Abstract
[Background] Virus integration into the host genome is one of the critical steps that lead to the progression of the precancerous lesion into cancer. This study aims to identify integration sites of two High Risk-HPV types (16 and 52 ) in women with cervical intraepithelial neoplasia(CIN) and cervical cancer in Shanghai and to study the relationship between integration and occurrence of cervical cancer. This is the first time HPV52 integration sites were studied.
[Methods] HPV DNA integrated status of totally 19 clinical samples including 13 single HPV16 infective and 6 single HPV52 infective samples with CIN or cervical squamous cell carcinoma (SCC) were detected by ligation-mediated PCR (DIPS-PCR)and DNA sequences. In total, 12 published nested primers were used in PCR for HPV16 and nine new nested-PCR primer sets were specifically designed for HPV52 viral-cellular junction analysis. All the results were analyzed by NCBI BLAST and NCBI-Map Viewer.
[Result]HPV16 integration rates in SCC samples is 60% (3/5), in CINII-III is 60% (3/5) and in CINI is 0%(0/3).Ten integration events were found in human genomes from HPV16 infected samples, including 3 SCC and 3 CINII-III. Single integration site was found in all three samples. Each of two samples has two integration sites.. And three integration sites were found in only one sample. HPV DNA integrated into SCAI, NAALADL2, KALRN and SNX25 gene distributed in human chromosomes 3, 4, 9, 17 and 22 respectively. Both chromosome 3 and 22 were integrated twice. And 100% of the integration sites located in the intron of the genes. In the eight integration sites identified, seven (87.5%) occurred near the common fragile sites of human genomes. Our results also shown that there were thirteen disruption sites in HPV genomes, in which six of them were happened at HPV16 E1, two at E2, two at L1, one at L2, one at E5 gene, and one happened at at E1 gene diffused with HPV16 E2 gene. The integration sites of HPV52 were also identified successfully.
[Conclusion]Our observations suggest that integration is a late events in HPV16 infection patients with CIN. All the HPV16 integration sites are in the intron area of human genome, two of them are cancer associated genes. These integration coexist with host genomic abnormal including translocation, breakage, recombination, deletions and chromosomal translocations. It is possible that HPV16 DNA integration plays a key role in tumor development.
References:
[1] Li J, Kang LN, Qiao YL. Review of the cervical cancer disease burden in mainland China. Asian Pac J Cancer Prev l2011;12: 1149-53.
[2] Li H, Yang Y, Zhang R, Cai Y, Yang X, Wang Z, Li Y, Cheng X, Ye X, Xiang Y, Zhu B. Preferential sites for the integration and disruption of human papillomavirus 16 in cervical lesions. J Clin Virol l2013;56: 342-7.
[3] De Sanjose S, Quint WG, Alemany L, et al. Human papillomavirusgenotype attribution in invasive cervical cancer: A retrospectivecross-sectional worldwide study. Lancet Oncol 2010; 11:1048–1056.
[4] Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med l2003;348: 518-27.
[5] Bao YP, Li N, Smith JS, Qiao YL. Human papillomavirus type distribution in women from Asia: a meta-analysis. Int J Gynecol Cancer l2008;18: 71-9.
[6] Natphopsuk S, Settheetham-Ishida W, Pientong C, Sinawat S, Yuenyao P, Ishida T, Settheetham D. Human papillomavirus genotypes and cervical cancer in northeast Thailand. Asian Pac J Cancer Prev l2013;14: 6961-4.
[7] Lee EH, Um TH, Chi HS, Hong YJ, Cha YJ. Prevalence and distribution of human papillomavirus infection in Korean women as determined by restriction fragment mass polymorphism assay. J Korean Med Sci l2012;27: 1091-7.
[8] Quek SC, Lim BK, Domingo E, Soon R, Park JS, Vu TN, Tay EH, Le QT, Kim YT, Vu BQ, Cao NT, Limson G, Pham VT, Molijn A, Ramakrishnan G, Chen J. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical intraepithelial neoplasia across 5 countries in Asia. Int J Gynecol Cancer l2013;23: 148-56.
[9] Frame FM, Rogoff HA, Pickering MT, Cress WD, Kowalik TF. E2F1 induces MRN foci formation and a cell cycle checkpoint response in human fibroblasts. Oncogene l2006;25: 3258-66.
[10] Pickering MT, Kowalik TF. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene l2006;25: 746-55.
[11] Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, Kerem B. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell l2011;145: 435-46.
[12] Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, Zur HH. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature l1985;314: 111-4.
[13] Wells SI, Francis DA, Karpova AY, Dowhanick JJ, Benson JD, Howley PM. Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways. EMBO J l2000;19: 5762-71.
[14] Sastre-Garau X, Favre M, Couturier J, Orth G. Distinct patterns of alteration of myc genes associated with integration of human papillomavirus type 16 or type 45 DNA in two genital tumours. J Gen Virol l2000;81: 1983-93.
