Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.
Keywords: 5-Aza, 5-aza-2′deoxycitidine; AKT, Protein kinase B; AraC, Cytosine arabinoside; CCNA1, Cyclin A1; CCND2, Cyclin D2; CDK4, Cyclin-dependent kinase 4; CDKN1A, Cyclin-dependent kinase inhibitor 1A (p21, Cip1); DNA methylation; DNMT, DNA methyltransferase; EIF2AK2, Eukaryotic translation initiation factor 2-αkinase 2; FASN, Fatty acid synthase; GSK-3, Glycogen synthase kinase 3; Gene expression; HM450, HumanMethylation450; HNSCC, Head and neck squamous cell cancer; Head and neck squamous cell cancer (HNSCC); IGFBP3, Insulin-like growth factor-binding protein 3; ILK, Integrin linked kinase; IPA, Ingenuity pathway analysis; IRF1, Interferon regulatory factor 1; KLF4, Kruppel-like factor 4; KRT19, Keratin 19, LIPG, Endothelial lipase; LXR, Liver X receptor; MGMT, O6-methylguanine DNA methyltransferase; NFATC2, Nuclear factor of activated t-cells cytoplasmic 2; PCNA, Proliferating cell nuclear antigen; PTEN, Phosphatase and tensin homolog; RXR, Retinoid X receptor; Radiation resistance; SAM, S-Adenosylmethionine; SOCS3, Suppressor of cytokine signaling 3; STAT1, Signal transducers and activators of transcription 1; TCGA, The Cancer Genome Atlas; The Cancer Genome Atlas (TCGA); VHL, Von Hippel–Lindau tumor suppressor; dmCpG, differentially methylated CpG; hTERT, human telomerase reverse transcriptase.