Article

Human papillomavirus type 16 E6/E7 upregulation of nucleophosmin is important for proliferation and inhibition of differentiation.

Centre for Cancer Research and Cell Biology, Queen's University, Belfast BT9 7BL, United Kingdom.
Journal of Virology (Impact Factor: 5.08). 03/2010; 84(10):5131-9. DOI: 10.1128/JVI.01965-09
Source: PubMed

ABSTRACT The E6 and E7 oncoproteins of high-risk human papillomaviruses (HPVs) are together sufficient to cause cellular transformation. Nucleophosmin (NPM) was identified as a protein with increased levels in two-dimensional (2-D) gel analysis of human foreskin keratinocytes (HFKs) expressing E7 following methylcellulose-induced differentiation. Analysis of NPM expression in E7-expressing cells and E6- and E7-expressing cells in culture and in organotypic rafts confirmed the increased levels observed in 2-D gel analysis. The elevated expression of NPM was determined to be posttranscriptional and was attributed to increased v-akt murine thymoma viral oncogene (AKT) activity in the E6- and E7-expressing cells. Depletion of NPM caused a reduction in the replicative capacity of E7- and E6/E7-expressing HFKs and an increase in markers of differentiation. Also, the p53 and pRb tumor suppressor levels are increased with the knockdown of NPM in E6/E7-expressing cells, and, interestingly, p14(ARF) is relocalized from the nucleolus to the nucleoplasm and cytoplasm in these cells. The results show for the first time that NPM is required for the proliferation and inhibition of differentiation observed in HPV E6- and E7-expressing primary cells.

0 Bookmarks
 · 
68 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50-p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.
    Journal of Experimental Medicine 06/2013; · 13.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status.
    Oncotarget 07/2014; · 6.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A number of epigenetic alterations occur in both the virus and host cellular genomes during human papillomavirus (HPV)-associated carcinogenesis, and investigations of such alterations, including changes in chromatin proteins and histone modifications, have the potential to lead to therapeutic epigenetic reversion. We report here that transformed HPV16 E6/E7-expressing primary human foreskin keratinocytes (HFKs) (E6/E7 cells) demonstrate increased expression of the PRC2 methyltransferase EZH2 at both the mRNA and protein levels but do not exhibit the expected increase in trimethylated H3K27 (H3K27me3) compared to normal keratinocytes. In contrast, these cells show a reduction in global H3K27me3 levels in vitro, as well as upregulation of the KDM6A demethylase. We further show for the first time that transformation with the HPV16 E6 and E7 oncogenes also results in an increase in phosphorylated EZH2 serine 21 (P-EZH2-Ser21), mediated by active Akt, and in a downregulation of the PRC1 protein BMI1 in these cells. High-grade squamous cervical intraepithelial lesions also showed a loss of H3K27me3 in the presence of increased expression of EZH2. Correlating with the loss of H3K27me3, E6/E7 cells exhibited derepression of specific EZH2-, KMD6A-, and BMI1-targeted HOX genes. These results suggest that the observed reduction in H3K27me3 may be due to a combination of reduced activities/levels of specific polycomb proteins and increases in demethylases. The dysregulation of multiple chromatin proteins resulting in the loss of global H3K27me3 and the transcriptional reprogramming in HPV16 E6/E7-infected cells could provide an epigenetic signature associated with risk and/or progression of HPV16-associated cancers, as well as the potential for epigenetic reversion in the future.
    Journal of Virology 08/2011; 85(21):10999-1006. · 5.08 Impact Factor

Full-text (2 Sources)

View
9 Downloads
Available from
May 21, 2014