Upregulation of vesicle-associated membrane protein 7 in breast cancer tissues

Abstract

BACKGROUND:

Vesicle-associated membrane protein 7 (VAMP7) plays oncogenic roles in cancers. However, its clinical significance in breast cancer (BC) tissues remains unknown.

OBJECTIVE:

To elucidate the clinical implications of VAMP7, as well as its involvement in the tumor microenvironment and molecular pathways of breast cancer.

METHODS:

BC ( $n=$ 100) and non-cancerous breast tissues ( $n=$ 100) were collected for an immunohistochemical experiment (1:200). The protein expression level of VAMP7 was determined by using a semi-quantitative scoring method. High-throughput RNA-sequencing data of BC tissues were analyzed to confirm the mRNA expression trend of VAMP7. Additionally, the largest BC prognosis cohort data were collected to mine the potential impact VAMP7 has on BC progression. The association between VAMP7 and the microenvironment of BC was evaluated by using a CIBERSORT algorithm. Moreover, we explored the co-expressed molecular mechanisms of VAMP7 in BC by calculating Pearson correlation coefficients and overexpressed genes. Finally, the biological mechanism underlying the relationship between VAMP7 and the key pathways was also explored using gene set enrichment analysis (GSEA). Potential therapeutic strategies were predicted targeting VAMP7.

RESULTS:

VAMP7 protein was significantly over-expressed in BC tissue than that in controls ( $p<$ 0.001). Compared with 459 normal breast tissues and 113 non-cancerous breast tissues, the expression level of VAMP7 mRNA was significantly increased in 1111 BC tissues. CD₄+T cells, macrophages, and naïve B cells had a higher infiltration rate in BC tissues with high VAMP7 expression, while regulatory T cells and CD₈+T cells had a lower infiltration rate. Over-expressed VAMP7 was associated with macrophages activation and transition from M1 to M2 polarization. Upregulated VAMP7 could predicted poorer OS, DMFS, PPS, and RFS outcomes. Upregulated VAMP7 co-expressed genes were significantly enriched in the cell cycle checkpoints. GSEA confirmed that over-expressed VAMP7 are markedly associated with functional enrichment in cell cycle related categories, including mitotic spindle, G2M checkpoint, and E2F targets. KU-55933 was predicted as a putative therapeutic drug for BC targeting VAMP7.

CONCLUSIONS:

VAMP7 was upregulated in BC tissue and correlated with poor prognosis of BC patients. VAMP7 may promote BC progression by targeting the cell cycle pathway.

Keywords

Breast cancer vesicle-associated membrane protein 7 immunohistochemistry molecular mechanisms

1. Introduction

Breast cancer (BC) is a frequently occurring cancerous tumor globally. It originates from the ductal epithelial cells and lobular epithelial cells of the breast. Because of the physiological differences between men and women, 99% of BC mainly occurs in women, and only about 1% occurs in men. The GLOBOCAN Project 2020 demonstrates that BC is currently the most prevalent type of cancer in women worldwide, causing significant illness and death. Approximately 2.3 million new cases of BC are diagnosed each year, accounting for 11.7% of all cancer cases globally [1]. BC pathogenesis involves molecular mutations, tumor cell heterogeneity, immune and metabolic dysregulation, and interactions with microenvironments, etc. Among them, changes in various oncogenes and tumor suppressor genes may promote the occurrence and progression of BC [2, 3, 4, 5]. Despite significant advancements in detection and treatment strategies for BC [6, 7], the cure rate remains limited. One of the most important reasons is that we still lack a comprehensive and accurate understanding of the molecular mechanistic details of BC. Therefore, an in-depth exploration of the possible carcinogenic factors of BC may contribute to the detection and management of BC worldwide [8]. The discovery of molecular targets of BC progression may also provide new ideas in tumor progression monitoring and even targeted treatment for BC.

