Abstract
Objective: Tumor-secreted exosomes are essential in driving tumor progression by releasing multivesicular bodies (MVBs), which are endosome organelles containing intraluminal vesicles (ILVs), as exosomes. However, the mechanisms controlling MVB biosynthesis, trafficking, and exosome exocytosis are not fully understood.
Methods: We examined the expression of vesicle-associated membrane protein 2 (VAMP2) and synaptosome-associated protein 25 (SNAP25) in head and neck cancer (HNC) tumor tissues using clinical analysis. Techniques, like nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and exosome quantification, were used to study the roles of VAMP2 and SNAP25 in exosome secretion. Experiments involving overexpression or depletion of VAMP2 and SNAP25 were performed to observe effects on exosome release, MVB trafficking, and cellular behavior.
Results: Clinical data showed that VAMP2 and SNAP25 were significantly upregulated in HNC tissues and linked to tumor progression. Increased expression of these proteins boosted exosome secretion and enhanced HNC cell tumorigenicity. Conversely, depleting VAMP2 or SNAP25 impaired MVB release, causing intracellular buildup of MVBs and ILVs, and reduced exosome secretion, which was reversible by exosome degradation methods. Blocking exosome secretion by inhibiting MVB-plasma membrane fusion weakened tumorigenic capacities but triggered a compensatory Ras-related protein Rab-11A-myosin9-phosphatidylserine (PS) externalization pathway, releasing MVB-like extracellular vesicle (EV) clusters.
Conclusions: VAMP2 and SNAP25 are key to exosome secretion and HNC tumor progression. Suppressing this pathway activated a compensatory Rab11a-myosin9-PS mechanism that sustained EV release. A dual-targeting strategy that blocks both VAMP2/SNAP25-mediated exosome secretion and the Rab11a-myosin9-PS pathway may improve tumor cell eradication, offering a potential treatment for HNC and other cancers.
keywords
Introduction
Head and neck cancer (HNC) is one of the most common types of solid tumors with approximately 890,000 new cases worldwide each year1. Most patients are distributed in Europe, the Americas, and Australia (World Health Organization, 2020). HNC consists of a group of cancers, including neoplasms of the thyroid, larynx, nasopharynx, and oral and maxillofacial regions2. Although extensive advances have been made in HNC treatment, including surgery, chemo-radiotherapy, targeted therapy, and immunotherapy, the HNC 5-year survival rate increased slightly from 52.7% to 63.5% in recent decades3. Nevertheless, 5.55% of HNC patients died from HNC worldwide in 2020 (https://www.cancer.net/cancer-types/head-and-neck-cancer/statistics), leading to a significant clinical challenge4. Hence, it is urgent to explore novel therapeutic approaches in the clinical management of patients with HNC.
VAMP2/SNAP25 promotes the aggressive progression of head and neck cancer by affecting the fate of multivesicular bodies. Part 1: In the preliminary study, the association between VAMP2/SNAP25 and exosome secretion was revealed through online data analysis and experimental verification. Part 2: VAMP2 and SNAP25 primarily drive the malignant progression of HNC cells by promoting exosome secretion at the cellular and animal levels. Mechanistically, VAMP2 binds to these multivesicular bodies (MVBs) and interacts with SNAP25 at the membrane after the exocyst complex transports MVBs to the plasma membrane (PM). This interaction forms a “zipper-like” structure that mediates the fusion between MVB membranes and PM, ultimately facilitating exosome secretion. Part 3: If the secretion of exosomes is inhibited by inhibiting VAMP2/SNAP25 expression, MVBs will be released into the extracellular space in the form of MVB-like EV clusters, alleviating the damage caused by MVB accumulation. EV, extracellular vesicle; HNC, head and neck cancer; MVBs, multivesicular bodies; PM, plasma membrene; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2.
Exosomes, with a particle size distribution between 30 and 150 nm, are vesicles outside the cell and are produced by all types of cells5,6. The role of exosomes in cancer research has gained popularity recently due to the important role of exosomes in intercellular communication using transmitting proteins, microRNAs (miRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), DNAs, and mRNAs7,8. Recent reports have indicated that tumor cells actively produce and secrete excessive exosomes when compared to normal cells9,10. These tumor-derived exosomes (TDEs) act as critical mediators of cell-to-cell communication and promote various tumorigenic properties, such as tumor invasion, proliferation, angiogenesis, chemoresistance, and radiation resistance10,11. Yet the machinery of abnormally enhanced biogenesis and secretion of TDEs in tumor cells remains elusive.
Exosomes are initially formed by the endosomal system and the biogenesis of exosomes is a complex and elaborate mechanism12,13. Endosomes are produced by cell membrane internalization. Subsequently, the invagination of the endosomal membrane forms several small vesicles [multivesicular bodies (MVBs) and intraluminal vesicles (ILVs)]. ILVs are secreted as exosomes into the extracellular space after MVBs fuse with the plasma membrane (PM)14,15. Our group previously reported that the exocyst has a vital role in docking MVBs and assembling an exocyst complex with Exo70 and Sec3 in the HNC PM16. Yet, the mechanism of how the exocyst mediates MVB membrane fusion with the PM is unknown.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) is a multi-protein complex modulating the fusion of two biomass membranes17,18. Recent reports have demonstrated that SNARE could have an important role in exosome secretion19–21. The synaptosomal-associated protein of 25 kDa (SNAP25), a classic member of the SNARE family, is located on the PM. SNAP25 can combine with vesicle-associated membrane protein 2 (VAMP2), another classic SNARE component, forming a complex22. Additional research on these two proteins has focused on neurotransmitter release23,24 but the role of SNAP25 and VAMP2 in exosome is unknown, especially in HNC cells. In this study the functional roles and mechanistic relationships between SNAP25 and VAMP2 in the fusion of the MVB membrane and PM were investigated. We were surprised to find that SNAP25 and VAMP2 knockdown and inhibition of exosome secretion resulted in the release of MVBs/ILVs into the extracellular compartment with other displays, thus slowing down the damage caused by the accumulation of MVBs/ILVs in the intracellular compartment. This finding may help to devise potential therapeutic approaches for anti-tumor growth and progression of HNC.
Methods and materials
Clinical samples
This research was supported by approval from the Clinical Research Ethics Committee of Anhui Medical University (Approval no. 20190327). Each sample was collected from patients who offered documented informed consent. The criteria for specimen collection were as follows: a primary diagnosis of laryngeal cancer requiring surgical resection; recurrence-free; metastasis-free; and on no medication. Twenty patient specimens were randomly selected, including cancer and para-cancerous tissues from male patients with a mean age of 63.75 years and an average weight of 58 kg.
Cell culture
NP69 (a cell line from normal human nasopharyngeal epithelial cells), Detroit 562 (human hypopharyngeal squamous cell carcinoma lines), and CNE2 (a nasopharyngeal carcinoma cell line) were cultured in Roswell Park Memorial Institute (RPMI)-1640 cell culture medium (VivaCell, Shanghai, China) supplemented with 10% fetal bovine serum [FBS] (VivaCell, Shanghai, China) and 1% antibiotics (100 kU/L penicillin and 100 mg/L streptomycin; VivaCell, Shanghai, China)16. HN4 and FaDu are HNC cell lines derived from a patient with squamous cell carcinoma of the head and neck, which were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g/L of glucose (VivaCell, Shanghai, China) supplemented with 10% FBS and 1% antibiotics (100 kU/L penicillin and 100 mg/L streptomycin)25. NP69, CNE2 and HN4 cell lines were purchased from Procell Company (Wuhan, Hubei, China). Detroit 562 and FaDu cell lines were purchased from Fuheng Biology Company (Shanghai, China). The cells were cultured at 37°C in a humidity-controlled incubator with 5% CO2. We used an exosome-free medium (SBI, Palo Alto, CA, USA) to culture cells for exosome isolation. All cell lines were shown to be mycoplasma-free using a Myco-Lumi™ luminescent mycoplasma detection kit (Beyotime, Shanghai, China).
