Abstract
HPV is a non-lytic virus that infects and replicates within keratinocytes in the skin. Persistent infection with high risk human papillomavirus (HPV) such as HPV type 16 and 18 is associated with cervical cancer. Despite the expression of viral antigens during replication, the immune system is unable to efficiently clear HPV infection due to the presence of multiple evasion techniques deployed by this virus. E-cadherin loss is a common feature in high-risk HPV infected cells and is associated with a reduction in the number of Langerhans cells at the infection site. The ability of HVP16 to reduce E-cadherin expression is directly linked to the expression of E6 and E7.
We utilised HCT116 with stable HPV16 E6 expression to study mechanisms involved in E6 regulation of surface E-cadherin. We examined regulation of E-cadherin in different cell densities in the presence and absence of E6 protein. Our results suggest the role for post-translational regulation of surface E-cadherin by E6, when cells are at high density and in cell-to-cell contact. When cells are grown sparsely, E-cadherin was transcriptionally regulated by E6. We hypothesised that when cells were grown at high density, regulation of surface E-cadherin by HPV16 E6 occurred by affecting E-cadherin trafficking at the cell membrane. Analysis of E-cadherin endocytosis showed that there was reduced E-cadherin endocytosis in cells expressing E6. We observed up regulation of E-cadherin ubiquitination in E6 expressing cells, suggesting a role for the ubiquitination machinery in the E6-dependent reduction in surface E-cadherin.
Although there was no indication of E6 control over E-cadherin transcription in dense cultures, we did observe abnormal localisation of SNAI2 E-cadherin gene repressor, in E6 expressing cells. Expression of E6 increased SNAI2 nuclear accumulation, indicating a potential function for SNAI2 in the nucleus that enables E6 control over E-cadherin. Upon further examination, we observed an increase in SNAI2 ubiquitination in E6 expressing cells, which suggests that similar to E-cadherin, SNAI2 is also regulated by E6 by ubiquitination. Treatment of cells with the proteosomal inhibitor MG132 revealed an inverse relationship between SNAI2 levels and its ubiquitination status in E6 cells, indicatinga novel degradation pathway that is important in E6 dependent SNAI2 regulation.
Here, we have shown that E-cadherin regulation by HPV16 E6 in dense HCT116 cells occurs at the post-translational level. Our overall data revealed a role of the ubiquitination pathway as a potential mechanism utilised by HPV 16 E6 to regulate surface E-cadherin. A similar ubiquitination pathway might also be employed by E6 to regulate SNAI2 protein expression. Although we have yet to determine the association between E6 dependent SNAI2 abnormalities and E-cadherin regulation, our findings suggest a contribution of protein ubiquitination in controlling E-cadherin and SNAI2 trafficking in the presence of HPV16 E6. These findings require further exploration to determine the specific factors involved in ubiquitination of these proteins. Once identified, these factors maybe useful as targets to combat E6 down-regulation of E-cadherin and to facilitate development of better therapeutic treatments for cervical cancer.