Mechanism of HPV E6 oncogene mediated silencing of Syntenin-2 gene expression and role of Syntenin-2 in skin homeostasis.
Virology
Final Report Abstract
Human papillomavirus (HPV) of genus betapapillomavirus (betaHPV, e.g. HPV8) have been implicated in the development of squamous cell carcinomas of the skin in Epidermodysplasia verruciformis (EV) patients and immunosuppressed organtransplant-recipients. Our lab recently showed that the E6 oncoprotein of HPV8 inhibits the expression of the nuclear PDZ-domain-protein Syntenin-2 at mRNA level. Knockdown of Syntenin-2 in primary human keratinocytes led to inhibition of differentiation and increase in proliferation capacity. We now could show that decreased Syntenin-2 protein expression in N/TERT-8E6 correlates with promoter hyper-methylation and the differentiation state of the E6 expressing keratinocytes. As Syntenin-2 is known to bind with high affinity to nuclear phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2) we hypothesized that PI(4,5)P2 levels may change due to HPV8-E6 mediated Syntenin-2 repression. We could demonstrate that absence of Syntenin-2 was paralleled with increased levels of PI(4,5)P2 / protein complexes in HPV8-E6 positive keratinocytes grown in monolayer cultures in low-calcium containing media. In addition, elevated PI(4,5)P2 levels were also found in HPV8-E6 positive organotypic skin cultures and in skin papillomas of HPV8-E6 transgenic mice. We also found strong nuclear staining for PI(4,5)P2 in HPV8 positive skin cancers of EV patients and HPV16 positive cervical tumor and cancer tissue. No staining for PI(4,5)P2 was found in normal human skin, merkel cell polyomavirus (MCPyV)- positive merkel cell carcinomas (MCC) and melanomas indicating that PI(4,5)P2 upregulation may be specifically be associated with HPV related keratinocytic cancer. PI(4,5)P2 is synthesized by either the action of phosphatidylinositol-4-phosphate-5- kinase type I (PIP5KI) or by phosphatidylinositol-5-phosphate 4-kinase type II (PIP4KII). Comparing HPV8-E6 positive and negative cells, we found an upregulation of all three isoforms of PIP4KII and PIP5KI. The knockdown of kinase isoforms did not result in significant changes of PI(4,5)P2 linked protein bands, indicating overlapping functions of kinase isoforms in nuclear PI(4,5)P2 generation. By further exploring the mechanism of PI(4,5)P2 generation in E6 cells, we observed that a) the epidermal lipid chaperone fatty acid binding protein 5 (FABP5) is overexpressed in HPV8-E6 expressing cells, b) E6 binds to the PI(4,5)P2 5’-phosphatase OCRL-1 (inactivation of OCRL-1 is known to lead to PI(4,5)P2 accumulation) and c) E6 binds to phosphatidylinositol (PI) (a precursor molecule for PI(4,5)P2 generation). The use of HPV8-E6 mutants will clarify the direct involvement of these three factors in the increase of PI(4,5)P2 levels in HPV8-E6 cells. Finally we could show that in HPV8-E6 cells PI associates with H3K4me3 and that PI(4,5)P2 localizes to H3K4me3 in HPV8 positive human skin cancers. In conclusion, our work led to the identification of a new oncogenic pathway in which HPV targets nuclear phosphoinositides, thereby affecting histone regulation, which may contribute to HPV mediated carcinogenesis.
Publications
- HPV8-E6 mediates silencing of Syntenin-2 and increases nuclear phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) levels in transformed skin keratinocytes. 30th International Papillomavirus Conference, Lisabon 17-21.9.2015
Benjamin Marx
- The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8. J Gen Virol. 2016 Feb;97(2):463-72
Heuser S, Hufbauer M, Marx B, Tok A, Majewski S, Pfister H, Akgül B
(See online at https://doi.org/10.1099/jgv.0.000363)