Cell transfection
HN4 or CNE2 cells were transiently transfected with human-specific SNAP25 or VAMP2 siRNA (sequences listed in Table S1). These siRNAs were designed and acquired from Biomics Company (Nantong, Jiangsu, China). Sec10, Sec3, and Exo70 siRNAs were cited in our previous report16. Lipofectamine 3000 reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were used to transfect siRNA into cells according to the manufacturer’s instructions. Transfected cells were cultured for at least 48 h before subsequent experiments.
Western blotting
The whole-cell lysates were extracted using RIPA lysis buffer [150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8.0), and 1× protease inhibitor cocktail (Biosharp, Hefei, Anhui, China)]. A bicinchoninic acid (BCA) kit (Beyotime) was used to measure protein concentration. The samples (loading normalized to 20 μg protein) were fractionated using 15% SDS-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride (PVDF) membrane. The following primary antibodies were used: anti-CD63 (1:250; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-SNAP25 (1:250; Zenbio, Chengdu, Sichuan, China); anti-VAMP2 (1:250; Zenbio); anti-Exo70 (1:200; Affinity Biosciences, Changzhou (Liyang), Jiangsu, China); anti-Sec3 (1:200; Affinity Biosciences); and anti-Sec10 (1:200; Affinity Biosciences). The immunoreactive bands were detected using enhanced chemiluminescence (Tanon, Shanghai, China) and a chemiluminescence imaging system (iBright CL1000; Invitrogen, Thermo Fisher Scientific) was used for image acquisition.
Co-immunoprecipitation
Co-immunoprecipitation (Co-IP) assays were performed as previously described16. Briefly, HN4 cells were lysed on ice using protein lysis buffer, sonicated, and centrifuged at 10,000 × g at 4°C for 20 min. CD63, SNAP25, VAMP2, Sec3, Sec10, and Exo70 proteins were immunoprecipitated by incubating 800 μg of isolated total proteins with 5 μg of anti-CD63, anti-SNAP25, anti-VAMP2, anti-Sec3, anti-Sec10, and anti-Exo70 antibody/pre-immune IgG, respectively, overnight on a shaking platform at 4°C. Protein A/G Magnetic Beads (MedChemExpress, Monmouth Junction, New Jersey, USA) were added, followed by incubation at 4°C for 3 h. The immunoprecipitates were thrice-rinsed with lysis buffer before adding the loading buffer. The samples were denatured at 100°C for 10 min and evaluated using gel electrophoresis, blotting, or transfer to a membrane, and selective immunodetection of an immobilized antigen.
Immunofluorescence
HN4 cells were seeded onto a cover slip (Servicebio, Wuhan, China) for 24 h and subsequently fixed in 4% paraformaldehyde (Servicebio) for 20 min at room temperature (20–25°C). Then, the cells were infiltrated with 0.2% Triton X-100 and 3% bovine serum albumin dissolved in phosphate-buffered solution (PBS) for 30 min at room temperature to block non-specific binding. Next, the cells were thrice-washed with PBS and incubated overnight with anti-CD63 primary antibody at 4°C in a moist chamber. After three washes with PBS, the cells were incubated at room temperature for 2 h with a fluorescent secondary antibody (donkey anti-rabbit IgG Alexa Fluor 488, 1:500; Invitrogen, Thermo Fisher Scientific). Fluorescence images were acquired using a confocal laser scanning microscope-LSM980 (Zeiss, Oberkochen, Germany).
Immunohistochemistry
Previously described procedures were used for immunochemistry (IHC)21. Briefly, the tumor tissue samples were soaked in 4% paraformaldehyde for 2–3 d, then dehydrated in ethanol, inserted in paraffin, and cut into 5-μm pieces. The slides were subsequently paraffinized and re-hydrated. Next, the sections were boiled in citrate buffer using a microwave oven for antigen recovery. An IHC kit (ZsBio, Beijing, China) was used for subsequent analysis, following the manufacturer’s instructions. The sections were incubated with primary antibodies (anti-SNAP25 or anti-VAMP2) at 4°C overnight. Hematoxylin was used to label the cell nuclei. Histologic images were obtained using an Olympus BX51 microscope mounted with a digital camera (Tokyo, Japan). Particles that stained brown in the cells were considered antibody-positive.
Annexin V-FITC/PI-stained fluorescence-activated cell sorter
Cells were harvested by trypsinization, washed with cold PBS three times, and centrifuged at 2000 × g for 5 min at 4°C. The cells were then resuspended in 195 μL of Annexin V-fluorescein isothiocyanate (FITC) binding buffer plus 10 μL propidium iodide (PI) and 5 μL Annexin V-FITC (Beyotime, Shanghai, China), and incubated at room temperature for 20 min in the dark. Finally, 300 μL of PBS was added before loading in the fluorescence-activated cell sorter [FACS] (FACSCanto II; Becton, Dickinson, and Company, Franklin Lakes, New Jersey, USA).
Annexin V-FITC/DAPI-stained fluorescence microscopy
HN4 cells were seeded on 18-mm coverslips for 24 h after treatment. Then, after discarding the cell medium and washing with PBS three times at room temperature, Annexin V-FITC conjugate and 5 μL of FITC-labeled Annexin [195 μL] (Beyotime, Shanghai, China) were added and mixed lightly, followed by nuclear staining using PI (Beyotime) for 20 min at room temperature. The stained cells were analyzed under a fluorescence microscope (Zeiss).
Cell cycle analysis
Cells were seeded in 6-well plates (6 × 105 cells/well). After treatment, the cells were fixed at 75% ethanol at 4°C overnight. After washing in PBS three times, the fixed cells were collected and stained with PI and RNase (Beyotime) using a Cell Cycle and Apoptosis Analysis kit (Beyotime) according to the manufacturer’s instructions and subjected to flow cytometry. The results were analyzed using FlowJo 7.6 (Becton, Dickinson, and Company).
Cell counting kit-8 assay
Cells were seeded onto 96-well culture plates (6 × 103 cells/well). After 24 h, 10 μL of cell counting kit-8 (CCK-8) in 100 μL of cell culture medium was added to each well and the cells were cultured for 4 h at 37°C in 5% CO2. The absorption was measured at 450 nm using FlexStation 3.0 (Molecular Devices, San Jose, CA, USA) and the quantity of formazan dye produced (absorbance of 450 nm) was positively correlated with the number of viable cells.
Exosome isolation and cell count
The cells were cultured in an exosome-free medium (SBI). The supernatant was collected after 48 h by centrifugation at 2000 × g for 30 min at 4°C. Total Exosome Isolation Reagent (Invitrogen, Thermo Fisher Scientific) was used to isolate the exosomes according to the manufacturer’s instructions. A 0.22-μm polyethersulfone (PES) membrane filter (NEST, Wuxi, Jiangsu, China) was used before detecting exosome diameter and concentration. Then, the cells were digested and resuspended in PBS and detected with an automated cell counter (Invitrogen, Thermo Fisher Scientific). Exosome quantity was determined by the total protein concentration, which was detected using a bicinchoninic acid (BCA) protein concentration detection kit (Beyotime). The exosomes were supplemented to the medium at 20 μg/mL concentration and incubated for 48 h16.
