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: 4.65). 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.

Full-text

Available from: Dennis J Mccance, Apr 17, 2015
0 Followers
 · 
83 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.
    PLoS ONE 05/2014; 9(5):e96136. DOI:10.1371/journal.pone.0096136 · 3.53 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; · 6.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Since p14(ARF)and HPV16 E6/E7 oncoproteins are important regulators participating the p53/Rb pathways, genetic variations of p14(ARF) may modify tumor HPV16 status and survival of HPV16-positive SCCOP patients. We determined tumor HPV16 status and expression of p14/p53, and genotyped p14(ARF) rs3731217 and rs3088440 polymorphisms in 552 incident SCCOP patients. We found that patients having variant genotypes for each p14(ARF) polymorphism were approximately twice or three times as likely to have HPV16-positive tumors compared with patients with corresponding common homozygous genotype, and such an association was particularly pronounced in patients with variant genotypes of both polymorphisms. After definitive chemoradiotherapy, patients having p14(ARF) rs3731217 TG/GG variant genotypes had significantly better overall, disease-specific, and disease-free survival than those having TT genotype, respectively. Multivariable analysis found that patients with p14(ARF) rs3731217 TT genotype had an approximately 7, 11, and 3-fold increased risk for death overall, death due to SCCOP, and recurrence than those with TG/GG variant genotypes, respectively. Furthermore, such significantly prognostic effect was also found when survival analysis was limited to HPV16-positive patients. Additionally, potentially functional relevance of the two variants was characterized to explore the genotype-phenotype correlation. Our findings indicate p14(ARF) variants may predict tumor HPV16-positive SCCOP patients and survival.
    Carcinogenesis 10/2013; DOI:10.1093/carcin/bgt336 · 5.27 Impact Factor