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. 2014 Sep 10;17(3):407–418. doi: 10.1093/neuonc/nou219

VAMP8 facilitates cellular proliferation and temozolomide resistance in human glioma cells

Yuanyuan Chen 1,†, Delong Meng 1,†, Huibo Wang 1,†, Ruochuan Sun 1, Dongrui Wang 1, Shuai Wang 1, Jiajun Fan 1, Yingjie Zhao 1, Jingkun Wang 1, Song Yang 1, Cong Huai 1, Xiao Song 1, Rong Qin 1, Tao Xu 1, Dapeng Yun 1, Lingna Hu 1, Jingmin Yang 1, Xiaotian Zhang 1, Haoming Chen 1, Juxiang Chen 1, Hongyan Chen 1, Daru Lu 1
PMCID: PMC4483099  PMID: 25209430

Abstract

Background

Malignant glioma is a common and lethal primary brain tumor in adults. Here we identified a novel oncoprotein, vesicle-associated membrane protein 8 (VAMP8), and investigated its roles in tumorigenisis and chemoresistance in glioma.

Methods

The expression of gene and protein were determined by quantitative PCR and Western blot, respectively. Histological analysis of 282 glioma samples and 12 normal controls was performed by Pearson's chi-squared test. Survival analysis was performed using the log-rank test and Cox proportional hazards regression. Cell proliferation and cytotoxicity assay were conducted using Cell Counting Kit-8. Autophagy was detected by confocal microscopy and Western blot.

Results

VAMP8 was significantly overexpressed in human glioma specimens and could become a potential novel prognostic and treatment-predictive marker for glioma patients. Overexpression of VAMP8 promoted cell proliferation in vitro and in vivo, whereas knockdown of VAMP8 attenuated glioma growth by arresting cell cycle in the G0/G1 phase. Moreover, VAMP8 contributed to temozolomide (TMZ) resistance by elevating the expression levels of autophagy proteins and the number of autophagosomes. Further inhibition of autophagy via siRNA-mediated knockdown of autophagy-related gene 5 (ATG5) or syntaxin 17 (STX17) reversed TMZ resistance in VAMP8-overexpressing cells, while silencing of VAMP8 impaired the autophagic flux and alleviated TMZ resistance in glioma cells.

Conclusion

Our findings identified VAMP8 as a novel oncogene by promoting cell proliferation and therapeutic resistance in glioma. Targeting VAMP8 may serve as a potential therapeutic regimen for the treatment of glioma.

Keywords: autophagy, glioma, proliferation, TMZ resistance, VAMP8

As one of the most notorious of malignancies, glioblastoma multiforme (GBM), accounts for 45.2% of all malignant primary brain tumors.1 Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients averages a mere 14 months after diagnosis.2

Soluble N-ethylmaleimide-sensitive factor (NSF) receptor (SNARE) is a superfamily of small proteins with more than 35 members in mammals, varying in size and primary structure.3 As an essential mechanism for cellular activities, SNARE has been observed in the progression of various tumors.4,5 Vesicle-associated membrane protein 8 (VAMP8) was first identified as an endosomal SNARE that participates in diverse biological functions including endosomal fusion,6–8 the exocytosis of GLUT4 and insulin,9,10 regulation of exocytosis in secretory cells,11 sequential granule-to-granule fusion,11 and autophagy.12,13 Nevertheless, nobody has reported its involvement in tumor progression.

Temozolomide (TMZ) is the most commonly used chemotherapeutic drug in clinical trials for glioma. However, a large number of patients are resistant to TMZ, which greatly compromise the clinical treatment. Thus, research on TMZ resistance is of great importance for ameliorating the therapeutic efficacy and alleviating the suffering of patients.

Autophagy is a dynamic process of protein degradation that is essential for survival, differentiation, development, and homeostasis.14 Despite evidence demonstrating the role of autophagy in modifying TMZ resistance in GBM,15–17 it remains incompletely understood, and clarification is needed.

In this study, we first analyzed the expression and prognostic value of VAMP8 in The Cancer Genome Atlas (TCGA) and a cohort of 282 glioma patients, and then investigated its roles on tumorigenisis and TMZ resistance in glioma. Our findings revealed that the dysregulation of VAMP8 is a potential component of glioma pathogenesis and TMZ resistance, which might become a new therapeutic target for patients with glioma.