[15] Pett MR, Alazawi WO, Roberts I, Dowen S, Smith DI, Stanley MA, Coleman N. Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes. Cancer Res l2004;64: 1359-68.
[16] Winder DM, Pett MR, Foster N, Shivji MK, Herdman MT, Stanley MA, Venkitaraman AR, Coleman N. An increase in DNA double-strand breaks, induced by Ku70 depletion, is associated with human papillomavirus 16 episome loss and de novo viral integration events. J Pathol l2007;213: 27-34.
[17] Cullen AP, Reid R, Campion M, Lorincz AT. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J Virol l1991;65: 606-12.
[18] Luft F, Klaes R, Nees M, Durst M, Heilmann V, Melsheimer P, von Knebel DM. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer l2001;92: 9-17.
[19] Peter M, Stransky N, Couturier J, Hupe P, Barillot E, de Cremoux P, Cottu P, Radvanyi F, Sastre-Garau X. Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma. J Pathol l2010;221: 320-30.
[20] Matovina M, Sabol I, Grubisic G, Gasperov NM, Grce M. Identification of human papillomavirus type 16 integration sites in high-grade precancerous cervical lesions. Gynecol Oncol l2009;113: 120-7.
[21] Wentzensen N, Ridder R, Klaes R, Vinokurova S, Schaefer U, Doeberitz M. Characterization of viral-cellular fusion transcripts in a large series of HPV16 and 18 positive anogenital lesions. Oncogene l2002;21: 419-26.
[22] Wentzensen N, Vinokurova S, von Knebel DM. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res l2004;64: 3878-84.
[23] Ferber MJ, Thorland EC, Brink AA, Rapp AK, Phillips LA, McGovern R, Gostout BS, Cheung TH, Chung TK, Fu WY, Smith DI. Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma. Oncogene l2003;22: 7233-42.
[24] Thorland EC, Myers SL, Gostout BS, Smith DI. Common fragile sites are preferential targets for HPV16 integrations in cervical tumors. Oncogene l2003;22: 1225-37.
[25] Ziegert C, Wentzensen N, Vinokurova S, Kisseljov F, Einenkel J, Hoeckel M, von Knebel DM. A comprehensive analysis of HPV integration loci in anogenital lesions combining transcript and genome-based amplification techniques. Oncogene l2003;22: 3977-84.
[26] Peter M, Rosty C, Couturier J, Radvanyi F, Teshima H, Sastre-Garau X. MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors. Oncogene l2006;25: 5985-93.
[27] Kraus I, Driesch C, Vinokurova S, Hovig E, Schneider A, von Knebel DM, Durst M. The majority of viral-cellular fusion transcripts in cervical carcinomas cotranscribe cellular sequences of known or predicted genes. Cancer Res l2008;68: 2514-22.
[28] Dall KL, Scarpini CG, Roberts I, Winder DM, Stanley MA, Muralidhar B, Herdman MT, Pett MR, Coleman N. Characterization of naturally occurring HPV16 integration sites isolated from cervical keratinocytes under noncompetitive conditions. Cancer Res l2008;68: 8249-59.
[29] Matovina M, Sabol I, Grubisic G, Gasperov NM, Grce M. Identification of human papillomavirus type 16 integration sites in high-grade precancerous cervical lesions. Gynecol Oncol l2009;113: 120-7.
[30] Zhang C, Zhang S, Zhang Z, He J, Xu Y, Liu S. ROCK has a crucial role in regulating prostate tumor growth through interaction with c-Myc. Oncogene l2013.
[31] Feng B, Li K, Zhong H, Ren G, Wang H, Shang Y, Bai M, Liang J, Wang X, Fan D. RhoE Promotes Metastasis in Gastric Cancer through a Mechanism Dependent on Enhanced Expression of CXCR4. PLoS One l2013;8: e81709.
[32] Dziuba N, Ferguson MR, O'Brien WA, Sanchez A, Prussia AJ, McDonald NJ, Friedrich BM, Li G, Shaw MW, Sheng J, Hodge TW, Rubin DH, Murray JL. Identification of cellular proteins required for replication of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses l2012;28: 1329-39.
[33] Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, Zur HH. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature l1985;314: 111-4.
[34] von Knebel DM, Bauknecht T, Bartsch D, Zur HH. Influence of chromosomal integration on glucocorticoid-regulated transcription of growth-stimulating papillomavirus genes E6 and E7 in cervical carcinoma cells. Proc Natl Acad Sci U S A l1991;88: 1411-5.
[35] Jeon S, Allen-Hoffmann BL, Lambert PF. Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J Virol l1995;69: 2989-97.
[36] Schmitz M, Driesch C, Jansen L, Runnebaum IB, Durst M. Non-random integration of the HPV genome in cervical cancer. PLoS One l2012;7: e39632.
