Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival

Rivera-Peña,Bianca; Folawiyo,Oluwasina; Turaga,Nitesh; Rodríguez-Benítez,Rosa J.; Felici,Marcos E.; Aponte-Ortiz,Jaime A.; Pirini,Francesca; Rodríguez-Torres,Sebastián; Vázquez,Roger; López,Ricardo; Sidransky,David; Guerrero-Preston,Rafael; Báez,Adriana

doi:10.3892/ol.2024.14223

Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival

Authors:
- Bianca Rivera-Peña
- Oluwasina Folawiyo
- Nitesh Turaga
- Rosa J. Rodríguez-Benítez
- Marcos E. Felici
- Jaime A. Aponte-Ortiz
- Francesca Pirini
- Sebastián Rodríguez-Torres
- Roger Vázquez
- Ricardo López
- David Sidransky
- Rafael Guerrero-Preston
- Adriana Báez
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Affiliations: Department of Biology, University of Puerto Rico, San Juan 00925, Puerto Rico, Department of Otolaryngology‑Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA, Department of General Social Sciences, Faculty of Social Sciences, University of Puerto Rico, San Juan 00925, Puerto Rico, Oral Health Division, Puerto Rico Department of Health, San Juan 00927, Puerto Rico, Department of General Surgery, University of Puerto Rico School of Medicine, San Juan 00936, Puerto Rico, Biosciences Laboratory, IRCCS Instituto Romagnolo per lo Studio dei Tumori ‘Dino Amadori’, Meldola I-47014, Italy, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, Department of Pharmacology, University of Puerto Rico School of Medicine, San Juan 00936, Puerto Rico
Published online on: January 8, 2024 https://doi.org/10.3892/ol.2024.14223
Article Number: 89
Copyright: © Rivera-Peña et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Differentially methylated regions (DMRs) can be used as head and neck squamous cell carcinoma (HNSCC) diagnostic, prognostic and therapeutic targets in precision medicine workflows. DNA from 21 HNSCC and 10 healthy oral tissue samples was hybridized to a genome‑wide tiling array to identify DMRs in a discovery cohort. Downstream analyses identified differences in promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions associated with tumor differentiation, nodal involvement and survival. Genome‑wide DMR analysis showed 2,565 DMRs common to the three subsites. A total of 738 DMRs were unique to laryngeal cancer (n=7), 889 DMRs were unique to oral cavity cancer (n=10) and 363 DMRs were unique to pharyngeal cancer (n=6). Based on the genome‑wide analysis and a Gene Ontology analysis, 10 candidate genes were selected to test for prognostic value and association with clinicopathological features. TIMP3 was associated with tumor differentiation in oral cavity cancer (P=0.039), DAPK1 was associated with nodal involvement in pharyngeal cancer (P=0.017) and PAX1 was associated with tumor differentiation in laryngeal cancer (P=0.040). A total of five candidate genes were selected, DAPK1, CDH1, PAX1, CALCA and TIMP3, for a prevalence study in a larger validation cohort: Oral cavity cancer samples (n=42), pharyngeal cancer tissues (n=25) and laryngeal cancer samples (n=52). PAX1 hypermethylation differed across HNSCC anatomic subsites (P=0.029), and was predominantly detected in laryngeal cancer. Kaplan‑Meier survival analysis (P=0.043) and Cox regression analysis of overall survival (P=0.001) showed that DAPK1 methylation is associated with better prognosis in HNSCC. The findings of the present study showed that the HNSCC subsites oral cavity, pharynx and larynx display substantial differences in aberrant DNA methylation patterns, which may serve as prognostic biomarkers and therapeutic targets.

