1887

Abstract

The oncogenic E6 proteins produced by high-risk human papillomaviruses (HPVs) are invariably expressed in cervical carcinomas and are multifunctional proteins capable of affecting host-cell proliferation by binding and deregulating key host molecules such as p53. High-risk HPVs, including HPV16, have the unique ability to splice the E6 viral transcript, resulting in the production of a truncated E6 protein known as E6*I whose precise biological function is unclear. This study explored the changes in gene expression of the cervical cancer C33A cell line stably expressing HPV16 E6*I (16E6*I) and observed the upregulation of ten genes. Two of these genes were aldo-keto reductases (AKR1Cs), AKR1C1 and AKR1C3, which have been implicated in drug resistance. The results demonstrated that expression of 16E6*I, but not full-length E6, specifically increased transcript levels although it did not alter transcript levels. HPV16 E7 alone also had the ability to cause a moderate increase in at both mRNA and protein levels. Site-directed mutagenesis of 16E6*I revealed that transactivation activity was abolished in R8A, R10A and T17A 16E6*I mutants without altering their intracellular localization patterns. Loss of transactivation activity of the 16E6*I mutants resulted in a significant loss of AKR1C expression and a decrease in drug resistance. Analysis of the promoter revealed that, unlike the E6 protein, 16E6*I does not mediate transactivation activity solely through Sp1-binding sites. Taken together, it was concluded that 16E6*I has a novel function in upregulating expression of and, in concert with E7, has implications for drug treatment in HPV-mediated cervical cancer.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.038265-0
2012-05-01
2020-09-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/93/5/1081.html?itemId=/content/journal/jgv/10.1099/vir.0.038265-0&mimeType=html&fmt=ahah

References

  1. Badaracco G., Savarese A., Micheli A., Rizzo C., Paolini F., Carosi M., Cutillo G., Vizza E., Arcangeli G., Venuti A.. 2010; Persistence of HPV after radio-chemotherapy in locally advanced cervical cancer. Oncol Rep23:1093–1099[PubMed]
    [Google Scholar]
  2. Borbély A. A., Murvai M., Kónya J., Beck Z., Gergely L., Li F., Veress G.. 2006; Effects of human papillomavirus type 16 oncoproteins on survivin gene expression. J Gen Virol87:287–294 [CrossRef][PubMed]
    [Google Scholar]
  3. Chen J., Adikari M., Pallai R., Parekh H. K., Simpkins H.. 2008; Dihydrodiol dehydrogenases regulate the generation of reactive oxygen species and the development of cisplatin resistance in human ovarian carcinoma cells. Cancer Chemother Pharmacol61:979–987 [CrossRef][PubMed]
    [Google Scholar]
  4. Chen J., Emara N., Solomides C., Parekh H., Simpkins H.. 2010; Resistance to platinum-based chemotherapy in lung cancer cell lines. Cancer Chemother Pharmacol66:1103–1111 [CrossRef][PubMed]
    [Google Scholar]
  5. Chien C.-W., Ho I.-C., Lee T.-C.. 2009; Induction of neoplastic transformation by ectopic expression of human aldo-keto reductase 1C isoforms in NIH3T3 cells. Carcinogenesis30:1813–1820 [CrossRef][PubMed]
    [Google Scholar]
  6. Cricca M., Venturoli S., Leo E., Costa S., Musiani M., Zerbini M.. 2009; Molecular analysis of HPV 16 E6I/E6II spliced mRNAs and correlation with the viral physical state and the grade of the cervical lesion. J Med Virol81:1276–1282 [CrossRef][PubMed]
    [Google Scholar]
  7. de la Cruz-Hernández E., García-Carrancá A., Mohar-Betancourt A., Dueñas-González A., Contreras-Paredes A., Pérez-Cardenas E., Herrera-Goepfert R., Lizano-Soberón M.. 2005; Differential splicing of E6 within human papillomavirus type 18 variants and functional consequences. J Gen Virol86:2459–2468 [CrossRef][PubMed]