Vesicle-associated membrane protein 7 (VAMP7) has the potential to become a valuable molecule in breast carcinogenesis. Synaptic vesicle docking and/or fusion with presynaptic membrane involve a protein complex comprising synaptobrevin, syntaxins, and synaptosomal-associated protein 25 (SNAP25). There are several members of the VAMP/synaptobrevin family, including VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, VAMP7, and VAMP8 [9, 10, 11, 12, 13, 14]. VAMP7, also called synaptobrevin 7 (Gene ID: 6845) located in Xq28 and Yq12 with eight exons [10, 12, 15, 16].

Currently, the clinical significance of VAMP7 expression in BC tissues has not been reported. Three existing publications on VAMP7 and BC only mentioned VAMP7 expression in BC MDA-MB-231 cells [10, 17, 18]. Thus, this study leveraged clinical BC tissues to detect the protein expression of VAMP7. Further, the mRNA expression of VAMP7 was calculated in parallel with gene microarray and RNA-sequencing (RNA-seq) data to explore the inferred role of VAMP7 in breast carcinogenesis.

2. Methods

2.1 The in-house protein level of VAMP7 in breast cancer tissues with immunohistochemistry

2.1.1 Clinical specimen

The tissue samples for the in-house immunohistochemical experiment were selected from the surgical excision and needle aspiration samples of patients diagnosed with non-specific invasive BC by the Pathology Department of the First Affiliated Hospital of Guangxi Medical University from January 2018 to December 2018. To meet the immunohistochemistry inclusion criteria, the following requirements were applied: (1) the cases should be adult females $\geqslant$ 18 years, and (2) the histological types of the patients should be consistent with WHO classification. The patients were diagnosed with non-specific invasive BC with ER, PR, HER-2, and Ki-67 being tested, (3) the patients underwent breast-related examination or surgical resection in our hospital, including classic radical surgery, modified radical surgery, simple mastectomy, and partial mastectomy, (4) the patients underwent surgery without prior radiotherapy, chemotherapy, Traditional Chinese Medicine, endocrine therapy, or biological targeted therapy. The patients had complete mobility and followed the doctor’s advice for routine rehabilitation therapy after surgery, (5) the clinical medical records and pathological data available were intact, the tissue had no extensive bleeding or necrosis, and the structure of the sample was clear and distinguishable, and (6) the cases complied with relevant criteria of the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. All included subjects signed informed consent voluntarily. The exclusion criteria were as follows: (1) female $<$ 18 years or male, (2) the patient’s clinical medical records were incomplete or the cases could not be evaluated for clinical TNM stage, or the pathological sections were not tested for ER, PR, HER-2, or Ki-67, (3) patients with other types of breast tumors or the tumors were metastatic, (4) the patient had received therapies before surgery, and (5) the tissue sample was not sufficient for the immunohistochemical test.

According to the expression of ER, PR, and HER-2, BC tissues were divided into four molecular subtypes by using the standards as follows, (1) Luminal-A was defined as positive ER and/or PR, negative HER-2, and Ki-67 $<$ 15%, (2) Luminal-B type was divided into HER-2 negative type and HER-2 positive type. Luminal-B (HER-2 negative) type was defined as positive ER and/or PR, negative HER-2, Ki-67 $\geqslant$ 14%. Luminal-B (HER-2 positive) type was defined as positive ER and/or PR, positive HER-2, and Ki-67 expression at any level, (3) HER-2 overexpression type was defined as positive HER-2 and negative ER and PR, and (4) the triple-negative type was defined as negative ER and PR and negative HER-2.