Size exclusion chromatography
Different sizes of extracellular vehicles (EVs) were separated using methods described in our previous study26. Briefly, 1 mL of the cell supernatant (filtered through a 0.22 μm filter) was loaded onto the prepared column and eluted with PBS. Fractions were collected with each fraction containing 100 μL. The final EV samples were harvested and stored at −80°C for further analysis.
Transmission electron microscopy
HN4 cells were washed in PBS and fixed with glutaraldehyde (2.5%) at 4°C overnight. The samples were then washed in PBS, dehydrated, and sectioned using a Leica ultramicrotome (50–70 nm; Leica, city, Germany). The sections were stained with uranyl acetate (2%) for 10 min, followed by immersion in a lead staining solution for 5 min before examination under a transmission electron microscope [TEM] (FEI, Hillsboro, OR, USA) at a voltage of 120 kV. TEM images were captured using a CCD digital camera (FEI) and analyzed using soft imaging (Olympus).
Nanoparticle tracking analysis
The number and size distribution of exosomes were determined using NanoSight NS300 (Malvern, city, UK). Cell supernatants were collected and separated into 1.5-mL centrifuge tubes. Then, the samples were resuspended in PBS without particles (range, 1–10 × 108 particles/mL). Next, a 0.22-μm pore size filter (Millipore, Darmstadt, Germany) was used to filter the above diluent. Finally, light scattering and Brownian motion properties were used to obtain the particle size distribution and the number of particles in liquid suspensions. These data were analyzed using nanoparticle tracking analysis (NTA) 2.3 build 17 software (Malvern, England).
Protein-protein interaction network analysis
The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes [STRING] (http://string-db.org) and the GeneMANIA (http://genemania.org) online database. For this study, PPI networks for SNAP25, VAMP2, Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8) were built by STRING and the GeneMANIA database27,28.
Xenograft mouse model
All animal experiments were approved by the Ethics Committee of Anhui Medical University (Approval no. 20190340). BALB/c nude mice (7 weeks old, Guangdong Medical Lab Animal Center, Foshan, Guangdong, China) were used to study tumor growth after VAMP2 or SNAP24 knockdown expression. In brief, 1 × 106 HN4 cells were suspended in 50 μL of DMEM medium and mixed with 50 μL of matrix gel (MedChemExpress) on ice. Then, a 100-μL mixture was injected subcutaneously into mice. HN4 xenograft mice were randomly divided into three groups. After two weeks, siVAMP2 or siSNAP25 were wrapped with Lipofectamine 3000 and injected intratumorally into tumor tissues. The control group used Lipofectamine 3000 wrapped scramble. siRNA treatment was performed for 2 weeks with injections every 3 d. The size of tumors was observed after siRNA treatment.
Statistical analysis
Data are presented as the mean values ± standard error of the mean (SEM) based on three independent experiments. Parametric data were analyzed using Student’s t-test, while group comparisons were performed using one- or two-way ANOVA, as appropriate. A P-value < 0.05 indicated statistical significance.
Results
VAMP2 and SNAP25 were highly expressed and promoted the malignant progression of HNC
Before releasing exosomes, it was first necessary to examine the docking of MVBs to the PM, then MVBs were fused to the PM. When MVBs dock with the PM, the v-SNARE proteins on the surface of the MVBs and the t-SNARE proteins on the PM pair up to form a functional SNARE complex. This interaction induces the fusion of the MVBs with the PM, resulting in the release of exosomes29. We previously reported that the exocyst mediates the transport of MVBs to facilitate juxtaposition with the PM in HNC cells16. Therefore, to investigate the process of MVB fusion with the PM in HNC cells, PPI analysis was performed using the STRING and GeneMANIA databases. Our analyses revealed that VAMP2, a v-SNARE protein, and SNAP25, a t-SNARE protein, interact with exocyst complex subunits, including Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8; Figure S1A and S1B). Co-IP analysis verified that the principal exocyst components (Sec10, Exo70, and Sec3) interact with SNAP25 and VAMP2 in HNC cells (Figure S1C–S1G). Interestingly, our findings demonstrated that VAMP2 and SNAP25 exhibit higher expression in human HNC cell lines (HN4, FaDu and Detroit 562) compared to human nasopharyngeal epithelial NP69 cells (Figure 1A–1C). Consistently, IHC analysis showed that VAMP2 and SNAP25 expression was significantly higher in laryngeal carcinoma (LC) tissues than in adjacent non-tumor tissues (Figure 1D–1G). Of note, VAMP2 and SNAP25 expression was progressively upregulated with increases ranging from 1.70- to 3.01-fold for VAMP2 and from 2.03- to 2.79-fold for SNAP25 across stages 1–4 of LC, respectively (Table 1). To estimate the role of VAMP2 and SNAP25 in tumor progression of HNC cells, we examined the proliferation and apoptosis of HN4 and CNE2 cells after knockdown of VAMP2 or SNAP25. The results indicated that cell proliferation in HN4 cells was significantly inhibited by 1.81- and 1.21-fold, respectively, while cell proliferation in FaDu cells was significantly inhibited by 1.51- and 1.71-fold following knockdown of VAMP2 or SNAP25, respectively. Conversely, experiments involving the overexpression of VAMP2 and SNAP25 reached the same conclusion (Figure 1H–1M). Moreover, depletion of VAMP2 or SNAP25 significantly promoted apoptosis across multiple cell lines. Specifically, the apoptotic rates increased by 1.53- and 1.86-fold in HN4 cells, 2.69- and 1.97-fold in FaDu cells, and 1.94- and 2.24-fold in CNE2 cells, respectively (Figures 1N–1U and S1H, S1I). Furthermore, knockdown of VAMP2 or SNAP25 in HN4 cells significantly inhibited the capacity for tumor growth in the xenograft HNC mouse model, as previously described (Figure 1V–1X). These results confirmed that high expression of VAMP2 and SNAP25 proteins is associated with enhanced tumorigenicity of HNC cells.
Demographic and clinical characteristics of LC patients
Elevated VAMP2 and SNAP25 promote the malignant progression of head and neck cancer. (A) Representative Western blotting images of VAMP2 and SNAP25 in NP69, HN4, FaDu, and Detroit 562 cells. (B) Statistical results of VAMP2 expression in NP69, HN4, FaDu, and Detroit 562 cells. (C) Statistical results of SNAP25 expression in NP69, HN4, FaDu, and Detroit 562 cells. (D) Immunohistochemistry typical pictures of VAMP2 staining in laryngocarcinoma (LC) tissue and adjacent normal tissues. (E) Data summary of immunohistochemistry VAMP2 staining. (F) Immunohistochemistry typical pictures of SNAP25 staining in laryngocarcinoma (LC) tissue and adjacent normal tissues. (G) Data summary of immunohistochemistry SNAP25 staining. Grade 1: Well differentiated (low grade), Grade 2: Moderately differentiated (intermediate grade), Grade 3: Poorly differentiated (high grade), Grade 4: Undifferentiated (high grade). Scale bar, 50 μm. (H, I) Data from CCK-8 are summarized after knockdown of VAMP2 (H) or SNAP25 (I) against siCtrl in HN4 cells. (J, K) Data from CCK-8 are summarized after knockdown of VAMP2 (J) or SNAP25 (K) against those of siCtrl in FaDu cells. (L, M) Data from CCK-8 are summarized after overexpressing VAMP2 or SNAP25 against Ctrl in HN4 (L) and FaDu (M) cells. (N, O) Representative apoptosis images (N) and statistical results (O) of HN4 cells after knockdown of VAMP2 and SNAP25 expression. (P, Q) Representative apoptosis images (P) and statistical results (Q) of FaDu cells after knockdown of VAMP2 and SNAP25 expression. (R, S) Representative apoptosis images (R) and statistical results (S) of HN4 cells after overexpressing VAMP2 and SNAP25. (T, U) Representative apoptosis images (T) and statistical results (U) of FaDu cells after knockdown of VAMP2 and SNAP25 expression. (V) Representative pictures of the tumor tissues after knockdown of VAMP2 or SNAP25 expression. (W) Statistical results of tumor volume. (X) Statistical results of tumor weight against body weight. *P < 0.05, ***P < 0.001 compared to control using Student’s t-test. CCK-8, Cell Counting Kit-8; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2.
