1887

Abstract

We detected the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001061
2018-05-01
2024-11-08
Loading full text...

Full text loading...

/deliver/fulltext/jgv/99/5/631.html?itemId=/content/journal/jgv/10.1099/jgv.0.001061&mimeType=html&fmt=ahah

References

  1. Brickman C, Palefsky JM. Human papillomavirus in the HIV-infected host: epidemiology and pathogenesis in the antiretroviral era. Curr HIV/AIDS Rep 2015; 12:6–15 [View Article][PubMed]
    [Google Scholar]
  2. Silverberg MJ, Lau B, Justice AC, Engels E, Gill MJ et al. Risk of anal cancer in HIV-infected and HIV-uninfected individuals in North America. Clin Infect Dis 2012; 54:1026–1034 [View Article][PubMed]
    [Google Scholar]
  3. Hoots BE, Palefsky JM, Pimenta JM, Smith JS. Human papillomavirus type distribution in anal cancer and anal intraepithelial lesions. Int J Cancer 2009; 124:2375–2383 [View Article][PubMed]
    [Google Scholar]
  4. Steinau M, Unger ER, Hernandez BY, Goodman MT, Copeland G et al. Human papillomavirus prevalence in invasive anal cancers in the United States before vaccine introduction. J Low Genit Tract Dis 2013; 17:397–403 [View Article][PubMed]
    [Google Scholar]
  5. Genther SM, Sterling S, Duensing S, Münger K, Sattler C et al. Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J Virol 2003; 77:2832–2842 [View Article][PubMed]
    [Google Scholar]
  6. Fehrmann F, Klumpp DJ, Laimins LA. Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation. J Virol 2003; 77:2819–2831 [View Article][PubMed]
    [Google Scholar]
  7. Regan JA, Laimins LA. Bap31 is a novel target of the human papillomavirus E5 protein. J Virol 2008; 82:10042–10051 [View Article][PubMed]
    [Google Scholar]
  8. Kotnik Halavaty K, Regan J, Mehta K, Laimins L. Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation. Virology 2014; 452-453:223–230 [View Article][PubMed]
    [Google Scholar]
  9. Chang JL, Tsao YP, Liu DW, Huang SJ, Lee WH et al. The expression of HPV-16 E5 protein in squamous neoplastic changes in the uterine cervix. J Biomed Sci 2001; 8:206–213 [View Article][PubMed]
    [Google Scholar]
  10. Cavuslu S, Starkey WG, Kell B, Best JM, Cason J. Detection of human papillomavirus type 16 in microtitre plate based immuno-enzymatic assays: use to determine E5 gene expression in cervical carcinomas. Clin Diagn Virol 1996; 5:215–218 [View Article][PubMed]
    [Google Scholar]
  11. Shirasawa H, Tomita Y, Kubota K, Kasai T, Sekiya S et al. Transcriptional differences of the human papillomavirus type 16 genome between precancerous lesions and invasive carcinomas. J Virol 1988; 62:1022–1027[PubMed]
    [Google Scholar]
  12. Um SH, Mundi N, Yoo J, Palma DA, Fung K et al. Variable expression of the forgotten oncogene E5 in HPV-positive oropharyngeal cancer. J Clin Virol 2014; 61:94–100 [View Article][PubMed]
    [Google Scholar]
  13. Sahab Z, Sudarshan SR, Liu X, Zhang Y, Kirilyuk A et al. Quantitative measurement of human papillomavirus type 16 e5 oncoprotein levels in epithelial cell lines by mass spectrometry. J Virol 2012; 86:9465–9473 [View Article][PubMed]
    [Google Scholar]
  14. Park JS, Hwang ES, Park SN, Ahn HK, Um SJ et al. Physical status and expression of HPV genes in cervical cancers. Gynecol Oncol 1997; 65:121–129 [View Article][PubMed]
    [Google Scholar]
  15. Cullen AP, Reid R, Campion M, Lörincz AT. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J Virol 1991; 65:606–612[PubMed]
    [Google Scholar]
  16. Matsukura T, Koi S, Sugase M. Both episomal and integrated forms of human papillomavirus type 16 are involved in invasive cervical cancers. Virology 1989; 172:63–72 [View Article][PubMed]
    [Google Scholar]
  17. Straight SW, Herman B, McCance DJ. The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes. J Virol 1995; 69:3185–3192[PubMed]
    [Google Scholar]
  18. Suprynowicz FA, Krawczyk E, Hebert JD, Sudarshan SR, Simic V et al. The human papillomavirus type 16 E5 oncoprotein inhibits epidermal growth factor trafficking independently of endosome acidification. J Virol 2010; 84:10619–10629 [View Article][PubMed]
