Interactive effects of CDKN2B‐AS1 gene polymorphism and habitual risk factors on oral cancer

Abstract Oral squamous cell carcinoma (OSCC) is a common malignant disease associated with a high mortality rate and heterogeneous disease aetiology. Cyclin dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B‐AS1), is a long noncoding RNA that has been shown to act as a scaffold, sponge, or signal hub to promote carcinogenesis. Here, we attempted to assess the effect of CDKN2B‐AS1 single‐nucleotide polymorphisms (SNPs) on the susceptibility to OSCC. Five CDKN2B‐AS1 SNPs, including rs564398, rs1333048, rs1537373, rs2151280 and rs8181047, were analysed in 1060 OSCC cases and 1183 cancer‐free controls. No significant association of these five SNPs with the risk of developing OSCC was detected between the case and control group. However, while examining the clinical characteristics, patients bearing at least one minor allele of rs1333048 (CA and CC) were more inclined to develop late‐stage (stage III/IV, adjusted OR, 1.480; 95% CI, 1.129–1.940; p = 0.005) and large‐size (greater than 2 cm in the greatest dimension, adjusted OR, 1.347; 95% CI, 1.028–1.765; p = 0.031) tumours, as compared with those homologous for the major allele (AA). Further stratification analyses demonstrated that this genetic correlation with the advanced stage of disease was observed only in habitual betel quid chewers (adjusted OR, 1.480; 95% CI, 1.076–2.035; p = 0.016) or cigarette smokers (adjusted OR, 1.531; 95% CI, 1.136–2.063; p = 0.005) but not in patients who were not exposed to these major habitual risks. These data reveal an interactive effect of CDKN2B‐AS1 rs1333048 with habitual exposure to behavioural risks on the progression of oral cancer.


| INTRODUC TI ON
Oral tumorigenesis is a complex process, during which inherited factors are interconnected with numerous environmental risks in the predisposition to this disease. 1In addition to a plethora of genetic components that control DNA repair, cell proliferation and apoptosis, 2 several acquired risks for oral cancer, including viral infection 3 and habitual consumption of tobacco, alcohol and areca nuts, [4][5][6] have been recognised.Moreover, other causes of oral cancer may involve but not limited to poor oral hygiene, periodontitis, 7 rehearsal micro traumatismes caused by sharp edges of decayed teeth and chronic infections and inflammation. 8These etiological parameters unveil that oral cancer is of a multifactorial nature that, to some extent, affects or is altered by a unique microbial milieu in the oral cavity.This notion has been currently supported by extensive investigations of shifts in the makeup and function of oral microbiota, 9 implicating a causal relationship between oral microbiome and tumorigenesis via direct metabolism of carcinogens and induction of inflammation. 10Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer [11][12][13] and associated with high mortality 14 despite the availability of current treatment options.Taking such heterogeneous nature of disease aetiology into consideration, all risk parameters mentioned above seem to be intertwined and required to assess the incidence and prognosis of OSCC.
6][17][18] Until now, a considerable number of lncRNA genes are found to be linked to a myriad of pathological conditions, 19 including malignant diseases.Among them, cyclin dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1), a lncRNA initially identified in a genome-wide association study of type 2 diabetes and cardiovascular diseases, 20 has been proposed as a promising therapeutic target or prognostic marker for various malignancies, as it is aberrantly expressed in diverse types of tumours. 21Functional investigations suggested that CDKN2B-AS1 promoted carcinogenesis by modulating the expression of tumour suppressor genes, CDKN2A/CDKN2B, in cis, 22,23 indicating a therapeutic potential for cancers.5][26][27] Intriguingly, CDKN2B-AS1 was shown to promote cancer cell survival and glucose metabolism in acute myeloid leukaemia through suppressing the expression of a key regulator of glucose uptake, adiponectin receptor 1. 28 These observations collectively reveal that CDKN2B-AS1, behaving as a scaffold, sponge or signal hub, could orchestrate cancer progression via genomic targeting, transcriptional regulation, epigenetic mechanisms and antisense interference.
Recently, studies using genome-wide or targeted gene approaches have unveiled a genetic association of CDKN2B-AS1 single-nucleotide polymorphism (SNP) with the predisposition to some cancer types.Specifically, CDKN2B-AS1 variants, rs10757274 and rs1333040, were linked to a lower likelihood of developing stage III-IV tumours in papillary thyroid cancer. 29CDKN2B-AS1 rs2151280 has been shown to affect susceptibility to basal cell carcinoma 30 and progression-free survival in multiple myeloma. 31reover, rs1412832 polymorphism demonstrated a genome-wide significant association with increased risk of pancreatic cancer. 32KN2B-AS1 rs1011970 was reported as a risk variant for melanoma and correlated with reduced expression of CDKN2B-AS1, 33 whereas rs11515 polymorphism was connected to breast cancer and elevated expression of CDKN2B-AS1.34 In addition, associations of rs4977756 and rs1412829 with glioma reached a genome-wide threshold.[35][36][37] Nevertheless, the interactive effect of CDKN2B-AS1 gene polymorphisms and habitual risk factors on oral cancer remains unclear.Here, we conducted a case-control study to evaluate the impact of CDKN2B-AS1 SNPs (rs564398, rs1333048, rs1537373, rs2151280 and rs8181047) on the risk of OSCC.

