VMA21: unveiling a novel oncogene that facilitates immune evasion in triple-negative breast cancer through TCIRG1 protein stability regulation

Abstract

Background: VMA21 has been shown to be dysregulated in a number of cancers. However, no study has yet explored whether VMA21 is involved in the regulation of triple-negative breast cancer (TNBC), especially from the level of immune escape. Methods: The Gene Expression Omnibus (GEO) database was accessed to obtain the microarray dataset identified as GSE38959, which was then subjected to an analysis aimed at identifying genes that are differentially expressed (DEGs). The researchers examined the expression of VMA21 in TNBC cell lines. After knockdown of VMA21 in TNBC cells, cell proliferation, invasion, and migration were assessed by clone formation, cell scratch, and Transwell assay, respectively. The effect of VMA21 on immune cell function was explored by cell co-culture method, which was used to assess how TNBC cells with suppressed VMA21 expression affected CD8+ T cytotoxic potential and cytokine secretion. The effect of VMA21 on TCIRG1 protein stability and ubiquitination was assessed using immunoprecipitation. The effects of VMA21 knockdown on tumor xenograft growth and CD8+ T cell immune infiltration in mice were further evaluated. Results: VMA21 expression is significantly elevated across various cell lines of TNBC. Furthermore, the perturbation of VMA21 notably suppresses key cell viability parameters in TNBC cells, including their proliferation, invasiveness, and migratory abilities. The modulation of VMA21 in TNBC cells can lead to a substantial augmentation in CD8+ T cell effectiveness. VMA21 stabilizes TCIRG1 protein expression by inhibiting its ubiquitination degradation. Further, VMA21 is involved in regulating TNBC cell proliferation, invasion and immune escape by promoting TCIRG1 expression. Conclusions: VMA21 is able to regulate TCIRG1 protein stability by binding to TCIRG1 protein in the form of ubiquitination, which ultimately promotes the malignant behavior as well as immune escape of TNBC cells.

Introduction

Characterized by the absence of three critical protein receptors, triple-negative breast cancer (TNBC) distinguishes itself from other breast cancer types. These missing receptors are the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [1]. This receptor-negative profile renders TNBC unresponsive to hormone therapies and HER2-directed treatments, leading to fewer therapeutic options and generally unfavorable outcomes [2]. Moreover, TNBC is known for its rapid growth, high malignancy, and tendency for early metastasis, which contribute to its status as one of the most aggressive forms of breast cancer [3]. The phenomenon of immune escape is especially pertinent in TNBC, enabling tumor cells to avoid detection and destruction by the immune system [4]. This evasion is mediated through several strategies, such as altering the tumor microenvironment, upregulating immune checkpoint proteins, and employing immunosuppressive cell populations. These tactics collectively contribute to the cancer’s ability to spread and metastasize. Despite advancements in immunotherapy for various cancers, effective application in TNBC remains a significant challenge. Gaining a thorough understanding of TNBC’s immune escape mechanisms is essential for devising potent immunotherapeutic approaches. Research into the interactions between TNBC cells and the immune system, and the potential to bolster anti-tumor immunity by targeting these interactions, could yield novel treatment strategies for patients with TNBC. Consequently, the investigation into the molecular underpinnings and possible therapeutic targets of immune escape in TNBC holds significant promise for enhancing clinical interventions and outcomes for this disease.

In this study, pre-screening based on the GEO database revealed that VMA21 expression was up-regulated in TNBC tissues. VMA21, known as Vacuolar Membrane Protein 21, is a protein expressed in tumor cells that has attracted much attention in oncology research in recent years [5]. WEI et al. [6] showed that up-regulated VMA21 was detected in cervical cancer cells, and reduction of VMA21 effectively inhibited cervical cancer cell proliferation and stimulated apoptosis. XUE et al. [7] found that VMA21 promoted the growth, migration and invasiveness of ovarian cancer cells. Although there is no direct evidence of the relationship between VMA21 and TNBC, considering the complexity of TNBC and the diversity of immune escape mechanisms, VMA21 may play an anti-tumor role by affecting the tumor microenvironment or directly acting on TNBC cells. Therefore, in conjunction with the existing research progress, the aim of this paper is to explore the immune escape mechanism of triple-negative breast cancer and the potential role of VMA21 in this process, and to provide a theoretical basis for further research and clinical application.

