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. 2016 Dec;2:70-77.
doi: 10.1016/j.pvr.2016.04.001. Epub 2016 Apr 7.

The High-Risk HPV E6 Target Scribble (hScrib) Is Required for HPV E6 Expression in Cervical Tumour-Derived Cell Lines

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Free PMC article

The High-Risk HPV E6 Target Scribble (hScrib) Is Required for HPV E6 Expression in Cervical Tumour-Derived Cell Lines

Christian Kranjec et al. Papillomavirus Res. .
Free PMC article

Abstract

The ability of high-risk HPV E6 oncoproteins to target cellular proteins which harbor PDZ domains is believed to play an important role in the virus life cycle and to influence the ability of these viruses to bring about malignant transformation. Whilst many of these PDZ proteins are potential tumour suppressors, involved in the control of cell polarity and cell-contact, recent studies suggest that mislocalisation or overexpression might result in the emergence of oncogenic functions. This has been shown most clearly for two E6 targets, hDlg and hScrib. In this study we show that hScrib plays such a role in HeLa cells, where its expression is required for maintaining high levels of HPV-18 E6 protein. Loss of hScrib has no effect on E6 stability but results in lower levels of E6 transcription and a reduced rate of E6 translation. We further show that, in the context of cervical tumour-derived cell lines, both hScrib and E6 cooperate in the activation of the S6 kinase signaling pathway, and thereby contribute towards maintaining high rates of protein translation. These results indicate that the residual hScrib that is present within HPV transformed cells is pro-oncogenic, and highlights the dual functions of E6 cell polarity targets.

Keywords: HPV E6; Protein translation; S6 kinase; hScrib.

Figures

Fig. 1.
Fig. 1
hScrib regulates the expression of HPV-18 E6 in HeLa cells. Panel A. HPV-positive HeLa cells were transfected with siRNA Luciferase or siRNA against the indicated E6 PDZ substrates. Cells were grown for 72 h prior to harvesting and the expression patterns of HPV-18 E6, hDlg, hScrib, TIP2, p53, E6AP and α-actinin (to monitor the protein loading) were assessed by western blot analysis. Note that Lanes 1 and 2 have been spliced but all samples were run on the same gel. Panel B. Band intensities were determined using the OptiQuant quantification program. E6 levels were normalized to 100% relative to siLuciferase-transfected HeLa cells. Standard deviations are also shown. Panel C. The silencing of hScrib was performed as in A but using two different siRNAs specific for hScrib. The expression levels of HPV-18 E6, hScrib and α-actinin to monitor the protein loading, were assessed by western blot analysis. Panel D. Strips showing levels of HPV16 E6 detection in CaSki and SiHa cells following transfection with siRNA luciferase (si Luc) or si Scrib. Arrows indicate the position of the internal positive control and the HPV-16 E6 specific band. The bottom panels show the quantification of the band intensities.
Fig. 2.
Fig. 2
Loss of hScrib reduces total HPV-18 E6 protein levels. HeLa cells were transfected with siRNA Luciferase or siRNA hScrib and after 72 h the cells were harvested and fractionated into cytosolic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions. These were then analysed by western blotting for HPV-18 E6, p53 and hScrib. Also shown are the loading controls for each fraction.
Fig. 3.
Fig. 3
Loss of hScrib does not affect HPV-18 E6 protein stability. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA against hScrib, 72 h after transfection, cells were treated with cycloheximide for 5 different time points: 0, 15, 30, 60 and 120 minutes prior to harvesting. The expression levels of HPV-18 E6, p53, hScrib, and α-actinin to monitor the protein loading, were assessed by western blot. The collated results from 3 independent experiments are shown in panel B. Band intensities were determined using the OptiQuant quantification program. The E6 levels in siLuciferase and siScrib transfected cells were normalized to 100% at time 0. Standard deviations are also shown.
Fig. 4.
Fig. 4
Loss of hScrib decreases the levels of HPV-18 E6 mRNA. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA HPV-18 E6/E7 or siRNA hScrib. 72 h after transfection, cells were harvested and the expression patterns of: pRB as a surrogate marker for E7 expression, p53 to monitor E6/E7 loss, hScrib and α-actinin, to monitor the protein loading, were assessed by western blot. Panel B. Band intensities of hypo-phosphorylated and hyper-phosphorylated pRB were determined using the OptiQuant quantification program. Levels of pRB expression are expressed as the % of hyper- and hypo-phosphorylated pRB relative to siLuciferase-transfected control cells. Standard deviations are also shown. Panel D. The graph shows the result of quantitative real time pcr for the levels of E6 mRNA, using GAPDH as the control. The bars show the relative levels of E6 expression where siLuc is used as the reference. Note the greater than 60% reduction in E6 mRNA following transfection with either siE6/E7 or siScrib. The numbers represent the means from 3 independent experiments and standard deviations are shown. Panel C shows the accompanying western blot verifying ablation of hScrib expression in a representative assay.
Fig. 5.
Fig. 5
hScrib regulates the translation of HPV-18 E6 in HeLa cells. Panel A. HeLa cells were transfected with siRNA against Luciferase or siRNA against hScrib. 72 h after transfection, cells were treated with cycloheximide for 6 h. Prior to harvesting the cells were washed three times with PBS to remove the cycloheximide and protein translation was left to recover in complete medium for 4 different time points: 0.5, 1, 3 and 5 h. The collated results from 3 independent experiments are shown in panel B. Band intensities were determined using the OptiQuant quantification program. The E6 levels in siLuciferase and siScrib transfected cells were normalized to 100% at time 0. Standard deviations are also shown.
Fig. 6.
Fig. 6
The expression of hScrib in HeLa cells maintains high levels of total PDK1 and S6 kinase. HeLa cells were transfected with siRNA Luciferase, siRNA hScrib or siRNA E6. 72 h after transfection cells were harvested and the patterns of expression of total and phosphorylated levels of PDK1 and S6 kinase as well as those of hScrib, HPV-18 E6 and α-actinin as a loading control, were assessed by western blot. Panel B. Protein band intensities relative to the experiment shown in panel A were determined using the OptiQuant quantification program. Levels of the indicated proteins were normalized to 100% relative to siLuciferase-transfected HeLa cells.

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