Abstract

Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world. Abnormal methylation can be one reason for this cancer. This study aimed to investigate the DNA promotor methylation status of cancer-associated genes (ATM, APC, CDO1, RB1, TP53, and WIF1) in patients with HNSCC.

Methods: Bisulfite conversion and methylation-sensitive high-resolution melting were used to analyze the DNA methylation levels in normal and tumor tissues in 44 patients.

Results: Significant differences in DNA methylation were observed between tumor and normal tissues for CDO1 and WIF1 genes in all subjects and subgroups (p < 0.05). In the T3 subgroup, a significant correlation was found between CDO1 gene methylation and age in normal tissue. The same correlation was detected for the WIF1 gene methylation in tumor tissue samples in the subgroup with T3 and normal tissue samples in the subgroup with T4 (p < 0.05). In all genes, no significant differences were found between the patient subgroups (T2, T3, T4 stage, primary/recurrent lesion, non-keratinizing/keratinizing SCC, age before/after 50, and smokers/non-smokers).

Conclusion: Changes in the expressions of CDO1 and WIF1 genes can affect the mechanisms of the occurrence and development of HNSCC. Methylation in the ATM, APC, RB1, and TP53 genes is not specific to HNSCC.

Introduction

Epigenetic gene regulation changes (such as DNA methylation, histone modification, chromatin remodeling, and non-coding RNA) and DNA mutation are the basis of the molecular mechanisms of cancerogenesis and tumorigenesis. Therefore, studying epigenetic markers for diagnosis, prognosis, and cancer prevention is particularly interesting. Detection of DNA methylation levels has recently been used as biomarkers for early diagnosis and prognosis to identify target genes for drug therapy.

The hypomethylation of single CpG dinucleotides has been one of the first changes found in tumor cells1. A large part of the investigation has been carried out during DNA methylation in the promoter region of various genes2, 3. There are changes in the methylation levels of general and tissue-specific cell genes in the ontogenesis in utero and post-natal periods.

The non-mutagenic etiology of cancer, such as disturbance of DNA methylation, is influenced by age, diet, transplacental, environmental, and occupational exposure. Moreover, its dual ability to be reversible and transgenerational demands a specific approach to interpreting results. Age-related DNA methylation is associated with different diseases4 and sex hormone status in some cancer types5. There is a significant gap in understanding the association between the age-expected DNA methylation level and cancer6. In particular, no data exist regarding pre- and postmenopausal differences in DNA methylation levels and their effects on cancer development.

The most intensive processes are in the genome of active proliferating cells, such as epithelial cells. Therefore, epithelial cells in various cancers are convenient models for studying epigenetic malformation. Head and neck squamous cell carcinoma (HNSCC) is an example of oncopathology with epithelial tissue damage.

HNSCC corresponds to the majority of cases (~90%) of head and neck cancers (HNCs)7. HNCs are rated 6–7 in global prevalence among cancers, with 700,000 newly registered cases and 470,000 deaths a year8. The therapeutic options for HNCs include surgical treatment, radiotherapy, and chemotherapy. The management tactics should be personalized by a multidisciplinary board comprising an oncological surgeon, a chemotherapist, a radiotherapist, a psychologist, a rehabilitation specialist, and a dentist9, 10.

Despite the advanced diagnostics, cancer screening, and HPV vaccination programs, 60–70% of the cases are diagnosed as late as during stage III–IV, associated with low life expectancy and high risks of recurrence11. Therefore, finding new potential targets for therapy is crucial.

The main risk factor for HNSCC is smoking. In some publications, it has been shown that smokers have a 10-fold higher risk of developing the disease than non-smokers. Smoking, in combination with frequent alcohol consumption, increases the risk more than 35-fold12, 13, 14. Other risk factors include ultraviolet and ionizing radiation, various toxic compounds, a weakened immune system, a diet low in vitamins A and B, age over 40, male, and some viral infections15.

DNA methylation also varies due to several factors, including age and disease status. Silenced genes are often methylated, while active genes remain largely unmethylated16, 17. DNA methylation is the most studied epigenetic mechanism in HNSCC. It allows us to observe changes in methylation patterns in a whole genome and individual genes that can be used to identify new disease biomarkers18. Various biomaterials can be used for studies, including liquid biopsy samples, which provide a noninvasive alternative for early cancer detection. In particular, Zhou C. studied 27 aberrantly methylated genes with altered expression and showed that FAM135B methylation is a favorable independent prognostic marker for overall survival in patients with HNSCC19.

We previously investigated the DNA methylation status of some tumor-associated genes (CDO1, MEST, RASSF1A, RASSF2, RASSF5, and WIF1) in patients with HNSCC20. We observed significant differences in DNA methylation levels between tumor and normal tissues in the CDO1 and WIF1 genes in all patient groups and subgroups (larynx and other cancers, SCC keratinizing and non-keratinizing, primary and recurrent tumor, and smokers and non-smokers)21. The methylation level in the CDO1 gene in tumor tissue was significantly increased in the T4 and T3 stage subgroups over the T2 stage22.

For further research, we investigated another group of tumors and normal tissue from patients with HNSCC. We chose other cancer-associated genes: ATM, APC, RB1, and TP53. We also used these earlier studied genes (CDO1 and WIF1), as they had shown notable results in other patients (Table 1). The selected genes play a considerable role in the regulation of cell proliferation, differentiation, and apoptosis, the disruption of which can lead to oncopathology.

This study aimed to investigate the DNA promotor methylation status of cancer-associated genes (ATM, APC, CDO1, RB1, TP53, and WIF1) in patients with HNSCC.

Table 1.

Characteristics of studied genes

Symbol Gene name Location Exon count Gene ID Transcripts MIM Gene type Gene function References APC GS; DP2; DP3; BTPS2; DESMD; DP2.5; PPP1R46 Adenomatous Polyposis Coli 5q22.2 20 5624 NM_000038.6 611731 protein-coding tumor suppressor 37; 39 ATM AT1; ATA; ATC; ATD; ATE; ATDC; TEL1; TELO1 Ataxia-telangiectasia-mutated 11q22.3 67 472 NM_000051.4 607585 protein-coding cell cycle regulation. 42-44 CDO1 (CDO-I) Cysteine dioxygenase type 1 5q22.3 9 1036 NM_001323565.2 603943 protein-coding tumor suppressor 26-28 TP53 (P53; BCC7; LFS1; BMFS5; TRP53) Tumor Protein P53 17p13.1 11 7157 NM_000546.6. 191170 protein-coding tumor suppressor 45-50 RB1 (RB; pRb; OSRC; pp110; p105-Rb; PPP1R130; p110-RB1) Retinoblastoma 1 13q14.2 27 5925 NM_000321.3... 614041 protein-coding cell cycle regulator, tumor suppressor 51-53 WIF1 (WIF-1) WNT inhibitory factor 1 12q14.3