HPV-16 E6 and E7 expression drives transcription of p14(ARF) and p16(INK4a) prior to Intragenic methylation of CDKN2A.
Main Article Content
Abstract
Human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinomas (OPSCC), associated with improved patient outcome, exhibit increased expression of CDKN2A tumor suppressor p16(INK4a), while inactivation of the CDKN2A locus is frequently observed in HPV negative HNSCC. We recently identified a novel region of intragenic DNA hypermethylation within the CDKN2A locus that correlates with RNA expression of two CDKN2A transcripts and improved patient outcome in oropharyngeal and laryngeal SCC. Given the importance of CDKN2A expression in OPSCC, we chose an in vitro model using primary keratinocytes transduced with HPV-16 oncogenes E6 and E7 to study the relationship between CDKN2A methylation and expression. RNA levels for CDKN2A transcripts, p14(ARF) and p16(INK4a), and intragenic CDKN2A methylation were assessed in the primary cells and after transduction with E6 and E7 at two time points. CDKN2A expression increased early post-transduction, while there was a clear delay in intragenic methylation. Methylation eventually increased suggesting that intragenic methylation is not required for transcription at the CDKN2A locus, and that methylation may be a consequence of open and active chromatin.
Keywords:
CDKN2A, p16, HPV, HNSCC, DNA methylation, transcription
Article Details
How to Cite
BEN-DAYAN, Miriam M et al.
HPV-16 E6 and E7 expression drives transcription of p14(ARF) and p16(INK4a) prior to Intragenic methylation of CDKN2A..
Medical Research Archives, [S.l.], v. 8, n. 5, may 2020.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/2113>. Date accessed: 27 apr. 2024.
doi: https://doi.org/10.18103/mra.v8i5.2113.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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28. Schlecht NF, Brandwein-Gensler M, Nuovo GJ, et al. A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer. Mod Pathol. 2011;24(10):1295-1305.
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30. Alam H, Sehgal L, Kundu ST, Dalal SN, Vaidya MM. Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells. Mol Biol Cell. 2011;22(21):4068-4078.
31. Fuchs E, Nowak JA. Building epithelial tissues from skin stem cells. Cold Spring Harb Symp Quant Biol. 2008;73:333-350.
32. Watt FM. Involucrin and other markers of keratinocyte terminal differentiation. J Invest Dermatol. 1983;81(1 Suppl):100s-103s.
33. Wong CW, LeGrand CF, Kinnear BF, et al. In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics. Sci Rep. 2019;9(1):18561.
34. Jones DL, Alani RM, Munger K. The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev. 1997;11(16):2101-2111.
35. Pei XF, Sherman L, Sun YH, Schlegel R. HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium. Carcinogenesis. 1998;19(8):1481-1486.
36. Zehbe I, Richard C, DeCarlo CA, et al. Human papillomavirus 16 E6 variants differ in their dysregulation of human keratinocyte differentiation and apoptosis. Virology. 2009;383(1):69-77.
37. Salazar CR, Smith RV, Garg MK, et al. Human papillomavirus-associated head and neck squamous cell carcinoma survival: a comparison by tumor site and initial treatment. Head Neck Pathol. 2014;8(1):77-87.
38. Faraji F, Zaidi M, Fakhry C, Gaykalova DA. Molecular mechanisms of human papillomavirus-related carcinogenesis in head and neck cancer. Microbes Infect. 2017;19(9-10):464-475.
39. Hsu CH, Peng KL, Jhang HC, et al. The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription. Oncogene. 2012;31(18):2335-2349.
40. Tsao SW, Mok SC, Fey EG, et al. Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs). Exp Cell Res. 1995;218(2):499-507.
41. Johnson GA, Burghardt RC, Newton GR, Bazer FW, Spencer TE. Development and characterization of immortalized ovine endometrial cell lines. Biol Reprod. 1999;61(5):1324-1330.
42. Hung SC, Yang DM, Chang CF, et al. Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes. Int J Cancer. 2004;110(3):313-319.
43. Turker MS. Gene silencing in mammalian cells and the spread of DNA methylation. Oncogene. 2002;21(35):5388-5393.
44. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004;4(12):988-993.