VAMP2 and SNAP25 regulation of exosome secretion affects HNC cell proliferation and apoptosis
Mounting evidence has suggested that TDEs accelerate tumor progression by promoting angiogenesis, proliferation, and metastasis27,28,30. Interestingly, VAMP2 and SNAP25 are essential factors required for mediating membrane fusion. Consequently, to elucidate the role of VAMP2 and SNAP25 in exosome secretion, an siRNA-mediated gene knockdown approach was used to silence VAMP2 or SNAP25 (Figure S2A–S2H). NTA revealed that depletion of either VAMP2 or SNAP25 led to a significant reduction in exosome secretion in HN4, FaDu, and CNE2 cells (Figures 2A–2D and S2I, S2J). These findings indicated that the overexpression of VAMP2 and SNAP25 has a critical role in facilitating exosome secretion in HNC cells, suggesting that VAMP2 and SNAP25 are essential components of the exosome secretion pathway mediated by the exocyst complex. To evaluate the potential impact of VAMP2 and SNAP25 on HNC tumor progression through enhanced exosome secretion, the proliferation and apoptosis rates of HN4 cells following exposure to varying levels of exosome secretions, which are modulated by the knockdown of either SNAP25 or VAMP2, were determined. If VAMP2 and SNAP25 facilitate tumor progression through the upregulation of exosome secretion, then knockdown would lead to a reduced count of exosomes in the cell culture supernatant. Consequently, when a separate group of HNC cells was cultured with this modified supernatant, a significant attenuation in cell proliferation was observed compared to cells cultured with supernatant from the non-knockdown control group, as previously reported16. To this end, supernatants from VAMP2- or SNAP25-knockdown cells were collected, then treated in another HN4 group. The results showed that an equal volume of supernatant with VAMP2-knockdown inhibited HN4 cell proliferation by nearly one-half compared to control, while treatment of HN4 cells with supernatants obtained from SNAP25-knockdown suppressed HN4 cell proliferation 1.4-fold (Figure 2E–2J). To clarify that VAMP2 and SNAP25 can alter the malignant biological behaviors of tumors by modulating the quantity of exosome secretion, VAMP2- and SNAP25-overexpressing HNC cell lines were treated with the exosome secretion inhibitor, GW4869. The results demonstrated that inhibiting exosome secretion can reverse the promoting effects of VAMP2 and SNAP25 on tumor malignant progression (Figure S3A–S3D). To ascertain whether VAMP2 and SNAP25 facilitate tumor progression through the regulation of exosome quantity rather than the content, equal amounts of exosomes were collected from HN4 cells, which were treated with siVAMP2 or SNAP25, and the progression of the cells after co-incubation with another group of HN4 cells was observed. The results indicated that the proliferation of HN4 cells was unaffected when treated with equal quantities of exosomes obtained from cells subjected to VAMP2 or SNAP25 knockdown (Figure 2K–2P). In addition, proteomic analysis was performed on exosomes from the supernatants of HN4 and FaDu cells with varying VAMP2 and SNAP25 expression, revealing no significant differences in the types of proteins within the exosomes (Figure S3E and S3F). This finding suggested that depletion of VAMP2 or SNAP25 decreased exosome secretion without altering the internal composition of the exosomes. Finally, to evaluate the cell proliferation rate, the parental HNC cells were co-cultured with exosome-depleted supernatant from HN4 cells, which were subjected to siRNA-mediated knockdown of SNAP25 or VAMP2. The results obtained from the CCK-8 assay and cell cycle analysis indicated that the proliferation of HN4 cells did not exhibit notable differences across the three experimental groups. The findings indicated that components of the supernatant, excluding exosomes, do not influence cell proliferation (Figure 2Q–2V). We also found that reduced expression of VAMP2 or SNAP25 encouraged apoptosis due to a decrease in exosome secretion (Figures 2W–2Y and S3G–S3N). These results implied that depletion of VAMP2 or SNAP25 reduced the secretion of exosomes, which in turn downregulated the influence of TDEs in enhancing HNC cell survival.
VAMP2 or SNAP25 contributes to HNC cell proliferation and apoptosis by regulating exosome secretion. (A) Typical nanoparticle tracking analysis (NTA) traces and exosome quantification after VAMP2 knockdown by transfecting HN4 cells. (B) Typical NTA traces and exosome quantification after VAMP2 knockdown by transfecting FaDu cells. (C) Typical NTA traces and exosome quantification after SNAP25 knockdown by transfecting HN4 cells. (D) Typical NTA traces and exosome quantification after SNAP25 knockdown by transfecting FaDu cells. (E) CCK-8 quantification assay after treatment with an equal volume of siCtrl and siVAMP2 cell supernatant. (F, G) Representative pictures (F) of cell cycle and statistical results (G) after treatment with an equal volume of siCtrl and siVAMP2 cell supernatant. (H) CCK-8 quantification assay after treated with an equal volume of siCtrl and siSNAP25 cell supernatant. (I, J) Representative pictures (I) of cell cycle and statistical results (J) after treatment with an equal volume of siCtrl and siSNAP25 cell supernatant. (K) CCK-8 quantification assay after treatment with an equal quantity of exosomes derived from siCtrl and siVAMP2 cells. (L, M) Representative pictures (L) of cell cycle and statistical results (M) after treatment with an equal quantity of exosomes derived from siCtrl and siVAMP2 cells. (N) CCK-8 quantification assay after treatment with an equal quantity of exosomes derived from siCtrl and siVAMP2 cells. (O, P) Representative pictures (O) of cell cycle and statistical results (P) after treatment with an equal quantity of exosomes derived from siCtrl and siVAMP2 cells. (Q) CCK-8 quantification assay after treatment with an equal volume of siCtrl and siVAMP2 cell supernatant in which exosomes were removed. (R, S) Representative pictures (R) of cell cycle and statistical results (S) after treatment with an equal volume of siCtrl and siVAMP2 cell supernatant in which exosomes were removed. (T) CCK-8 quantification assay after treatment with an equal volume of siCtrl and siSNAP25 cell supernatant in which exosomes were removed. (U, V) Representative pictures (U) of cell cycle and statistical results (V) after treatment with an equal volume of siCtrl and siSNAP25 cell supernatant in which exosomes were removed. (W) Representative apoptosis images and statistical results of HN4 cells after treatment with an equal volume of siCtrl, siVAMP2, and siSNAP25 cell supernatant. (X) Representative apoptosis images and statistical results of HN4 cells after treatment with an equal quantity of exosomes from siCtrl, siVAMP2, and siSNAP25 cells. (Y) Representative apoptosis images and statistical results of HN4 cells after treatment with an equal volume of exosomal separation supernatant from siCtrl, siVAMP2, and siSNAP25 cells. CCK-8, Cell Counting Kit-8; NTA, Nanoparticle Tracking Analysis; OD, optical density; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.