    [Google Scholar]
  8. Deng H. B., Adikari M., Parekh H. K., Simpkins H.. 2004; Ubiquitous induction of resistance to platinum drugs in human ovarian, cervical, germ-cell and lung carcinoma tumor cells overexpressing isoforms 1 and 2 of dihydrodiol dehydrogenase. Cancer Chemother Pharmacol54:301–307 [CrossRef][PubMed]
    [Google Scholar]
  9. Dey A., Atcha I. A., Bagchi S.. 1997; HPV16 E6 oncoprotein stimulates the transforming growth factor-β1 promoter in fibroblasts through a specific GC-rich sequence. Virology228:190–199 [CrossRef][PubMed]
    [Google Scholar]
  10. Filippova M., Johnson M. M., Bautista M., Filippov V., Fodor N., Tungteakkhun S. S., Williams K., Duerksen-Hughes P. J.. 2007; The large and small isoforms of human papillomavirus type 16 E6 bind to and differentially affect procaspase 8 stability and activity. J Virol81:4116–4129 [CrossRef][PubMed]
    [Google Scholar]
  11. Gewin L., Galloway D. A.. 2001; E box-dependent activation of telomerase by human papillomavirus type 16 E6 does not require induction of c-myc . J Virol75:7198–7201 [CrossRef][PubMed]
    [Google Scholar]
  12. Gewin L., Myers H., Kiyono T., Galloway D. A.. 2004; Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex. Genes Dev18:2269–2282 [CrossRef][PubMed]
    [Google Scholar]
  13. Hasan U. A., Bates E., Takeshita F., Biliato A., Accardi R., Bouvard V., Mansour M., Vincent I., Gissmann L.. other authors 2007; TLR9 expression and function is abolished by the cervical cancer-associated human papillomavirus type 16. J Immunol178:3186–3197[PubMed][CrossRef]
    [Google Scholar]
  14. Howie H. L., Katzenellenbogen R. A., Galloway D. A.. 2009; Papillomavirus E6 proteins. Virology384:324–334 [CrossRef][PubMed]
    [Google Scholar]
  15. Hsu N.-Y., Ho H.-C., Chow K.-C., Lin T.-Y., Shih C.-S., Wang L.-S., Tsai C.-M.. 2001; Overexpression of dihydrodiol dehydrogenase as a prognostic marker of non-small cell lung cancer. Cancer Res61:2727–2731[PubMed]
    [Google Scholar]
  16. Huertas-Salgado A., Martín-Gámez D. C., Moreno P., Murillo R., Bravo M. M., Villa L., Molano M.. 2011; E6 molecular variants of human papillomavirus (HPV) type 16: an updated and unified criterion for clustering and nomenclature. Virology410:201–215 [CrossRef][PubMed]
    [Google Scholar]
  17. Ji Q., Aoyama C., Nien Y.-D., Liu P. I., Chen P. K., Chang L., Stanczyk F. Z., Stolz A.. 2004; Selective loss of AKR1C1 and AKR1C2 in breast cancer and their potential effect on progesterone signaling. Cancer Res64:7610–7617 [CrossRef][PubMed]
    [Google Scholar]
  18. Ji Q., Chang L., Stanczyk F. Z., Ookhtens M., Sherrod A., Stolz A.. 2007; Impaired dihydrotestosterone catabolism in human prostate cancer: critical role of AKR1C2 as a pre-receptor regulator of androgen receptor signaling. Cancer Res67:1361–1369 [CrossRef][PubMed]
    [Google Scholar]
  19. Kösel S., Burggraf S., Engelhardt W., Olgemöller B.. 2007; Increased levels of HPV16 E6*I transcripts in high-grade cervical cytology and histology (CIN II+) detected by rapid real-time RT-PCR amplification. Cytopathology18:290–299 [CrossRef][PubMed]
    [Google Scholar]
  20. Kumar A., Zhao Y., Meng G., Zeng M., Srinivasan S., Delmolino L. M., Gao Q., Dimri G., Weber G. F.. other authors 2002; Human papillomavirus oncoprotein E6 inactivates the transcriptional coactivator human ADA3. Mol Cell Biol22:5801–5812 [CrossRef][PubMed]
    [Google Scholar]
  21. Laochariyakul P., Ponglikitmongkol M., Mankhetkorn S.. 2003; Functional study of intracellular P-gp- and MRP1-mediated pumping of free cytosolic pirarubicin into acidic organelles in intrinsic resistant SiHa cells. Can J Physiol Pharmacol81:790–799 [CrossRef][PubMed]
    [Google Scholar]