2.1.2 Immunohistochemical experiments

BC ( $n=$ 100) and non-cancerous breast tissues ( $n=$ 100) were included in the current study by using the aforementioned inclusion and exclusion criteria. For the immunohistochemical experiment, the first antibody was a concentrated rabbit anti-human polyclonal antibody VAMP7 (working concentration: 1:200, reagent number ab193200, Abcam Biotechnology, UK). The main experimental steps were as follows: after antigen repair, the endogenous peroxidase activity was blocked by using a 3% H₂O₂ solution. The diluted primary antibody was pipetted onto the surface of the tissue completely and then incubated in a wet box at 37^∘C for 90 minutes. Afterward, the secondary antibody was added. Other steps were performed according to the standard procedures from the experiment instructions. For each staining, both positive and negative controls were established. Brain tissue was used as the positive control for VAMP7, while the primary antibody was replaced with PBS buffer for the negative control.

2.1.3 Evaluation of immunohistochemistry of VAMP7 in breast tissues

Referring to the interpretation methods of IHC staining previously reported [19], the VAMP7 protein expression levels in different breast tissues were quantified by using a semi-quantitative scoring method. The optical density (IOD) and positive signal area (Area) were intercepted by using the Imageproplus v6.0 software, and the average optical density (AOD) of each case was calculated according to the formula IOD/Area. The first quartile (Q1) and third quartile (Q3) values of AOD in VAMP7 were calculated by using all the included tissue samples. The IHC staining scores of VAMP7 were determined by using the formula as follows, (1) the staining intensity I score was recorded according to the AOD level: AOD $<$ Q1 was one point for no or low staining of the cells; Q1 $<$ AOD $<$ Q3 was two points for moderate staining; AOD $>$ Q3 was three points for strong staining; (2) the P score was scored according to the positive cell rate: no staining was scored as zero, $P<$ 10% was one point, 10% $\leqslant$ $P<$ 50% was two points, 50% $\leqslant$ $P<$ 80% was three points, and $\geqslant$ 80% was four points; and (3) the final total score (Q) $=$ P $\times$ I. BC patients were divided into the low VAMP7 expression group (Q $<$ 4) and the high expression group (Q $\geqslant$ 4).

2.2 The in-silico mRNA level of VAMP7 in breast cancer tissues with RNA-sequencing

2.2.1 High-throughput RNA-sequencing data of breast cancer tissues

To further compare the clinical value of VAMP7 from the mRNA level, we calculated the VAMP7 mRNA expression level in BC tissues using high-throughput techniques. In this study, the dataset of TCGA with the largest sample size and the most complete clinicopathological parameters was selected for data mining (https://portal.gdc.cancer.gov/). Hence, the high-throughput RNA-sequencing data of 459 GTEx normal breast tissues (https://gtexportal.org/home/), 113 TCGA non-cancerous breast tissues, and 1111 TCGA BC tissues were obtained in the current study. GTEx and TCGA TPM RNA-sequencing data (TCGA-GTEx) were combined and their batch effect was removed. Log₂(TPM $+$ 1) conversion was performed on TCGA-GTEx RNA-sequencing data to compare the relative expression levels of VAMP7 in BC and non-cancerous breast tissues.

2.2.2 Multicenter prognosis cohort of breast cancer

In addition to the clinical value of VAMP7 in the onset of BC, we also wondered whether it will also yield some impact on BC progression and prognosis. The global BC prognosis cohort data were collected based on the Kaplan-Meier Plotter platform (https://kmplot.com/analysis/). The study considered several survival measures, including overall survival, post-progression survival, relapse-free survival, and distant metastasis-free survival. A Cox proportional hazards regression model was constructed and hazard ratios (HR) were calculated.

2.2.3 The relationship between VAMP7 and tumor immune infiltration in breast cancer

The potential association between VAMP7 and the microenvironment of BC was evaluated by using the CIBERSORT algorithm. We categorized patients based on their levels of VAMP7 expression (high vs. low) and analyzed differences in immune cell fractions between these groups. Furthermore, we explored the relationship between VAMP7 dysregulation and the tumor associated macrophages within the tumor microenvironment using gene set enrichment analysis (GSEA) using a clusterprofiler package (Version 4.7.1.003).