| Subjects
In this study, 1060 male patients with oral cancer and 1183 cancerfree male controls were recruited to assess the impact of CDKN2B-AS1 gene polymorphisms on the risk of developing OSCC, with the approval by the institutional review board of Chung Shan Medical University Hospital in Taichung, Taiwan (CS1-21151).Subjects, enrolled from 2012 to 2021, provided informed written consent at enrolment.Clinical staging and tumour differentiation of OSCC patients were evaluated by a pathologist and rated according to the TNM staging system of the American Joint Committee on Cancer (AJCC). 38Males who had neither self-reported history of cancer of any site nor oral precancerous diseases, such as oral submucous fibrosis, leukoplakia, erythroplakia, verrucous hyperplasia, etc., were enrolled to the control cohort.Information regarding age and habitual risk factors (betel quid chewing, cigarette smoking and alcohol drinking) was obtained from each participant by using a questionnaire.Betel quid chewing and alcohol drinking are defined as behavioural use of areca nut-related products and alcoholic beverages, respectively.Cigarette smoking is defined as current smoking of at least one cigarette per day during the latest 3 months.

| CDKN2B-AS1 SNPs genotyping
0][41][42][43] DNA from the whole blood was isolated by using QIAamp DNA Blood Mini kit (Qiagen). 44,45Evaluation of allelic discrimination for these five loci was carried out by using the TaqMan assay with an ABI StepOne™ Real-Time PCR System (Applied Biosystems) and subsequently analysed by SDS version 3.0 software (Applied Biosystems).

| Statistical analysis
Demographic and clinical parameters between OSCC cases and controls were compared by using Mann-Whitney U test.Relationship of genotype frequencies with the risk or clinical status of OSCC was explored by multiple logistic regression models followed by adjustment for potential confounding variables.The differences of CDKN2B-AS1 levels in the head and neck squamous cell carcinoma (HNSCC) dataset from The Cancer Genome Atlas (TCGA) were compared by Mann-Whitney U test.Data were calculated by using SAS software (v9.4,2013; SAS Institute Inc., Cary, NC).The threshold of difference or association was set by a p value of < 0.05.

| Subject characteristics
The detail of OSCC characters are also presented in the Table 1.In this case-control study, 1060 male patients with OSCC and 1183 cancer-free male controls were recruited to investigate the association of CDKN2B-AS1 gene polymorphisms with the development of oral tumorigenesis.Demographic and clinical features of both study cohorts were evaluated (Table 1).In compliance with the observations from other studies conducted in Central and Southeast Asia, 14,46 significant differences in the frequencies of cigarette smoking, alcohol consumption and betel quid chewing were detected between the case and control group.Among the case group, nodal spread and distal metastasis occurred in 33.3% and 0.7% of patients, respectively.A total of 84.8% of tumours were moderately or poorly differentiated in our study.

| Association of CDKN2B-AS1 SNP with the progression of oral cancer
To test the possible association of CDKN2B-AS1 gene polymorphisms with the development of OSCC, five SNPs of CDKN2B-AS1 gene (rs564398, rs1333048, rs1537373, rs2151280 and rs8181047) were genotyped in this study.The distribution of genotype and allele frequencies for each SNP in OSCC patients and cancer-free controls was examined.No deviation (p > 0.05) from Hardy-Weinberg equilibrium in the controls was detected for all SNPs tested.We failed to observe any significant association of these SNPs with the risk of oral cancer between the case and control group (Table 2).Furthermore, we evaluated the correlations of polymorphic genotypes of CDKN2B-AS1 with clinicopathological characteristics of OSCC patients.We observed that patients who carry at least one minor allele (C) of rs1333048 (CA and CC) were more prone to develop latestage (stage III/IV, AOR, 1.480; 95% CI, 1.129-1.940;p = 0.005) and large-size (greater than 2 cm in the greatest dimension, AOR, 1.347; 95% CI, 1.028-1.765;p = 0.031) tumours as compared with those homologous for the major allele (AA) and (Table 3).These results reveal a potential link of CDKN2B-AS1 gene polymorphisms with the disease progression but not the occurrence in oral cancer.