The objective of this research was to conduct an in-depth investigation into the function of VMA21 in TNBC and to elucidate the mechanisms through which it exerts its regulatory effects. Through examining the expression profile of VMA21 within TNBC cells and assessing its impact on the biological behaviors of tumor cell proliferation, invasion, and evasion of the immune system, this study aims to uncover the pivotal role that VMA21 plays in the progression of TNBC. The findings could lay the groundwork for the development of novel therapeutic strategies, ultimately enhancing the treatment outcomes and the quality of life for patients afflicted with TNBC.

Materials and methods

Cell culture

The human TNBC cell lines, including MCF-7, MDA-MB-231, BT-20, and MDA-MB-468, along with the normal human mammary epithelial cells MCF-10A, are maintained at the Medical Research and Experiment Center of Shanxi Province Cancer Hospital. Afterward, the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher, USA) that had been supplemented with 10% Fetal Bovine Serum (FBS, ThermoFisher, USA) and a 1% mixture of Penicillin and Streptomycin antibiotics (Solarbio, Beijing, China), with the antibiotics being present at a concentration of 10 mg per milliliter. The cells were maintained under culture conditions at a temperature of 37°C in an incubator calibrated to a CO₂ concentration of 5%. Post-revival, once the cells exhibited robust growth, they were enzymatically dissociated with trypsin and plated into 6-well plates. Upon reaching a confluence of approximately 90% to 95% under standard culture conditions, they were processed for subsequent analyses of cellular behavior. Specifically, MDA-MB-231 and BT-20 cells were chosen to develop a VMA21 knockdown model. Additionally, an in vitro co-culture system was established using Transwell chambers, where MDA-MB-231/BT-20 cells were placed in the upper compartment and CD8+ T cells in the lower, allowing for direct cell-cell interaction.

Bioinformatics analysis

Using the GEO public database, we extracted gene expression profiles and pertinent clinical data specific to TNBC from the platform GPL4133, within the dataset GSE38959. Subsequently, we utilized the Limma R package to analyze the preprocessed data, which had undergone background correction and normalization. We identified differentially expressed genes (DEGs) by applying a threshold of |log Fold Change| ≥0.5 and adjusting for statistical significance with P<0.05. Further exploration of the VMA21 protein’s co-expression network was conducted using the STRING database, a repository that consolidates protein-protein interaction data from a variety of sources, such as experimental evidence, scientific literature, and computational predictions. Additionally, we sourced the expression levels of VMA21 in TNBC clinical tissues from the TCGA database. To elucidate the relationship between VMA21 and various immunological events within TNBC, we performed an analysis leveraging the TISIDB database. This approach allowed us to gain insights into the immunological interactions associated with VMA21 in the context of TNBC.

Genetic overexpression and knockdown

Utilizing the NCBI database, we obtained the mRNA sequence for the VMA21 gene (gene ID: 203547) and, in conjunction with the coding sequence (CDS) of the target gene, designed and synthesized three distinct shRNA constructs aimed at VMA21 for use in subsequent experimental applications. The sequence of sh-VMA21#1: 5’-GTGAAGGCAAACAGGATTAAA-3’, sh-VMA21#2: 5’-GTTCGAGGAAGCTCCATTTAA-3’, sh-VMA21#3: 5’-CACCGTGCCTGGCCTAATAAT-3’. Additionally, the full-length sequence of TCIRG1 was cloned into the pcDNA3.1 vector to create a plasmid expression system.

The human TNBC cell lines MDA-MB-231 and BT-20 were subjected to transfection with VMA21 shRNA constructs (sh-VMA21#1, sh-VMA21#2, sh-VMA21#3) as well as with the pcDNA-TCIRG1 plasmid, using the Lipofectamine^®3000 transfection reagent according to the manufacturer’s protocol. Following the transfection process, the cells were incubated at 37°C in an atmosphere containing 5% CO₂ for a specified duration to allow for gene expression manipulation. After the designated time period, cells from all experimental groups were harvested and prepared for the continuation of the study.

Clone formation assay

Cells exhibiting robust growth from each group were plated into 12-well plates at a density of 1×10³ cells per well, with triplicate wells maintained for each group. The cells were then cultured in a controlled environment incubator set at 37°C with 5% CO₂, and the culture medium was refreshed every 3 to 4 days. Colony formation became evident after approximately two weeks, marked by the formation of visible cell colonies that were macroscopic in size. Thereafter, the cells were collected and fixed with a 4% paraformaldehyde solution for a period of 30 minutes. This was followed by staining with 0.1% crystal violet for 15 minutes to visualize the colonies. To remove any unbound stain and nonspecific materials, phosphate-buffered saline (PBS, Gibco, USA) buffer was employed for washing, followed by drying. Ultimately, images of the stained colonies were captured, and the number of colonies was enumerated to assess the cloning efficiency.