VAMP2-SNAP25 binding mediates exocyst-dependent MVB-plasma membrane fusion
To determine whether VAMP2 and SNAP25-mediated the fusion of MVB with PM and thereby promote exosome secretion in HNC cells, the MVB markers, CD9, CD63, and CD81, were selected to detect whether MVBs can bind to VAMP2 and SNAP25 using Co-IP assays. The results confirmed a physical interaction among MVBs, VAMP2, and SNAP25 in the normal culture environment (Figure S4A). With downregulations of the exocyst subunits (mainly Sec10, Sec3, and Exo70), there was a noticeable increase in the separation between MVBs and PM, surpassing the gap observed after siRNA-induced reduction of VAMP2 or SNAP25 (Figure 3A–3C). The protein interactions can further determine the order of MVB transport in the cell. Therefore, to determine the temporal order of exocyst, VAMP2, and SNAP25 involvement in MVB transport in HN4 cells, we assessed the interactions between CD63-positive MVBs and these trafficking factors. The Co-IP and immunofluorescence data revealed a substantial reduction in the interaction and co-localization between CD63 and VAMP2 or SNAP25 following knockdown of exocyst subunits (Figures 3D–3L and S4B, S4C). Upon downregulation of VAMP2 or SNAP25, the interaction between CD63 and the primary exocyst subunits (Exo70, Sec10, and Sec3) remained largely unchanged. Notably, VAMP2 reduction appeared to negatively influence the association between CD63 and SNAP25. Conversely, SNAP25 silencing did not significantly impact the interaction between CD63 and VAMP2 (Figure 3M–3V). The findings indicated that the exocyst complex facilitates the transport of MVBs to the PM. At the PM, the VAMP2 protein associated with the MVBs, subsequently engaging in the interaction with the SNAP25 protein present on the PM. Finally, the complex comprised of VAMP2 and SNAP25 orchestrated the merging of the MVB membrane with the PM. This process governs the release of ILVs from MVBs as exosomes into the extracellular environment in HNC cells (Figure 3W).
Exocyst transports MVBs to the plasma membrane, then delivers MVBs to VAMP2, which binds to SNAP25 and promotes secretion of exosomes in HNC cells. (A) Representative fluorescent images of the relative distance between CD63 and PM after knocking down the expression of Exo70, Sec3, Sec10, VAMP2, or SNAP25. (B, C) Representative images (B) and statistical results (C) of the relative distances between MVBs and PM after knockdown of Exo70, Sec3, Sec10, VAMP2, or SNAP25 expression. (D–L) The interactions between CD63 and SNAP25 or VAMP2 after the knockdown of exocyst subunits (Exo70, Sec3, or Sec10). (M–V) The interactions between CD63 and exocyst subunits or SNAP25 or VAMP2 after SNAP25 or VAMP2 inhibition (low expression). (W) Modeling of VAMP2- and SNAP25-mediated exosome secretion in HNC cells. Exo70, Exocyst complex component 7; Lys, whole-cell lysates; MVB, multivesicular body; PM, plasma membrene; Pre-immu, pre-immune serum; Sec10, Exocyst complex component 5; Sec3, Exocyst complex component 1; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2. *P < 0.05, **P < 0.01 compared to siCtrl using Student’s t-test.
VAMP2/SNAP25 knockdown induces the accumulation of MVBs/ILVs leading to damage in HNC cells
The primary roles of VAMP2 and SNAP25 are to facilitate membrane fusion. VAMP2 contributes a helical structure from the vesicle membrane that interacts with the three helices presented by SNAP25 on the PM, creating a “zipper” configuration that initiates the membrane fusion process31. To determine whether VAMP2 and SNAP25 influence exosome release by mediating the fusion of MVB membranes with the PM, the expression of CD63, a marker for late endosomes and MVBs, was investigated using immunofluorescence staining. Downregulation of VAMP2 or SNAP25 led to a rise in CD63-positive MVBs, which were distinguished by larger sizes and more intense fluorescence (Figure 4A and 4B). This finding suggested that there is an intracellular buildup of exosomes that are unable to be secreted, resulting in a decrease in exosome release subsequent to the knockdown of VAMP2 or SNAP25. Furthermore, western blot analysis showed that CD63 expression was significantly elevated in HN4 cells after knockdown of VAMP2 or SNAP25 compared to the scrambled control (Figure 4C–4E). Electron microscopy showed a significant increase in the number of MVBs per 100 μm2 and ILVs per MVB. In contrast to the marked decrease in extracellular exosomes, a notable increase in ILVs and MVBs was detected in HN4 cells subjected to VAMP2 or SNAP25 knockdown compared to control cells (Figure 4F–4H). These findings indicated that depleting VAMP2 or SNAP25 diminished exosome secretion by disrupting the fusion of MVBs with the PM, resulting in the intracellular buildup of tumor-secreted exosomes.
Knockdown of VAMP2 or SNAP25 leads to MVBs/ILVs accumulation and enhancing proliferation and reducing apoptosis by clearing these MVBs/ILVs. (A–E) The expression of CD63 after the HN4 cells were transfected with siCtrl or SNAP25 or VAMP2 siRNA. (A) CD63 immunofluorescence in HN4 cells shows accumulation after transfection with SNAP25 or VAMP2 siRNA compared to siCtrl. Scale bar, 50 μm. (B) Quantitative analysis of CD63 fluorescence intensity. (C) Images of typical Western blotting for CD63 expression. (D, E) CD63 quantification after knockdown of SNAP25 (D) or VAMP2 (E). β-tubulin was used for normalization. (F–H) Number of MVBs and ILVs after transfection with siCtrl, SNAP25 siRNA, or VAMP2 siRNA. (F) Typical electron microscopy images. (G) The number of MVBs per 100 μm2. (H) The number of ILVs per MVB. (I, J) Typical cell cycle images (I) and statistical results (J) of HN4 cells after knockdown of VAMP2 and SNAP25 expression and treated by FBS-free medium for 96 h. (K, L) Typical cell cycle images (K) and statistical results (L) of FaDu cells after knockdown of VAMP2 and SNAP25 expression and treated by FBS-free medium for 96 h. (M, N) Typical apoptosis images (M) and statistical results (N) of HN4 cells after knockdown of VAMP2 and SNAP25 expression and treated by FBS-free medium for 96 h. (O, P) Typical apoptosis images (O) and statistical results (P) of FaDu cells after knockdown of VAMP2 and SNAP25 expression and treated by FBS-free for 96 h. FBS, fetal bovine serum; ILV, intraluminal vesicle; MVB, multivesicular body; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2. *P < 0.05, **P < 0.01, ***P < 0.001 compared to siCtrl using Student’s t-test.
Autophagy has a vital role in the degradation of exosomes32. To explore the impact of accumulated MVBs and ILVs on tumor cell proliferation and apoptosis, autophagy was promoted in HN4 cells by using a FBS-free medium, a condition recognized for activating autophagic pathways16. Following induction, an accumulation of ILVs and MVBs was noted due to the knockdown of VAMP2/SNAP25. Activated autophagy under serum starvation mitigated the build-up of ILVs/MVBs that was prompted by depletion of VAMP2 or SNAP25 (Figure S5A–S5D). To further assess if the accumulated ILVs/MVBs were degraded through activation of autophagy instead of other means, the tumor-secreted exosomes were isolated from the supernatant of HN4 cells with or without siRNA-mediated knockdown of VAMP2 or SNAP25. As shown in Figure S5E–S5H, the exosome concentration was not significantly different among HN4 cells with VAMP2 or SNAP25 knockdown compared to the scrambled control cells after the autophagic effect was activated via FBS-free culture. These findings showed that the intracellular exosomes, also known as ILVs, can be consumed upon autophagy activation. To determine the effect of knockdown of VAMP2 or SNAP25, accumulated MVBs and ILVs on HNC cells, the proliferation and apoptosis of HNC cells were examined by depleting accumulated MVBs and ILVs. The results showed that there was no significant change in cell proliferation and apoptosis after knockdown of VAMP2 or SNAP25 expression under FBS-free treatment (Figures 4I–4P and S5I–S5L). These results implied that downregulation of VAMP2 or SNAP25 in HNC cells triggered an accumulation of MVBs and ILVs, which correlates with decreased cell proliferation and an increase in apoptosis rates.
