Multi-Omics Characterization of Genome-Wide Abnormal DNA Methylation Reveals FGF5 as a Diagnosis of Nasopharyngeal Carcinoma Recurrence After Radiotherapy

Abstract

Background: Aberrant expression and mutations in the fibroblast growth factor (FGF) family play crucial roles in cell differentiation, growth, and migration, contributing to tumor progression across various cancers. Nasopharyngeal carcinoma (NPC), a malignancy prevalent in East Asia, is primarily treated with radiotherapy; however, radioresistance remains a major challenge, leading to recurrence and poor outcomes. While FGFs are known to activate signaling pathways such as MAPK, PI3K/AKT, and JAK/STAT to promote cancer progression, the specific role of individual FGFs in NPC radioresistance remains unclear. Emerging evidence highlights FGF5 as a key player in NPC progression, metastasis, and radioresistance, underscoring its potential as a therapeutic target to overcome treatment resistance and improve clinical outcomes.

Methods: We analyzed single nucleotide variation (SNV) data, gene expression, and DNA methylation patterns using cancer datasets, including TCGA and GTEx, to investigate FGF5 expression. Differentially expressed genes (DEGs) were identified and interpreted using functional enrichment analysis, while survival analysis and gene set enrichment analysis (GSEA) were conducted to identify clinical correlations. DNA methylation patterns were specifically assessed using the HumanMethylation850 BeadChips on tissue samples from nine recurrent and nine non-recurrent NPC patients. Functional assays, including cell viability, migration, invasion, and clonogenic survival assays, were performed to evaluate the effects of FGF5 on NPC cell behavior in vitro and in vivo.

Results: FGF5 showed elevated SNV frequencies across multiple cancers, particularly in HNSC and NPC. DNA methylation analysis revealed an inverse relationship between FGF5 expression and methylation levels in recurrent NPC tumors. Functional assays demonstrated that FGF5 enhances migration, invasion, and radioresistance in NPC cells. High FGF5 expression was associated with reduced distant metastasis-free survival (DMFS) and increased radioresistance, highlighting its role in metastatic progression and recurrence.

Conclusions: FGF5 plays a significant role in the progression and recurrence of nasopharyngeal carcinoma. Its elevated expression correlates with increased migration, invasion, and radioresistance as well as reduced distant metastasis-free survival. These findings suggest that FGF5 contributes to the metastatic and recurrence potential of NPC, making it a potential target for therapeutic intervention in treating these cancers.

Keywords: FGF5; multi-omics characterization; nasopharyngeal carcinoma.

Figure 1

Genomic alterations and expression landscape of the FGF gene family in pan-cancer and head and neck cancer. (a) Oncoplot showing the single nucleotide variant profile of the FGF gene family across pan-cancer. (b) Summary of SNV classes in the FGF gene family. (c) Chromosomal locations of the FGF gene family members. (d) Correlation heatmap of FGF gene family expression across cancer types.

Figure 2

Epigenetic regulation of FGF5 and its role in NPC recurrence post-radiotherapy. (a) Heatmap of methylation signal intensity in NPC patient samples. (b,c) Biological processes (BP), cellular components (CC), and molecular functions (MF) (b) and KEGG pathway (c) enrichment analysis of differentially methylated genes in NPC samples. (d) Gene set enrichment analysis (GSEA) of differentially methylated genes in NPC samples. (e) Boxplot comparing FGF5 methylation signals between recurrence and non-recurrence NPC samples. (f) KEGG enrichment analysis of genes associated with high or low FGF5 methylation signals. (g) Protein–protein interaction network of the FGF gene family; **, p < 0.01.

Figure 3

Clinical relevance and functional analysis of FGF5 in NPC. (a) Correlation profile between methylation levels and mRNA expression of FGF genes. (b) Boxplot comparing TPM values of FGF5 in TCGA head and neck cancer samples and adjacent normal tissues. (c) Boxplot comparing methylation signals of the FGF gene family between patients with and without distant metastasis. (d) Heatmap of FGF gene family expression across normal tissues from the GTEx database. (e) Pan-cancer expression heatmap of the FGF gene family in TCGA datasets, with red indicating upregulation and blue indicating downregulation. (f) RT-PCR analysis of FGF5 mRNA expression in NP69 (normal nasopharyngeal epithelial) and NPC cell lines. (g) Boxplots comparing FGF gene family expression between TCGA head and neck cancer samples and normal tissues; ***, p < 0.001, *, p < 0.05.

Figure 4

Prognostic value and functional impact of FGF5 in NPC progression and metastasis. (a) Bubble heatmap showing the correlation between FGF gene family expression and disease-free interval (DFI) in the TCGA pan-cancer cohort. (b–e) Kaplan–Meier survival analysis of distant metastasis-free survival (DMFS), overall survival (OS), disease-free survival (DFS), and relapse-free survival (RRFS) in NPC patients stratified by high or low FGF5 expression. (f–h) CCK-8 assays evaluating the proliferation ability of HONE-1 (f) and 5-8F (g) cells transfected with vector or FGF5-overexpression plasmid and relative mRNA expression of FGF5 (h). (i) Transwell assays assessing migration and invasion capacity of 5-8F and HONE-1 cells transfected with empty vector or FGF5 overexpression plasmid. (j) Transwell assays evaluating migration and invasion capacity of 5-8F and HONE-1 cells transfected with scrambled control or si-FGF5; ***, p < 0.001, ns, non-significant.

Figure 5

FGF5 enhances radioresistance and modulates radiation response in NPC. (a) qPCR analysis of FGF5 mRNA expression in NPC cell lines 6 h after irradiation (IR) at indicated doses. GAPDH was used as a loading control. (b) qPCR analysis of FGF5 mRNA expression at indicated time points after 6 Gy irradiation. GAPDH was used as a loading control. (c) Validation of FGF5 overexpression in HONE-1 and 5-8F NPC cell lines using qRT-PCR. GAPDH was used as an endogenous control. (d) Clonogenic assays and survival fraction curves of SUNE1 and HONE1 cells stably transfected with FGF5 or empty vector plasmids after exposure to indicated irradiation doses. (e–g) In vivo tumor growth analysis: (e) Subcutaneous xenograft tumors retrieved from mice on day 25 after inoculation, (f) tumor weight measurements, and (g) tumor volume measurements over 25 days in xenografts with or without FGF5 depletion; ***, p < 0.001.