Transwell assay

Matrigel was uniformly spread on the underside of the top chamber in a Transwell (Corning, USA) apparatus and then incubated at 37°C for 30 minutes to create an even layer. Following this, cells from each experimental group were suspended in 200 μL of DMEM devoid of serum to a density of 50,000 cells per well, after which they were introduced into the Matrigel-coated upper chamber. The bottom chamber was replenished with 600 μL of DMEM supplemented with 20% FBS. Post a 48-hour incubation period, the cells were immobilized using 4% paraformaldehyde for 30 minutes at ambient temperature, followed by staining with 0.1% crystal violet for 15 minutes. Cells on the upper surface were carefully wiped away to reveal those that had traversed to the lower surface. The migrated cells were subsequently observed under a light microscope at 10x magnification, and their quantity was assessed for subsequent data analysis.

Scratch-wound assay

A total of 8×10⁵ stably transfected cells were plated into 6-well plates for culture. After a 24-hour incubation period, a wound was introduced using the tip of a 200 μL pipette. Following the initial procedure, the wells were carefully rinsed with PBS to eliminate any cells that had become dislodged. Once the washing was complete, fresh culture medium was introduced to the wells to sustain the ongoing growth and maintenance of the remaining cells. To assess the cells’ migratory capacity, photographic documentation was taken at both 0- and 36-hours post-wounding. The rate of wound closure was then determined by calculating the change in wound area over time.

Cytotoxicity assay

Cytotoxicity of T cells was assessed using the LDH Cytotoxicity Kit, in accordance with the manufacturer’s protocol (Elabscience, Wuhan, China). CD8+ T cells were extracted directly from the initial sample without any alteration, utilizing the CD8+ T Cell Isolation Kit. Subsequently, the total number of cells was ascertained through the application of the Vi-CELL XR cell viability analyzer, a product of Beckman Coulter. For the co-culture experiment, 5,000 TNBC cells were plated in triplicate in a 96-well flat-bottom plate and subsequently co-cultured with 50,000 CD8+ T cells. The cytotoxicity was calculated using the formula: Cytotoxicity = [1 - (OD_Case - OD_{effector cell})/OD_{target cell}] × 100%, where OD represents the optical density measured at a specific wavelength, indicative of the amount of lysis in the sample.

Clinical tissue collection

Twenty-eight patients with primary TNBC admitted to the Department of Breast and Thyroid Surgery of Shanxi Province Cancer Hospital between May 2021 and December 2023 were collected. General clinical data of the patients including gender, age, AFP, tumor size and number of tumors were also collected. Inclusion criteria: The specimens collected were primary breast cancer patients with complete clinicopathologic data, and none of the patients received any kind of preoperative treatment to avoid interfering with the experimental results. Patients diagnosed with triple-negative breast cancer, i.e., negative expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). Exclusion criteria: all were confirmed to be of breast origin after detailed clinical laboratory tests upon admission and were not combined with primary tumors from other sites. There were serious comorbidities or complications.

The study was approved by the Ethics Committee of Shanxi Province Cancer Hospital and all patients signed an informed consent form.

Immunohistochemical staining

Sections of tumor tissue underwent a series of preparation steps including removal of paraffin, rehydration, antigen retrieval, and blocking with serum. This was followed by the application of primary antibodies against VMA21 and CD8 (both sourced from Abcam, USA), subsequent incubation with their respective secondary antibodies, a washing process, development of color using DAB (Diaminobenzidine), counterstaining with hematoxylin, and finally, the mounting of the tissue sections. The quantification of cells expressing VMA21 and CD8 was conducted by counting the number of positively stained cells within three randomly chosen high-magnification fields for each section. This examination was carried out using an inverted fluorescence microscope model IX53 (Olympus, Tokyo, Japan).