MVBs are released directly into the extracellular space to counteract the intracellular accumulation of MVBs caused by VAMP2/SNAP25 knockdown
MVBs/ILVs significantly accumulated intracellularly, indicating that autophagy was not activated in HNC cells after VAMP2/SNAP25 knockdown. Western blotting showed no significant increase in the expression of LC3-II, a key molecule for autophagy activation, after knockdown of VAMP2/SNAP25 (Figure 5A). Fluorescence co-localization results also showed that CD63, an MVB marker, did not co-localize with autophagosomes and lysosomes after knockdown of VAMP2/SNAP25 (Figure S6). To investigate the trajectories of intracellularly accumulated MVBs/ILVs, TEM was utilized to examine the distribution pattern of MVBs/ILVs. The results revealed a large vesicle resembling MVB structures at the cell membrane or the surrounding area with a size of approximately 1 μm after knocking down VAMP2 or SNAP25 expression (Figure 5B). To determine whether the knockdown of VAMP2 or SNAP25 led to increased secretion of large vesicles, NTA was utilized to analyze the number of large EVs in cell supernatants. The results demonstrated that knockdown of VAMP2 or SNAP25 led to an increase in the secretion of vesicles approximately 1 μm in size (Figure 5C). To determine whether the secreted large vesicles were MVBs, the MVB marker protein, CD63, was used for staining. The results demonstrated that the secreted large vesicles clearly carried CD63 protein (Figure 5D and 5E). To further validate these findings based on the findings of previous studies, the VAMP2 protein was shown to bind to MVBs when MVBs approached the PM. Knocking down SNAP25 inhibited MVB fusion with the PM, which should result in MVBs being secreted into the extracellular space, carrying the VAMP2 protein that MVBs possess. Therefore, the expression of VAMP2 protein in EVs was detected from cell supernatants. The results showed that knocking down SNAP25 significantly increased the expression of VAMP2 protein in EVs (Figure 5F). These results suggested that knocking down VAMP2 or SNAP25 causes MVBs/ILVs in HNC cells to be secreted as a whole into the extracellular space. These secreted MVBs are referred to as MVB-like EV clusters.
VAMP2 or SNAP25 knockdown induces MVB-like EV cluster release. (A) Representative Western blotting images and statistical results of LC3-II/LC3-I in HN4 cells after knockdown of VAMP2 or SNAP25 expression. (B) Typical TEM images in HN4 cells after knockdown of VAMP2 or SNAP25 expression. (C) Typical trace and statistical results of particle size distributions in HN4 cells after knockdown of VAMP2 and SNAP25 expression. (D) Representative images of cells chemically stained for CD63 after knockdown of VAMP2 or SNAP25. (E) Immunofluorescence staining to determine the distribution of CD63 after knockdown of VAMP2 or SNAP25. (F) Representative Western blot images and statistics of VAMP2 expression in EVs from cell supernatants. EV, extracellular vesicle; EXO, exosomes; MVB, multivesicular body; SNAP25, Synaptosome-associated protein of 25; TEM, transmission electron microscopy; VAMP2, Vesicle-associated membrane protein 2. *P < 0.05 compared to siCtrl using Student’s t-test.
MVBs-like EV clusters are secreted extracellularly through the Rab11a-myosin9-phosphatidylserine exocytic pathway after VAMP2/SNAP25 knockdown
Large vesicles secreted into the extracellular space involves membrane dynamics. Previous morphologic observations showed that MVB release into the extracellular space is accompanied by membrane blebbing, a process linked to phosphatidylserine (PS) exposure33,34. Interestingly, PS externalization was significantly enhanced after knocking down VAMP2 or SNAP25 expression (Figure 6A). To determine the specific mechanism by which MVB-like EV clusters are released into the extracellular space, a proteomic approach was used to identify proteins in HNC cells associated with Rab11a, a key molecule involved in MVB transport. The results revealed that 37 proteins were found to interact with Rab11a. Among these proteins, myosin9 (MYH9) was identified as an essential factor required for maintaining the cytoskeleton (Figure 6B). The Co-IP results in HN4 and FaDu cells showed a clear interaction between Rab11a and myosin9, demonstrating the specific molecular interaction of the Rab11a-myosin9-PS pathway (Figure 6C). As a member of the myosin protein family, the connection to PS suggested a potential role in cellular processes involving PS. These results suggested that Rab11a, myosin9, and PS externalization maybe regulate MVBs-like EV cluster release to the extracellular space. To determine this hypothesis, VAMP2 or SNAP25 expression was knocked down first, followed by either suppression of Rab11a and myosin9 expression or PS externalization inhibition to observe the resulting PS exposure. The results showed that further suppression of Rab11a or myosin9 expression, significantly increased PS externalization. In contrast, inhibition of PLSCR1 protein expression significantly inhibited PS exposure (Figure 6D and 6E). Further suppression of Rab11a or myosin9 expression, or inhibition of PS externalization, significantly reduced the release of MVB-like EV clusters (Figure 6F and 6G). These results suggested that the release of MVB-like EV clusters is mainly mediated via the Rab11a-myosin9-PS externalization pathway after reducing VAMP2 or SNAP25 expression.
Rab11a-myosin9-phosphatidylserine exocytic mediates the release of MVB-like EV cluster after knockdown of VAMP2/SNAP25. (A) Representative images and statistics of the number of positive cells for phosphatidylserine (PS) ectopics after knockdown of VAMP2 or SNAP25 expression. (B) Intersection of two cases of interacting proteins with Rab11a and specific names of proteins that interact with Rab11a. (C) The interactions between Rab11a and myosin9 through Co-IP in HN4 and FaDu cells. (D) Representative images and statistics of the number of positive cells for PS ectopics after knockdown of VAMP2 and Rab11a, myosin9, or PLSCR1. (E) Representative images and statistics of the number of positive cells for PS ectopics after knockdown of SNAP25 and Rab11a, myosin9, or PLSCR1. (F) Typical trace and statistical results of particle size distributions in HN4 cells after knockdown of VAMP2 and Rab11a, myosin9, or PLSCR1. (G) Typical trace and statistical results of particle size distributions in HN4 cells after knockdown of SNAP25 and Rab11a, myosin9, or PLSCR1. Co-IP, co-immunoprecipitation; EV, extracellular vesicle; MVB, multivesicular body; PS, phosphatidylserine; Rab11a, Ras-related protein Rab-11A; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2. *P < 0.05 compared to siCtrl using Student’s t-test.
MVBs-like EV clusters release alleviates the damage caused by accumulation of MVBs/ILVs resulting from low VAMP2 or SNAP25 expression
To determine the impact of MVB-like EV clusters on the malignant progression of HNC cells, after knockdown of VAMP2 or SNAP25 protein, the expression of Rab11a and myosin9 or PS externalization was inhibited and the proliferation, invasion, and migration of HN4, FaDu, and CNE2 cells were examined. The results showed that knockdown of VAMP2/SNAP25 expression significantly inhibited the proliferation, invasion, and migration of HN4, FaDu, and CNE2 cells. However, after further inhibiting the secretion of MVB-like EV clusters by simultaneously knocking down Rab11a and myosin9 expression or inhibiting the PS externalization process, the proliferation, invasion, and migration of HNC cells was more significantly inhibited (Figures 7A–7F and S7A–S7C). These results suggested that the release of MVB-like EV clusters alleviated cellular damage caused by exosome secretion inhibition, thereby mitigating the adverse effects of impaired exosome release.