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1	Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68:394–424. 2018. View Article : Google Scholar : PubMed/NCBI
2	O'Rorke MA, Ellison MV, Murray LJ, Moran M, James J and Anderson LA: Human papillomavirus related head and neck cancer survival: A systematic review and meta-analysis. Oral Oncol. 48:1191–1201. 2012. View Article : Google Scholar : PubMed/NCBI
3	Báez A: Genetic and environmental factors in head and neck cancer genesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 26:174–200. 2008. View Article : Google Scholar : PubMed/NCBI
4	Saracci R: The interactions of tobacco smoking and other agents in cancer etiology. Epidemiol Rev. 9:175–193. 1987. View Article : Google Scholar : PubMed/NCBI
5	Blot WJ, McLaughlin JK, Winn DM, Austin DF, Greenberg RS, Preston-Martin S, Bernstein L, Schoenberg JB, Stemhagen A and Fraumeni JF Jr: Smoking and drinking in relation to oral and pharyngeal cancer. Cancer Res. 48:3282–3287. 1988.PubMed/NCBI
6	Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C, Curado MP, Dal Maso L, Daudt AW, Fabianova E, Fernandez L, et al: Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: Pooled analysis in the International Head and Neck Cancer Epidemiology Consortium. J Natl Cancer Inst. 99:777–789. 2007. View Article : Google Scholar : PubMed/NCBI
7	Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, Zahurak ML, Daniel RW, Viglione M, Symer DE, et al: Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 92:709–720. 2000. View Article : Google Scholar : PubMed/NCBI
8	Mork J, Lie AK, Glattre E, Hallmans G, Jellum E, Koskela P, Møller B, Pukkala E, Schiller JT, Youngman L, et al: Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck. N Engl J Med. 344:1125–1131. 2001. View Article : Google Scholar : PubMed/NCBI
9	Báez A, Almodóvar JI, Cantor A, Celestin F, Cruz-Cruz L, Fonseca S, Trinidad-Pinedo J and Vega W: High frequency of HPV16-associated head and neck squamous cell carcinoma in the Puerto Rican population. Head Neck. 26:778–784. 2004. View Article : Google Scholar : PubMed/NCBI
10	Rivera-Peña B, Ruíz-Fullana FJ, Vélez-Reyes GL, Rodriguez-Benitez RJ, Marcos-Martínez MJ, Trinidad-Pinedo J and Báez A: HPV-16 infection modifies overall survival of Puerto Rican HNSCC patients. Infect Agent Cancer. 11:472004. View Article : Google Scholar : PubMed/NCBI
11	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2017. CA Cancer J Clin. 67:7–30. 2017. View Article : Google Scholar : PubMed/NCBI
12	Park A, Alabaster A, Shen H, Mell LK and Katzel JA: Undertreatment of women with locoregionally advanced head and neck cancer. Cancer. 125:3033–3039. 2019. View Article : Google Scholar : PubMed/NCBI
13	Katzel JA, Merchant M, Chaturvedi AK and Silverberg MJ: Contribution of demographic and behavioral factors on the changing incidence rates of oropharyngeal and oral cavity cancers in northern California. Cancer Epidemiol Biomarkers Prev. 24:978–984. 2015. View Article : Google Scholar : PubMed/NCBI
14	Dayyani F, Etzel CJ, Liu M, Ho CH, Lippman SM and Tsao AS: Meta-analysis of the impact of human papillomavirus (HPV) on cancer risk and overall survival in head and neck squamous cell carcinomas (HNSCC). Head Neck Oncol. 2:152010. View Article : Google Scholar : PubMed/NCBI
15	Shiboski CH, Schmidt BL and Jordan RC: Tongue and tonsil carcinoma: Increasing trends in the U.S. population ages 20–44 years. Cancer. 103:1843–1849. 2005. View Article : Google Scholar : PubMed/NCBI
16	American Joint Committee on Cancer, . AJCC Cancer Staging Manual. 8. New York; Springer: 2016
17	Baliga S, Yildiz VO, Bazan J, Palmer JD, Jhawar SR, Konieczkowski DJ, Grecula J, Blakaj DM, Mitchell D, Henson C, et al: Disparities in survival outcomes among Racial/Ethnic minorities with head and neck squamous cell cancer in the united states. Cancers (Basel). 15:17812023. View Article : Google Scholar : PubMed/NCBI
18	Suárez E, Calo WA, Hernández EY, Diaz EC, Figueroa NR and Ortiz AP: Age-standardized incidence and mortality rates of oral and pharyngeal cancer in Puerto Rico and among Non-Hispanics Whites, Non-Hispanic Blacks, and Hispanics in the USA. BMC Cancer. 9:1292009. View Article : Google Scholar : PubMed/NCBI