In vivo experiments

A total of twelve female BALB/c nude mice, aged between 4 to 6 weeks and weighing between 18 to 22 grams, were obtained from the Institute for Laboratory Animal Research, Shanxi Province Cancer Hospital. These specific pathogen-free (SPF) nude mice were housed in a clean-grade animal room maintained at a room temperature of about 24°C and humidity of about 50%. Mice were randomly assigned into two groups: the NC shRNA group and the VMA21 shRNA#2 group, with six mice per group. Each mouse was then injected with 0.2 mL of a cell suspension containing either non-targeting shRNA (NC shRNA group) or VMA21 shRNA#2 stably transfected MDA-MB-231 cells at a concentration of 5×10⁷ cells/mL, in the left flank area. The development of the tumors was monitored and documented every four days over a period of four weeks. After 32 days, the mice were sacrificed by cervical dislocation, and the tumours were surgically excised, photographed, weighed and subjected to paraffin-embedded sectioning. Throughout the study, the dimensions of the tumors were measured at regular intervals using calipers. On the final day, the tumors were weighed using an electronic scale to determine their mass. All procedures involving the use of animals were reviewed and approved by the Management Committee for the Use of Laboratory Animals at the Institute for Laboratory Animal Research, Shanxi Province Cancer Hospital, ensuring adherence to ethical standards and guidelines for animal research.

Western blotting

The tumor samples were rinsed twice using cold PBS and then lysed in a solution of RIPA buffer from Roche Diagnostics, which included a cocktail of protease inhibitors. Following this, the protein content was precisely determined using the BCA protein assay kit provided by Thermo Fisher Scientific. Protein samples of equal concentration were loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. The initial phase of electrophoresis was conducted at a voltage of 70 V for 30 minutes, which was then increased to 120 V for an additional 90 minutes. The separated protein bands were transferred to PVDF membranes at a constant current of 300 mA for a period of 2 hours. After the transfer, the membranes were incubated in a blocking solution containing 5% skim milk for 2 hours to prevent non-specific binding. Next, the membranes were exposed to HRP-labeled secondary antibodies, specifically anti-rabbit IgG derived from goats, for 1 hour at room temperature to amplify the detection signal. β-actin was utilized as an internal loading control to normalize the protein levels. The relative intensities of the protein bands were quantified and analyzed using ImageJ software, which facilitated the assessment of the specific bands of interest.

RT-qPCR

Tumor tissue samples were gathered from each experimental group. To isolate the total cellular RNA, Trizol reagent from ThermoFisher Scientific (USA) was applied following the manufacturer’s guidelines for RNA extraction. The purity and integrity of the extracted RNA were confirmed by measuring the absorbance ratio at 260 nm to 280 nm, ensuring it fell within the acceptable range of 1.8 to 2.0. The isolated RNA was then converted into complementary DNA (cDNA) using a commercially available reverse transcription kit from Qiagen (Germany). The synthesized cDNA served as a template for the amplification of the gene using a Bio-Rad CFX90 Real-Time PCR system. The PCR efficiency was optimized to be within the range of 90-110%, which corresponds to a slope of the standard curve between 3.1 and 3.6. GAPDH was utilized as an endogenous control gene to normalize the gene expression levels. The relative quantification of PD-L1 expression was determined using the comparative CT (2-ΔΔCT) method, which accounts for differences in the initial amount of target gene and normalizes it to the reference gene.

Statistical analysis

Utilizing Graphpadprism8 for statistical analysis, data were processed and results are reported as the average value ± standard error of the mean (SEM), based on a minimum of three distinct experiments. For the comparison of two sample averages, the Student’s t-test was the chosen method. One-way analysis of variance (ANOVA) was used for comparisons amongst multiple groups, repeated measures ANOVA was used for comparisons at different timepoints and the SNK-Q test was used for pairwise comparisons. A difference of P<0.05 was considered to indicate statistical significance.

Results

Database-based mining reveals that VMA21 is associated with immune infiltration in TNBC

Screening for DEGs dysregulated in TNBC based on the GSE38959 database revealed that the top 20 DEGs were OGN, AGR3, APOD, DCN, CPE, EGR1, PIGR, PIP, LEP, CXCL12, MUCL1, TMSB10, TOP2A, CKS2, FN1, HORMAD1, VMA21, CXCL9, TMEM65, S100A9, of which VMA21 caught our attention (Figure 1A). Subsequently, a search in the TCGA database revealed that VMA21 was significantly highly expressed in TNBC and correlated with TNBC staging (Figure 1B, 1C). In addition, analysis of the immune correlation between VMA21 and TNBC based on the database showed that VMA21 was significantly associated with immune cells and behaviors in TNBC, including NK cell, Macrophage, Endothelial cell, T cell CD8T+, T cell CD4T+, microenvironment score, etc (Figure 1D, 1E). The above suggests that VMA21 is significantly up-regulated in TNBC expression and may be associated with immune infiltration.

