated lymphangiogenesis and angiogenesis [32]. While, M1 macrophages have signi cant antitumour effects [16]. Our results showed that the expression level of SYT4 was positively related to the molecular markers of TAMs (CCL2 and IL10) and M2 macrophages (CD163, VSIG4 and MS4A4A) in STAD, while the expression of SYT4 showed a negative relationship with these markers in LGG. In addition, the markers of M1 macrophages had a strong correlation with the expression of SYT4 in LGG. However, the correlation was weak in STAD. These results reveal the potential regulatory role of SYT4 in TAM polarization. Therefore, we speculated that SYT4 might affect the prognosis of cancer patients through TAMs.
In addition, we also examined the correlation between SYT4 expression and Treg cell markers. Treg cells have been shown to be associated with poor prognosis [33]. Activated TGF-β existed widely in the TME, and inhibited the activity of NK cells and CTLs, the proliferation of Teff cells and the production of cytokines, as well as inhibited the differentiation of Teff cells into Th1 and Th17 cells, aiding the migration and metastasis of tumour cells [34]. Moreover, current research has determined that CCR8+ regulatory T cells are drivers of immunosuppression, which can help the immune escape of tumours [35]. In our study, we found that the Treg cell marker TGFB1 had the highest correlation coe cient (cor = 0.38) with SYT4 expression levels, and other Treg cells markers, such as CCR8, FOXP3 and STAT5B, also had signi cantly positive correlations with SYT4 expression in STAD. However, the Treg cells markers CCR8 and TGFB1 showed a negative association with the expression level of SYT4 in LGG. These ndings suggested that the molecular markers of Treg cells are positively correlated with the expression of SYT4 in STAD and that high expression of SYT4 is related to the poor prognosis of STAD patients. It is worth noting that the research by Yiftah Barshesheta et al. [35] showed that certain ligands of CCL1 could induce Ca2+ ux by binding to CCR8 and enhance the inhibitory activity of Treg cells. CCR8 could also induce melanoma cells to enter the lymph nodes by binding to CCL1 secreted by the lymph node sinusoidal endothelial cells, causing lymphatic metastasis [36]. A recent study found that SYT4 regulates Ca2+ in ux through TRPM1, leading to melanin production and axon elongation in alpaca melanocytes, which suggests that the growth and metastasis of melanoma is controlled by the suppression of SYT4 expression in melanoma cells [12]. As a membrane protein that can regulate calcium ux, whether SYT4 plays an important role in the binding of CCR8 and CCL1 and whether this role is related to Treg cells require further research.
In addition, we also considered the relationship between the markers of DCs and SYT4 expression in STAD and LGG. DCs in promote tumour metastasis by increasing Treg cells and reducing the cytotoxicity of CD8+ T cells [37]. We found that the relationship between DC markers, including HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, BDCA-1, BDCA-4 and CD11c, and SYT4 expression levels showed a negative correlation in LGG. However, the expression levels of HLA-DPB1, HLA-DQB1, HLA-DRA and HLA-DPA1 did not show a signi cant correlation with SYT4 in STAD,, while the expression levels of BDCA-1, BDCA-4 and CD11c showed a signi cantly positive correlation with SYT4. The results suggest that SYT4 might be a potential factor affecting tumour metastasis through DCs.
In addition, we also found that SYT4 was associated with B lymphocyte in ltration in STAD and LGG. B cell receptors on the surface of B lymphocytes bind to the tumour antigen, resulting in the processing and presentation the tumour antigen; The response of T lymphocytes to the tumour can then be activated, which plays a very important role in the tumour immunity [38]. We found that the relationship between the surface markers (CD19 and CD79a) and the expression level of SYT4 showed a signi cant positive correlation in STAD. However, in LGG, it opposite correlation was observed. These ndings together indicate that SYT4 plays an important role in the recruitment and regulation of in ltrating immune cells in LGG and STAD. However, our study had some limitations. First, we explored multiple databases, and biases might exist in their data. Second, due to the different sources of data, some results were contradictory. Third, our study was a bioinformatics analysis of SYT4, and the results were not veri ed in vivo or in vitro. Fourth, causal inferences cannot be made from our current research results, and follow-up prospective studies are needed.

Conclusions
In general, SYT4 had the opposite effect on the prognosis of patients with STAD and LGG. And SYT4 has different effects on immune in ltration in these two tumours. Therefore, SYT4 might be a potential prognostic and tumour immune-related biomarker in STAD and LGG.

Declarations Ethics approval and consent to participate
The human data in this study were all from the online databases, and thus, their use did not require ethical approval.

Consent for publication
Not applicable

Competing interests
The authors declare that they have no competing interests.  LGG: Brain lower-grade glioma; BC: Breast cancer; TAM: Tumour-associated macrophage; Th: T helper; Tfh: Follicular helper T; Treg: Regulatory T; Cor: R value of Spearman's correlation; None: Correlation without adjustment; Purity: Correlation adjusted by purity. *p < .01; **p < .001; ***p < .0001. LGG: Brain lower-grade glioma; TAM: tumour-associated macrophage; Cor: R value of Spearman's correlation; *p < 0.01; **p < 0.001; ***p < 0.0001. Figure 1 The expression levels of SYT4 in cancers. A. The data from Oncomine show the differences between the expression levels of SYT4 in tumours and normal tissues. B. The data from TIMIER show the expression level of SYT4 in different cancers.