Materials and Methods

Patient Samples

Patients with histologically confirmed glioma were consecutively recruited between March 1998 and August 2010 from the Department of Neurosurgery at Changzheng Hospital in Shanghai, China. Patients were excluded if they had a prior history of malignancy and previous radiotherapy or chemotherapy for unknown disease conditions. In total, 282 eligible patients were recruited for this study and 12 normal samples were obtained as controls from individuals who were trauma outpatients with no prior pathologically detectable condition. Written informed consent was provided by all participants. All specimens were handled anonymously according to the ethical standards. This study was carried out with the approval of the Ethical Review Committee of Fudan University.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from surgically resected tissues using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. First-strand cDNA was synthesized using ReverTra Ace (Toyobo). The primers used for PCR are shown in Supplementary Table S1. The reaction was run using the ABI 7900HT Sequence Detection System (Applied Biosystems) in the presence of SYBR-Green dye (Toyobo). Each sample was tested in triplicate, and a melting curve analysis of each sample was used to check the specificity of amplification. The relative gene expression level was calculated using the comparative CT method.

Western Blot

The tissue and cell samples were homogenized in lysis buffer containing a cocktail of protease inhibitors (Sigma) and 1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged at 12 000 rpm for 10 minutes, and the supernatant was heated at 100°C for 10 minutes before loading onto a 12%–15% sodium dodecyl sulfate-polyacrylamide gel. Then, the resolved proteins were transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat milk in tris-buffered saline and tween 20 for 1 hour at room temperature and blotted with primary antibodies, followed by incubation with horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgM as the secondary antibody (Epitomics). Anti-alpha–tubulin antibody (1:1000; Abcam) was used as a loading control. The bound antibodies were detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore). The primary antibodies were LC3, Beclin1, ATG5, VPS34 (1:1000; CST), VAMP8, and STX17 (1:1000; Abcam).

Immunohistochemistry, Tissue Microarray Analysis, and Evaluation of the Degree of Immunoreactivity

The construction of the tissue microarray (TMA) and the following immunohistochemistry and TMA analysis were performed by Shanghai Biochip Company, Ltd. Each slide was read and scored independently by 2 pathologists in a blinded fashion. The evaluation of the staining density and intensity has been described previously.18,19 Briefly, the total score of staining was the product of the scores for intensity and density. Low VAMP8 expression, indicating weakly positive cases (−), had a score of 0 or 1, while high VAMP8 expression indicated strongly positive cases (+) with a score of ≧2.

Cell Culture and Lentivirus Infection

The human malignant glioma cell lines U251 and U87 were obtained from the Japanese Cancer Research Resources Bank. The human glioma progenitor cell line SU320 was provided by Dr. Qiang Huang (Soochow University). The glioma cell lines were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO2. The maintenance of the glioma neurosphere line has been described previously.20 To generate cell lines overexpressing VAMP8, we cloned VAMP8 from one clinical sample into PCDH-copGFP vector (System Biosciences). To establish cell lines in which VAMP8 expression would be stably knocked down, we ligated sequences targeting VAMP8 mRNA (Supplementary Table S1) into PLVX-mU6-GFP. Notably, shLuc21 and PCDH-copGFP empty vector (EV) served as controls for PLVX-mU6-shVAMP8 and PCDH-VAMP8-copGFP, respectively. To generate VAMP8 transcripts that were resistance to shRNA, we mutated the binding sequence of shVAMP8-2 (Supplementary Table S1) while keeping protein sequence unchanged. The procedures of packaging and infecting with lentivirus were done according to the previous study.22,23

Colony Formation, Cell Proliferation, and Cytotoxicity Assay

Colony formation was performed, as described previously.24 Cell proliferation and cytotoxicity assay were analyzed by Cell Counting Kit 8 (Dojindo) according to the manufacturer's instructions. Each dose was done in triplicate, and the experiments were done at least 3 times. Cell viability was calculated as a percentage of the optical density value of the control group in each time point.25

Cell Cycle Analysis

Cell cycle analysis was performed, as described previously.24 Cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with 70% ice-cold ethanol. Fixed cells were resuspended in PBS containing 25 mg/mL propidium iodide and 10 mg/mL RNase and incubated for 30 minutes in the dark before being analyzed by flow cytometry.