Figure 1.

Database-based mining reveals that VMA21 is associated with immune infiltration in TNBC. A: The GSE38959 dataset was analyzed to produce a volcano plot illustrating differentially expressed genes (DEGs). B: A heatmap depicting the expression levels of VMA21 in TNBC was sourced from the TCGA database. C: Violin plots were generated to represent VMA21 expression across various stages of TNBC, as obtained from a TCGA database search. D, E: The relationship between VMA21 and diverse immune events in TNBC was investigated using data from the Tumor Immune System Interaction (TISIDB) database.

Silencing of VMA21 hindered the processes of cell multiplication and invasiveness in TNBC cells

Immunohistochemical examination was conducted to evaluate and compare the VMA21 protein expression across clinical samples representing diverse stages of TNBC. According to the data illustrated in Figure 2A, there was a progressive rise in the levels of VMA21 as the cancer advanced through its various stages. This overexpression pattern was also observed in various TNBC cell lines (Figure 2B, 2C).

Figure 2.

Silencing of VMA21 hindered the processes of cell multiplication and invasiveness in TNBC cells. A: Immunohistochemistry was employed to evaluate the presence and expression levels of VMA21 across various stages of clinical tissues affected by TNBC. B: RT-qPCR was performed to assess the expression level of VMA21 in TNBC cell lines (MCF-7, MDA-MB-231, BT-20, and MDA-MB-468) and breast cancer epithelial cells. C: Western blotting was performed to assess the expression level of VMA21 in TNBC cell lines (MCF-7, MDA-MB-231, BT-20, and MDA-MB-468) and breast cancer epithelial cells. **P<0.01, ***P<0.001 vs MCF-10A group. D: RT-qPCR assessment of mRNA VMA21 levels in TNBC cells transfected with VMA21 shRNA#1, VMA21 shRNA#2, and VMA21 shRNA#3. E: Western blotting assessment of protein VMA21 levels in TNBC cells transfected with VMA21 shRNA#1, VMA21 shRNA#2, and VMA21 shRNA#3. F: Clone formation detection of proliferation of TNBC cells transfected with VMA21 shRNA#2. G, H: Cell scratch experiment detection of migration of TNBC cells transfected with VMA21 shRNA#2. I, J: Transwell assay detection of invasion of TNBC cells transfected with VMA21 shRNA#2. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs NC shRNA group. n=6.

Subsequently, to appraise the influence of VMA21 on the oncogenic properties of TNBC cells, we established MDA-MB-231 and BT-20 cell lines with stable VMA21 knockdown. Notably, the VMA21 shRNA#2 exhibited the most effective silencing capacity, with VMA21 shRNA#3 also showing strong performance. To minimize the potential for off-target effects, both constructs were advanced to the subsequent phases of our investigation (Figure 2D, 2E).

Subsequent cellular experiments demonstrated that the suppression of VMA21 significantly impeded the proliferation, migration, and invasiveness of the TNBC cell lines, as shown in Figure 2F-J. Taken together, these results indicate that the regulation of VMA21 expression can serve as an effective strategy to inhibit the aggressive behavior of TNBC cells.

Knockdown of VMA21 inhibits immune escape of TNBC cells

Following on from this, the impact of VMA21 on the functionality of immune cells was explored through a cell co-culture approach. This method was utilized to evaluate how TNBC cells with suppressed VMA21 expression influence the cytotoxic potential, and cytokine secretion profile of CD8+ T cells. Initially, cytotoxicity assays revealed a pronounced elevation in the cytotoxicity of CD8+ T cells following the VMA21 knockdown, as shown in Figure 3A. Additionally, ELISA data demonstrated a notable increase in the secretion levels of key immunological cytokines, such as IFN-γ, IL-2, and TNF-α, by CD8+ T cells after the VMA21 knockdown, which is detailed in Figure 3B and 3C. Collectively, these findings imply that the modulation of VMA21 in TNBC cells can lead to a substantial augmentation in CD8+ T cell effectiveness.

Figure 3.

Knockdown of VMA21 inhibits immune escape of TNBC cells. A: LDH assay detection of the cytotoxicity of CD8+ T cells co-cultured in TNBC cells after transfection with VMA21 shRNA#2. B, C: ELISA detection of the immune-related cytokine secretion in CD8+ T cells co-cultured in TNBC cells after transfection with VMA21 shRNA#2. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs NC shRNA group. n=6.