  22. Liu Y., Chen J. J., Gao Q., Dalal S., Hong Y., Mansur C. P., Band V., Androphy E. J.. 1999; Multiple functions of human papillomavirus type 16 E6 contribute to the immortalization of mammary epithelial cells. J Virol73:7297–7307[PubMed]
    [Google Scholar]
  23. Liu X., Yuan H., Fu B., Disbrow G. L., Apolinario T., Tomaic V., Kelley M. L., Baker C. C., Huibregtse J., Schlegel R.. 2005; The E6AP ubiquitin ligase is required for transactivation of the hTERT promoter by the human papillomavirus E6 oncoprotein. J Biol Chem280:10807–10816 [CrossRef][PubMed]
    [Google Scholar]
  24. López-Ocejo O., Viloria-Petit A., Bequet-Romero M., Mukhopadhyay D., Rak J., Kerbel R. S.. 2000; Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner. Oncogene19:4611–4620 [CrossRef][PubMed]
    [Google Scholar]
  25. Münger K., Phelps W. C., Bubb V., Howley P. M., Schlegel R.. 1989; The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol63:4417–4421[PubMed]
    [Google Scholar]
  26. Muñoz N., Bosch F. X., de Sanjosé S., Herrero R., Castellsagué X., Shah K. V., Snijders P. J., Meijer C. J.. International Agency for Research on Cancer Multicenter Cervical Cancer Study Group 2003; Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med348:518–527 [CrossRef][PubMed]
    [Google Scholar]
  27. Nees M., Geoghegan J. M., Hyman T., Frank S., Miller L., Woodworth C. D.. 2001; Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-κB-responsive genes in cervical keratinocytes. J Virol75:4283–4296 [CrossRef][PubMed]
    [Google Scholar]
  28. Oh S. T., Kyo S., Laimins L. A.. 2001; Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol75:5559–5566 [CrossRef][PubMed]
    [Google Scholar]
  29. Pallai R., Simpkins H., Chen J., Parekh H. K.. 2010; The CCAAT box binding transcription factor, nuclear factor-Y (NF-Y) regulates transcription of human aldo-keto reductase 1C1 (AKR1C1) gene. Gene459:11–23 [CrossRef][PubMed]
    [Google Scholar]
  30. Penning T. M., Burczynski M. E., Jez J. M., Hung C.-F., Lin H.-K., Ma H., Moore M., Palackal N., Ratnam K.. 2000; Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1–AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem J351:67–77 [CrossRef][PubMed]
    [Google Scholar]
  31. Penning T. M., Jin Y., Steckelbroeck S., Lanisnik Rizner T., Lewis M.. 2004; Structure–function of human 3α-hydroxysteroid dehydrogenases: genes and proteins. Mol Cell Endocrinol215:63–72 [CrossRef][PubMed]
    [Google Scholar]
  32. Peralta-Zaragoza O., Bermúdez-Morales V., Gutiérrez-Xicotencatl L., Alcocer-González J., Recillas-Targa F., Madrid-Marina V.. 2006; E6 and E7 oncoproteins from human papillomavirus type 16 induce activation of human transforming growth factor β1 promoter throughout Sp1 recognition sequence. Viral Immunol19:468–480 [CrossRef][PubMed]
    [Google Scholar]
  33. Pim D., Banks L.. 1999; HPV-18 E6*I protein modulates the E6-directed degradation of p53 by binding to full-length HPV-18 E6. Oncogene18:7403–7408 [CrossRef][PubMed]
    [Google Scholar]
  34. Pim D., Massimi P., Banks L.. 1997; Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth. Oncogene15:257–264 [CrossRef][PubMed]
    [Google Scholar]
  35. Pim D., Tomaic V., Banks L.. 2009; The human papillomavirus (HPV) E6* proteins from high-risk, mucosal HPVs can direct degradation of cellular proteins in the absence of full-length E6 protein. J Virol83:9863–9874 [CrossRef][PubMed]
    [Google Scholar]
  36. Rampias T., Sasaki C., Weinberger P., Psyrri A.. 2009; E6 and E7 gene silencing and transformed phenotype of human papillomavirus 16-positive oropharyngeal cancer cells. J Natl Cancer Inst101:412–423 [CrossRef][PubMed]
    [Google Scholar]
  37. Rižner T. L., Šmuc T., Rupreht R., Šinkovec J., Penning T. M.. 2006; AKR1C1 and AKR1C3 may determine progesterone and estrogen ratios in endometrial cancer. Mol Cell Endocrinol248:126–135 [CrossRef][PubMed]