[37] Iliopoulos D, Guler G, Han SY, Druck T, Ottey M, McCorkell KA, Huebner K. Roles of FHIT and WWOX fragile genes in cancer. Cancer Lett l2006;232: 27-36.
[38] Ozeri-Galai E, Bester AC, Kerem B. The complex basis underlying common fragile site instability in cancer. Trends Genet l2012;28: 295-302.
[39] Matovina M, Sabol I, Grubisic G, Gasperov NM, Grce M. Identification of human papillomavirus type 16 integration sites in high-grade precancerous cervical lesions. Gynecol Oncol l2009;113: 120-7.
[40] Wentzensen N, Vinokurova S, von Knebel DM. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res l2004;64: 3878-84.
[41] Lukusa T, Fryns JP. Human chromosome fragility. Biochim Biophys Acta l2008;1779: 3-16.
[42] Schwartz M, Zlotorynski E, Kerem B. The molecular basis of common and rare fragile sites. Cancer Lett l2006;232: 13-26.
[43] Peter M, Rosty C, Couturier J, Radvanyi F, Teshima H, Sastre-Garau X. MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors. Oncogene l2006;25: 5985-93.
[44] Klimov E, Vinokourova S, Moisjak E, Rakhmanaliev E, Kobseva V, Laimins L, Kisseljov F, Sulimova G. Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences. BMC Cancer l2002;2: 24.
[45] Brandt DT, Baarlink C, Kitzing TM, Kremmer E, Ivaska J, Nollau P, Grosse R. SCAI acts as a suppressor of cancer cell invasion through the transcriptional control of beta1-integrin. Nat Cell Biol l2009;11: 557-68.
[46] Brauchle M, Yao Z, Arora R, Thigale S, Clay I, Inverardi B, Fletcher J, Taslimi P, Acker MG, Gerrits B, Voshol J, Bauer A, Schubeler D, Bouwmeester T, Ruffner H. Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation. PLoS One l2013;8: e60549.
[47] Fintha A, Gasparics A, Fang L, Erdei Z, Hamar P, Mozes MM, Kokeny G, Rosivall L, Sebe A. Characterization and role of SCAI during renal fibrosis and epithelial-to-mesenchymal transition. Am J Pathol l2013;182: 388-400.
[48] Tonkin ET, Smith M, Eichhorn P, Jones S, Imamwerdi B, Lindsay S, Jackson M, Wang TJ, Ireland M, Burn J, Krantz ID, Carr P, Strachan T. A giant novel gene undergoing extensive alternative splicing is severed by a Cornelia de Lange-associated translocation breakpoint at 3q26.3. Hum Genet l2004;115: 139-48.
[49] Brumby AM, Goulding KR, Schlosser T, Loi S, Galea R, Khoo P, Bolden JE, Aigaki T, Humbert PO, Richardson HE. Identification of novel Ras-cooperating oncogenes in Drosophila melanogaster: a RhoGEF/Rho-family/JNK pathway is a central driver of tumorigenesis. Genetics l2011;188: 105-25.
[50] Du Y, Zou Y, Yu W, Shi R, Zhang M, Yang W, Duan J, Deng Y, Wang X, Lu Y. Expression pattern of sorting Nexin 25 in temporal lobe epilepsy: a study on patients and pilocarpine-induced rats. Brain Res l2013;1509: 79-85.
[51] Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, Kerem B. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell l2011;145: 435-46.
[52] You J. Papillomavirus interaction with cellular chromatin. Biochim Biophys Acta l2010;1799: 192-9.
[53] Dey A, Chitsaz F, Abbasi A, Misteli T, Ozato K. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci U S A l2003;100: 8758-63.
[54] Angulo M, Carvajal-Rodriguez A. Evidence of recombination within human alpha-papillomavirus. Virol J l2007;4: 33.
[55] Chen Z, Schiffman M, Herrero R, Desalle R, Anastos K, Segondy M, Sahasrabuddhe VV, Gravitt PE, Hsing AW, Burk RD. Evolution and taxonomic classification of human papillomavirus 16 (HPV16)-related variant genomes: HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. PLoS One l2011;6: e20183.
[56] Chen Z, Terai M, Fu L, Herrero R, DeSalle R, Burk RD. Diversifying selection in human papillomavirus type 16 lineages based on complete genome analyses. J Virol l2005;79: 7014-23.
[57] Schiffman M, Rodriguez AC, Chen Z, Wacholder S, Herrero R, Hildesheim A, Desalle R, Befano B, Yu K, Safaeian M, Sherman ME, Morales J, Guillen D, Alfaro M, Hutchinson M, Solomon D, Castle PE, Burk RD. A population-based prospective study of carcinogenic human papillomavirus variant lineages, viral persistence, and cervical neoplasia. Cancer Res l2010;70: 3159-69.
[58] Ganguly N. Human papillomavirus-16 E5 protein: oncogenic role and therapeutic value. Cell Oncol (Dordr) l2012;35: 67-76.