19	Fernandez AF, Assenov Y, Martin-Subero JI, Balint B, Siebert R, Taniguchi H, Yamamoto H, Hidalgo M, Tan AC, Galm O, et al: A DNA methylation fingerprint of 1628 human samples. Genome Res. 22:407–419. 2012. View Article : Google Scholar : PubMed/NCBI
20	Taby R and Issa JP: Cancer epigenetics. CA Cancer J Clin. 60:376–392. 2010. View Article : Google Scholar : PubMed/NCBI
21	Ibáñez de Cáceres I and Cairns P: Methylated DNA sequences for early cancer detection, molecular classification and chemotherapy response prediction. Clin Transl Oncol. 9:429–437. 2007. View Article : Google Scholar : PubMed/NCBI
22	Calmon MF, Colombo J, Carvalho F, Souza FP, Filho JF, Fukuyama EE, Camargo AA, Caballero OL, Tajara EH, Cordeiro JA and Rahal P: Methylation profile of genes CDKN2A (p14 and p16), DAPK1, CDH1, and ADAM23 in head and neck cancer. Cancer Genet Cytogenet. 173:31–37. 2007. View Article : Google Scholar : PubMed/NCBI
23	Demokan S and Dalay N: Role of DNA methylation in head and neck cancer. Clin Epigenetics. 2:123–150. 2011. View Article : Google Scholar : PubMed/NCBI
24	Ovchinnikov DA, Cooper MA, Pandit P, Coman WB, Cooper-White JJ, Keith P, Wolvetang EJ, Slowey PD and Punyadeera C: Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients. Transl Oncol. 5:321–326. 2012. View Article : Google Scholar : PubMed/NCBI
25	Herman JG and Baylin SB: Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 349:2042–2054. 2003. View Article : Google Scholar : PubMed/NCBI
26	Esteller M: Epigenetics in cancer. N Engl J Med. 358:1148–1159. 2008. View Article : Google Scholar : PubMed/NCBI
27	Lleras RA, Smith RV, Adrien LR, Schlecht NF, Burk RD, Harris TM, Childs G, Prystowsky MB and Belbin TJ: Unique DNA methylation loci distinguish anatomic site and HPV status in head and neck squamous cell carcinoma. Clin Cancer Res. 19:5444–5455. 2013. View Article : Google Scholar : PubMed/NCBI
28	Bernabe RD: INK4a/ARF/INK4b tumor suppressor locus: Its potential role in head and neck cancer tumorigenesis (Doctoral dissertation) (order no. 3221909). ProQuest Dissertations and Theses Global. 2006.Available from. https://search.proquest.com/docview/304984294?accountid=44820
29	Rivera-Peña B and Báez A: Abstract 4793: Aberrant methylation of CDH1 correlates with poor prognosis in patients with head and neck squamous cell carcinoma (abstract). In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research. Cancer Res. 71 (Suppl 8):4793. 2011. View Article : Google Scholar
30	Irizarry RA, Ladd-Acosta C, Carvalho B, Wu H, Brandenburg SA, Jeddeloh JA, Wen B and Feinberg AP: Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res. 18:780–790. 2008. View Article : Google Scholar : PubMed/NCBI
31	Jaffe AE, Murakami P, Lee H, Leek JT, Fallin MD, Feinberg AP and Irizarry RA: Bump hunting to identify differentially methylated regions in epigenetic epidemiology studies. Int J Epidemiol. 41:200–209. 2012. View Article : Google Scholar : PubMed/NCBI
32	Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, Hou L and Lin SM: Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics. 11:5872010. View Article : Google Scholar : PubMed/NCBI
33	The Cancer Genome Atlas Network, . Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 517:576–582. 2015. View Article : Google Scholar : PubMed/NCBI
34	Aryee MJ, Jaffe AE, Corrada-Bravo H, Ladd-Acosta C, Feinberg AP, Hansen KD and Irizarry RA: Minfi: A flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics. 30:1363–1369. 2014. View Article : Google Scholar : PubMed/NCBI
35	Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV and Laird PW: MethyLight: A high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28:E322000. View Article : Google Scholar : PubMed/NCBI
36	Rettori MM, de Carvalho AC, Longo AL, de Oliveira CZ, Kowalski LP, Carvalho AL and Vettore AL: TIMP3 and CCNA1 hypermethylation in HNSCC is associated with an increased incidence of second primary tumors. J Transl Med. 11:3162013. View Article : Google Scholar : PubMed/NCBI
37	Li LC and Dahiya R: MethPrimer: Designing primers for methylation PCRs. Bioinformatics. 18:1427–1431. 2002. View Article : Google Scholar : PubMed/NCBI