VMA21 stabilizes TCIRG1 protein expression by inhibiting its ubiquitination degradation

Next, this study aimed to analyze the specific mechanism of VMA21 involved in TNBC immune escape. Based on the STRING database, VMA21 was found to be potentially associated with TCIRG1 (Figure 4A). Accordingly, we examined the mRNA and protein levels of TCIRG1 in MDA-MB-231 and BT-20 cells after silencing VMA21. The findings depicted in Figure 4A indicate that the reduction of VMA21 expression led to a diminished level of TCIRG1 protein. However, no substantial differences were observed in the expression levels of TCIRG1 mRNA across the various experimental groups, as illustrated in Figure 4B and 4C. Subsequently, VMA21 shRNA#2 was transfected in MDA-MB-231 and BT-20 cells, and then the cells were treated with CHX, a selective inhibitor of protein synthesis, and a proteasome inhibitor (MG132) in order to observe the degrading effect of VMA21 on TCIRG1 protein. The data revealed a marked enhancement in the degradation of the TCIRG1 protein within the group treated with VMA21 shRNA#2 when juxtaposed with the group treated with the NC shRNA. Further, after the addition of MG132, it could be observed that the ability of silencing VMA21 to induce TCIRG1 protein degradation was obviously and significantly blocked (Figure 4D). Besides, this study further examined whether VMA21 overexpression in MDA-MB-231 and BT-20 cells had any effect on the ubiquitination level of TCIRG1 protein. The results displayed that the amount of ubiquitinated protein in TCIRG1 protein precipitates was remarkably reduced after overexpression of VMA21 (Figure 4E), suggesting that VMA21 may stabilize the expression of TCIRG1 proteins by inhibiting their ubiquitinated degradation. Interestingly, we searched database for immunohistochemical staining of TCIRG1 protein in TNBC clinical tissues and showed that TCIRG1 was positive in more than 75% of TNBC patients (Figure 4F), which further confirmed our results.

Figure 4.

VMA21 stabilizes TCIRG1 protein expression by inhibiting its ubiquitination degradation. A: STRING database-based discovery of VMA21 and TCIRG1 as contingent proteins. B: Western blotting detection of TCIRG1 protein expression levels in TNBC cells transfected with VMA21 shRNA#2. C: RT-qPCR detection of TCIRG1 mRNA expression levels in TNBC cells transfected with VMA21 shRNA#2; *P<0.05, **P<0.01, ***P<0.001 vs NC shRNA group. D: Western blotting detection of TCIRG1 protein degradation after interference with VMA21 expression in TNBC cells; *P<0.05, **P<0.01, ***P<0.001. E: Ubiquitination level of TCIRG1 protein after overexpression of VMA21 in TNBC using immunoprecipitation assay. F: Database-based search for immunohistochemical staining of TCIRG1 in TNBC clinical and normal tissues. Data are shown as mean ± SEM. n=6.

VMA21 is involved in regulating TNBC cell proliferation, invasion by promoting TCIRG1 expression

Subsequently, MDA-MB-231 and BT-20 cells were co-transfected with VMA21 shRNA#2 and a plasmid expressing TCIRG1 (pcDNA-TCIRG1). The cellular functional assays revealed that the suppressive impact of VMA21 knockdown on cell proliferation, migration, and invasive capabilities in both MDA-MB-231 and BT-20 cell lines was substantially mitigated by the enforced expression of TCIRG1 (Figure 5A-D). Furthermore, co-culture experiments involving TNBC cells and CD8+ T cells demonstrated that elevated intracellular levels of TCIRG1 significantly counteracted the enhanced cytotoxicity and immune-related cytokine secretion in CD8+ T cells that were induced by VMA21 knockdown (Figure 6A-C).

Figure 5.

VMA21 is involved in regulating TNBC cell proliferation, invasion by promoting TCIRG1 expression. A: Western blotting detection of TCIRG1 protein levels in TNBC cells transfected with VMA21 shRNA#2 and pcDNA-TCIRG1. B: Clone formation detection of proliferation of TNBC cells transfected with VMA21 shRNA#2 and pcDNA-TCIRG1. C: Cell scratch experiment detection of migration of TNBC cells transfected with VMA21 shRNA#2 and pcDNA-TCIRG1. D: Transwell assay detection of invasion of TNBC cells transfected with VMA21 shRNA#2 and pcDNA-TCIRG1. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs NC shRNA group. #P<0.05, ##P<0.01, ###P<0.001 vs VMA21 shRNA#2+pcDNA-3.1 group. n=6.