2.2.4 VAMP7 co-expressed genes and their pathways in breast cancer

The TCGA-GTEx TPM expression matrix was used to identify genes that were differentially expressed in BC, which offers the largest cohort with RNA-sequencing worldwide. The identification criteria of BC overexpressed genes were: log₂foldchange $>$ 1 and $p$ .adjusted $<$ 0.05 using a limma package (Version 3.52.4). At the same time, the Pearson correlation coefficients (PCCs) of VAMP7 and other genes were calculated in batches from the log₂(TPM $+$ 1) expression matrix of TCGA BC tissues. The intersection of BC overexpressed genes and VAMP7 co-expressed genes was taken, and the clusterprofiler expansion package was used to perform gene Reactome pathway enrichment analysis.

2.3 Gene set enrichment analysis of VAMP7 in breast cancer

In the breast cancer dataset, all genes were ranked according to their differential expression between two groups: the VAMP7 low-expression group and the VAMP7 high-expression group. Subsequently, GSEA was conducted to determine the enrichment score for hallmark gene sets within the collection. The enrichment score was calculated based on the distribution of ranked genes contained in each gene set. This analysis helps identify potential associations between VAMP7 expression levels and specific biological pathways or functions relevant to breast cancer. Finally, we mined putative therapeutic drugs targeting VAMP7 protein using Enrichr (https://maayanlab.cloud/Enrichr/).

2.4 Statistical analysis

All the statistical analyses in the present study were accomplished by using SPSS (Version 22.0) and R (Version 4.2.1) software. BC patients were separated into different groups according to their clinicopathological parameters. The expression status of VAMP7 was compared in each group by using a Chi-square test. A statistical significance level of $p<$ 0.05 was deemed significant.

3. Results

3.1 Overexpression of the protein level of VAMP7 in breast cancer tissues with in-house immunohistochemistry

3.1.1 Summary of the clinical parameters of included cases

A total of 100 cases of BC were included based on specific criteria, with 62 cases having paired adjacent breast tissue and 38 cases having unpaired non-cancerous breast tissue (Table 1). The patients’ ages ranged from 20 to 77 years, with a median age of 45.65 years. As this was a retrospective study, HER-2 immunohistochemistry showed a total of 19 cases of 2+, among which 6 cases were tested for HER-2 gene amplification by in situ hybridization. Another 13 cases were not; thus, they were excluded when considering the molecular types. When examining the molecular types of BC, 87 cases were analyzed to investigate the correlation with VAMP7 expression.

Table 1
Basic clinicopathological information of the included breast cancer patients

Parameters	Sample size	Proportion (%)
Histological type
Invasive ductal carcinoma	95	95
Invasive lobular carcinoma	5	5
Histopathological grade
Grade I	19	19
Grade II	56	56
Grade III	25	25
Pathological stage
T1 ( $\leqslant$ 2CM)	29	29
T2 (2–5 CM)	60	60
T3 ( $>$ 5 CM)	11	11
Regional lymph node (pN)		0
pN0	43	43
pN1	27	27
pN2	26	26
pN3	4	4
Distant metastasis (M)
M0	100	100
M1	0	0
Clinical stage		0
Stage I	16	16
Stage II	52	52
Stage III	27	27
Stage IV	5	5
Molecular subtypes
Luminal-A	22	22
Luminal-B (HER-2 negative)	20	20
Luminal-B (HER-2 positive)	16	16
HER-2 overexpression	18	18
Triple-negative	11	11

Figure 1.

Negative expression of VAMP7 protein in non-BC tissue. A: 100 $\times$ ; B: 400 $\times$ .

Figure 2.

VAMP7 protein expression in BC tissue. A–B: strong positive; C–D: median positive; E–F: negative. A, C, E: 100 $\times$ ; B, D, F: 400 $\times$ .

Figure 3.