38	Valle B, Rodriguez-Torres S, Kuhn E, Díaz-Montes T, Parrilla-Castellar E, Lawson F, Folawiyo O, Ili-Gangas C, Brebi-Mieville P, Eshleman J, et al: HIST1H2BB and MAGI2 methylation and somatic mutations as precision medicine biomarkers for diagnosis and prognosis of high-grade serous ovarian cancer. Cancer Prev Res (Phila). 13:783–794. 2020. View Article : Google Scholar : PubMed/NCBI
39	Guerrero-Preston R, Valle BL, Jedlicka A, Turaga N, Folawiyo O, Pirini F, Lawson F, Vergura A, Noordhuis M, Dziedzic A, et al: Molecular triage of premalignant lesions in Liquid-Based cervical cytology and circulating Cell-Free DNA from urine, using a panel of methylated human papilloma virus and host genes. Cancer Prev Res (Phila). 9:915–924. 2016. View Article : Google Scholar : PubMed/NCBI
40	Guerrero-Preston R, Michailidi C, Marchionni L, Pickering CR, Frederick MJ, Myers JN, Yegnasubramanian S, Hadar T, Noordhuis MG, Zizkova V, et al: Key tumor suppressor genes inactivated by ‘greater promoter’ methylation and somatic mutations in head and neck cancer. Epigenetics. 9:1031–1046. 2014. View Article : Google Scholar : PubMed/NCBI
41	Anglim PP, Galler JS, Koss MN, Hagen JA, Turla S, Campan M, Weisenberger DJ, Laird PW, Siegmund KD and Laird-Offringa IA: Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer. Mol Cancer. 7:622008. View Article : Google Scholar : PubMed/NCBI
42	Müller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, Marth C and Widschwendter M: DNA methylation in serum of breast cancer patients: An independent prognostic marker. Cancer Res. 63:7641–7665. 2003.PubMed/NCBI
43	Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, Peters JH, DeMeester SR, DeMeester TR, Skinner KA and Laird PW: Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res. 61:3410–3418. 2001.PubMed/NCBI
44	van Asperen CJ, Brohet RM, Meijers-Heijboer EJ, Hoogerbrugge N, Verhoef S, Vasen HF, Ausems MG, Menko FH, Gomez Garcia EB, Klijn JG, et al: Cancer risks in BRCA2 families: Estimates for sites other than breast and ovary. J Med Genet. 42:711–719. 2005. View Article : Google Scholar : PubMed/NCBI
45	Seiwert TY, Zuo Z, Keck MK, Khattri A, Pedamallu CS, Stricker T, Brown C, Pugh TJ, Stojanov P, Cho J, et al: Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas. Clin Cancer Res. 21:632–641. 2015. View Article : Google Scholar : PubMed/NCBI
46	Califano J, Van Der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S, Corio R, Lee D, Greenberg B, Koch W and Sidransky D: Genetic progression model for head and neck cancer: Implications for field cancerization. Cancer Res. 56:2488–2492. 1996.PubMed/NCBI
47	Cai F, Xiao X, Niu X and Zhong Y: Association between promoter methylation of DAPK gene and HNSCC: A meta-analysis. PLoS One. 12:e01731942017. View Article : Google Scholar : PubMed/NCBI
48	Ngan HL, Liu Y, Fong AY, Poon PHY, Yeung CK, Chan SSM, Lau A, Piao W, Li H, Tse J, et al: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling. Life Sci Alliance. 3:e2019005452020. View Article : Google Scholar : PubMed/NCBI
49	Cilona M, Locatello LG, Novelli L and Gallo O: The mismatch repair system (MMR) in head and neck carcinogenesis and its role in modulating the response to immunotherapy: A critical review. Cancers (Basel). 12:30062020. View Article : Google Scholar : PubMed/NCBI
50	Meng RW, Li YC, Chen X, Huang YX, Shi H, Du DD, Niu X, Lu C and Lu MX: Aberrant Methylation of RASSF1A closely associated with HNSCC, a Meta-Analysis. Sci Rep. 6:207562016. View Article : Google Scholar : PubMed/NCBI
51	Moon SM, Kim SA, Yoon JH and Ahn SG: HOXC6 is deregulated in human head and neck squamous cell carcinoma and modulates Bcl-2 expression. J Biol Chem. 287:35678–35688. 2012. View Article : Google Scholar : PubMed/NCBI
52	Carla C, Daris F, Cecilia B, Francesca B, Francesca C and Paolo F: Angiogenesis in head and neck cancer: A review of the literature. J Oncol. 2012:3584722012. View Article : Google Scholar : PubMed/NCBI
53	Mineta H, Miura K, Ogino T, Takebayashi S, Misawa K, Ueda Y, Suzuki I, Dictor M, Borg A and Wennerberg J: Prognostic value of vascular endothelial growth factor (VEGF) in head and neck squamous cell carcinomas. Br J Cancer. 83:775–781. 2000. View Article : Google Scholar : PubMed/NCBI
54	Iwai S, Katagiri W, Kong C, Amekawa S, Nakazawa M and Yura Y: Mutations of the APC, beta-catenin, and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma. J Cancer Res Clin Oncol. 131:773–782. 2005. View Article : Google Scholar : PubMed/NCBI
