Analysis of MUC6 polymorphisms on the clinicopathologic characteristics of Asian patients with oral squamous cell carcinoma

Abstract Head and neck squamous cell carcinomas (HNSCCs) are generally associated with tobacco consumption, alcohol abuse or both. Mucins (MUCs) are high‐molecular‐weight glycoproteins produced by many epithelial tissues. Many studies have indicated that MUCs play an important role in cancer metastasis. MUC6 expression has been observed in gastric and oncocytic phenotypes and plays an important role during cancer progression. We found that levels of MUC6 are lower in Asian HNCC patients and affect the disease‐free survival of HNCC patients. Next, we investigated the combined effect of MUC6 polymorphisms and exposure to environmental carcinogens on the susceptibility to and clinicopathological characteristics of HNCC. Three single‐nucleotide polymorphisms (SNPs) of MUC6 (rs7481521, rs6597947 and rs61869016) were analysed using real‐time PCR. After adjusting for other co‐variants, we found that carrying a CC genotype at MUC6 rs6597947 led to a lower risk of developing oral squamous cell carcinoma (OSCC) than wild‐type carriers among non‐betel‐quid chewers. Moreover, male oral cancer patients who carried the AA + CC genotype at MUC6 rs6597947 had a lower risk of lymph node metastasis than other genotypes, suggesting a significant functional compromise and decompensated disease. Therefore, our findings suggest that genetic variations in MUC6 may correlate to OSCC and indicate the progression in OSCC patients.


| INTRODUC TI ON
Head and neck squamous cell carcinomas (HNSCCs) are a group of biologically similar cancers originating in the oral cavity, nasopharynx, oropharynx, hypopharynx and larynx, which histologically present, mainly, as squamous cell carcinomas. HNSCC is the sixth most common malignancy worldwide, and one of the most common types of HNSCC is oral squamous cell carcinoma (OSCC). [1][2][3][4] Despite significant advances in combined multidisciplinary diagnoses and treatments, only 30%-50% of HNCC patients worldwide survive more than 5 years after initial diagnosis. 5 This poor prognosis is due to the occurrence of distant metastases and local recurrence. 6 OSCC occurs as a result of various genetic changes due to long-term exposure to environmental carcinogens. The main risk factors for OSCC are chronic inflammation, tobacco use, alcohol consumption, betel-nut chewing and viral infection. Mucin (MUC) is the main component of any mucus secretion, which provides the mucus with its biophysiochemical properties as a function of its characteristics and a degree of glycosylation. 7,8 Moreover, mucins play a role in both physiological and pathological conditions. [9][10][11][12][13] Aberrant expression of mucins can lead to a loss in epithelial cell polarity and promote epithelial-mesenchymal transition (EMT), leading to increased cell motility and invasion, a crucial step in tumorigenesis. 9,14,15 Reports suggest that MUC1 modulates the impact of hypoxia in head and neck squamous cell carcinoma (HNSCC) cells by regulating HIF-1α. 16 MUCs can be found either membrane-bound or secreted with or without gel formation. Among the membrane-bound MUCs, MUC1, 4, 15 and 16 have received the most attention, whereas MUC2, 5B, 5AC, 6 and 19 have received the most attention among the gel-forming MUCs. 9,17,18 Over the last decade, numerous independent works have evaluated the clinical significance of MUC protein expression in cancer as well as its ability to predict outcomes. [19][20][21][22] Despite these efforts, the findings from these studies have been inconsistent and no definitive conclusion has yet been reached. One of the primary elements of the mucus barrier in the stomach is Mucin 6 (MUC6), which is produced by the pyloric gland cells of the gastric sinus and the mucus neck cells in the lower layer of the gastric mucosa. Both gastric and cancer cell types exhibit MUC6 expression. In our previous study, we found that genetic variations in MUC6 may correlate to hepatocellular carcinoma and indicate the progression in hepatocellular carcinoma patients. 23 Research has revealed that the methylation of the MUC6 promoter may cause a considerable decrease in MUC6 expression in gastric cancer and drive its progression. 21 Furthermore, we found low MUC6 expression reduced the risk of OSCC. However, the detailed role of the tissue expression of mucins in OSCC tumour cells is not well understood.
Studies have revealed several genetic predisposition factors that could play a role in HNSCC. One of the most prevalent forms of DNA sequence variations is single-nucleotide polymorphisms (SNPs), which have been demonstrated to have the capability to predict the probability of developing cancer. [24][25][26] SNPs can change the expression or function of proteins, affecting the development of cancer. A connection was discovered between the expression of MUC6 SNPs and the onset of chronic atrophic gastritis and hepatocellular carcinoma. 27 However, the exact role of MUC6 SNPs in cancer progression and development in Taiwanese OSCC patients remains poorly investigated. In addition, there is a strong correlation between the incidence of oral squamous cell carcinoma and gender in Taiwan.
The occurrence ratio between males and females is approximately 9:1, which may be related to the fact that the majority of betel-nut chewers are male. Therefore, the current study aims to investigate the correlations between three MUC6 SNPs (rs61869016, located in the 5′-UTR; rs6597947, located in the 5′-UTR and rs7481521, located in the exon) and Taiwanese male patients with OSCC and their cancer prognosis. Data on gender and age, as well as habits of betel-quid chewing, cigarette smoking and alcohol consumption, were gathered from each participant. The consumption of more than two alcoholic beverages per day by a subject was classified as alcohol consumption. a significant functional compromise and decompensated disease. Therefore, our findings suggest that genetic variations in MUC6 may correlate to OSCC and indicate the progression in OSCC patients.