The mRNA expression level of VAMP7 was increased in breast cancer tissues. (A) Breast cancer tissues (B) Luminal A breast cancer tissues (C) Luminal B breast cancer tissues (D) HER2+ breast cancer tissues (E) Triple-negative breast cancer tissues. VAMP7 mRNA was overexpressed in breast cancer tissues compared with normal breast tissues. Higher mRNA expression levels of VAMP7 could be found in Luminal A, Luminal B, and triple-negative breast cancer tissues rather than HER2+ breast cancer.

Figure 4.

The discriminatory ability of VAMP7 mRNA overexpression in breast cancer tissues. (A) Breast cancer tissues (B) Luminal A breast cancer tissues (C) Luminal B breast cancer tissues (D) HER2+ breast cancer tissues (E) Triple-negative breast cancer tissues.

3.1.2 Detection of VAMP7 protein expression level in BC and non-cancer tissues based on IHC

Excluding the sections with no obvious BC or para-cancer breast tissue, there were 95 BC sections and 91 non-BC remained for the statistics. VAMP7 protein showed various staining levels. There were 8 cases of low expression (8.4%) and 87 cases of high expression (91.6%) in BC samples, 34 cases of low expression (37.4%), and 57 cases of high expression (62.6%) in control breast samples. VAMP7 protein was significantly over-expressed in BC tissue than that in controls ( $p<$ 0.001) (Figs 1 and 2). The VAMP7 protein was found to be significantly upregulated in BC patients aged $\geqslant$ 50 years (100%) compared to those aged $<$ 50 years (86%; $p=$ 0.021). However, the correlation between VAMP7 protein expression and other clinicopathological parameters was insignificant, including nationality, histological grade, molecular typing, and TNM stage (data not shown).

3.2 Upregulation of the mRNA level of VAMP7 in breast cancer tissues with in-silico high throughput data

Compared with 459 normal breast tissues and 113 non-cancerous breast tissues, the expression level of VAMP7 mRNA was significantly increased in 1111 BC tissues (Fig. 3). Additionally, a significantly higher mRNA level of VAMP7 was found in Luminal A/B and triple-negative BC tissues, rather than HER2+ ones. Finally, the discriminatory ability of VAMP7 mRNA upregulation in BC tissues was exhibited in Fig. 4.

3.3 Differences in the levels of immune and stromal cell infiltration in breast cancer tissue

According to the accumulation histogram (Fig. 5), the immune cell infiltration differed from VAMP7 high-expression BC tissues to VAMP7 low-expression BC tissues. The BC group exhibiting high VAMP7 expression showed a significantly larger infiltration proportion of immune cells compared to the group with low VAMP7 expression. Moreover, through statistical tests and visual analysis of the phenotypic differences of 12 kinds of immune cells (Fig. 6), the results showed that CD₄+T cells, macrophages, and naïve B cells had a higher infiltration rate in BC tissues with high VAMP7 expression, while regulatory T cells and CD₈+T cells had a lower infiltration rate. In-depth GSEA demonstrated a significant association between the overexpression of VAMP7 and the activation of macrophages, as well as their polarization towards the M1 phenotype (Fig.7). Additionally, VAMP7 was predicted to play a role in the transition from M1 to M2 polarization in macrophages (Fig. 7).

Figure 5.

Accumulation histogram of immune cells in breast cancer tissue.

Figure 6.

Violin plot of immune infiltration levels according to VAMP7 expression in breast cancer tissues.

Figure 7.

Potential role of VAMP7 in modulating the activation and polarization of macrophage in breast cancer. Over-expressed VAMP7 was significantly enriched in macrophage activation and macrophage polarization.

3.4 The mRNA level of VAMP7 as a prognostic marker in breast cancer

In light of the abnormal upregulation of VAMP7 in BC, we further explored its prognostic value in global BC cohorts. Upregulated VAMP7 was associated with poorer OS, DMFS, PPS, and RFS outcomes (Fig. 8). Moreover, VAMP7 served as an independent risk predictor for BC patients (HRs $>$ 1).

