The Role of HPV E7 in Maintenance of Cell Transformation

Basukala, Om (2019). The Role of HPV E7 in Maintenance of Cell Transformation. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00010bfd

Abstract

A hallmark feature of cervical cancers caused by the Human Papillomavirus is the continued and high-level expression of viral oncoproteins. These viral oncoproteins play a significant role in induction of malignancy by targeting several critical cell control pathways. One of the potentially important elements in this process is phosphorylation of E7 by Casein Kinase II (CKII) which induces S-phase entry and modulates association with pRB and other pocket proteins. Phosphorylation of E7 has also been known to enhance E7’s ability to interact with other cellular targets and thereby increasing the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on the importance of this site in E7, once the tumour cells have become fully transformed. To determine this, we have generated genome edited cell lines at CKII phospho-acceptor site of HPV-18 E7 in C4-1 cervical tumour derived cells using CRISPR/Cas9. We show that the HPV-18 E7 S32A/S34A mutation abolishes CKII phosphorylation. Genome edited cells with this mutation continue to proliferate but are more slow-growing than wild type cells, reach lower saturation densities and are susceptible to low nutrient conditions. The invasive ability of these cells is markedly reduced, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. Using proteomic analysis, we identify Vangl1, planar cell polarity protein, as a novel CKII phospho-dependent interactor of E7. The steady levels of Vangl1 are downregulated in genome edited cells and it co-operates with HPV-16 E7 in BRK cell transformation assays, indicating Vangl1 being important for E7 transforming activity. Finally, I also show that HPV-E7 interacts with PTPN21, a non-receptor tyrosine phosphatase, via c-terminal region of E7. The interaction leads to destabilization of PTPN21 by high-risk E7, suggesting a potential role of E7 in affecting post-translation modifications of its targets. Thus, HPV E7 continue to play an important role in maintenance of transformed phenotype and targeting E7 phosphorylation could be a potential therapeutic strategy in HPV-induced malignancy.

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