Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV-positive oropharyngeal squamous cell carcinoma

Abstract

Background: Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well-studied investigation about virus-host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins.

Materials and methods: High-throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing.

Results: HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD-L1 expression demonstrated increased integration sites (p = .024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty-nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process.

Conclusions: HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus-host interactions, enhancing our understanding of HPV-related carcinogenesis mechanisms.

Keywords

host genomic alterations / HPV / oropharyngeal squamous cell carcinoma / viral integration

References

[1] EI-Naggar AK, Chan JKC, Grandis JR, Takata T, Slootweg PJ. WHO Classification of Head and Neck Tumours. International Agency for Research on Cancer. 2017e-pub ahead of print.

[2] Cillo AR, Kurten CHL, Tabib T, et al. Immune landscape of viral- and carcinogen-driven head and neck cancer. Immunity. 2020;52(1):183-199.

[3] Gillison ML, Akagi K, Xiao W, et al. Human papillomavirus and the landscape of secondary genetic alterations in oral cancers. Genome Res. 2019;29(1):1-17.

[4] Taberna M, Mena M, Pavon MA, Alemany L, Gillison ML, Mesia R. Human papillomavirus-related oropharyngeal cancer. Ann Oncol. 2017;28(10):2386-2398.

[5] Xu S, Sun B, Zhou R, et al. Evaluation of p16 as a surrogate marker for transcriptionally active human papillomavirus status of oropharyngeal squamous cell carcinoma in an eastern Chinese population. Oral Surg Oral Med Oral Pathol Oral Radiol. 2020;129(3):236-245.e232.

[6] Global Burden of Disease Cancer C, Fitzmaurice C, Allen C, et al. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis for the global burden of disease study. JAMA Oncol. 2017;3(4):524-548.

[7] Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med. 2010;363(1):24-35.

[8] Tinhofer I, Budach V, Saki M, et al. Targeted next-generation sequencing of locally advanced squamous cell carcinomas of the head and neck reveals druggable targets for improving adjuvant chemoradiation. Eur J Cancer. 2016;57:78-86.

[9] Leeman JE, Li Y, Bell A, et al. Human papillomavirus 16 promotes microhomology-mediated end-joining. Proc Natl Acad Sci USA. 2019;116(43):21573-21579.

[10] Zhao LH, Liu X, Yan HX, et al. Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma. Nat Commun. 2016;7:12992.

[11] Xu M, Zhang WL, Zhu Q, et al. Genome-wide profiling of Epstein-Barr virus integration by targeted sequencing in Epstein-Barr virus associated malignancies. Theranostics. 2019;9(4):1115-1124.

[12] Hu Z, Zhu D, Wang W, et al. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nat Genet. 2015;47(2):158-163.

[13] Pang J, Nguyen N, Luebeck J, et al. Extrachromosomal DNA in HPV-mediated oropharyngeal cancer drives diverse oncogene transcription. Clin Cancer Res. 2021;27(24):6772-6786.

[14] Symer DE, Akagi K, Geiger HM, et al. Diverse tumorigenic consequences of human papillomavirus integration in primary oropharyngeal cancers. Genome Res. 2022;32(1):55-70.

[15] Akagi K, Symer DE, Mahmoud M, et al. Intratumoral heterogeneity and clonal evolution induced by HPV integration. Cancer Discov. 2023;13(4):910-927.

[16] Rossi NM, Dai J, Xie Y, et al. Extrachromosomal amplification of human papillomavirus episomes is a mechanism of cervical carcinogenesis. Cancer Res. 2023;83(11):1768-1781.

[17] Cancer Genome Atlas Research N, Albert Einstein College of M, Analytical Biological S, Barretos Cancer H, Baylor College of M, Beckman Research Institute of City of H, et al. Integrated genomic and molecular characterization of cervical cancer. Nature. 2017;543(7645):378-384.

[18] Xu SM, Shi CJ, Xia RH, et al. Analysis of immunological characteristics and genomic alterations in HPV-positive oropharyngeal squamous cell carcinoma based on PD-L1 expression. Front Immunol. 2021;12:798424.

[19] Warburton A, Redmond CJ, Dooley KE, et al. HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression. PLoS Genet. 2018;14(1):e1007179.