    [Google Scholar]
  19. Zhang B, Srirangam A, Potter DA, Roman A. HPV16 E5 protein disrupts the c-Cbl-EGFR interaction and EGFR ubiquitination in human foreskin keratinocytes. Oncogene 2005; 24:2585–2588 [View Article][PubMed]
    [Google Scholar]
  20. Schrevel M, Gorter A, Kolkman-Uljee SM, Trimbos JB, Fleuren GJ et al. Molecular mechanisms of epidermal growth factor receptor overexpression in patients with cervical cancer. Mod Pathol 2011; 24:720–728 [View Article][PubMed]
    [Google Scholar]
  21. Mathur SP, Mathur RS, Young RC. Cervical epidermal growth factor-receptor (EGF-R) and serum insulin-like growth factor II (IGF-II) levels are potential markers for cervical cancer. Am J Reprod Immunol 2000; 44:222–230 [View Article][PubMed]
    [Google Scholar]
  22. Soonthornthum T, Arias-Pulido H, Joste N, Lomo L, Muller C et al. Epidermal growth factor receptor as a biomarker for cervical cancer. Ann Oncol 2011; 22:2166–2178 [View Article][PubMed]
    [Google Scholar]
  23. Santini J, Formento JL, Francoual M, Milano G, Schneider M et al. Characterization, quantification, and potential clinical value of the epidermal growth factor receptor in head and neck squamous cell carcinomas. Head Neck 1991; 13:132–139 [View Article][PubMed]
    [Google Scholar]
  24. Kalyankrishna S, Grandis JR. Epidermal growth factor receptor biology in head and neck cancer. J Clin Oncol 2006; 24:2666–2672 [View Article][PubMed]
    [Google Scholar]
  25. LH, Chetty R, Moore MJ. Epidermal growth factor receptor expression in anal canal carcinoma. Am J Clin Pathol 2005; 124:20–23 [View Article][PubMed]
    [Google Scholar]
  26. Ajani JA, Wang X, Izzo JG, Crane CH, Eng C et al. Molecular biomarkers correlate with disease-free survival in patients with anal canal carcinoma treated with chemoradiation. Dig Dis Sci 2010; 55:1098–1105 [View Article][PubMed]
    [Google Scholar]
  27. Leechanachai P, Banks L, Moreau F, Matlashewski G. The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Oncogene 1992; 7:19–25[PubMed]
    [Google Scholar]
  28. Pim D, Collins M, Banks L. Human papillomavirus type 16 E5 gene stimulates the transforming activity of the epidermal growth factor receptor. Oncogene 1992; 7:27–32[PubMed]
    [Google Scholar]
  29. Straight SW, Hinkle PM, Jewers RJ, McCance DJ. The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes. J Virol 1993; 67:4521–4532[PubMed]
    [Google Scholar]
  30. Genther Williams SM, Disbrow GL, Schlegel R, Lee D, Threadgill DW et al. Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high-risk human papillomavirus oncogene. Cancer Res 2005; 65:6534–6542 [View Article][PubMed]
    [Google Scholar]
  31. Palefsky JM, Holly EA, Ralston ML, Jay N. Prevalence and risk factors for human papillomavirus infection of the anal canal in human immunodeficiency virus (HIV)-positive and HIV-negative homosexual men. J Infect Dis 1998; 177:361–367 [View Article][PubMed]
    [Google Scholar]
  32. Akagi K, Li J, Broutian TR, Padilla-Nash H, Xiao W et al. Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability. Genome Res 2014; 24:185–199 [View Article][PubMed]
    [Google Scholar]
  33. Wang-Johanning F, Lu DW, Wang Y, Johnson MR, Johanning GL. Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicolaou tests using real-time polymerase chain reaction analysis. Cancer 2002; 94:2199–2210 [View Article][PubMed]
    [Google Scholar]
  34. Klaes R, Woerner SM, Ridder R, Wentzensen N, Duerst M et al. Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res 1999; 59:6132–6136[PubMed]
    [Google Scholar]
  35. Middleton K, Peh W, Southern S, Griffin H, Sotlar K et al. Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers. J Virol 2003; 77:10186–10201 [View Article][PubMed]
    [Google Scholar]
  36. Griffin H, Soneji Y, van Baars R, Arora R, Jenkins D et al. Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM. Mod Pathol 2015; 28:977–993 [View Article][PubMed]
    [Google Scholar]