55	Leethanakul C, Patel V, Gillespie J, Pallente M, Ensley JF, Koontongkaew S, Liotta LA, Emmert-Buck M and Gutkind JS: Distinct pattern of expression of differentiation and growthrelated genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene. 19:3220–3224. 2000. View Article : Google Scholar : PubMed/NCBI
56	Paska AV and Hudler P: Aberrant methylation patterns in cancer: A clinical view. Biochem Med (Zagreb). 25:161–176. 2015. View Article : Google Scholar : PubMed/NCBI
57	Hao X, Luo H, Krawczyk M, Wei W, Wang W, Wang J, Flagg K, Hou J, Zhang H, Yi S, et al: DNA methylation markers for diagnosis and prognosis of common cancers. Proc Natl Acad Sci USA. 114:7414–7419. 2017. View Article : Google Scholar : PubMed/NCBI
58	Chen D, Wang M, Guo Y, Wu W, Ji X, Dou X, Tang H, Zong Z, Zhang X and Xiong D: An aberrant DNA methylation signature for predicting the prognosis of head and neck squamous cell carcinoma. Cancer Med. 10:5936–5947. 2021. View Article : Google Scholar : PubMed/NCBI
59	Stadler ME, Patel MR, Couch ME and Hayes DN: Molecular biology of head and neck cancer: Risks and pathways. Hematol Oncol Clin North Am. 22:1099–1124. 2008. View Article : Google Scholar : PubMed/NCBI
60	Bialik S and Kimchi A: The death-associated protein kinases: Structure, function, and beyond. Annu Rev Biochem. 75:189–210. 2006. View Article : Google Scholar : PubMed/NCBI
61	Gade P, Manjegowda SB, Nallar SC, Maachani UB, Cross AS and Kalvakolanu DV: Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1. Mol Cell Biol. 34:4033–448. 2014. View Article : Google Scholar : PubMed/NCBI
62	Li C, Wang L, Su J, Zhang R, Fu L and Zhou Y: mRNA expression and hypermethylation of tumor suppressor genes apoptosis protease activating factor-1 and death-associated protein kinase in oral squamous cell carcinoma. Oncol Lett. 6:280–286. 2013. View Article : Google Scholar : PubMed/NCBI
63	Jayaprakash C, Varghese VK, Bellampalli R, Radhakrishnan R, Ray S, Kabekkodu SP and Satyamoorthy K: Hypermethylation of Death-associated protein kinase (DAPK1) and its association with oral carcinogenesis-An experimental and meta-analysis study. Arch Oral Biol. 80:117–129. 2017. View Article : Google Scholar : PubMed/NCBI
64	Melchers LJ, Clausen MJ, Mastik MF, Slagter-Menkema L, van der Wal JE, Wisman GB, Roodenburg JL and Schuuring E: Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma. Epigenetics. 10:850–860. 2015. View Article : Google Scholar : PubMed/NCBI
65	Wei DM, Liu DY, Lei DP, Jin T, Wang J and Pan XL: Aberrant methylation and expression of DAPk1 in human hypopharyngeal squamous cell carcinoma. Acta Otolaryngol. 135:70–78. 2015. View Article : Google Scholar : PubMed/NCBI
66	van Kempen PM, van Bockel L, Braunius WW, Moelans CB, van Olst M, de Jong R, Stegeman I, van Diest PJ, Grolman W and Willems SM: HPV-positive oropharyngeal squamous cell carcinoma is associated with TIMP3 and CADM1 promoter hypermethylation. Cancer Med. 3:1185–1196. 2014. View Article : Google Scholar : PubMed/NCBI
67	Stephen JK, Chen KM, Shah V, Havard S, Kapke A, Lu M, Benninger MS and Worsham MJ: DNA hypermethylation markers of poor outcome in laryngeal cancer. Clin Epigenetics. 1:61–69. 2010. View Article : Google Scholar : PubMed/NCBI
68	Strzelczyk JK, Krakowczyk Ł and Owczarek AJ: Aberrant DNA methylation of the p16, APC, MGMT, TIMP3 and CDH1 gene promoters in tumours and the surgical margins of patients with oral cavity cancer. J Cancer. 9:1896–1904. 2018. View Article : Google Scholar : PubMed/NCBI
69	Schmezer P and Plass C: Epigenetic aspects in carcinomas of the head and neck. HNO. 56:594–602. 2008.(In German). View Article : Google Scholar : PubMed/NCBI
70	Singh P, Ravanan P and Talwar P: Death associated protein kinase 1 (DAPK1): A regulator of apoptosis and autophagy. Front Mol Neurosci. 9:462016. View Article : Google Scholar : PubMed/NCBI
71	Humphries MJ and Newham P: The structure of cell-adhesion molecules. Trends Cell Biol. 8:78–83. 1998. View Article : Google Scholar : PubMed/NCBI
72	Riethmacher D, Brinkmann V and Birchmeier C: A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc Natl Acad Sci USA. 92:855–859. 1995. View Article : Google Scholar : PubMed/NCBI