[20] Parfenov M, Pedamallu CS, Gehlenborg N, et al. Characterization of HPV and host genome interactions in primary head and neck cancers. Proc Natl Acad Sci USA. 2014;111(43):15544-15549.

[21] Li W, Zeng X, Lee NP, et al. HIVID: an efficient method to detect HBV integration using low coverage sequencing. Genomics. 2013;102(4):338-344.

[22] Alexandrov LB, Nik-Zainal S, Wedge DC, et al. Signatures of mutational processes in human cancer. Nature. 2013;500(7463):415-421.

[23] Simpson BS, Pye H, Whitaker HC. The oncological relevance of fragile sites in cancer. Commun Biol. 2021;4(1):567.

[24] Simpson BS, Camacho N, Luxton HJ, et al. Genetic alterations in the 3q26.31-32 locus confer an aggressive prostate cancer phenotype. Commun Biol. 2020;3(1):440.

[25] Kudo-Saito C, Ishida A, Shouya Y, et al. Blocking the FSTL1-DIP2A axis improves anti-tumor immunity. Cell Rep. 2018;24(7):1790-1801.

[26] Bal E, Park HS, Belaid-Choucair Z, et al. Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas. Nat Med. 2017;23(10):1226-1233.

[27] Kaminskiy Y, Kuznetsova V, Kudriaeva A, Zmievskaya E, Bulatov E. Neglected, yet significant role of FOXP1 in T-cell quiescence, differentiation and exhaustion. Front Immunol. 2022;13:971045.

[28] Kumar SU, Balasundaram A, Cathryn RH, et al. Whole-exome sequencing analysis of NSCLC reveals the pathogenic missense variants from cancer-associated genes. Comput Biol Med. 2022;148:105701.

[29] Cai H, Agersnap SN, Sjogren A, et al. In vivo application of CRISPR/Cas9 revealed implication of foxa1 and foxp1 in prostate cancer proliferation and epithelial plasticity. Cancers (Basel). 2022;14(18).

[30] Koneva LA, Zhang Y, Virani S, et al. HPV integration in HNSCC correlates with survival outcomes, immune response signatures, and candidate drivers. Mol Cancer Res. 2018;16(1):90-102.

[31] Akagi K, Li J, Broutian TR, et al. Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability. Genome Res. 2014;24(2):185-199.

[32] Fan J, Fu Y, Peng W, et al. Multi-omics characterization of silent and productive HPV integration in cervical cancer. Cell Genom. 2023;3(1):100211.

[33] Lerman MA, Almazrooa S, Lindeman N, Hall D, Villa A, Woo SB. HPV-16 in a distinct subset of oral epithelial dysplasia. Mod Pathol. 2017;30(12):1646-1654.

[34] Liu M, Han Z, Zhi Y, et al. Long-read sequencing reveals oncogenic mechanism of HPV-human fusion transcripts in cervical cancer. Transl Res. 2023;253:80-94.

[35] Cao CH, Liu R, Lin XR, et al. LRP1B mutation is associated with tumor HPV status and promotes poor disease outcomes with a higher mutation count in HPV-related cervical carcinoma and head & neck squamous cell carcinoma. Int J Biol Sci. 2021;17(7):1744-1756.

[36] Ni S, Hu J, Duan Y, et al. Down expression of LRP1B promotes cell migration via RhoA/Cdc42 pathway and actin cytoskeleton remodeling in renal cell cancer. Cancer Sci. 2013;104(7):817-825.

[37] Cowin PA, George J, Fereday S, et al. LRP1B deletion in high-grade serous ovarian cancers is associated with acquired chemotherapy resistance to liposomal doxorubicin. Cancer Res. 2012;72(16):4060-4073.

[38] Gao G, Kasperbauer JL, Tombers NM, Cornell MD, Smith DI. Prognostic significance of decreased expression of six large common fragile site genes in oropharyngeal squamous cell carcinomas. Transl Oncol. 2014;7(6):726-731.

[39] Pim D, Massimi P, Banks L. Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth. Oncogene. 1997;15(3):257-264.

[40] Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer. 2010;10(8):550-560.

[41] Ren S, Gaykalova DA, Guo T, et al. HPV E2, E4, E5 drive alternative carcinogenic pathways in HPV positive cancers. Oncogene. 2020;39(40):6327-6339.