45. Laurson J, Khan S, Chung R, Cross K, Raj K. Epigenetic repression of E-cadherin by human papillomavirus 16 E7 protein. Carcinogenesis. 2010;31(5):918-926.
46. Chalertpet K, Pakdeechaidan W, Patel V, Mutirangura A, Yanatatsaneejit P. Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation. Cancer Sci. 2015;106(10):1333-1340.
47. Na Rangsee N, Yanatatsaneejit P, Pisitkun T, Somparn P, Jintaridth P, Topanurak S. Host proteome linked to HPV E7-mediated specific gene hypermethylation in cancer pathways. Infect Agent Cancer. 2020;15:7.
2. Gillison ML, D'Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. Journal of the National Cancer Institute. 2008;100(6):407-420.
3. Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;100(4):261-269.
4. Ben-Dayan MM, MacCarthy T, Schlecht NF, et al. Cancer as the Disintegration of Robustness: Population-Level Variance in Gene Expression Identifies Key Differences Between Tobacco- and HPV-Associated Oropharyngeal Carcinogenesis. Arch Pathol Lab Med. 2015;139(11):1362-1372.
5. Chung CH, Zhang Q, Kong CS, et al. p16 protein expression and human papillomavirus status as prognostic biomarkers of nonoropharyngeal head and neck squamous cell carcinoma. J Clin Oncol. 2014;32(35):3930-3938.
6. Salazar CR, Anayannis N, Smith RV, et al. Combined P16 and human papillomavirus testing predicts head and neck cancer survival. Int J Cancer. 2014;135(10):2404-2412.
7. Pyeon D, Newton MA, Lambert PF, et al. Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus–Positive and Human Papillomavirus–Negative Head/Neck and Cervical Cancers. Cancer research. 2007;67(10):4605-4619.
8. Paschos K, Allday MJ. Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends in Microbiology. 2010;18(10):439-447.
9. Lawrence MS, Sougnez C, Lichtenstein L, et al. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517(7536):576-582.
10. Schlecht NF, Ben-Dayan M, Anayannis N, et al. Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma. Cancer Med. 2015;4(3):342-353.
11. Lleras RA, Smith RV, Adrien LR, et al. Unique DNA methylation loci distinguish anatomic site and HPV status in head and neck squamous cell carcinoma. Clin Cancer Res. 2013;19(19):5444-5455.
12. Sartor MA, Dolinoy DC, Jones TR, et al. Genome-wide methylation and expression differences in HPV(+) and HPV(-) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis. Epigenetics. 2011;6(6):777-787.
13. Marsit CJ, Christensen BC, Houseman EA, et al. Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head and neck squamous cell carcinoma. Carcinogenesis. 2009;30(3):416-422.
14. von Knebel Doeberitz M, Prigge ES. Role of DNA methylation in HPV associated lesions. Papillomavirus Res. 2019;7:180-183.
15. Au Yeung CL, Tsang WP, Tsang TY, Co NN, Yau PL, Kwok TT. HPV-16 E6 upregulation of DNMT1 through repression of tumor suppressor p53. Oncol Rep. 2010;24(6):1599-1604.
16. Burgers WA, Blanchon L, Pradhan S, de Launoit Y, Kouzarides T, Fuks F. Viral oncoproteins target the DNA methyltransferases. Oncogene. 2007;26(11):1650-1655.
17. Jones PA, Baylin SB. The Epigenomics of Cancer. Cell. 2007;128(4):683-692.
18. Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 2012;13(7):484-492.
19. Murtha M, Esteller M. Extraordinary Cancer Epigenomics: Thinking Outside the Classical Coding and Promoter Box. Trends Cancer. 2016;2(10):572-584.
20. Maunakea AK, Nagarajan RP, Bilenky M, et al. Conserved Role of Intragenic DNA Methylation in Regulating Alternative Promoters. Nature. 2010;466(7303):253-257.
21. Kempster S, Phillips WA, Baindur-Hudson S, Thomas RJS, Dow C, Rockman SP. Methylation of exon 2 of p16 is associated with late stage oesophageal cancer. Cancer Letters. 2000;150(1):57-62.
22. Ye X, Mo M, Xu S, et al. The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging. BMC Cancer. 2018;18.
23. Spitzwieser M, Entfellner E, Werner B, et al. Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients. BMC Cancer. 2017;17.