Subcutanous Xenograft Models

Pooled populations of transduced U87 cells were used for subcutaneous tumor growth experiments, as described previously.26 Briefly, U87 cells (1 × 106 in 200 μL PBS) that overexpressed or suppressed VAMP8 and their corresponding control cells were injected subcutaneously into the right flank of nude mice (n = 6), respectively. After the xenografts became visible, the longest and shortest diameters of the xenografts were measured using a digital caliper periodically. Tumor volumes were calculated using the following formula: volume = 1/2 × width2 × length.27 The groups of xenografts were harvested when the length of the largest xenograft reached 2 cm. At the end of the experiments, tumors were fixed and sectioned for histological and immunological analyses.

Confocal Microscopy

Confocal microscopy was performed, as described previously.28,29 Briefly, 48 hours after being transiently transfected with mCherry-LC3, cells were treated with 100 μM TMZ for 48 hours. Twenty-four–hour treatment of 50 nM rapamycin served as positive control. Then, the cells were fixed in 4% paraformaldehyde for 30 minutes, washed twice with 1 × PBS, and analyzed with the LSM700 confocal microscope (Carl Zeiss).

Statistical Analysis

All experiments were performed in triplicate with means and standard deviation subjected to Student t test or ANOVA for multivariate analysis in SPSS Statistics 17.0. Analysis of survival was performed using Kaplan-Meier analysis and Cox regression analysis in SPSS Statistics 17.0. ( *, **, or *** indicate P < .05, P < .01, or P < .001, respectively, and “ns” indicates not significant.)

Results

VAMP8 is Elevated in Glioma Tissues

To identify deregulated genes in glioma, we initially analyzed the expression profile from TCGA30 and the correlations between these aberrantly expressed genes and the overall survival (OS) of GBM patients. Consequently, we found that VAMP8 expression was significantly enhanced in 92.67% (392/423) of the GBM samples when compared with the normal brain controls (Fig. 1A). In addition, when the clinical data of these samples were taken into account, it was shown that VAMP8 was an independent prognostic factor for glioblastoma patients (P = .005; Fig. 1B and Supplementary Table S2).

Fig. 1.

Fig. 1.

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Expression and prognostic value of VAMP8 in glioma. (A) The expression profile of VAMP8 in 423 GBM patients in TCGA. Each point represents the log2 ratio of VAMP8 expression to the mean of normal controls. (B) Correlation between VAMP8 level and overall survival by Kaplan-Meier analysis of GBM patients in TCGA with high (n = 264) or low (n = 159) VAMP8 expression (P = .005). (C) The relative expression of VAMP8 in 11 glioma samples compared with 5 normal controls. Each bar represents the log2 ratio of VAMP8 expression in each glioma sample to the mean of 5 normal controls. (D) Representative image of VAMP8 expression in glioma tissues and normal controls. Scale bars are depicted in figures.

We next measured the expression of VAMP8 in a cohort of 11 glioma samples and 5 normal controls via quantitative RT-PCR. Consistent with the findings from TCGA data, VAMP8 was found to be markedly increased in 90.91% (10/11) of the glioma samples (Fig. 1C) compared with the normal controls. Moreover, we performed histological analysis of a TMA containing 282 glioma samples and 12 normal controls. The results showed that 67.73% (191/282) of the glioma samples exhibited stronger immunoreactive scores than those of the normal controls (6/12) (P < .001, Fig. 1D). Together, these results suggest that VAMP8 is frequently overexpressed in glioma.

VAMP8 Serves as a Potential Novel Prognostic and Treatment-predictive Marker for Glioma Patients

To further examine the correlation between VAMP8 levels and clinical prognosis, we performed Kaplan-Meier analysis and observed that glioma patients with high VAMP8 expression had an unfavorable OS (log rank test, P = .007; Fig. 2A) and a shorter progression-free survival (PFS) (log rank test, P = .011; Fig. 2B) than those with low VAMP8 expression. The median OS of the patients with high and low VAMP8 expression were 23 months (95% CI, 17.758–28.242) and 54 months (95% CI, 28.296–70.704), respectively. Moreover, we found that high-grade glioma (HGG) with high VAMP8 expression also predicted a worse OS (log rank test, P = .001; Fig. 2C) and a shorter PFS (log rank test, P = .003; Fig. 2D) than those with low VAMP8 expression. In addition, multivariate Cox regression analysis identified VAMP8 as an independent prognostic factor for glioma patients, higher levels of which predicted poorer survival (Supplementary Table S3).