Figure 6.

VMA21 is involved in regulating TNBC immune escape by promoting TCIRG1 expression. A: LDH assay detection of the cytotoxicity of CD8+ T cells co-cultured in TNBC cells after transfection with VMA21 shRNA#2 and pcDNA-TCIRG1. B, C: ELISA detection of the immune-related cytokine secretion in CD8+ T cells co-cultured in TNBC cells after transfection with VMA21 shRNA#2 and pcDNA-TCIRG1. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs NC shRNA group. #P<0.05, ##P<0.01, ###P<0.001 vs VMA21 shRNA#2+pcDNA-3.1 group. n=6.

The depletion of VMA21 effectively impedes the proliferation and the ability of ectopically implanted mouse TNBC hormone tumors to evade immune surveillance

Moving forward, the impact of VMA21 knockdown on the growth of tumor xenografts in mice was further assessed. The findings indicated that VMA21 knockdown had a pronounced inhibitory effect on tumor growth in mice bearing transplanted tumors, as demonstrated by a notable decrease in both tumor volume and weight (Figure 7A-C). Furthermore, immunohistochemical analysis was utilized to evaluate the influence of VMA21 knockdown on the infiltration of CD8+ T cells within the tumor tissues of nude mice. As shown in Figure 7D, there was a diminished expression of VMA21 in the hormonal tissues of the nude mice post-knockdown, while the presence of CD8+ T cells was markedly elevated (Figure 7D). To summarize, the knockdown of VMA21 was found to effectively suppress the growth and impede the immune evasion capabilities of ectopic mouse TNBC rhabdomyosarcoma.

Figure 7.

The depletion of VMA21 effectively impedes the proliferation and the ability of ectopically implanted mouse TNBC hormone tumors to evade immune surveillance. A: Representative images of transplanted tumors in nude mice. B: Tumor growth curves indicated by the mean tumor volume of VMA21 knockdown in xenograft tumors derived from MDA-MB-231 cells. C: Tumor mass. D: Immunohistochemical assessment of the effect of knockdown of VMA21 on VMA21 expression and CD8+ T cell infiltration in nude mouse hormonal tissues. **P<0.01, ***P<0.001 vs NC shRNA group.

Discussion

TNBC is a distinct subtype of breast cancer distinguished by the absence of three key receptor proteins: ER, PR, and HER2. This unique profile poses significant therapeutic challenges, as TNBC does not respond to hormone therapies or HER2-targeted treatments that are effective for other breast cancer types. The scarcity of effective targeted therapies for TNBC contributes to its generally unfavorable prognosis, characterized by a higher propensity for invasion and metastasis compared to other breast cancer subtypes [8]. In recent years, with the development of immunotherapy techniques, new therapeutic options have been provided for TNBC patients. Immunotherapy targets tumor cells by stimulating or bolstering the patient’s immune system, emerging as a significant research avenue within the realm of TNBC treatment [9]. In this study, we screened DEGs with abnormal expression in TNBC based on the GSE38959 database and found that the top 20 DEGs were OGN, AGR3, APOD, DCN, CPE, EGR1, PIGR, PIP, LEP, CXCL12, MUCL1, TMSB10, TOP2A, CKS2, and FN1, HORMAD1, VMA21, CXCL9, TMEM65, and S100A9. Subsequently, a search of the TCGA database revealed that VMA21 was significantly highly expressed in TNBC and correlated with TNBC staging. In addition, database-based analysis of the immune correlation between VMA21 and TNBC showed that VMA21 was significantly correlated with immune cells and immune behaviors in TNBC, suggesting that the expression of VMA21 was up-regulated in TNBC, which might be related to immune infiltration. Screening of DEGs in TNBC by the GEO database is of far-reaching significance for scientific research and clinical treatment. First, such screening can help researchers identify molecules that play key roles in the development of TNBC, thus providing clues to understanding the complex molecular mechanisms of the disease. Second, the screened DEGs may become potential biomarkers for early diagnosis of the disease, assessment of treatment efficacy, and prognosis prediction. In addition, DEGs may point out targets for new therapeutic approaches, providing the possibility of developing new drugs or immunotherapeutic strategies against TNBC [10-12].