73	Pećina-Slaus N: Tumor suppressor gene E-cadherin and its role in normal and malignant cells. Cancer Cell Int. 3:172003. View Article : Google Scholar : PubMed/NCBI
74	Starska K, Forma E, Lewy-Trenda I, Papież P, Woś J and Bryś M: Diagnostic impact of promoter methylation and E-cadherin gene and protein expression levels in laryngeal carcinoma. Contemp Oncol (Pozn). 17:263–271. 2013.PubMed/NCBI
75	Fan CC, Wang TY, Cheng YA, Jiang SS, Cheng CW, Lee AY and Kao TY: Expression of E-cadherin, Twist, and p53 and their prognostic value in patients with oral squamous cell carcinoma. J Cancer Res Clin Oncol. 139:1735–1744. 2013. View Article : Google Scholar : PubMed/NCBI
76	Fujii R, Imanishi Y, Shibata K, Sakai N, Sakamoto K, Shigetomi S, Habu N, Otsuka K, Sato Y, Watanabe Y, et al: Restoration of E-cadherin expression by selective Cox-2 inhibition and the clinical relevance of the epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma. J Exp Clin Cancer Res. 33:402014. View Article : Google Scholar : PubMed/NCBI
77	Shen Z, Zhou C, Li J, Deng H, Li Q and Wang J: The association, clinicopathological significance, and diagnostic value of CDH1 promoter methylation in head and neck squamous cell carcinoma: A meta-analysis of 23 studies. Onco Targets Ther. 9:6763–6773. 2016. View Article : Google Scholar : PubMed/NCBI
78	McGaughran JM, Oates A, Donnai D, Read AP and Tassabehji M: Mutations in PAX1 may be associated with Klippel-Feil syndrome. Eur J Hum Genet. 11:468–474. 2003. View Article : Google Scholar : PubMed/NCBI
79	Wallin J, Eibel H, Neubüser A, Wilting J, Koseki H and Balling R: Pax1 is expressed during development of the thymus epithelium and is required for normal T-cell maturation. Development. 122:23–30. 1996. View Article : Google Scholar : PubMed/NCBI
80	Xu J, Xu L, Yang B, Wang L, Lin X and Tu H: Assessing methylation status of PAX1 in cervical scrapings, as a novel diagnostic and predictive biomarker, was closely related to screen cervical cancer. Int J Clin Exp Pathol. 8:1674–1681. 2015.PubMed/NCBI
81	Dietrich S and Gruss P: Undulated phenotypes suggest a role of Pax-1 for the development of vertebral and extravertebral structures. Dev Biol. 167:529–548. 1995. View Article : Google Scholar : PubMed/NCBI
82	Su D, Ellis S, Napier A, Lee K and Manley NR: Hoxa3 and pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis. Dev Biol. 236:316–329. 2001. View Article : Google Scholar : PubMed/NCBI
83	Lorincz AT: Virtues and Weaknesses of DNA methylation as a test for cervical cancer prevention. Acta Cytol. 60:501–512. 2016. View Article : Google Scholar : PubMed/NCBI
84	Juodzbalys G, Kasradze D, Cicciù M, Sudeikis A, Banys L, Galindo-Moreno P and Guobis Z: Modern molecular biomarkers of head and neck cancer. Part I. Epigenetic diagnostics and prognostics: Systematic review. Cancer Biomark. 17:487–502. 2016. View Article : Google Scholar : PubMed/NCBI
85	Huang YK, Peng BY, Wu CY, Su CT, Wang HC and Lai HC: DNA methylation of PAX1 as a biomarker for oral squamous cell carcinoma. Clin Oral Investig. 18:801–808. 2014. View Article : Google Scholar : PubMed/NCBI
86	Sun R, Juan YC, Su YF, Zhang WB, Yu Y, Yang HY, Yu GY and Peng X: Hypermethylated PAX1 and ZNF582 genes in the tissue sample are associated with aggressive progression of oral squamous cell carcinoma. J Oral Pathol Med. 49:751–760. 2020. View Article : Google Scholar : PubMed/NCBI
87	Cheng SJ, Chang CF, Ko HH, Lee JJ, Chen HM, Wang HJ, Lin HS and Chiang CP: Hypermethylated ZNF582 and PAX1 genes in mouth rinse samples as biomarkers for oral dysplasia and oral cancer detection. Head Neck. 40:355–368. 2018. View Article : Google Scholar : PubMed/NCBI
88	Morandi L, Gissi D, Tarsitano A, Asioli S, Gabusi A, Marchetti C, Montebugnoli L and Foschini MP: CpG location and methylation level are crucial factors for the early detection of oral squamous cell carcinoma in brushing samples using bisulfite sequencing of a 13-gene panel. Clin Epigenetics. 9:852017. View Article : Google Scholar : PubMed/NCBI
89	Cheng SJ, Chang CF, Ko HH, Liu YC, Peng HH, Wang HJ, Lin HS and Chiang CP: Hypermethylated ZNF582 and PAX1 genes in oral scrapings collected from cancer-adjacent normal oral mucosal sites are associated with aggressive progression and poor prognosis of oral cancer. Oral Oncol. 75:169–177. 2017. View Article : Google Scholar : PubMed/NCBI