Inhibition of MVB-like EV cluster release through suppression of Rab11a-myosin9-phosphatidylserine exocytic signaling pathway further inhibits malignant progression in HNC cells. (A) The statistics of CCK-8 results after knockdown of VAMP2/SNAP25 and Rab11a, myosin9, or PLSCR1 in HN4 cells. (B) The statistics of CCK-8 results after knockdown of VAMP2/SNAP25 and Rab11a, myosin9, or PLSCR1 in FaDu cells. (C) Representative images and statistical results of HN4 cell invasion after knockdown of VAMP2/SNAP25 and Rab11a, myosin9, or PLSCR1 in FaDu cells. (D) Representative images and statistical results of FaDu cell invasion after knockdown of VAMP2/SNAP25 and Rab11a, myosin9, or PLSCR1 in FaDu cells. (E) Representative images and statistical results of HN4 cell migration after knockdown of VAMP2/SNAP25 and Rab11a, myosin9, or PLSCR1 in FaDu cells. (F) Representative images and statistical results of FaDu cell migration after knockdown of VAMP2/SNAP25 and Rab11a, myosin9, or PLSCR1 in FaDu cells. CCK-8, Cell Counting Kit-8; HNC, head and neck cancer; Rab11a, Ras-related protein Rab-11A; SNAP25, Synaptosome-associated protein of 25; VAMP2, Vesicle-associated membrane protein 2. *P < 0.05 compared to siCtrl or siVAMP2 or siSNAP25 using Student’s t-test.
Discussion
Exosomes are crucial for local and distant cellular communication35. Emerging evidence has indicated that abnormal exosome biogenesis in tumor cells leads to heightened exosome secretion, which contributes to alterations in the TME. This remodeling facilitates adaptation, survival, and progression in cancer cells36–38. Despite the significant clinical implications, the molecular mechanisms underlying tumor exosome biogenesis remain largely elusive. In this study, the final stage of exosome secretion (the fusion of MVBs with the PM) was investigated. However, upon inhibition of this process, HNC cells released MVBs as MVB-like EV clusters extracellularly to alleviate damage caused by restricted exosome secretion. The key findings from the current investigation were as follows: (1) VAMP2 and SNAP25 were upregulated in HNC cells compared to adjacent non-tumor tissue, which contributed to enhanced exosome secretion and consequently promotes malignant tumor progression; (2) VAMP2 interacted with SNAP25 to facilitate the exocyst-dependent fusion of MVBs with the PM; (3) knockdown of VAMP2 and SNAP25 led to an accumulation of MVBs and ILVs, correlating with decreased cell proliferation and increased apoptosis; (4) knockdown of VAMP2 and SNAP25 led to MVB-like EV cluster release in HNC cells; (5) Rab11a-myopsin9-PS externalization mediates the release of MVB-like EV clusters; and (6) blocking exosome secretion while inhibiting MVB-like EV cluster release significantly increased HNC cell death. The current study offers new insights into the functions of VAMP2 and SNAP25 in exosome biogenesis, aiming to illuminate the complete exosome transport and secretion pathway in HNC cells, thus presenting a novel view on the intricacies of exosome release dynamics. Furthermore, a potential theoretical groundwork for how HNC cells self-rescue through the secretion of MVB-like EV clusters when exosome secretion is restricted was provided.
VAMP2 or SNAP25 has been identified as the key regulatory factor in tumor development and progression of prostate cancer, glioblastoma, ovarian cancer, and lung adenocarcinoma39. However, the expression and role of VAMP2 and SNAP 25 in HNC cells are unknown. In this study both VAMP2 and SNAP25 were shown to be critical molecules driving malignant progression in HNC. Importantly, the current study demonstrated that upregulated VAMP2 or SNAP25 is crucial for promoting exosomal secretion in HNC cells. Moreover, TDEs, which are rich in mRNA, miRNA, lncRNA, DNA, proteins, and various metabolites, can profoundly influence the behavior of recipient cells, thereby considerably facilitating tumor progression40,41. For example, chronic myeloid leukemia (CML)-derived exosomes have been shown to stimulate tumor cell proliferation in vitro and in vivo, primarily through the action of TGF-β1 contained within these CML-derived exosomes42. Exosomes secreted by gastric cancer (GC) cells have been shown to enhance GC cell proliferation in vitro with the extent of proliferation being dose-dependent43. When exosome secretion is inhibited, tumor cells are observed to undergo apoptosis44. This study showed that VAMP2 and SNAP25 knockdown significantly promoted apoptosis and reduced the proliferation of HN4 cells. This finding could be attributed to the reduced exosome numbers and altered exosome content. The alteration of exosome contents was not explored because the components in exosomes are complex and it is difficult to focus on exactly what components have a major role. The current study was sufficient to demonstrate that VAMP2 and SNAP25 promote malignant tumor progression mainly by altering the quantity of exosomes secreted.
Exosome secretion involves several critical cellular processes. Collective evidence has indicated that formation of MVBs and ILVs is frequently linked with the endosomal sorting complex required for transport (ESCRT)45. However, there is also evidence supporting the existence of alternative ESCRT-independent pathways for MVB and ILV formation46. Indeed, the formation of MVBs and ILVs is categorized into two distinct processes (ESCRT-dependent and -independent) each giving rise to unique exosome subpopulations with the respective biogenic origins47. In addition, several Rab GTPases, such as Rab11, Rab27, and Rab35, have been identified to have pivotal roles in exosome secretion, especially in mediating the transport of MVBs to the PM48. However, the mechanism underlying Rab GTPase-associated delivery of MVBs to the cell membrane remains unclear. Our previous study reported that the exocyst complex mediates the delivery of MVBs to the PM via Rab11a in HNC16. In addition, the exocyst is crucial for the anchoring of MVBs to the PM. Nevertheless, the precise mechanisms governing the fusion of MVBs with the PM are not fully understood. It has been documented that SNARE proteins facilitate the fusion process between MVBs and the PM49. For example, activation of SNAP23, a component of the SNARE complex, has been shown to significantly enhance exosome release21. VAMP7, another SNARE component, has been implicated in the fusion of MVBs with the PM, facilitating the release of exosomes into the extracellular space21. The fusion of MVBs with the PM represents the final step in the secretion of exosomes50. The mechanisms underlying endosomal secretion have been thoroughly investigated across various cell types, including tumors. Furthermore, the SNARE complex has a crucial role in the process of membrane. However, the specific subunits of SNAREs that mediate MVB fusion to the PM are unknown in HNC cells. In the present study interactions between the exocyst complex and the SNAP25 and VAMP2 proteins were observed in HNC cells. SNAP25 and VAMP2 come together to create a complex that constitutes the central machinery for intracellular membrane fusion, a process that is particularly vital for neurotransmitter release31.
Hemi-fusion stalk formation and fusion pore formation are induced when VAMP2 binds to SNAP25. The complex formed by VAMP2 and SNAP25 acts like a ‘zipper’ to mediate the fusion of the MVB membrane and the PM51. Eventually, MVB content is released into the extracellular space. Similar to this process, exocysts also deliver MVBs to the VAMP2 protein, which then binds to SNAP25 and induces MVB membrane fusion with the PM. Finally, the exosomes are secreted into the extracellular space. There may also be other proteins involved in this process, such as syntaxin 4, that assist in the mediation of membrane fusion52. However, considering the complexity of introducing one more molecule, we simply chose VAMP2 on the membrane of MVBs and SNAP25 protein on the PM, which is sufficient to illustrate the process of membrane fusion.