| Joint effect of CDKN2B-AS1 rs1333048 and habitual risk factors on OSCC progression
As a genetic association of CDKN2B-AS1 rs1333048 with OSCC progression was noted, we further tested whether there is any combined effect of rs1333048 and three major habitual risks   5).However, this association was not seen in patients who were not exposed to betel quid chewing (Table 4) or cigarette smoking (Table 5).Our data indicate that habitual exposure to behavioural risks in combination with CDKN2B-AS1 polymorphisms might influence OSCC progression.

| Clinical insights of CDKN2B-AS1 in head and neck cancer
Since a genetic impact of CDKN2B-AS1 on OSCC was seen, its clinical relevance was explored by conducting extra surveys using public datasets.We showed an increase of CDKN2B-AS1 expression in patients with head and neck squamous cell carcinoma (HNSCC) in The Cancer Genome Atlas (TCGA) dataset (Figure 1).Furthermore, elevated CDKN2B-AS1 levels were associated with lymph node metastasis in HNSCC patients.These results reflect a link of CDKN2B-AS1 induction to HNSCC progression.but not with the disease occurrence was demonstrated in oral cancer.Further stratification analyses revealed that this correlation of rs1333048 genotypes with the advanced stage of disease was only seen in habitual betel quid or cigarette users but not in patients free of these major habitual risks.Our results unveil an interactive effect of CDKN2B-AS1 rs1333048 with habitual exposure to behavioural risks on OSCC progression.

| DISCUSS ION
Like several SNPs of CDKN2B-AS1, rs1333048 was extensively documented for its correlation with many types of malignant diseases, such as breast, 47 thyroid 29 and prostate cancer. 48wever, instead of conferring the susceptibility to oral malignancies, we observed a joint effect of rs1333048 with habitual risk factors on OSCC progression to the advanced stage of disease.
Intriguingly, a genetic association of rs1333048 with periodontitis has been reported 49 and successfully replicated in additional cohorts, 50,51 indicating a potential link of CDKN2B-AS1 variants to oral health.Periodontitis is an inflammatory condition of the oral mucosa and epidemiologically associated with some chronic inflammation-driven comorbidities, including oral cancer. 52In addition, rs1333048 has been found to be associated with the levels of highly sensitive C-reactive protein (hsCRP), which is a biomarker for systemic inflammation. 53These evidence together with our findings led us to tentatively postulate a mechanistic linkage between the aberration of tissue-specific CDKN2B-AS1 functions and overlapping disease phenotypes of chronic inflammation. 54Although the functional analysis of rs1333048 remains unavailable, several diseased associated CDKN2B-AS1 SNPs were shown to affect its free energy of folding, activity of enhancer regions, capacity of miRNA sponge and access to chromatins, leading to predicted alterations in secondary structure, abundance or downstream functionality. 55Collectively, our findings highlight an interplay between CDKN2B-AS1 variants and habitual risks in the promotion of tissue-specific inflammatory responses, potentiating the progression of oral cancer.

