Human papillomavirus type 16 E6/E7 upregulation of nucleophosmin is important for proliferation and inhibition of differentiation.
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.
- SourceAvailable from: Massimo Tommasino[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
- [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
- [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
JOURNAL OF VIROLOGY, May 2010, p. 5131–5139
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 10
Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin
Is Important for Proliferation and Inhibition of Differentiation?
Rachel McCloskey,1Craig Menges,1† Alan Friedman,2Daksha Patel,1and Dennis J. McCance1*
Centre for Cancer Research and Cell Biology, Queen’s University, Belfast BT9 7BL, United Kingdom,1and Proteomics Center,
University of Rochester, 575 Elmwood Avenue, Rochester, New York 146422
Received 16 September 2009/Accepted 23 February 2010
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 methylcellu-
lose-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, p14ARFis 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.
The E6 and E7 oncoproteins of human papillomavirus type
16 (HPV-16) have been shown to cause immortalization of
primary human keratinocytes and are expressed in malignant
cancers caused by HPV-16 infection (27, 28). E6 is best known
for its ability to bind and degrade the tumor suppressor p53,
whereas E7 can inactivate the pRb family of tumor suppressors
(2, 3, 26). E6 is one of the earliest genes expressed during HPV
infection and has been shown to bind sites at both the C
terminus and the DNA binding domain of p53. Degradation is
mediated by the ubiquitin ligase E6-associated protein (E6-
AP/UBC3A), leading to degradation of p53 via the 26S pro-
teasome (14, 34). Another mechanism by which E6 inhibits p53
activity is by binding to p300/CBP and inhibiting the coactiva-
tion of p53-dependent gene transcription (30).
E7 can bind to and inactivate the pRb family of tumor
suppressors, Rb, p107, and p130 (5). These proteins play a
major role in regulating the cell cycle, transcriptional repres-
sion, and tumor suppression (7, 11). E7 has the ability to
override normal cell cycle activities by binding to the hypo-
phosphorylated form of Rb, prematurely pushing cells into the
S phase and resulting in disruption of differentiation. Recent
data have indicated the role of E7 in pRb-independent mech-
anisms that target other cellular proteins and disrupt their
normal function (1).
In an attempt to identify other significant targets of E7 we
carried out a two-dimensional (2-D) gel analysis of proteins
from E7-expressing primary human foreskin keratinocytes
(HFKs) during methylcellulose-induced differentiation. Nu-
cleophosmin (NPM) was identified as a protein showing in-
creased levels compared to the vector control cells. NPM is a
nucleolar phosphoprotein that is abundant in tumor and pro-
liferating cells (9, 21). Although it is localized in the nucleoli,
NPM has the ability to shuttle between the nucleus and cyto-
plasm and can bind and chaperone proteins to alter their
cellular localization (4). Regarded as a proto-oncogene, NPM
is overexpressed in a range of cancers and is used as a marker
for colon, gastric, and ovarian cancers, with increased levels of
NPM correlating with tumor progression (8). It is also the most
frequently mutated gene in acute myeloid leukemia (AML),
with approximately 35% of patients showing an abnormality in
the gene (9). NPM functions through sustaining ribosome bio-
genesis, inhibiting apoptosis and disrupting differentiation, and
upregulation of NPM in cells leads to an increase in prolifer-
ation (4). In this report, we provide the first evidence of a role
for NPM in HPV-mediated proliferation and inhibition of dif-
ferentiation. We show that NPM is upregulated by E7 at the
protein level through the ability of E7 to deregulate v-akt
murine thymoma viral oncogene (AKT) and that this upregu-
lation is required for proliferation of cells and for the inhibi-
tion of differentiation.
MATERIALS AND METHODS
Plasmids and siRNAs. The pBabe (puro), pBabe-E6stopE7 (E7), and pBabe
E6/E7 retroviral constructs used were described previously (10). pSuper-retro
constructs expressing short-hairpin RNAs (shRNA) against no known annotated
gene (shScr) were cloned as previously described (31), as were the pSuper-retro
constructs expressing shRNAs targeting Rb and p53(15). The following se-
quences were used for shRNAs targeting NPM: forward, 5?-CCA GTG GTC
TTA AGG TTG AAG TGT GG-3?; reverse, 5?-TCC AGA TAT ACT TAA
GAG TTT CAC ATC CTC CTC C-3?. Before transfection into ?NYX-GP
packaging cells, all retroviral plasmid constructs were sequenced. Small interfer-
ing RNAs (siRNAs) targeting AKT (SignalSilence 6211, 6510, and 6511) were
purchased from Cell Signaling. siRNAs targeting NPM (sense, UGA UGA AAA
UGA GCA CCA G) and a Scrambled control (ACG GUA ACA GUC ACU
GAG C) were designed and purchased from Darmacon.
* Corresponding author. Mailing address: Centre for Cancer Re-
search and Cell Biology, Queen’s University, Belfast BT9 7BL, United
Kingdom. Phone: 44 2890972184. Fax: 44 2890972776. E-mail: d
† Present address: Fox Chase Cancer Centre, Philadelphia, PA
?Published ahead of print on 17 March 2010.
Cell culture. Primary human foreskin keratinocytes (HFKs) were isolated
from neonatal foreskin and transduced with retrovirus produced using the
?NYX-GP packaging line as previously described (10).