It has been published that inhibition of exosome secretion resulting in intracellular accumulation of MVBs/ILVs, excluding the HNC53,54. However, the intracellular role of these accumulated MVBs/ILVs is unclear. Recently, autophagy, a deeply conserved biological process that controls cellular homeostasis55, has been reported to regulate exosome biogenesis, including exosome secretion56. Fader et al. reported that autophagy induction inhibits secretion of the exosome, which is associated with the interaction of MVBs with the autophagic pathway56. In this study fusion of MVBs with the PM was impeded by knockdown of VAMP2 or SNAP25 expression. This process led to an accumulation of MVBs and ILVs in HN4 cells. Nevertheless, when autophagy was induced using a serum-free medium, the built-up exosomes were subsequently degraded through the autophagic pathway. This finding could be attributed to the autophagosome fusion with MVB and this eventual degradation by lysosomes57. In addition, the reduced cell proliferation and increased apoptosis resulting from the low expression of VAMP2 and SNAP25 were markedly mitigated after the accumulated exosomes were depleted. This observation suggested that inhibiting exosome secretion in HNC cells not only disrupts cell-to-cell communication via exosomes but also impairs the cells that are prevented from secreting. This dual mechanism offers promising targets and strategies for cancer therapy.
With advances in technology, various approaches have been developed for tumor treatment. However, tumor cells also develop protective mechanisms in response to these treatments. For example, Zhang et al. reported that VPS35 within tumor cells senses reactive oxygen species (ROS), suppresses mitochondrial translation, and ultimately reduces the sensitivity of tumor cells to chemotherapy drugs58. In the current study similar findings were observed. When the secretion of exosomes in HNC cells was inhibited, MVBs were shown to accumulate within the cytoplasm. However, instead of activating autophagy to eliminate these accumulated MVBs, the cells activated alternative pathways to directly secrete the MVBs into the extracellular space as MVB-like EV clusters, thereby alleviating cellular damage caused by the restriction of exosome secretion. This finding provided direction for studies in which blocking exosome secretion does not completely halt tumor progression.
MVB-like EV clusters were first identified in colorectal cancer cells by Valcz59, who showed that these structures frequently exhibit partial or complete extracellular positioning at the interface between the tumor cell membrane and the stromal matrix. In addition, these particles present MVB-like aggregates of ALIX/CD63-positive EV clusters released by migrating tumor cells as a whole59. In the current study we identified large-sized EVs that exhibit structural similarities to MVBs, containing small EVs and CD63-positive components. MVB-like EV clusters may be structurally more complex, contain more small vesicles, and may have advantages in signaling or cargo volume. For example, the high efficiency of large EVs in transmitting messages is illustrated by the finding of Soukup et al. that large EVs have an important role in prion trafficking and propagation compared to the widely studied small EVs60. Therefore, inhibiting the secretion of large-sized EVs, such as MVB-like EV clusters, could potentially inhibit tumor progression. However, the mechanism by which MVB-like EV clusters are secreted into the extracellular space is unclear.
Rab proteins bind GTP and undergo hydrolysis, utilizing the GTP-GDP cycle to regulate vesicle fusion. Rab GTPases depend on motor proteins, like myosins, for vesicle transport. Specifically, Li and colleagues found that Rab10 relies on myosin Va to mediate vesicle trafficking into migrasomes61. Our findings showed that Rab11a associates with myosin9, serving a role in the membrane-mediated release of MVB-like EV clusters. The release of MVB-like EV clusters involves the outward expansion of specific membrane regions, leading to vesicle-like protrusions. This process is termed membrane blebbing62. Interestingly, membrane blebbing is closely associated with the externalization of PS63,64. In addition, myosin interacts with and triggers the activation of PS, facilitating the functional role of PS65,66. The current study demonstrated that the Rab11a-myosin9 interaction mediates the release of MVB-like EV clusters, primarily through the externalization of PS. Our findings provide novel insights into the mechanisms underlying the release of MVB-like EV clusters, advancing our understanding of this process. However, due to the relatively small sample size of this study, it is difficult to comprehensively reflect the overall diversity and complexity characteristics, thereby limiting the generalizability and extrapolation value of the research conclusions. Corollary studies could expand the sample size to further validate and extend the findings of this research.
Conclusions
This research has shown that VAMP2 interacts with SNAP25 to facilitate exosomal release by mediating the fusion of MVBs with the PM in HNC cells. However, inhibiting this process activates the Rab11a-myosin9 signaling pathway, enabling PS externalization and the subsequent release of MVBs as MVB-like EV clusters into the extracellular space, thereby alleviating cellular damage in HNC cells caused by impaired exosome secretion. This study provides a comprehensive characterization of MVB trafficking within cells. Furthermore, a novel potential therapeutic target that simultaneously inhibits tumor-derived exosome secretion and MVB-like EV cluster release, which may ultimately hinder tumor progression, was identified (Figure 8).
The fate of VAMP2/SNAP25-associated MVBs in HNC cells. VAMP2/SNAP25 mediates the release of MVBs into the extracellular environment through exosome-mediated transport (A). In HNC, highly expressed VAMP2 insert into MVB membranes, which are transported and anchored to the plasma membrane via the exocyst complex. Subsequently, VAMP2 interacts with SNAP25, an elevated protein on the plasma membrane, forming a “zipper-like” structure that facilitates MVB membrane and plasma membrane fusion. This process enables the release of ILVs from MVBs as exosomes into the extracellular space. Therefore, inhibiting VAMP2/SNAP25 expression can block the secretion of tumor exosomes, thereby suppressing cancer progression. However, after VAMP2/SNAP25 expression is suppressed, MVBs form MVB-like extracellular vesicle clusters that are released into the extracellular environment (B). When VAMP2/SNAP25 is knocked down, Rab11a proteins on the MVB membrane bind to myosin9, which then interacts with PS. During the PS exocytosis process, myosin9-Rab11a pulls the entire MVBs out of the cell. This alternative exocytosis pathway effectively mitigates cellular damage caused by blocked exosome secretion. HNC, head and neck cancer; VAMP2, vesicle-associated membrane protein 2; SNAP25, synaptosome associated protein 25; Rab11a, Ras-related protein Rab-11A; MVBs, multivesicular bodies; ILVs, intraluminal vesicles; Rab11a, Ras-related protein in brain 11A; PS, phosphatidylserine; siRNA, small interfering RNA.
Supporting Information
Conflict of interest statement
No potential conflicts of interest are disclosed.
Author contributions
Conceived the project and designed and supervised the research: Juan Du.
Designed, performed, and analyzed the experiments: Suwen Bai, Hexing Sun, Rong Liu, Minghua Wang.
Contributed data or analysis tools: Minghua Wang, Yumei Luo.
Provided laryngocarcinoma tissues and sample storage: Wenjun Zhang, Jing Wu.
Wrote and gave suggestions on the paper: Suwen Bai, Hexing Sun, David W. Chan, Yuan Wei.
Data availability statement
The data generated in this study are available upon request from the corresponding author.
Acknowledgments
We sincerely thank Yuqi Dang, Shuchen Han, Yanheng Yao, Xinyue Zhang, and Chenxi Zhang for assistance with cell culture experiments, model establishment, fluorescence staining supervision, and clinical sample collection during the revision phase. We are also grateful to Ye Zhang and Xiaoqiang Yao for their helpful feedback on the revised manuscript and assistance in polishing the final draft. Their technical expertise and supportive contributions are greatly appreciated.
- Received September 16, 2025.
- Accepted March 17, 2026.
- Copyright: © 2026, The Authors
This work is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License.
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