Date Published: September 20, 2016
Publisher: Public Library of Science
Author(s): Ketaki Ganti, Paola Massimi, Joaquin Manzo-Merino, Vjekoslav Tomaić, David Pim, Martin P. Playford, Marcela Lizano, Sally Roberts, Christian Kranjec, John Doorbar, Lawrence Banks, Paul Francis Lambert.
A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.
Human Papillomaviruses (HPVs) are the causative agents of a large number of human malignancies, chief among which is cervical cancer, with over 500,000 reported cases worldwide annually [1,2]. There are currently more than 150 known types of HPVs, but not all of them are etiological agents of carcinomas. The cancer-causing HPVs are classified as “high-risk” types and these include HPV-16 and HPV-18, among others . A hallmark of HPV induced-malignancy is the continued expression of the viral oncoproteins E6 and E7 throughout the course of tumour development [4,5]. Inhibiting the expression of either oncoprotein in cells derived from cervical tumours results in cell growth arrest and induction of apoptosis, demonstrating a continued requirement for E6 and E7 in the maintenance of the transformed phenotype . Both viral oncoproteins act cooperatively, where E7 reprograms the infected cell to enter S phase by targeting, in part, the pRb family members, thus allowing the E2F family of transcription factors to transactivate various cell cycle genes [7–9]. The E6 oncoprotein complements the action of E7 by curbing the cell’s pro-apoptotic response to unscheduled DNA replication and targets pro-apoptotic proteins such as p53  and Bak  for proteasome-mediated degradation via the action of the E6AP ubiquitin ligase . However the ability of both E6 and E7 to contribute to cancer development depends upon a large number of other important interactions. In the case of the high-risk E6 oncoproteins a typical example is interaction with cellular PDZ (PSD-95/DLG/ZO-1) domain containing proteins.
In this study we have identified a novel activity of the high risk HPV E6 oncoproteins, linking them to the modulation of endosomal transport pathways. This appears to be mediated, at least in part, through a direct interaction between the high risk HPV E6 oncoproteins and the cellular SNX27, a protein that controls cargo fate determination in endocytic recycling. One consequence of this interaction is modulation of the endocytic transport of the glucose transporter GLUT-1, which subsequently affects the amount of glucose uptake in HPV-positive tumour cells. These results suggest that E6 can directly affect the nutrient balance in HPV infected cells, through modulation of endocytic recycling pathways to maintain sufficient nutrients for cell survival during the HPV life cycle and in progression to malignancy.