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Oncological research at the Jean-Uhrmacher-Institute

Approximately 600.000 new cases of head and neck squamous cell carcinoma (HNSCC) have been estimated to occur worldwide in 2011, ranking them in sixth position of all carcinomas 1-3. Risk factors for the development of HNSCC include environmental factors, excessive tobacco- and alcohol use, as well as human papillomavirus (HPV) infections. Particularly oropharyngeal squamous cell carcinomas (OSCC) are associated with HPV16 4. This group of virally induced carcinomas shows distinct clinicopathological and molecular characteristics that differ from alcohol- and tobacco induced carcinomas 4-6. Studies that have assessed the prevalence of HPV-induced OSCC report frequencies ranging from 20% to up to 90%  with solid indications that their incidence and is increasing 5,7-9. Apparently, these patients often present with an advanced stage of the disease, nevertheless, they await a better prognosis 10. It is our research goal to discover new diagnostic and pharmaceutical strategies supporting treatment options for HNSCC in general and especially in OSCC.

  1. Parkin, D. M., Bray, F., Ferlay, J. & Pisani, P. Global cancer statistics, 2002. CA: A Cancer Journal for Clinicians 55, 74–108 (2005).
  2. Global cancer statistics. 61, 69–90 (2011).
  3. Kamangar, F., Dores, G. M. & Anderson, W. F. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. Journal of Clinical Oncology 24, 2137–2150 (2006).
  4. Olthof, N. C. et al. Next-generation treatment strategies for human papillomavirus-related head and neck squamous cell carcinoma: where do we go? Rev. Med. Virol. (2011). doi:10.1002/rmv.714
  5. Hafkamp, H. C. et al. Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas. Int J Cancer 122, 2656–2664 (2008).
  6. Klussmann, J. P. et al. Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 15, 1779–1786 (2009).
  7. Mellin, H. et al. Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer. Int J Cancer 102, 152–158 (2002).
  8. Koskinen, W. J. et al. Prevalence and physical status of human papillomavirus in squamous cell carcinomas of the head and neck. Int J Cancer 107, 401–406 (2003).
  9. Begum, S., Cao, D., Gillison, M., Zahurak, M. & Westra, W. H. Tissue distribution of human papillomavirus 16 DNA integration in patients with tonsillar carcinoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 11, 5694–5699 (2005).
  10. Fakhry, C. et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. Journal of the National Cancer Institute 100, 261–269 (2008).

Karzinome im Kopf-Hals-Bereich (Head and Neck Squamous Cell Carcinoma, HNSCC) gehören weltweit zu den häufigsten Krebserkrankungen. Krankheitsauslösend sind häufig langjähriger Konsum von Tabak und Alkohol, sowie Infektionen mit Humanen Papillomviren (HPV) [1]. Insbesondere der Oropharynx ist von HPV-Infektionen betroffen (Oropharyngeal Squamous Cell Carcinoma, OPSCC). Mittlerweile kann von mehr als 50% HPV-indizierter OPSCC ausgegangen werden. Weltweit ist eine kontinuierliche Zunahme HPV-positiver OPSCC zu beobachten und zukünftig könnten diese sogar die Inzidenz von Karzinomen der Cervix uteri übertreffen [2]. Die meisten OPSCC Patienten werden in Deutschland operiert, oft zusätzlich kombiniert mit Radio(-Chemo)-Therapie.

 

Zitate:

  1. C. U. Hübbers and B. Akgül, “HPV and cancer of the oral cavity.,” Virulence, vol. 6, no. 3, pp. 244–248, 2015.
  2. A. K. Chaturvedi, E. A. Engels, R. M. Pfeiffer, B. Y. Hernandez, W. Xiao, E. Kim, B. Jiang, M. T. Goodman, M. Sibug-Saber, W. Cozen, L. Liu, C. F. Lynch, N. Wentzensen, R. C. Jordan, S. Altekruse, W. F. Anderson, P. S. Rosenberg, and M. L. Gillison, “Human Papillomavirus and Rising Oropharyngeal Cancer Incidence in the United States,” Journal of Clinical Oncology, vol. 29, no. 32, pp. 4294–4301, Nov. 2011.

Human papillomaviruses are small DNA-Viruses which can be devided in more than 200 subtypes. In common, they cause benign papillomas or warts. But a groop of so called ‚high risk’ types (especially HPV16 and 18) can induce malignant neoplasias as cervical carcinomas and HNSCC 1. Todays knowledge about the events during the malignant tronsformation from an acute HPV-infection to a tumor , research was done especially on the cervical situation. Here, HPVs infect the basal layer of proliferating epidermal or mucosal cells via microlesions of skin or mucosa. During an accute infection, viral and cellular DNA replicates independently (episomal virus) 2,3. Except for some limited expression of the viral proteins E5, E6 and E7, viral gene expression is largely suppressed in these cells through the viral proteins E1 and E2. While the infection progresses, HPV spreads laterally or migrates into the suprabasal differentiating cell layers by cell division. In the latter cells, late viral gene expression is started resulting in replication of the circular genome and production of structural proteins. In the upper cell layers, viral particles are assembled and released.