Fig. 2.

Fig. 2.

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VAMP8 predicts OS and PFS in glioma patients and the patients who received chemotherapy. (A and B) Kaplan-Meier analysis of the correlations between different VAMP8 levels and OS (A) or PFS (B) in 267 glioma patients. (C and D) Kaplan-Meier analysis of the correlations between different VAMP8 levels and OS (C) or PFS (D) in 162 high-grade glioma (HGG) patients. (E and F) Kaplan-Meier analysis of the correlations between different VAMP8 levels and OS (E) or PFS (F) in 173 glioma patients who received chemotherapy. (G and H) Kaplan-Meier analysis of the correlations between different VAMP8 levels and OS (G) or PFS (H) in 116 HGG patients who received chemotherapy.

We next assessed the prognostic value of VAMP8 in patients who received TMZ-based therapy. Kaplan-Meier analysis revealed that glioma patients with high VAMP8 expression displayed a worse response to TMZ therapy compared with patients having low VAMP8 expression (OS, 21 vs 54 mo; HR, 1.888; 95% CI, 1.182–3.017; log rank test, P = .006; Fig. 2E) (PFS, 19 vs 42 mo; HR, 1.656; 95% CI, 1.085–2.527; P = .017; Fig. 2F). Furthermore, a worse response to TMZ therapy was noted for HGG patients with high VAMP8 expression compared with patients with low VAMP8 expression (OS, 15 vs 30 mo; HR, 2.195; 95% CI, 1.298–3.712; P = .002; Fig. 2G) ( PFS, 11 vs 26 mo; HR, 1.995; 95% CI, 1.230–3.235; P = .004; Fig. 2H). In addition, multivariate Cox regression analysis showed that VAMP8 could be an independent predictor of TMZ-based therapeutic efficacy for patients with glioma (Supplementary Table S4). Taken together, these results suggest that VAMP8 could function as a prognostic and treatment-predictive marker for glioma patients.

VAMP8 Regulates Glioma Proliferation in Vitro via Cell Cycle Regulation

Because VAMP8 was significantly upregulated in glioma, we postulated that VAMP8 might promote glioma growth. To test our hypothesis, we constructed lentivirus vectors to stable overexpress or knock Kitdown the expression of VAMP8. Altered expression of VAMP8 in U87 cells was confirmed by Western blot analysis (Fig. 3A). shVAMP8-2 and shVAMP8-3 were chosen for the following study according to their knockdown efficiency. Then, the effect of VAMP8 on cellular proliferation was performed in U251 and U87 cells using Cell Counting Kit 8 assay, which showed that forced expression of VAMP8 significantly stimulated the proliferation compared with the control groups, whereas knockdown of VAMP8 could suppress cellular growth in both cell lines (Fig. 3B). Similar results were also seen in glioma progenitor cells (Supplementary Fig. S1A and S1B). Colony formation assays consistently showed that overexpression of VAMP8 dramatically increased the number of colonies compared with the control groups (P < .01, Fig. 3C), while depletion of VAMP8 reduced the colony numbers in both cell lines (P < .05, Fig. 3C).

Fig. 3.

Fig. 3.

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VAMP8 regulates glioma proliferation in vitro. (A) Western blot analysis of VAMP8 in U87 cells with VAMP8 overexpression and depletion. (B) Proliferation curves of U251 and U87 cells with VAMP8 overexpression and knockdown. (C) Colony formation assays for U251 and U87 cells with VAMP8 overexpression and knockdown. Values are means ± SD from 3 independent experiments. (D) Cell cycle analysis of U251 and U87 cells with abrogation of VAMP8. (E and F) Growth curves, tumor weights, and their representative images of tumors that were VAMP8 overexpressed (E) or depleted (F) as well as the corresponding controls. Values are means ± SD of the tumor weights (mg). (G) Immunohistochemistry of Ki-67 in VAMP8-overexpressing or knocked-down tumors and its controls. EV and shLuc served as controls. Scale bars: 25 μm.

