gms | German Medical Science

Human Papilloma Virus (HPV) associated carcinomas show a fundamentally different DNA methylation profile compared to HPV-negative carcinomas. This could already be demonstrated for cervical carcinoma as well as for head and neck squamous cell carcinoma (HNSCC). Methylation of the promoter of tumor suppressor genes leads to gene silencing. Therefore, HPV-dependent methylation may play an important role in tumor development and progression. The methylation of the gene promoter is achieved through the addition of a methyl group to the 5 position of cytosine in CG rich regions. The detailed mechanisms by which HPV causes this methylation are still unknown.

We aimed to analyze differences in methylation status of two tumor suppressor genes (CyclinA1 and Transcriptionfactor(TCF)-21) by methylation-specific PCR and expression of methyltransferases (DNMT1, DNMT3a and DNMT3b) by quantitative RT-PCR between HPV16-positive and HPV16-negative head and neck squamous cell carcinoma (fresh-frozen tissue samples of N=88). Furthermore, we developed a basic model of oropharyngeal carcinoma by immortalizing benign, HPV-negative, oral keratinozytes (HOK) by infection with HPV16. Methylation status of tumor suppressor genes as well as expression of methyltransferases was compared between the HPV-negative and the HPV16-positive HOK.

For the fresh-frozen tissue samples we could show that the methylation of CyclinA1 as well of TCF21 was much more frequent in HPV16-positive tumors. Compared to the HPV16-negative samples the HPV16-positive samples demonstrated a 2-fold increased expression rate of all examined methyltransferases. Correlating the expression of the two major HPV-oncogenes E6 and E7 (quantitative RT-PCR) with those of the methyltransferases we were able to ascertain that the higher the expression of E6 the higher the expression of all methyltransferases. Regarding E7 only the methyltransferases DNMT1 and DNMT3b showed such a positive correlation. For both oncogenes, the methyltransferase DNMT3b showed the greatest dependency. In contrast, the immortalization of the HOK cells by HPV16 did not lead to a detectable methylation of CyclinA1 or TCF21 and only the expression of the methyltransferase DNMT1 showed a weak increase by the factor 1.2.

Taken together, we could demonstrate a clear association between the methylation of tumor suppressor genes and HPV16 infection most likely orchestrated by the HPV16 oncogenes and the host methyltransferases. According to our data the oncogene E6 and the methyltransferase DNMT3b seem to play a major role here. Unfortunately, these findings could not be underpinned or further resolved by our basic tumor model, at least until now.