24. Wijetunga NA, Belbin TJ, Burk RD, et al. Novel epigenetic changes in CDKN2A are associated with progression of cervical intraepithelial neoplasia. Gynecol Oncol. 2016;142(3):566-573.
25. Arechederra M, Daian F, Yim A, et al. Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer. Nat Commun. 2018;9(1):3164.
26. Ben-Dayan MM, Ow TJ, Belbin TJ, et al. Nonpromoter methylation of the CDKN2A gene with active transcription is associated with improved locoregional control in laryngeal squamous cell carcinoma. Cancer Med. 2017;6(2):397-407.
27. Lleras RA, Adrien LR, Smith RV, et al. Hypermethylation of a cluster of Kruppel-type zinc finger protein genes on chromosome 19q13 in oropharyngeal squamous cell carcinoma. Am J Pathol. 2011;178(5):1965-1974.
28. Schlecht NF, Brandwein-Gensler M, Nuovo GJ, et al. A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer. Mod Pathol. 2011;24(10):1295-1305.
29. Wojdacz TK, Dobrovic A, Hansen LL. Methylation-sensitive high-resolution melting. Nat Protoc. 2008;3(12):1903-1908.
30. Alam H, Sehgal L, Kundu ST, Dalal SN, Vaidya MM. Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells. Mol Biol Cell. 2011;22(21):4068-4078.
31. Fuchs E, Nowak JA. Building epithelial tissues from skin stem cells. Cold Spring Harb Symp Quant Biol. 2008;73:333-350.
32. Watt FM. Involucrin and other markers of keratinocyte terminal differentiation. J Invest Dermatol. 1983;81(1 Suppl):100s-103s.
33. Wong CW, LeGrand CF, Kinnear BF, et al. In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics. Sci Rep. 2019;9(1):18561.
34. Jones DL, Alani RM, Munger K. The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev. 1997;11(16):2101-2111.
35. Pei XF, Sherman L, Sun YH, Schlegel R. HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium. Carcinogenesis. 1998;19(8):1481-1486.
36. Zehbe I, Richard C, DeCarlo CA, et al. Human papillomavirus 16 E6 variants differ in their dysregulation of human keratinocyte differentiation and apoptosis. Virology. 2009;383(1):69-77.
37. Salazar CR, Smith RV, Garg MK, et al. Human papillomavirus-associated head and neck squamous cell carcinoma survival: a comparison by tumor site and initial treatment. Head Neck Pathol. 2014;8(1):77-87.
38. Faraji F, Zaidi M, Fakhry C, Gaykalova DA. Molecular mechanisms of human papillomavirus-related carcinogenesis in head and neck cancer. Microbes Infect. 2017;19(9-10):464-475.
39. Hsu CH, Peng KL, Jhang HC, et al. The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription. Oncogene. 2012;31(18):2335-2349.
40. Tsao SW, Mok SC, Fey EG, et al. Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs). Exp Cell Res. 1995;218(2):499-507.
41. Johnson GA, Burghardt RC, Newton GR, Bazer FW, Spencer TE. Development and characterization of immortalized ovine endometrial cell lines. Biol Reprod. 1999;61(5):1324-1330.
42. Hung SC, Yang DM, Chang CF, et al. Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes. Int J Cancer. 2004;110(3):313-319.
43. Turker MS. Gene silencing in mammalian cells and the spread of DNA methylation. Oncogene. 2002;21(35):5388-5393.
44. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004;4(12):988-993.
45. Laurson J, Khan S, Chung R, Cross K, Raj K. Epigenetic repression of E-cadherin by human papillomavirus 16 E7 protein. Carcinogenesis. 2010;31(5):918-926.
46. Chalertpet K, Pakdeechaidan W, Patel V, Mutirangura A, Yanatatsaneejit P. Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation. Cancer Sci. 2015;106(10):1333-1340.
47. Na Rangsee N, Yanatatsaneejit P, Pisitkun T, Somparn P, Jintaridth P, Topanurak S. Host proteome linked to HPV E7-mediated specific gene hypermethylation in cancer pathways. Infect Agent Cancer. 2020;15:7.