  37. Pirisi L, Creek KE, Doniger J, Dipaolo JA. Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA. Carcinogenesis 1988; 9:1573–1579 [View Article][PubMed]
    [Google Scholar]
  38. Niemeyer CM, Kang MW, Shin DH, Furlan I, Erlacher M et al. Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nat Genet 2010; 42:794–800 [View Article][PubMed]
    [Google Scholar]
  39. Hu D, Zhou J, Wang F, Shi H, Li Y et al. HPV-16 E6/E7 promotes cell migration and invasion in cervical cancer via regulating cadherin switch in vitro and in vivo. Arch Gynecol Obstet 2015; 292:1345–1354 [View Article][PubMed]
    [Google Scholar]
  40. Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci 2006; 110:525–541 [View Article][PubMed]
    [Google Scholar]
  41. Isaacson Wechsler E, Wang Q, Roberts I, Pagliarulo E, Jackson D et al. Reconstruction of human papillomavirus type 16-mediated early-stage neoplasia implicates E6/E7 deregulation and the loss of contact inhibition in neoplastic progression. J Virol 2012; 86:6358–6364 [View Article][PubMed]
    [Google Scholar]
  42. Walker F, Abramowitz L, Benabderrahmane D, Duval X, Descatoire V et al. Growth factor receptor expression in anal squamous lesions: modifications associated with oncogenic human papillomavirus and human immunodeficiency virus. Hum Pathol 2009; 40:1517–1527 [View Article][PubMed]
    [Google Scholar]
  43. Alvarez G, Perry A, Tan BR, Wang HL. Expression of epidermal growth factor receptor in squamous cell carcinomas of the anal canal is independent of gene amplification. Mod Pathol 2006; 19:942–949 [View Article][PubMed]
    [Google Scholar]
  44. Zuo JH, Zhu W, Li MY, Li XH, Yi H et al. Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin. J Cell Biochem 2011; 112:2508–2517 [View Article][PubMed]
    [Google Scholar]
  45. Lu Z, Jiang G, Blume-Jensen P, Hunter T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase. Mol Cell Biol 2001; 21:4016–4031 [View Article][PubMed]
    [Google Scholar]
  46. Zhen Y, Guanghui L, Xiefu Z. Knockdown of EGFR inhibits growth and invasion of gastric cancer cells. Cancer Gene Ther 2014; 21:491–497 [View Article][PubMed]
    [Google Scholar]
  47. Hanning JE, Groves IJ, Pett MR, Coleman N. Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR. Virol J 2013; 10:159 [View Article][PubMed]
    [Google Scholar]
  48. Akerman GS, Tolleson WH, Brown KL, Zyzak LL, Mourateva E et al. Human papillomavirus type 16 E6 and E7 cooperate to increase epidermal growth factor receptor (EGFR) mRNA levels, overcoming mechanisms by which excessive EGFR signaling shortens the life span of normal human keratinocytes. Cancer Res 2001; 61:3837–3843[PubMed]
    [Google Scholar]
  49. Sizemore N, Choo CK, Eckert RL, Rorke EA. Transcriptional regulation of the EGF receptor promoter by HPV16 and retinoic acid in human ectocervical epithelial cells. Exp Cell Res 1998; 244:349–356 [View Article][PubMed]
    [Google Scholar]
  50. Peto M, Tolle-Ersü I, Kreysch HG, Klock G. Epidermal growth factor induction of human papillomavirus type 16 E6/E7 MRNA in tumor cells involves two AP-1 binding sites in the viral enhancer. J Gen Virol 1995; 76:1945–1958 [View Article][PubMed]
    [Google Scholar]
  51. Rosenberger S, de-Castro Arce J, Langbein L, Steenbergen RD, Rösl F. Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation. Proc Natl Acad Sci USA 2010; 107:7006–7011 [View Article][PubMed]
    [Google Scholar]
  52. Wheeler CM, Yamada T, Hildesheim A, Jenison SA. Human papillomavirus type 16 sequence variants: identification by E6 and L1 lineage-specific hybridization. J Clin Microbiol 1997; 35:11–19[PubMed]
    [Google Scholar]
  53. Lambert PF, Ozbun MA, Collins A, Holmgren S, Lee D et al. Using an immortalized cell line to study the HPV life cycle in organotypic "raft" cultures. Methods Mol Med 2005; 119:141–155 [View Article][PubMed]
    [Google Scholar]
/content/journal/jgv/10.1099/jgv.0.001061
Loading
/content/journal/jgv/10.1099/jgv.0.001061
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error