To elucidate the mechanism by which VAMP8 regulates glioma cell growth, we examined whether growth inhibition was associated with specific cell cycle changes. The effects of VAMP8 knockdown on cell cycle progression of U87 and U251 cells were characterized by flow cytometric analysis. Compared with the control group, both VAMP8-depleted cells showed marked increases in the numbers of G0/G1 phases (Fig. 3D). Together, these results suggest that VAMP8 is a critical factor in regulating cell growth and cell cycle progression.

VAMP8 Favors Tumor Cell Growth in Vivo

To assess the effect of VAMP8 in vivo, we injected either VAMP8-transduced or -depleted (shVAMP8-2) U87 cells, together with the control cells (EV and shLuc) into nude mice subcutaneously. During the entire tumor-growth period, tumors from VAMP8-transduced cells grew faster in comparison with the control group. The average weight of tumors developed from VAMP8-overexpressing cells (1.98 ± 0.70 g) was also significantly heavier than those from the control cells (0.79 ± 0.43 g) (Fig. 3E). In contrast, tumors derived from VAMP8-depleted cells showed reduced growth rates and smaller tumor weights (0.44 ± 0.24 g) when compared with the control group (2.41 ± 1.14 g) (Fig. 3F). Immunostaining analysis of Ki-67 expression was then performed in resected tumor tissues. In comparison with that of the control groups, the positivity of Ki-67 in the VAMP8-overexpressing group was significantly increased, whereas the VAMP8-depleted group exhibited decreased staining (Fig. 3G). Together, these results suggest that VAMP8 facilitates glioma growth in vivo.

VAMP8 Mediates Resistance to Temozolomide in Glioma Cells

TMZ is the most widely used chemotherapeutic agent for the treatment of glioma. Given that VAMP8 was found to be an effective predictive marker for the efficacy of TMZ therapy in glioma, we then asked whether VAMP8 could modulate the sensitivity of glioma cells to TMZ. The VAMP8 or EV-transduced U87 and U251 cells were treated with different doses of TMZ for 72 hours, respectively. As shown in Fig. 4A and B, the viability of VAMP8-overexpressing cells was markedly higher than that of the control group after treatment with TMZ. Conversely, antagonizing VAMP8 in both cell lines conferred hypersensitivity to TMZ compared with the control cells (Fig. 4C and D). Similar results were also seen in glioma progenitor cells (Supplementary Fig. S1C and S1D). To further validate its regulation of TMZ sensitivity, VAMP8 was first depleted in VAMP8-transduced U87 cells before TMZ treatment. As shown in Fig. 4E, cells with abrogated VAMP8 reversed VAMP8-overexpressing cells’ resistance to TMZ. Then, we also infected VAMP8-suppressed U87 cells with VAMP8 transcripts that were mutated in shRNA matched sequence (VAMP8mut). We found that VAMP8mut cells markedly rescued the resistance to TMZ when compared with the control group in VAMP8-deprived cells (Fig. 4F). Together, these results suggest that VAMP8 might mediate the resistance to TMZ in glioma cells.

Fig. 4.

Fig. 4.

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VAMP8 mediates resistance to TMZ in glioma cells. (A–D) Cell viabilities of VAMP8-transduced U251 (A) and U87 (B), VAMP8-suppressed U251 (C), and U87 (D) under treatment of indicated doses of TMZ. (E) Cell viabilities of U87-overexpressing cells with further VAMP8 deletion under treatment of indicated doses of TMZ. (F) Cell viabilities of VAMP8-suppressed U87 cells transduced with VAMP8 transcripts that were mutated in shRNA matched sequence (VAMP8mut) under treatment of indicated doses of TMZ.