90	Hsu YW, Huang RL, Su PH, Chen YC, Wang HC, Liao CC and Lai HC: Genotype-specific methylation of HPV in cervical intraepithelial neoplasia. J Gynecol Oncol. 28:e562017. View Article : Google Scholar : PubMed/NCBI
91	Brain SD, Williams TJ, Tippins JR, Morris HR and MacIntyre I: Calcitonin gene-related peptide is a potent vasodilator. Nature. 313:54–56. 1985. View Article : Google Scholar : PubMed/NCBI
92	Vidal DO, Paixão VA, Brait M, Souto EX, Caballero OL, Lopes LF and Vettore AL: Aberrant methylation in pediatric myelodysplastic syndrome. Leuk Res. 31:175–181. 2007. View Article : Google Scholar : PubMed/NCBI
93	Martinelli CMDS, Lengert AVH, Cárcano FM, Silva ECA, Brait M, Lopes LF and Vidal DO: MGMT and CALCA promoter methylation are associated with poor prognosis in testicular germ cell tumor patients. Oncotarget. 8:50608–50617. 2016. View Article : Google Scholar : PubMed/NCBI
94	Brait M, Begum S, Carvalho AL, Dasgupta S, Vettore AL, Czerniak B, Caballero OL, Westra WH, Sidransky D and Hoque MO: Aberrant promoter methylation of multiple genes during pathogenesis of bladder cancer. Cancer Epidemiol Biomarkers Prev. 17:2786–2794. 2008. View Article : Google Scholar : PubMed/NCBI
95	Wang Y, Zhang D, Zheng W, Luo J, Bai Y and Lu Z: Multiple gene methylation of nonsmall cell lung cancers evaluated with 3-dimensional microarray. Cancer. 112:1325–1336. 2008. View Article : Google Scholar : PubMed/NCBI
96	Zhang B, Liu S, Zhang Z, Wei J, Qu Y, Wu K, Yang Q, Hou P and Shi B: Analysis of BRAF(V600E) mutation and DNA methylation improves the diagnostics of thyroid fine needle aspiration biopsies. Diagn Pathol. 9:452014. View Article : Google Scholar : PubMed/NCBI
97	Loyo M, Brait M, Kim MS, Ostrow KL, Jie CC, Chuang AY, Califano JA, Liégeois NJ, Begum S, Westra WH, et al: Asurvey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma. Int J Cancer. 128:1393–1403. 2011. View Article : Google Scholar : PubMed/NCBI
98	Ismail EA, El-Mogy MI, Mohamed DS and El-Farrash RA: Methylation pattern of calcitonin (CALCA) gene in pediatric acute leukemia. J Pediatr Hematol Oncol. 33:534–542. 2011. View Article : Google Scholar : PubMed/NCBI
99	Paixão VA, Vidal DO, Caballero OL, Vettore AL, Tone LG, Ribeiro KB and Lopes LF: Hypermethylation of CpG island in the promoter region of CALCA in acute lymphoblastic leukemia with central nervous system (CNS) infiltration correlates with poorer prognosis. Leuk Res. 30:891–894. 2006. View Article : Google Scholar : PubMed/NCBI
100	Ji M, Guan H, Gao C, Shi B and Hou P: Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC). BMC Cancer. 11:1472011. View Article : Google Scholar : PubMed/NCBI
101	Morán A, Fernández-Marcelo T, Carro J, De Juan C, Pascua I, Head J, Gómez A, Hernando F, Torres AJ, Benito M and Iniesta P: Methylation profiling in non-small cell lung cancer: Clinical implications. Int J Oncol. 40:739–746. 2012.PubMed/NCBI
102	Guerrero-Preston R, Soudry E, Acero J, Orera M, Moreno-López L, Macía-Colón G, Jaffe A, Berdasco M, Ili-Gangas C, Brebi-Mieville P, et al: NID2 and HOXA9 promoter hypermethylation as biomarkers for prevention and early detection in oral cavity squamous cell carcinoma tissues and saliva. Cancer Prev Res (Phila). 4:1061–1072. 2011. View Article : Google Scholar : PubMed/NCBI
103	Jithesh PV, Risk JM, Schache AG, Dhanda J, Lane B, Liloglou T and Shaw RJ: The epigenetic landscape of oral squamous cell carcinoma. Br J Cancer. 108:370–379. 2013. View Article : Google Scholar : PubMed/NCBI
104	Esteller M, Corn PG, Baylin SB and Herman JG: A gene hypermethylation profile of human cancer. Cancer Res. 61:3225–3229. 2001.PubMed/NCBI
105	Bachman KE, Herman JG, Corn PG, Merlo A, Costello JF, Cavenee WK, Baylin SB and Graff JR: Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers. Cancer Res. 59:798–802. 1999.PubMed/NCBI
106	Darnton SJ, Hardie LJ, Muc RS, Wild CP and Casson AG: Tissue inhibitor of metalloproteinase-3 (TIMP-3) gene is methylated in the development of esophageal adenocarcinoma: Loss of expression correlates with poor prognosis. Int J Cancer. 115:351–358. 2005. View Article : Google Scholar : PubMed/NCBI