K E Y W O R D S
lymph node metastasis, MUC6, oral squamous cell carcinoma, single-nucleotide polymorphisms If an individual smoked one cigarette per day in the past 3 months, they were considered to have a chronic smoking habit. The study received approval from the Institutional Review Board at Chung Shan Medical University Hospital.

| Comprehensive analyses of MUC6 from The Cancer Genome Atlas (TCGA)
UALCAN is a comprehensive, user-friendly and interactive web resource for analysing cancer omics data (http://ualcan.path.uab.edu/ index.html). UALCAN uses TCGA level 3 RNA-seq and clinical data from 31 cancer types. 28 Gene Expression Profile Interactive Analysis 2 (GEPIA2, http://gepia2.cance r-pku.cn/#index) is an updated version of GEPIA, used to analyse the RNA-sequencing expression data of 9736 tumours and 8587 normal samples from the TCGA and the GTEx project using a standard processing pipeline. 29 In this study, we used UALCAN and GEPIA2 for analyses of tumour/normal differential expression and overall survival of MUC6 expression in HNSCC patients.

| Selection of MUC6 polymorphisms
For this study, three SNPs in MUC6 (NM_005961.3) were selected from the International HapMap Project data. These SNPs were rs61869016, located in the 5′-UTR; rs6597947, located in the 5′-UTR and rs7481521, located in the exon of MUC6.

| Statistical analyses
To evaluate the differences in age and demographic characteristics between the control group and the OSCC patients, the Mann-Whitney U test was used. The odds ratios with 95% confidence intervals (CIs) were estimated using logistic regression models. A p value of <0.05 was considered significant. The data were analysed using SAS statistical software.

| RE SULTS
To investigate the clinical impact of MUC6 on HNSCC progression, we used UALCAN and GEPIA 2 to assess the relationship between the cellular levels of MUC6 of the controls and the HNSCC patients and the overall or disease-free survival of the HNSCC patients. Initially, however, there was no difference in MUC6 levels between the controls and the HNSCC patients (p = 0.1995; Figure 1A). Upon further analyses, we found that levels of MUC6 in the controls were significantly higher than in Asian HNSCC patients (p = 0.0499; Figure 1B).
Interestingly, it was found that expression of MUC6 affects the disease-free survival of HNSCC patients. The HNSCC patients with high MUC6 expression had significantly longer disease-free survival than those with low MUC6 expression ( Figure 1C). These results imply that the regulation of MUC6 might be involved in HNSCC progression.
To identify possible factors causing OSCC in clinical practice, a total of 1115 healthy controls and 837 male OSCC patients were recruited for this case-cohort study. According to our analyses of OSCC patients, we found significant differences in age (p < 0.001), betel-nut chewing (p < 0.001), smoking (p < 0.001) and alcohol consumption (p < 0.001) between the OSCC patients and the healthy group (Table 1).
To reduce the possibility of confounding by several environmental factors, AORs and their corresponding 95% CIs were estimated by multivariate logistic regression models after controlling for age, betel-quid chewing, cigarette smoking and alcohol drinking.
The genotype distributions and the associations between OSCC and MUC6 SNPs are presented in Table 2. The alleles with the highest frequency of distribution in MUC6 rs7481521, rs6597947 and rs61869016 were homozygous C/C, homozygous A/A and homozygous T/T, respectively, in the OSCC patients and the controls. However, individuals with the rs7481521, rs6597947 and rs61869016 polymorphisms showed no reduction in OSCC risk compared to the wild-type individuals.
According to recent research, betel-nut chewing is an important risk factor for OSCC progression. Therefore, genotyping and allele frequency data for MUC6 SNPs among betel-quid chewers are shown in Table 3. Unfortunately, the results showed no reduction in OSCC risk compared to the wild type among betel-quid chewers. Because the risk factor of betel-nut chewing is so influential, we removed this risk for further analyses. Surprisingly, the results showed that patients with the C/C genotype of rs6597947 SNP (OR = 0.394, 95% CI: 0.194-0.816, p = 0.012) had a reduced risk of OSCC compared to other genotypes among non-betel-quid chewers ( Table 4).
The ORs analysed by their 95% CIs were estimated using logistic regression models: >T2, multiple tumours of more than 5 cm or a tumour involving a major branch of the portal or hepatic vein(s); *p < 0.05 was considered statistically significant.
Moreover, we further compared the associations between the  (Table 5).

