Role of Spz1 in HPV-mediated carcinogenesis
Persistent infection with high-risk human papillomavirus (HR-HPV) is associated with genital and head and neck cancers, yet most HR-HPV infections have a benign course. The molecular mechanisms that lead to HR-HPV persistence and cellular transformation are not well understood. The HPV oncoprotein E7 plays a central role in viral survival and proliferation in host cells and it is partly responsible for the transformation of cells infected with HPV. Its most studied role is inducing a proliferative state in epithelial cells via degradation of the Retinoblastoma (Rb) protein, but it also interferes with other cellular activities such as DNA repair and immune responses to provide a suitable environment for viral replication. Previous work in the group identified the Spermatogenic Leucine Zipper 1 protein (Spz1) as a novel interactor of the high-risk HPV E7 protein. Spz1 is a protein that is not well studied and has been described as an oncogenic transcription factor linked to cellular proliferation and cancer progression. The aim of this project was to characterise and study the function of Spz1 within the cell and investigate its role in HPV-induced carcinogenesis through its interaction with E7. To achieve this, HaCaT and NIH3T3-derived cell lines stably overexpressing HPV-16 E7 and/or Spz1 were generated to study the effect of Spz1 on E7-induced transformation of cells in vitro. Cells overexpressing Spz1 and E7 had significantly reduced proliferation and impaired growth in suspension compared to cells that only expressed E7. Furthermore, Spz1 was found to have a protective effect against Rb degradation caused by E7 in experiments where Spz1, E7 and Rb proteins were transiently overexpressed. Cycloheximide chase assays showed increased half-life of Rb in presence of Spz1, regardless of E7 expression, suggesting its anti-oncogenic activities may go beyond HPV- induced carcinogenesis. Surprisingly, E7 levels and half-life were also increased by Spz1 in a dose-dependent manner, suggesting that, when in complex, Spz1 modulates E7 degradation too. Additionally, Spz1 was found to be secreted extracellularly via non-classical secretion pathway and cells that were in contact with extracellular Spz1 showed higher E7 and Rb levels than cells that did not. This showed the ability of exogenous Spz1 to enter cells and block E7 and protect Rb, making it a very interesting candidate for further research as an anti-oncogenic agent. The work presented in this study shows Spz1 is a novel secreted factor with a tumour suppressor role, as it binds to and partially blocks the activity of the E7 oncoprotein and independently stabilises the retinoblastoma protein. Overall, these results suggest that Spz1 may be used as a therapeutic against HPV-induced cancers and possibly other cancers with Rb mutations.