Autophagy is Responsible for VAMP8-induced Temozolomide Resistance

Autophagy is an evolutionarily conserved catabolic process in which portions of cytosol and organelles are sequestered into a double-membrane vesicle and delivered to the lysosome for bulk degradation.31 It serves as a protective mechanism to mediate the resistance of radio- and chemotherapy in tumors.32–35 Consistent with previous findings,16,33 we found that TMZ treatment induced autophagy in glioma cells (Fig. 5A). Moreover, previous reports had indicated that VAMP8 had a role in the process of autophagosome-lysosome fusion.12,13 In our study, we observed that VAMP8 levels were also significantly upregulated in U87 cells after TMZ treatment in a dose- and time-dependent manner (Fig. 5B). Similar results were also observed in U251 cells (data not shown). Hence, we asked whether autophagy might be involved in VAMP8-induced TMZ resistance in glioma cells. To ascertain this assumption, we first knocked down the expression of ATG5, a key regulator of autophagy, or STX17, another SNARE that interacts with VAMP8 during autophagosome-lysosome fusion13 in VAMP8-overexpressing cells by RNA interference (RNAi) before TMZ treatment. The efficiency of interference was detected by Western blot analysis (Fig. 5C). As expected, the resistance of TMZ induced by VAMP8 was dramatically compromised after ATG5 or STX17 deletion (Fig. 5D). In addition, we further silenced the expression of STX17 in VAMP8-depleted U87 cells before subsequent treatment of TMZ. As shown in Fig. 5E–G, a significant alteration of TMZ sensitivity was observed in control cells but not in VAMP8-depleted cells, which demonstrated that the changes of TMZ sensitivity in glioma cells were VAMP8 dependent.

Fig. 5.

Fig. 5.

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Autophagy involves the VAMP8-mediated TMZ resistance in glioma cells. (A) Western blot analysis of LC3 in U251 and U87 cells with indicated doses treatment of TMZ for 48 hours. RAPA, indicated as rapamycin, served as positive control. DMSO served as the control without TMZ treatment. (B) Western blot analysis of VAMP8 under TMZ exposure of indicated concentrations and time. (C) Western blot analysis of ATG5, STX17, and VAMP8 in ATG5- or STX17-depleted U87-overexpressing cells. (D) Cell viabilities of VAMP8-transduced U87 with siRNA-mediated ATG5 and STX17 knockdown under treatment of indicated concentrations of TMZ. (E–G) Cell viabilities of the control cells (E) or 2 VAMP8-deprived (F and G) U87 cells transfected with STX17-interfering siRNA under treatment of indicated doses of TMZ. (H) Western blot detection of autophagy-related proteins in VAMP8-transduced U87 and its control cells (EV). (I) Western blot detection of LC3 in VAMP8-deprived U87 and its control cells (shLuc) with or without the presence of 10 mM CQ for 24 hours prior to 100 μM TMZ treatment for 72 hours and its quantitative analysis of the relative amounts of LC3-II to loading control in each group mentioned above. (J) Western blot detection of LC3 in U87 that were VAMP8 depletion or VAMP8 and STX17 double suppression with or without treatments of 100 μM TMZ for 72 hours and its quantitative analysis of the relative amounts of LC3-II to loading control in each group mentioned above. siNC served as negative control.

Based on these findings, we then asked whether VAMP8 could activate autophagy in glioma cells. To validate this hypothesis, we detected the expression of 4 key autophagy proteins (VPS34, Beclin1, ATG5 and LC3) in VAMP8-overexpressing U87 cells. As shown in Fig. 5H, all of these proteins were dramatically elevated in VAMP8-overexpressing cells compared with the control cells. Moreover, we also transfected VAMP8-overexpressing U87 cells with mCherry-LC3, and the number of the LC3 puncta within each cell was counted to evaluate the level of autophagy in each group.31 We found that forced expression of VAMP8 increased the number of LC3 puncta, but it did not reach statistical significance. However, after a 48 hour exposure to TMZ, cells of both groups exhibited a dramatic boost in LC3 puncta, and VAMP8-overexpressing cells displayed significantly more LC3 puncta than the control group (Fig. 6A and B).

Fig. 6.

Fig. 6.

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VAMP8 increases the number of autophagosomes in glioma cells. (A) Confocal microscopy of VAMP8-transduced U87 cells with or without the presence of 100 μM TMZ treatment for 48 hours. EV served as control, and treatment of 50 nM rapamycin for 24 hours served as positive control. (B) Bar plot of the numbers of LC3 puncta within each cell in each group as indicated. Scale bars: 20 μm.