    [Google Scholar]
  38. Schmittgen T. D., Livak K. J.. 2008; Analyzing real-time PCR data by the comparative C T method. Nat Protoc3:1101–1108 [CrossRef][PubMed]
    [Google Scholar]
  39. Sedman S. A., Barbosa M. S., Vass W. C., Hubbert N. L., Haas J. A., Lowy D. R., Schiller J. T.. 1991; The full-length E6 protein of human papillomavirus type 16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture. J Virol65:4860–4866[PubMed]
    [Google Scholar]
  40. Selga E., Noé V., Ciudad C. J.. 2008; Transcriptional regulation of aldo-keto reductase 1C1 in HT29 human colon cancer cells resistant to methotrexate: role in the cell cycle and apoptosis. Biochem Pharmacol75:414–426 [CrossRef][PubMed]
    [Google Scholar]
  41. Shally M., Alloul N., Jackman A., Muller M., Gissmann L., Sherman L.. 1996; The E6 variant proteins E6I–E6IV of human papillomavirus 16: expression in cell free systems and bacteria and study of their interaction with p53. Virus Res42:81–96 [CrossRef][PubMed]
    [Google Scholar]
  42. Shirasawa H., Jin M. H., Shimizu K., Akutsu N., Shino Y., Simizu B.. 1994; Transcription-modulatory activity of full-length E6 and E6*I proteins of human papillomavirus type 16. Virology203:36–42 [CrossRef][PubMed]
    [Google Scholar]
  43. Šmuc T., Rizner T. L.. 2009; Expression of 17β-hydroxysteroid dehydrogenases and other estrogen-metabolizing enzymes in different cancer cell lines. Chem Biol Interact178:228–233 [CrossRef][PubMed]
    [Google Scholar]
  44. Stacey S. N., Jordan D., Snijders P. J., Mackett M., Walboomers J. M., Arrand J. R.. 1995; Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame. J Virol69:7023–7031[PubMed]
    [Google Scholar]
  45. Storrs C. H., Silverstein S. J.. 2007; PATJ, a tight junction-associated PDZ protein, is a novel degradation target of high-risk human papillomavirus E6 and the alternatively spliced isoform 18 E6. J Virol81:4080–4090 [CrossRef][PubMed]
    [Google Scholar]
  46. Tao M., Kruhlak M., Xia S., Androphy E., Zheng Z. M.. 2003; Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells. J Virol77:13232–13247 [CrossRef][PubMed]
    [Google Scholar]
  47. Tungteakkhun S. S., Filippova M., Fodor N., Duerksen-Hughes P. J.. 2010; The full-length isoform of human papillomavirus 16 E6 and its splice variant E6* bind to different sites on the procaspase 8 death effector domain. J Virol84:1453–1463 [CrossRef][PubMed]
    [Google Scholar]
  48. Ueda M., Hung Y.-C., Chen J.-T., Chiou S.-H., Huang H.-H., Lin T.-Y., Terai Y., Chow K.-C.. 2006; Infection of human papillomavirus and overexpression of dihydrodiol dehydrogenase in uterine cervical cancer. Gynecol Oncol102:173–181 [CrossRef][PubMed]
    [Google Scholar]
  49. Vaeteewoottacharn K., Chamutpong S., Ponglikitmongkol M., Angeletti P. C.. 2005; Differential localization of HPV16 E6 splice products with E6-associated protein. Virol J2:50 [CrossRef][PubMed]
    [Google Scholar]
  50. Wang J., Shou J., Chen X.. 2000; Dickkopf-1, an inhibitor of the Wnt signaling pathway, is induced by p53. Oncogene19:1843–1848 [CrossRef][PubMed]
    [Google Scholar]
  51. Wang H.-W., Lin C.-P., Chiu J. -H., Chow K.-C., Kuo K.-T., Lin C.-S., Wang L.-S.. 2007; Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin. Int J Cancer120:2019–2027 [CrossRef][PubMed]
    [Google Scholar]
  52. Zupanska A., Kaminska B.. 2002; The diversity of p53 mutations among human brain tumors and their functional consequences. Neurochem Int40:637–645 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.038265-0
Loading
/content/journal/jgv/10.1099/vir.0.038265-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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