Figure 8.

Prognostic value of VAMP7 mRNA overexpression in breast cancer patients. (A) Overall survival (B) Distant metastasis-free survival (C) Post-progression survival (D) Relapse-free survival.

3.5 Potential molecular pathways of VAMP7 in breast cancer

Based on the high throughput RNA-sequencing data, a total of 1606 upregulated genes and 1976 downregulated genes were identified from BC tissues (Fig. 9A, 9B). Additionally, a total of 5505 VAMP7 co-expressed genes were filtered from BC tissues (Fig. 9C). After the intersection, a total of 275 upregulated VAMP7 co-expressed genes were obtained, which were significantly enriched in the cell cycle pathways, such as cell cycle checkpoints, signal amplification from kinetochores, and signal amplification from unattached kinetochores via a MAD2 inhibitory signal (Fig. 9D). In-depth GSEA analysis showed that over-expressed VAMP7 was observed to be related to functional enrichment within mitotic spindle, G2M checkpoint, and E2F targets (Fig. 10) Finally, KU-55933 was predicted as a potential therapeutic agent targeting VAMP7.

Figure 9.

Potential co-expression mechanisms of VAMP7 mRNA in breast cancer. (A) Volcano plot and (B) heatmap of the differentially expressed genes in breast cancer tissues (C) Correlation diagram of the top 10 VAMP7 co-expression genes in breast cancer tissues (D) Reactome pathway of the upregulated VAMP7 co-expression gene in breast cancer tissues.

Figure 10.

Gene set enrichment analysis of VAMP7 in breast cancer. Over-expressed VAMP7 was correlated with functional enrichment within mitotic spindle, G2M checkpoint, and E2F targets, all of which are responsible for controlling cell cycle.

4. Discussion

VAMP7 is a vesicle-associated membrane protein-encoding gene that belongs to the VAMP gene family. Although VAMP7 was reported to promote tumor progression, its clinical implications and molecular function in the tumor microenvironment and signaling pathways remains unclear in BC. Herein, the present study confirmed the over-expression and poor prognosis of VAMP7 by integrating in-house IHC and external high throughput sequencing BC data. This is the first study to explore the relationship between VAMP7 overexpression and macrophage polarization, as well as functional enrichment of the cell cycle pathway.

In this information era, the utilization of big data in healthcare offers significant opportunities for enhancing clinical management of BC patients [20]. This study, based on a total of 1111 BC samples, showed that there was a significant correlation between high expression of VAMP7 and VAMP7 mRNA and breast carcinogenesis, which has a unique clinical value. We also found that VAMP7 mRNA expression levels differed among BCs with different molecular typing. VAMP7 mRNA levels were significantly higher in Luminal A/B and triple-negative BC tissues than in HER2+ BC tissues. Overexpressed VAMP mRNA was also differentially identified in BC tissues with higher sensitivity and specificity, which may suggest that we can target the differences in VAMP7 expression levels when treating BC patients with different molecular typing, and thus adopt personalized and targeted treatment regimens. It has been shown that VAMP7 promotes the formation of an invasive footprint and the decrease of its level can diminish the invasive ability of tumor cells [21]. Furthermore, the expression of VAMP7 has been implicated in the growth, invasiveness, and lung metastasis of breast cancer cells, exerting its effects through intricate mechanisms such as mediating the cellular trafficking of membrane type 1-matrix metalloproteinase [18, 22]. It is thus hypothesized that VAMP7 could be used as a potential target for treatment and provide a new direction for BC treatment, which provides support for the above speculation.