107	Smookler DS, Mohammed FF, Kassiri Z, Duncan GS, Mak TW and Khokha R: Tissue inhibitor of metalloproteinase 3 regulates TNF-dependent systemic inflammation. J Immunol. 176:721–725. 2006. View Article : Google Scholar : PubMed/NCBI
108	Mohammed FF, Smookler DS, Taylor SE, Fingleton B, Kassiri Z, Sanchez OH, English JL, Matrisian LM, Au B, Yeh WC and Khokha R: Abnormal TNF activity in Timp3-/-mice leads to chronic hepatic inflammation and failure of liver regeneration. Nat Genet. 36:969–977. 2004. View Article : Google Scholar : PubMed/NCBI
109	Zhang L, Zhao L, Zhao D, Lin G, Guo B, Li Y, Liang Z, Zhao XJ and Fang X: Inhibition of tumor growth and induction of apoptosis in prostate cancer cell lines by overexpression of tissue inhibitor of matrix metalloproteinase-3. Cancer Gene Ther. 17:171–179. 2010. View Article : Google Scholar : PubMed/NCBI
110	Ahonen M, Ala-Aho R, Baker AH, George SJ, Grénman R, Saarialho-Kere U and Kähäri VM: Antitumor activity and bystander effect of adenovirally delivered tissue inhibitor of metalloproteinases-3. Mol Ther. 5:705–715. 2002. View Article : Google Scholar : PubMed/NCBI
111	Spurbeck WW, Ng CY, Strom TS, Vanin EF and Davidoff AM: Enforced expression of tissue inhibitor of matrix metalloproteinase-3 affects functional capillary morphogenesis and inhibits tumor growth in a murine tumor model. Blood. 100:3361–3368. 2002. View Article : Google Scholar : PubMed/NCBI
112	Bian J, Wang Y, Smith MR, Kim H, Jacobs C, Jackman J, Kung HF, Colburn NH and Sun Y: Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3. Carcinogenesis. 17:1805–1811. 1996. View Article : Google Scholar : PubMed/NCBI
113	Arantes LM, de Carvalho AC, Melendez ME, Centrone CC, Góis-Filho JF, Toporcov TN, Caly DN, Tajara EH, Goloni-Bertollo EM and Carvalho AL; GENCAPO: Validation of methylation markers for diagnosis of oral cavity cancer. Eur J Cancer. 51:632–641. 2015. View Article : Google Scholar : PubMed/NCBI
114	Righini CA, de Fraipont F, Timsit JF, Faure C, Brambilla E, Reyt E and Favrot MC: Tumor-specific methylation in saliva: A promising biomarker for early detection of head and neck cancer recurrence. Clin Cancer Res. 13:1179–1185. 2007. View Article : Google Scholar : PubMed/NCBI
115	Zhang HZ, Shan CG, Huang AP and Wang JM: Characterization of gene methylation in human papillomavirus associated-head and neck squamous cell carcinoma. Genet Mol Res. 152016.doi: 10.4238/gmr.15038206.
116	Sartor MA, Dolinoy DC, Jones TR, Colacino JA, Prince ME, Carey TE and Rozek LS: Genome-wide methylation and expression differences in HPV(+) and HPV(−) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis. Epigenetics. 6:777–787. 2011. View Article : Google Scholar : PubMed/NCBI
117	Nakagawa T, Matsusaka K, Misawa K, Ota S, Takane K, Fukuyo M, Rahmutulla B, Shinohara KI, Kunii N, Sakurai D, et al: Frequent promoter hypermethylation associated with human papillomavirus infection in pharyngeal cancer. Cancer Lett. 407:21–31. 2017. View Article : Google Scholar : PubMed/NCBI
118	Nakagawa T, Kurokawa T, Mima M, Imamoto S, Mizokami H, Kondo S, Okamoto Y, Misawa K, Hanazawa T and Kaneda A: DNA Methylation and HPV-Associated Head and Neck Cancer. Microorganisms. 9:8012021. View Article : Google Scholar : PubMed/NCBI
119	Nakahara Y, Shintani S, Mihara M, Ueyama Y and Matsumura T: High frequency of homozygous deletion and methylation of p16(INK4A) gene in oral squamous cell carcinomas. Cancer Lett. 163:221–228. 2001. View Article : Google Scholar : PubMed/NCBI
120	Richards KL, Zhang B, Baggerly KA, Colella S, Lang JC, Schuller DE and Krahe R: Genome-wide hypomethylation in head and neck cancer is more pronounced in HPV-negative tumors and is associated with genomic instability. PLoS One. 4:e49412009. View Article : Google Scholar : PubMed/NCBI
121	van Kempen PM, Noorlag R, Braunius WW, Stegeman I, Willems SM and Grolman W: Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma: A systematic review. Epigenetics. 9:194–203. 2014. View Article : Google Scholar : PubMed/NCBI
122	Camuzi D, Buexm LA, Lourenço SQC, Esposti DD, Cuenin C, Lopes MdSA, Manara F, Talukdar FR, Herceg Z, Ribeiro Pinto LF, et al: HPV infection leaves a DNA methylation signature in oropharyngeal cancer affecting both coding genes and transposable elements. Cancers. 13:36212021. View Article : Google Scholar : PubMed/NCBI