To assess the impact of VMA21 on the malignant characteristics of TNBC cells, we developed stable MDA-MB-231 and BT-20 cell lines with targeted VMA21 suppression. Subsequent cellular analysis demonstrated that the reduction of VMA21 preeminently impeded the proliferation, migration, and invasive capabilities of both the MDA-MB-231 and BT-20 cell lines. Previous research has identified that lncRNA ZFPM2-AS1 enhances the proliferative, migratory, and invasive capabilities of lung adenocarcinoma cells through the upregulation of VMA21 expression, implicating VMA21 as a potentially significant molecular target for further investigation into the mechanisms underlying the aggressive behavior of lung adenocarcinoma [13]. The elevated expression level of VMA21 in colorectal cancer suggests that it may be associated with the development of colorectal cancer [14]. In addition, Wang et al. [15] found that knockdown of VMA21 had a similar disruptive effect on the biological processes of melanoma cells. Furthermore, the discovery of the VMA21p.93X mutation in follicular lymphoma reveals the importance of VMA21 in specific cancer types. This mutation affects V-ATPase assembly, which in turn may influence processes such as cellular autophagy, providing new insights into understanding the role of VMA21 in cancer [16]. Subsequently, we also found that knockdown of VMA21 in TNBC cells substantially increased the effectiveness of CD8+ T cells. Although the specific role of VMA21 in cancer immune escape was not explicitly discussed in the information I searched for, based on the available information, we can speculate that VMA21 may be associated with the above mechanisms, especially in regulating the tumor microenvironment, influencing the function of MHC I molecules, or participating in immune cell interactions.

For this research, utilizing the STRING database, we identified a potential link between VMA21 and TCIRG1. TCIRG1 is a protein known for its critical involvement in cellular metabolism and immune regulation, and it has been a focal point in cancer research within recent years [17-19]. TCIRG1’s abnormal expression is intimately connected to the genesis and advancement of diverse cancers. It exerts a pivotal influence on the regulation of metabolic pathways within malignant cells and significantly shapes the tumor’s immune microenvironment. Studies have shown that TCIRG1 may promote the growth and survival of tumor cells by affecting the aerobic glycolysis process, and at the same time, it may be involved in the mechanism by which tumor cells evade immune surveillance, thus playing a role in the immune escape of cancer [17]. In addition, the variation of TCIRG1 expression levels in different cancer samples suggests that it may serve as a potential biomarker for cancer diagnosis and prognostic assessment [20]. In some cases, high TCIRG1 expression is associated with tumor aggressiveness and poor prognosis, which provides new molecular targets for clinical treatment of cancer. With the in-depth study of TCIRG1 function, future therapeutic strategies may target the regulatory mechanism of TCIRG1 and develop new anticancer drugs to enhance therapeutic efficacy and improve the quality of patient survival. The present study further suggests that VMA21 may stabilize the expression of TCIRG1 protein by inhibiting its ubiquitinated degradation. The ubiquitin proteasome system is a tightly controlled pathway for the degradation of proteins, playing a vital role in maintaining cellular equilibrium [21]. It is central to numerous cellular functions, including the repair of DNA damage, the control of cell cycle checkpoints, and the regulation of immune responses [22]. In recent times, the system has become a focal point for cancer therapeutics due to its contribution to the initiation and progression of tumors [23,24]. The dysregulation of ubiquitin-mediated proteolysis of essential regulatory proteins, such as those that inhibit or promote tumor growth, can lead to unchecked proliferation of cells, avoidance of apoptosis, and the metastasis of cancer [25-27]. The above is supported by subsequent studies that the inhibitory effects of VMA21 knockdown on cell proliferation, migration and invasive ability were greatly attenuated by the forced expression of TCIRG1 in MDA-MB-231 and BT-20 cell lines. Besides, elevated intracellular TCIRG1 levels significantly counteracted the VMA21 knockdown-induced enhancement of CD8+ T cell proliferation, cytotoxicity, and secretion of immune-related cytokines.

In summary, the present study demonstrates for the first time that VMA21 is able to regulate TCIRG1 protein stability by binding to TCIRG1 protein in the form of ubiquitination, which ultimately promotes the malignant behavior as well as immune escape of TNBC cells. Although our findings provide an initial direction for the treatment of TNBC, there are some limitations of this study. First, the size of this study is relatively small, and the research team will subsequently collect more clinical samples to confirm our findings. In addition, only two TNBC cell lines were involved in this study, which is insufficient to confirm that VMA21 is of equal significance to TCIRG1 in all types of TNBC.

Disclosure of conflict of interest

None.