In response to the aberrant upregulation of VAMP7 levels in BC, we also further explored its prognostic value in a global BC cohort. Excitingly, we found that OS, DMFS, PPS, and RFS were significantly lower in BC patients with high VAMP7 expression than in the group with low VAMP7 expression, and the two were significantly different, suggesting that elevated VAMP7 expression levels are closely associated with poor prognosis, and high levels of VAMP7 expression may impact the prognosis of individuals with BC. It can be used as an independent prognostic factor and has great value for prognostic assessment. VAMP7 expression is linked to shorter survival in esophageal cancer and can serve as an independent prognostic factor [23]. In addition, it has also been pointed out that VAMP7 has prognostic value on the overall survival of gastric cancer and may be used as a diagnostic and prognostic biomarker for the treatment of gastric cancer [24]. Therefore, it is reasonable to believe that VAMP7 will be a powerful biomarker in BC prognosis and deserves further study in the clinic.

Compared with previous studies [10, 17, 18], this study not only verified the poor prognosis of VAMP7, but also revealed its association with the tumor microenvironment and the cell cycle pathway. In the present study, we highlighted the relationship between VAMP7 expression levels and clinicopathological parameters of BC, so how does VAMP7 regulate the cell cycle of BC cells? Based on the analysis of high-throughput RNA sequencing data, we found that the VAMP7 gene was significantly enriched in the cell cycle pathway. Our research offers a novel understanding of VAMP7’s molecular mechanisms during the BC cell cycle. Reactome pathway analysis revealed that the upregulated VAMP7 co-expressed genes were primarily enriched in processes such as cell cycle checkpoints, signal amplification from kinetochores, and sister chromatid cohesion in mitotic spindle formation involving EML4 and NUDC. Different upregulated genes regulate different processes of the cell cycle respectively, thus achieving regulation of transcription in cancer cells, affecting cancer cell metabolism and promoting cancer cell growth and development. Interestingly, our GSEA analysis provided further confirmation of the intimate association between VAMP7 and the cell cycle pathway. Our findings suggest that overexpression of VAMP7 may regulate the cell cycle by influencing the activities of the mitotic spindle, G2M checkpoint, and E2F targets. In addition, in-depth research has shown that in human MDA-MB-231 BC cells, VAMP7 can form a Nethylmaleimide-sensitive factor attachment protein receptor complex with SNAP23 and Syntaxin4, which plays a crucial role in BC cell invasion [10, 21]. VAMP7 can also transport membrane-type 1-matrix metalloproteinase to specific sites and activate protein hydrolases, thus degrading the extracellular matrix, which contributes to the spread and metastasis of cancer cells and maintains the basic functional mechanisms required for cancer cell invasion [17]. These are all based on the role of VAMP7 in the secretion of unconventional proteins. VAMP7 is mainly present in advanced nuclear endosomes and is involved in processes such as intracellular vesicle transport and merging of vesicles and plasma membrane, thus mediating the release of cancer cell secretions, becoming part of the tumor microenvironment, mediating interactions between tumor cells and host cells, and promoting tumor growth and development [25]. Furthermore, we predicted that KU-55933 may be a novel therapy by targeting VAMP7 in breast cancer, which was demonstrated to suppress BC proliferation and was able to induce cell cycle arrest [26, 27].

