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: 4.44). 03/2010; 84(10):5131-9. DOI: 10.1128/JVI.01965-09
Source: PubMed


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.

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Available from: Dennis J Mccance, Oct 06, 2015
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    • "ARF is considered to be a potential nucleolar integrator of growth signals, and Apicelli et al [17] recently proposed that basal ARF acts as a monitor of steady-state ribosome biogenesis and growth. ARF can be induced by protein E7 from the cottontail rabbit in E6/E7- or E7-immortalized rabbit keratinocytes [53] and by HPV16 E7 in E6/E7-expressing keratinocytes [52]. Our data show that E7 expression can induce p14ARF early after transduction of primary cervical keratinocytes, before their immortalization. "
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    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 · 3.23 Impact Factor
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    • "shER was provided by D. Picard (Geneva University, Geneva, Switzerland) siRNA for IKK or IKK was purchased from Ambion, and the sequence used was previously published (Accardi et al., 2011). siRNA for HPV16E6E7 or E7 was purchased from Dharmacon and previously published (Tang et al., 2006; McCloskey et al., 2010). The ISRE Luciferase minimal promoter was purchased from Stratagene. "
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    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; 210(7). DOI:10.1084/jem.20122394 · 12.52 Impact Factor
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    • "HPV-16 E6/E7 oncoproteins have been demonstrated to cause immortalisation of primary human keratinocytes and are expressed in malignant cancers [20]. Many studies have previously reported the ability of the HPV-16 E6/E7 oncoproteins to disrupt the normal process of differentiation of human foreskin keratinocytes [21] by targeting key tumour suppressors, such as p53 [22] and pRb [23], resulting in increased levels of cell survival proteins, such as Akt [24], and disruption of the cell cycle [25]. The HPV E2 protein functions as a repressor or an activator of early gene transcription, which regulates viral transcription and genome replication [26]. "
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    ABSTRACT: Background Human papillomavirus type 16 (HPV 16) E2 protein is a multifunctional DNA-binding protein. HPV 16 E2 regulates many biological responses, including DNA replication, gene expression, and apoptosis. The purpose of this study was to investigate the relationship among the receptor for globular heads of the human C1q (gC1qR) gene expression, HPV 16 E2 transfection and apoptosis regulation in human cervical squamous carcinoma cells (C33a and SiHa). Methods gC1qR expression was examined in C33a and SiHa cells using real-time PCR and Western blot analysis. Apoptosis of C33a and SiHa cells was assessed by flow cytometry. C33a and SiHa cell viability, migration and proliferation were detected using the water-soluble tetrazolium salt (WST-1) assay, a transwell assay and 3H-thymidine incorporation into DNA (3H-TdR), respectively. Results C33a and SiHa cells that were transfected with a vector encoding HPV 16 E2 displayed significantly increased gC1qR gene expression and p38 mitogen-activated protein kinase (p38 MAPK)/ c-jun N-terminal kinase (JNK) activation as well as up-regulation of cellular apoptosis, which was abrogated by the addition of gC1qR small interfering RNA (siRNA). Furthermore, the changes in C33a and SiHa cell viability, migration and proliferation that were observed upon HPV 16 E2 transfection were abrogated by SB203580 (a p38 MAPK inhibitor) or SP600125 (a JNK inhibitor) treatment. Conclusion These data support a mechanism whereby HPV 16 E2 induces apoptosis by silencing the gC1qR gene or inhibiting p38 MAPK/JNK signalling in cervical squamous cell carcinoma.
    Journal of Translational Medicine 05/2013; 11(1):118. DOI:10.1186/1479-5876-11-118 · 3.93 Impact Factor
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