Next, we examined whether the autophagic flux was also affected by VAMP8. To answer this question, we first treated VAMP8-depleted or control U87 cells with chloroquine (CQ), an autophagosome-lysosome fusion inhibitor, before TMZ exposure. We observed an obvious change in the expression level of LC3-II in control cells, indicating the successful sequestration of autophagosome-lysosome fusion. However, we did not detect significant change in VAMP8-depleted cells, which implied an impaired autophagic flux in VAMP8-depleted cells prior to CQ addition (Fig. 5I). To further verify this finding, we knocked down the expression of STX17 in VAMP8-depleted U87 cells before 72 hours of TMZ treatment. Similar to previous results, TMZ dramatically stimulated LC3-II expression in control cells but not in VAMP8-depleted cells (Fig. 5J), while knockdown of both STX17 and VAMP8 almost totally blocked the elevation of LC3-II when compared with VAMP8 knockdown alone (Fig. 5J). Collectively, these results suggest that VAMP8-induced TMZ resistance may, at least in part, be due to the activation of autophagy.

Discussion

VAMP8 is a SNARE that has been revealed in multiple essential cellular activities,9–11 yet nobody has reported its possible roles in tumor progression and clinical treatments. Our results first demonstrated the clinical significance of VAMP8 as well as its regulatory roles in glioma proliferation and TMZ sensitivity.

Sustained proliferation is one hallmark of cancer; however, few reports refer to the SNARE family in this aspect. Thus, our research first demonstrated a novel function of SNAREs in tumor development by revealing the regulatory role of VAMP8 in glioma progression. Interestingly, previous studies have pointed out that VAMP8 participated in the terminal step of cytokinesis, which is the abscission of the mid body of 2 daughter cells during cell division, the depletion of which could block the process.36,37 Our results agreed with these discoveries because facilitated cell division results in promoted cell proliferation, while blocking mid body abscission brings about cell-cycle arrest and attenuated cell growth.

Chemotherapy resistance is a serious problem that puzzles researchers and threatens patient survival. Temozolomide, the most frequently used chemotherapeutic agent, is also faced with this tricky situation.38,39 In this study, we found a new role for VAMP8 in modulating TMZ sensitivity of glioma cells, which agreed with our former speculation from clinical samples. Autophagy is a profound mechanism that cells employ to survive the outer environment.31,35 Recent studies have demonstrated that resistance to anticancer therapies, including radiation therapy, chemotherapy and targeted therapies, can be enhanced through upregulation of autophagy in different tumors.32–35 In addition, one paper has reported that VAMP8, together with its SNARE partners STX17 and SNAP29, mediated the fusion with autophagosomes and lysosomes. In the present study, we showed for the first time that VAMP8 was responsible for TMZ resistance by glioma cells, partly via autophagy. We observed that forced expression of VAMP8 could activate autophagy, whereas abrogation of VAMP8 suppressed the autophagic flux induced by TMZ. These findings were consistent with several reports indicating that autophagy is a protective mechanism allowing glioma cells to survive TMZ treatment.17,40 Notably, another paper suggested that inhibition of autophagy, after the association between LC3 and autophagosome membrane in glioma cells, was expected to raise the cytotoxicity of TMZ.33 Our data not only validated the speculation that the inhibition of autophagy at the autophagosome-lysosome fusion stage can resensitize TMZ-resistant U87 cells and augment cytotoxicity of TMZ, it also addressed one possible mechanism responsible for the autophagy-associated TMZ sensitivity.

In summary, the SNARE family exerts its functions on many parts of a cell. However, little was reported about its role in tumor biology. This study, exemplified by VAMP8, emphasized the critical functions of SNAREs in tumor progression as well as chemosensitivity and calls for more attention to this familiar yet mysterious family.

Supplementary Material

Supplementary material is available online at Neuro-Oncology (http://neuro-oncology.oxfordjournals.org/).

Funding

This work was supported by the National Natural Science Foundation of China (81372235, 81372706, 81071739 and 81201978).

Supplementary Material

Supplementary Data
supp_17_3_407__index.html (1.4KB, html)

Acknowledgments

We thank Dr. Qiang Huang and Dr. Xinliang Dai (Department of Neurosurgery, Soochow University, Suzhou, China) for providing the glioma progenitor cell SU3 and technical assistance.

Conflict of interest statement. None declared.

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supp_nou219_nou219supp_fig1.tif (531.8KB, tif)
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supp_nou219_nou219supp_table2.doc (42KB, doc)
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