Current studies of expression quantitative trait locus (eQTLs)
have demonstrated that most lncRNA gene expression is influenced by genetic variations. 56Through surveying the HNSCC data from TCGA, we noted a link of CDKN2B-AS1 induction with the cancer risk and lymphatic spread.Although it is unclear whether rs1333048 serves as an eQTL, specific genotypes of other CDKN2B-AS1 variants were found to manipulate the expression of CDKN2B-AS1 gene.For instance, rs10811656, rs1333049, rs2383208 and rs10811661 were found to increase or decrease CDKN2B-AS1 expression. 33,57These loci were located within a predicted enhancer region in the downstream of the CDKN2B-AS1 gene and distal to its exon 21.Due to the proximity of rs1333048 to these alleles located within a predicted or proven enhancer, it implies that CDKN2B-AS1 rs1333048 may alter its own expression to affect OSCC progression.In addition, another group of CDKN2B-AS1 variants only impact CDKN2A/B expression but not CDKN2B-AS1 expression. 58,59CDKN2A/B are well-known tumour suppressor genes that have been implicated in many hallmarks of oral cancer 60 and frequently altered in OSCC genome. 61,62Taken together, these SNPs may function as eQTLs in a cell or tissue type-specific manner to influence oral tumorigenesis.
Moreover, some disease associated CDKN2B-AS1 SNPs were reported to interfere with its RNA splicing and subsequently affect the stability of RNA isoforms. 63In lymphocytes, rs10757278 A allele was found to impede the skipping of exon 15, prompting the formation of a circular transcript that is repellent to RNase-mediated cleavage.This circular form of CDKN2B-AS1 RNA can elicit nucleolar stress and p53 activation, leading to the induction of apoptosis and inhibition of proliferation. 646][67] Whether the variation (A > C) affects its own expression or disrupts the interaction with PRC or microRNAs needs further explorations.
Moreover, our findings here may be restricted to particular ethnic populations if not replicated in other cohorts.
In conclusion, our results exhibit an association of CD-KN2B-AS1 rs1333048 with the development of late-stage tumours but not with the disease occurrence in oral cancer.This correlation of rs1333048 genotypes with the advanced stage of disease was only observed in habitual betel quid or cigarette users but not in patients without exposure to these major habitual risks.These findings suggest an interactive effect of CDKN2B-AS1 rs1333048 with habitual consumption of betel quid-related products or cigarettes on OSCC progression.

(
cigarette smoking, betel quid chewing and alcohol drinking) on clinicopathological features of OSCC.For patients who chewed betel quid (n = 775) or smoked (n = 885), a significant association TA B L E 1 Comparisons of clinical and demographic characteristics in patients with OSCC (n = 1060) and cancer-free controls (n = 1183).

) 1 .
875 (0.693-5.070)Note: Cell differentiation grade: grade I, well differentiated; grade II, moderately differentiated; grade III, poorly differentiated.The adjusted odds ratio (AOR) with their 95% confidence intervals were estimated by multiple logistic regression models after controlling for betel quid chewing and alcohol drinking.*p-Value < 0.05 as statistically significant.F I G U R E 1 CDKN2B-AS1 expression levels are increased and associated with clinicopathological parameters in head and neck squamous cell carcinoma (HNSCC).(A) Comparison of CDKN2B-AS1 expression between tumour and normal tissues.(B-D) Correlations of CDKN2B-AS1 expression with the clinical staging (B), lymphatic spread (C) and tumour size (D) of HNSCC from The Cancer Genome Atlas (TCGA) database.The total number of samples is given in brackets.p-Values are calculated with student's t-test.
Association of CDKN2B-AS1 genotypes/alleles with the risk of OSCC.Clinical statuses and genotypic frequencies of CDKN2B-AS1 rs1333048 in OSCC patients who are cigarette smokers or not cigarette smokers.
It is generally accepted that the development and progression of oral cancer are affected by a combination of etiological factors, both inherited and acquired.Here, an association of CDKN2B-AS1 variants, rs1333048, with the development of late-stage tumours TA B L E 2 Note: Cell differentiation grade: grade I, well differentiated; grade II, moderately differentiated; grade III, poorly differentiated.The adjusted odds ratio (AOR) with their 95% confidence intervals were estimated by multiple logistic regression models after controlling for betel quid chewing, cigarette smoking and alcohol drinking.*p-Value < 0.05 as statistically significant.TA B L E 4 Clinical statuses and genotypic frequencies of CDKN2B-AS1 rs1333048 in OSCC patients who are betel quid chewers or not betel quid chewers.Note: Cell differentiation grade: grade I, well differentiated; grade II, moderately differentiated; grade III, poorly differentiated.The adjusted odds ratio (AOR) with their 95% confidence intervals were estimated by multiple logistic regression models after controlling for cigarette smoking and alcohol drinking.*p-Value < 0.05 as statistically significant.TA B L E 5 199 (82.6%) 543 (84.3%) 1.135 (0.764-1.686) 47 (85.4%) 110 (91.7% OSCC; nevertheless, extra investigations are needed to address several limitations in the study.One is that quantitative definition for major habitual risks (betel nut chewing, alcohol drinking and cigarette smoking) was unavailable, which might underestimate the impacts of these environmental risks on the development and progression of oral cancer.Another issue is that the molecular mechanism underlying the role of rs1333048 in OSCC progression remains a puzzle, as the functionality of many other CDKN2B-AS1