  1. Psyrri, A. & Dimaio, D. Human papillomavirus in cervical and head-and-neck cancer. Nat Clin Prac Oncol5, 24–31 (2008).
  2. Hausen, zur, H. Human papillomaviruses and their possible role in squamous cell carcinomas. Curr Top Microbiol Immunol78, 1–30 (1977).
  3. Hausen, zur, H. Intracellular surveillance of persisting viral infections. Human genital cancer results from deficient cellular control of papillomavirus gene expression. Lancet2, 489–491 (1986).

During the progression of a persistant HPV16 infection, proliferating cells of the lower epithelial layers start to express viral genes. Replication of the viral genome still occures as extrachromosomal circular episomes, whereupon the expression of HPV-genes highly rushes. This effects especially the oncogenes E6 and E7. They bind and degrade p53 and Rb, respectively. This leads to a massive deregulation of the host-cell DNA synthesis and loss of cell cyle control with rising instability of the host genome 1-3. The consequences are abnormal centrosome numbers, misssegregations, and aneuploidies 1. But for the transition from dysplasia to invasive cancer integration of the viral DNA into the cellular host genome is needed. Therefore, the open reading frame of the viral E1/E2 region is disrupted and nonhomologous sequence-specific recombination at a single or a few chromosomal loci occures. Subsequently the internal viral transcription control of E6 and E7 gets lost so that their effects are increased 4,5. Furthermore, the integration results in the loss of the polyadenylation signal of genes in the E4 region. This leads to the formation of viral-cellular fusiontranskripts. Through the loss of AU-rich, mRNA-destabilising sequences in the 3’-LCR E6- and E7-fusiontranscripts are more stabile than episomal ocogenetranscripts 6. Even more, the direct context of the genomic integration site seems to influence the activity of the viral oncogeneexpression 2. In cervical carcinomas, integration sites are distributed through the whole genome and differ from clinical sample to sample 3,7. The involvmet of specific integration sites like the paradigm of the retroviral insertionmutagenesis can be excluded 8.

On the other hand, the HP-Virus shows a targeted deaktivation of cell cycle regulation. The viral oncogene E6 binds to p53 and degrades it, so that apoptosis induction is avoided. Oncoprotein E7 binds to retinoblastoma protein b (pRb) and subsequentially inactivates cell cycle control with unregulated cell cycle activity. Furthermore, E6 and E7 disrupt the regulation of the mitotic spindle apparatus. This leads to mistakes in seggregation of chromosomes, with broken, numeric and structural chromosome abnormalities, as well as inbalances of allels 9. Hence, they are not as pronounced as in HPV-negative HNSCC 10. The chromosomal instability is therefore an outstanding event in the development of HPV-induced tumors.

  1. Duensing, S. et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci USA97, 10002–10007 (2000).
  2. Knebel Doeberitz, von, M., Bauknecht, T., Bartsch, D. & Hausen, zur, H. Influence of chromosomal integration on glucocorticoid-regulated transcription of growth-stimulating papillomavirus genes E6 and E7 in cervical carcinoma cells. Proc Natl Acad Sci USA88, 1411–1415 (1991).
  3. Luft, F. et al. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer92, 9–17 (2001).
  4.  Kessis, T. D., Connolly, D. C., Hedrick, L. & Cho, K. R. Expression of HPV16 E6 or E7 increases integration of foreign DNA. Oncogene13, 427–431 (1996).
  5. Romanczuk, H. & Howley, P. M. Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. Proc Natl Acad Sci USA89, 3159–3163 (1992).
  6. Jeon, S. & Lambert, P. F. Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis. Proc Natl Acad Sci USA92, 1654–1658 (1995).
  7. Wentzensen, N., Vinokurova, S. & Knebel Doeberitz, von, M. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. 64, 3878–3884 (2004).
  8. Butel, J. S. Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease. Carcinogenesis21, 405–426 (2000).
  9. Thomas, J. T. & Laimins, L. A. Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint. J Virol72, 1131–1137 (1998).
  10. Klussmann, J. P. et al. Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research15, 1779–1786 (2009).

Während im HPV16-Genom insgesamt auffällig wenig Methylierungsstellen zu finden sind, können in der viralen Long Control Region LCR zwei Cluster identifiziert werden, die eine Bindung des viralen E2-Proteins regulieren. Die distalen E2-Bindungsstellen (E2BS) wirken sich dabei hyperaktivierend auf die Expression der viralen Onkogene E6 und E7 aus, während die proximalen E2BS eine Deaktivierung von E6 und E7 verhindern. Wir konnten insbesondere für die proximalen E2-Bindungsstellen deutlich unterschiedliche Methylierungsmuster erkennen. Eine Korrelation mit prognostischen Daten hat gezeigt, dass eine mittlere Methylierungsfrequenz (20-80% Methylierung) häufig mit einem intakten E2-Leseraster einher geht (d.h. nicht integrierte virale DNA). Bei hoher Methylierungshäufigkeit korreliert dies mit gemischtem Vorkommen von integrierten und episomalen viralen Kopien, bei der niedrigen Methylierungshäufigkeit mit einem unterbrochenem E2-Leseraster (integriertes Virus) (Reuschenbach, Huebbers et al., Cancer 2015). Somit könnte die Methylierungsanalyse ein guter Marker für die prognostisch relevante virale Integrationsanalyse sein.