Elevated VAMP8 expression promotes cervical cancer progression by enhancing autophagy via HIF-1 pathway

Abstract

Background: Cervical cancer, prevalent in low- and middle-income countries, is primarily caused by high-risk HPV16. Vesicle-Associated Membrane Protein 8 (VAMP8), involved in vesicle trafficking and autophagy, may influence HPV16-related cervical cancer progression.

Methods: VAMP8 expression was evaluated in cervical tissue specimens from patients with HPV16-positive lesions (including low- and high-grade squamous intraepithelial lesions and cancer) and HPV-negative normal controls using proteomics, qPCR, and immunohistochemistry. A Cox proportional hazards model for prognosis was developed using immunohistochemical data from a cohort of cervical cancer patients. The clinical significance of VAMP8 was further assessed using RNA-seq and clinical data from The Cancer Genome Atlas-Cervical Cancer (TCGA-CESC) cohort. The effects of VAMP8 on autophagy and tumor progression were examined in HPV16 E6/E7-immortalized cervical epithelial cells (Ect1/E6E7) and cervical cancer cell lines (SiHa, HeLa, C-33A) in vitro, and in a SiHa xenograft model in vivo. Transcriptomic analysis of Ect1/E6E7 and SiHa cells identified VAMP8-regulated pathways. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays in SiHa cells were used to confirm the regulation of the HIF-1 pathway.

Results: VAMP8 was upregulated in HPV16-positive samples, particularly in low-grade squamous intraepithelial lesions (LSIL). Elevated VAMP8 correlated with poor survival outcomes and advanced tumor stages. VAMP8 enhanced autophagy and reduced proliferation and invasiveness in HPV16-positive cervical cells but increased in established cancer cell lines. In vivo, VAMP8 overexpression promoted tumor growth and autophagy. The HIF-1 pathway emerged as a key regulatory axis of VAMP8, enhancing hypoxic responses and angiogenesis.

Conclusion: Elevated VAMP8 in HPV16-associated cervical cancer promotes tumor progression by enhancing autophagy via the HIF-1 pathway, suggesting its potential as a diagnostic and prognostic biomarker.

Keywords: Autophagy; Biomarker; Cervical cancer; HIF-1 pathway; HPV16; VAMP8.

Fig. 1

VAMP8 Expression analysis in cervical tissues and cells. A, B Proteomic analysis (A) and qPCR (B) of VAMP8 in HPV16-positive and HPV16-negative tissues. C qPCR measurement of VAMP8 mRNA levels in cervical cell lines. D Immunohistochemical staining of VAMP8 in postoperative specimens

Fig. 2

Prognostic analysis of low/high VAMP8 expression based on immunohistochemical staining of postoperative cervical cancer specimens. A, B Kaplan–Meier curves for overall survival (OS) (A) and disease-free survival (DFS) (B) in patients with low/high VAMP8 expression

Fig. 3

Analysis of VAMP8 expression in cervical cancer tissues and its clinical correlation based on the TCGA-CESC database. A Comparison of VAMP8 expression between normal and tumor tissues. B Receiver operating characteristic (ROC) curve illustrating the diagnostic performance of VAMP8. C VAMP8 expression across different histological subtypes (adenocarcinoma vs. squamous cell carcinoma). D-F VAMP8 expression in relation to pathological T stage (D), pathological M stage (E), and clinical stage (F)

Fig. 4

Correlation analysis between VAMP8 expression and immune cell infiltration in cervical cancer. A Correlation between VAMP8 expression and various immune cell types. The size and color of the circles represent the strength of correlation and significance, respectively. B Boxplots comparing immune cell enrichment scores for DC, aDC, Tcm, and NK cells between high and low VAMP8 expression groups. C, D Scatter plots illustrating the relationship between VAMP8 expression and the enrichment of DCs and NK cells

Fig. 5

VAMP8 modulation and autophagic vesicles in cervical cell lines. A qPCR analysis confirming VAMP8 knockdown and overexpression in Ect1/E6E7, SiHa, HeLa, and C-33A cell lines. shNC: negative control for knockdown; shVAMP8: VAMP8 knockdown; OE-NC: negative control for overexpression; OE-VAMP8: VAMP8 overexpression. B Transmission electron microscopy images of autophagic vesicles in VAMP8 knockdown and overexpression cell lines across the four cervical cell lines. Scale bar = 2 μm

Fig. 6

Effects of VAMP8 knockdown and overexpression on cell proliferation measured by CCK-8 assay. A–D Cell proliferation was assessed at 0, 24, 48, and 72 h in Ect1/E6E7, SiHa, HeLa, and C-33A cell lines, respectively

Fig. 7

Effects of VAMP8 knockdown and overexpression on cell migration and invasion in cervical cell lines. A-D Transwell migration and invasion assays were performed in Ect1/E6E7, SiHa, HeLa, and C-33A cell lines, with relative cell numbers quantified

Fig. 8

Flow cytometry analysis of cell cycle distribution and apoptosis in cervical cell lines. A Cell cycle distribution analysis after VAMP8 knockdown or overexpression. B Annexin V-APC/PI staining for apoptosis detection with VAMP8 knockdown or overexpression

Fig. 9

Effects of VAMP8 on tumor growth and autophagy in vivo. A Representative images of excised tumors from nude mice (n = 4 per group) at day 28 post-injection. B Tumor volume growth curve measured over 28 days. C Violin plots showing tumor weight comparison across groups. D Transmission electron microscopy images of tumor tissues displaying autophagic vesicles in different experimental conditions

Fig. 10

Analysis of VAMP8-regulated genes and enriched pathways in cervical cancer. A Volcano plots showing differentially expressed genes between VAMP8-overexpressing and control Ect1/E6E7 and SiHa cells. B Volcano plot of single-gene differential expression analysis for VAMP8 in the TCGA-CESC dataset. C Heatmap showing the correlation analysis between VAMP8 expression and other genes in the TCGA-CESC dataset. D Venn diagram depicting the overlap of genes identified through differential expression and correlation analyses across the two cell lines and TCGA data. E GO and KEGG enrichment analysis of the common genes, highlighting key biological processes and pathways

Fig. 11

Effects of VAMP8 overexpression on HIF-1 signaling, cell viability, and angiogenesis in SiHa cells. A Relative mRNA expression levels of HIF-1α, VEGFA, and ENO1 in VAMP8-overexpressing cells compared to controls. B Relative protein expression levels of HIF-1α, VEGFA, and ENO1 in VAMP8-overexpressing cells. C ChIP assay showing the relative enrichment of HIF-1α binding to the VEGFA and ENO1 promoters. D Luciferase reporter assay of HRE-linked activity in VAMP8-overexpressing cells. E Relative hypoxic cell viability at 24 h, 48 h, and 72 h. F Colony formation assay showing the relative number of colonies formed under hypoxia. G HUVEC tube formation assay displaying the total number of tubes and total tube length formed in the presence of conditioned media from VAMP8-overexpressing cells

Fig. 12

Schematic representation of VAMP8’s role in HPV16-associated cervical cancer progression