In the analysis described above, we have known the enrichment sites of VAMP7 in the cell cycle pathway and the role of VAMP7 in participating in exosomes, suggesting that VAMP7 may indirectly influence local infiltration of immune cells by affecting extracellular matrix components. After analyzing the results, it has been found that BC patients show high expression of VAMP7. This protein plays a crucial role in regulating unconventional protein secretion by being involved in the soluble N-ethylmaleimide-sensitive factor attachment proteins receptor process [25]. Therefore, we further speculate that the cancer cells of BC patients may chemotaxis the infiltration of some or several kinds of immune cells through the high expression of VAMP7, and affect the prognosis of the patients through the infiltration of related immune cells [28]. Surprisingly, although many studies have found a close association between certain genes or proteins and immune infiltration of BC, such as the RUNX family, which can affect the infiltration level of various immune cells through various mechanisms [29]; BC-associated 11-lncRNA, which is associated with the infiltration of immune cell subtypes [30]; and tumor mutational load, which can further affect the prognosis of BC by influencing immune infiltration. However, no relevant studies have mentioned the potential link between VAMP7 and immune infiltration in BC, so our study may be the first to explore its mechanism of action in BC from the perspective of immune infiltration, which is of great research value. Overall, the main infiltrating cells in BC tissues are T cells and NK cells, which is consistent with the findings of Xinhui Li et al. [29]. Treg cells have the role of regulating the immune response, and their main role in tumors is to suppress the immune response and help the immune escape effect of tumor cells, which is closely associated with poor patient prognosis. NK CD4+T and CD8+T, on the other hand, mainly inhibit the growth of tumor cells by enhancing the immune response of the body or directly killing the target cells, which is associated with prolonged survival of patients [31]. It is in this complex interaction mechanism that the body resists the development of tumors and affects the prognosis of patients. More importantly, in the present study, we speculated that overexpressed VAMP7 may be associated with macrophages activation and promoted the transition from M1 to M2 polarization. In BC, M2 polarized macrophages could result in tumor progression through multiple mechanisms [32, 33]. Therefore, VAMP7 may be one of the bridges between tumor microenvironment and BC, which may become a new target and prognostic marker in BC.

However, our limitations are also evident, such as small clinical data samples, inherent flaws in retrospective studies, lack of long-term prognostic assessments, and possible differences in outcomes due to varying patient treatment regimens. However, our results still reveal the upregulation of the expression level of VAMP7 mRNA and its proteins in BC, which will enrich our understanding of the interaction between VAMP7 and BC and provide a new direction for targeted therapy and prognostic evaluation of BC.

5. Conclusions

VAMP7 was over-expressed in BC tissue and may be used as a prognostic biomarker. We are the first to explore the impact of VAMP7 on the polarization of tumor associated macrophages and enrichment in the cell cycle pathway. The mechanism underlying VAMP7 and cell cycle in breast cancer deserves further exploration in the future.

Footnotes

Acknowledgments

The authors sincerely thank the technical support provided by Professor Gang Chen from Guangxi Key Laboratory of Medical Pathology. Additionally, they appreciate the people who contributed to The Cancer Genome Atlas.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

Guangxi Zhuang Autonomous Region Health Commission Self-financed Scientific Research Project (Z-A20230519).

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Upregulation of vesicle-associated membrane protein 7 in breast cancer tissues

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Methods

2.1 The in-house protein level of VAMP7 in breast cancer tissues with immunohistochemistry

2.1.1 Clinical specimen

2.1.2 Immunohistochemical experiments

2.1.3 Evaluation of immunohistochemistry of VAMP7 in breast tissues

2.2 The in-silico mRNA level of VAMP7 in breast cancer tissues with RNA-sequencing

2.2.1 High-throughput RNA-sequencing data of breast cancer tissues

2.2.2 Multicenter prognosis cohort of breast cancer

2.2.3 The relationship between VAMP7 and tumor immune infiltration in breast cancer

2.2.4 VAMP7 co-expressed genes and their pathways in breast cancer

2.3 Gene set enrichment analysis of VAMP7 in breast cancer

2.4 Statistical analysis

3. Results

3.1 Overexpression of the protein level of VAMP7 in breast cancer tissues with in-house immunohistochemistry

3.1.1 Summary of the clinical parameters of included cases

Table 1 Basic clinicopathological information of the included breast cancer patients

3.2 Upregulation of the mRNA level of VAMP7 in breast cancer tissues with in-silico high throughput data

3.3 Differences in the levels of immune and stromal cell infiltration in breast cancer tissue

5. Conclusions

Footnotes

Acknowledgments

Conflict of interest

Funding

References

Table 1
Basic clinicopathological information of the included breast cancer patients