Moderator of 4 Sessions
Presenter of 3 Presentations
CERVIX AND CERVICAL CANCER
BASIC MECHANISMS OF HPV CARCINOGENESIS
E6AP IS IMPORTANT FOR HPV E6’S ROLE IN REGULATING EPITHELIAL HOMEOSTASIS AND ITS LOSS IMPAIRS KERATINOCYTE COMMITMENT TO DIFFERENTIATION
Abstract
Introduction
E6AP is a conserved binding partner and cellular target of all Alpha group HPV E6 proteins, but its precise role in modulating keratinocyte phenotype and associated signalling pathways have not been defined.
Methods
NIKS and primary foreskin keratinocytes transduced with the FUCCI cell cycle reporter, were used in combination with EGFP/mCherry cell-cell competition assays to model the epithelial basal layer. Digital imaging approaches and molecular analysis were used complement these studies.
Results
The homeostasis functions of E6 are dependent on E6AP and NHERF1, with both proteins involved in regulating basal cell density and differentiation. Interestingly, both 16 and 11 E6 required E6AP and NHERF1 binding to promote YAP nuclear localisation and YAP-reponsive gene activation to stimulate cell proliferation. Deletion of E6AP delayed the onset of differentiation, as determined by K10 immunofluorescence and PCR/RNAseq analysis. Furthermore, cells lacking E6AP resembled those expressing E6, and showed activation of the same YAP-responsive genes. NHERF1 downregulation was also common to both E6-expressing and E6AP -/- cells, suggesting that E6 and E6AP differentially regulate NHERF1 level transcriptionally and at the protein level. NHERF1 is known to interact with signalling proteins including PTEN, YAP1, beta-catenin and frizzled. Knock down NHERF1 in NIKS cells, led to YAP nuclear localisation and the activation of downstream genes. Importantly, immunostaining studies on clinical material revealed that both E6AP and NHERF1 are clearly detectable in the human epithelial basal layer, and that their abundance decreases significantly following HPV infection.
Conclusions
E6AP-/- keratinocytes phenotypically resemble E6-expressing cells, with E6AP impairing keratinocyte differentiation commitment, which is critical for HPV E6’s ability to regulate epithelial homeostasis. We suspect that E6 modulates homeostasis by inhibiting E6AP function, which impairs NHERF1 function to activate the YAP pathway, and that this accentuates the established impact of E6 on commitment to differentiation is mediated through p53.