[42] Cancer Genome Atlas N. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517(7536):576-582.

[43] Refsland EW, Harris RS. The APOBEC3 family of retroelement restriction factors. Curr Top Microbiol Immunol. 2013;371:1-27.

[44] Vieira VC, Leonard B, White EA, et al. Human papillomavirus E6 triggers upregulation of the antiviral and cancer genomic DNA deaminase APOBEC3B. mBio. 2014;5(6).

[45] Chang HHY, Pannunzio NR, Adachi N, Lieber MR. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol. 2017;18(8):495-506.

[46] Moody CA, Laimins LA. Human papillomaviruses activate the ATM DNA damage pathway for viral genome amplification upon differentiation. PLoS Pathog. 2009;5(10):e1000605.

[47] Liu Q, Ma L, Jones T, Palomero L, et al. Subjugation of TGFbeta signaling by human papilloma virus in head and neck squamous cell carcinoma shifts DNA repair from homologous recombination to alternative end joining. Clin Cancer Res. 2018;24(23):6001-6014.

[48] Wallace NA, Khanal S, Robinson KL, Wendel SO, Messer JJ, Galloway DA. High-Risk alphapapillomavirus oncogenes impair the homologous recombination pathway. J Virol. 2017;91(20).

[49] Peter M, Stransky N, Couturier J, et al. Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma. J Pathol. 2010;221(3):320-330.

[50] Dooley KE, Warburton A, McBride AA. Tandemly integrated HPV16 can form a Brd4-dependent super-enhancer-like element that drives transcription of viral oncogenes. mBio. 2016;7(5).

[51] Schlecht NF, Burk RD, Adrien L, et al. Gene expression profiles in HPV-infected head and neck cancer. J Pathol. 2007;213(3):283-293.

[52] Slebos RJ, Yi Y, Ely K, et al. Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma. Clin Cancer Res. 2006;12:701-709. Pt 1.

[53] Garcia-Garcia A, Ceballos-Laita L, Serna S, et al. Structural basis for substrate specificity and catalysis of alpha1,6-fucosyltransferase. Nat Commun. 2020;11(1):973.

[54] Huang Y, Zhang HL, Li ZL, et al. FUT8-mediated aberrant N-glycosylation of B7H3 suppresses the immune response in triple-negative breast cancer. Nat Commun. 2021;12(1):2672.

[55] Bennett EP, Mandel U, Clausen H, Gerken TA, Fritz TA, Tabak LA. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology. 2012;22(6):736-756.

[56] Wu Q, Liu HO, Liu YD, et al. Decreased expression of hepatocyte nuclear factor 4alpha (Hnf4alpha)/microRNA-122 (miR-122) axis in hepatitis B virus-associated hepatocellular carcinoma enhances potential oncogenic GALNT10 protein activity. J Biol Chem. 2015;290(2):1170-1185.

[57] Sun J, Huang J, Lan J, et al. Overexpression of CENPF correlates with poor prognosis and tumor bone metastasis in breast cancer. Cancer Cell Int. 2019;19:264.

[58] Cao JY, Liu L, Chen SP, et al. Prognostic significance and therapeutic implications of centromere protein F expression in human nasopharyngeal carcinoma. Mol Cancer. 2010;9:237.

[59] Wu P, Ma D, Wu P. Oncogenic virus integration: moving toward clinical applications. Med. 2023;4(6):347-352.

[60] Tian R, Liu J, Fan W, et al. Gene knock-out chain reaction enables high disruption efficiency of HPV18 E6/E7 genes in cervical cancer cells. Mol Ther Oncolytics. 2022;24:171-179.

[61] Zhou L, Qiu Q, Zhou Q, et al. Long-read sequencing unveils high-resolution HPV integration and its oncogenic progression in cervical cancer. Nat Commun. 2022;13(1):2563.

[62] Cao C, Hong P, Huang X, et al. HPV-CCDC106 integration alters local chromosome architecture and hijacks an enhancer by three-dimensional genome structure remodeling in cervical cancer. J Genet Genomics. 2020;47(8):437-450.

[63] Zhi W, Wei Y, Lazare C, et al. HPV-CCDC106 integration promotes cervical cancer progression by facilitating the high expression of CCDC106 after HPV E6 splicing. J Med Virol. 2023;95(1):e28009.

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