| DISCUSS ION
SNPs, or single-nucleotide polymorphisms, are variations at the DNA level found in each human cell. These variations, influenced by environmental factors, contribute to the diversity of human phenotypes and can indicate a person's susceptibility to various diseases, including cancer. 30,31 To distinguish the impact of SNPs from other types of mutations, the frequency of each polymorphism must be higher than that of a single natural mutation.
Changes in gene function caused by mutations or gene polymorphisms can result in increased cancer risk and specific disease phenotypes. 32,33 The laboratory evidence shows that an abnormal increase in the expression of tumour-associated MUCs, mismatched features and shortened glycans is a common occurrence in various epithelial cancers. [34][35][36][37][38] However, clinical findings are not always consistent. While most research suggests that high MUC levels are associated with poorer clinicopathological parameters and a worse prognosis, there are some studies that have reached the opposite conclusion. [39][40][41][42] The role of MUC1 in the EMT process, which enables cancer cells to become invasive and metastatic, has been directly proven by several studies. The importance of MUC6 in cancer is well known, but its precise role in tumorigenesis remains disputed as both oncogenic and inhibitory effects have been demonstrated. 19,21,43 For example, the SNP rs7481521 of the MUC6 gene showed a significant decrease in risk for homozygous carriers and a clear relationship between the number of alleles and chronic atrophic gastritis. 27 Moreover, MUC6 expression is high in the early stages of pancreatic tumour progression, but it decreases or is lost in later stages. 44,45 However, the connection between MUC6 variations and risk factors for OSCC is still unclear. This study provides clarification on the role of MUC6 SNPs in OSCC susceptibility and other related conditions. Taiwan has a higher incidence of head and neck cancer due to betel-nut chewing and diet. Surprisingly, in our results, we observed that MUC6 rs6597947 (AC + CC genotype) had a significantly lowerlevel lymph node metastasis compared to the AA genotype in patients with OSCC ( Table 5). As shown in Table 4, patients with the

TA B L E 2
Odds ratios (ORs) and 95% confidence intervals (CIs) of oral cancer associated with MUC6 genotypic frequencies. The AORs with their 95% confidence intervals were estimated using multiple logistic regression models after controlling for age, cigarette smoking and alcohol drinking.
*p value <0.05 was considered statistically significant in Bold.

TA B L E 5
Odds ratios (ORs) and 95% confidence intervals (CIs) of clinical statuses associated with genotypic frequencies of MUC6 rs6597947 in male oral cancer patients.  The adjusted odds ratios (AORs) with their 95% confidence intervals were estimated using multiple logistic regression models after controlling for age, betel-quid chewing, cigarette smoking and alcohol drinking. AOR = 0.740 (0.575-0.953).

Variable
*p value <0.05 was considered statistically significant in Bold.
the detailed mechanisms of MUC6 SNPs in OSCC require future elucidation.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study are available on request from the corresponding author.

I N FO R M E D CO N S E NT
Informed consent was obtained from all subjects involved in the study.