Differentiation of HFK cell lines and induction of differentiation. pBabe shScr,
pBabe shRNA targeting NPM (shNPM), E6/E7 shScr, and E6/E7 shNPM HFK
cell lines were induced to differentiate by using organotypic rafts as previously
described (10). Raft cultures were harvested, fixed in 4% paraformaldehyde,
embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).
Bromodeoxyuridine (BrDU) (20 ?mol/liter) was added to the raft culture 12 h
before harvest to label DNA-synthesizing cells. Cell lines were also induced to
differentiate by suspension in 1.6% methylcellulose (29).
Mass spectrometric sequencing of proteins extracted from silver-stained poly-
acrylamide gels. Proteins were extracted from gel samples and digested into
tryptic peptides as previously described (35). The tandem mass spectrometry
(MS/MS) spectra were searched using SEQUEST7 and the Bioworks browser
(both from Thermo Corporation, San Jose, CA) and the publicly available
European Bioinformatics Institute nonredundant human.fasta sequence data-
base (http://www.ebi.ac.uk/IPI/IPIhuman.html) to determine possible sequence
correlations of known proteins.
Western blot analysis. Protein lysate concentrations were either 30 or 50 ?g
for all blots, as described previously (29). In this study, the following primary
antibodies were used: mouse monoclonal anti-Rb and mouse monoclonal anti-
p53 (BD PharMingen) (1:1.000); mouse monoclonal anti-B23 and rabbit poly-
clonal anti-C23 (1:1,000) and mouse monoclonal antiactin (1:20,000) (Santa Cruz
Biotechnology); mouse monoclonal anti-p14ARF(Neomarkers) (1:500); and rab-
bit polyclonal anti-K1 (Covance) (1:5,000). Secondary antibodies used in this
study were goat anti-mouse horseradish peroxidase (HRP) and goat anti-rabbit
HRP (Santa Cruz Biotechnology) (1:2,000). Luminescence was detected by ei-
ther Perkin-Elmer or Pierce enhanced chemiluminescence (ECL), and the signal
was detected using an Alpha Innotech FluorChem SP imaging system.
Real-time reverse transcription-PCR (RT-PCR) analysis. RNA was extracted
with a High Pure RNA isolation kit (Roche), according to the manufacturer’s
instructions. FastStart SYBR green Master (Roche) was used according to the
manufacturer’s instructions to amplify PCR products, and fluorescence was mon-
itored using a DNA engine Peltier thermal cycler (Bio-Rad) equipped with a
Chromo4 real-time PCR detection system (Bio-Rad). cDNA samples were di-
luted 1:10 and quantified by amplification using a series of dilutions of control
cDNA. The following cycling conditions were used: initial denaturation at 95°C
for 10 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, and 60°C for 60 s.
Expression levels were assessed in triplicate and normalized to ribosomal large
protein P0 (RPLP0) control levels. Graphs produced represent the combined
results of three independent replicate experiments.
Metabolic labeling for NPM half-life determinations. pBabe- and E6/E7-
expressing HFKs were pulse-labeled with 110 ?Ci/ml of EasyTag EXPRESS35S
protein-labeling mix–[35S]methionine-cysteine–2 mCi (74 MBq) stabilized aque-
ous solution (catalog no. NEG772002MC; Perkin Elmer). After 3 h, the cells
were washed and labeled and were media chased with fresh unlabeled media.
Cells were harvested at indicated time points thereafter, and immunoprecipita-
FIG. 1. Nucleophosmin levels are increased in E7- and E6/E7-expressing cells. HFKs were induced to differentiate by suspension in methyl-
cellulose or organotypic rafts. A 2-D gel (a) and a magnified portion (b) showing an increase in NPM (circled in red and indicated with red arrows,
respectively) in cells expressing E7 compared to pBabe control cells. (c and d) Western blots showing the expression of NPM in pBabe control cells
and E7-expressing cells during keratinocyte differentiation (c) or in cycling cells (d). (e) pBabe- and E7- and E6/E7-expressing organotypic rafts
stained for NPM and DAPI, confirming the findings of the 2-D gel analysis. (Merged images show overlays of DAPI and NPM staining images).
Scale bar, 100 ?m. (f) NPM staining of normal and CIN3 cervical sections. NPM is upregulated in the CIN3 lesions compared to the paired normal
tissue. Scale bar, 100 ?m.
5132MCCLOSKEY ET AL. J. VIROL.
tion (IP) and Western blotting were performed. Samples were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fol-
lowed by staining in colloidal Coomassie blue (Invitrogen), and gels were dried
under vacuum conditions (Bio-Rad Geldryer). Dried gels were exposed to a
Phosphoimager screen and read using a Fuji FLA-7000 analyzer. Bands were
normalized to protein levels in the immunoprecipitate.
Immunofluorescent staining. Paraffin-embedded organotypic raft sections
were deparaffinized with 2 washes in xylene and rehydrated with 4 washes in
step-down concentrations of ethanol. Antigen retrieval with citrate buffer (Dako)
was performed for 20 min in a steamer. The sections were then washed three
times in phosphate-buffered saline (PBS), and the primary antibody was applied.
The following primary antibodies (diluted in 10% fetal bovine serum) were used
for this study: rabbit polyclonal anti-keratin 1 (Covance) (1:4,000; no retrieval);
mouse monoclonal anti-B23 and rabbit polyclonal anti-C23 (Santa Cruz Biotech-
nology) (1:200); mouse monoclonal anti-BrDU (BD PharMingen) (for antigen
retrieval; 1:200); rabbit polyclonal anti-Ki67 (LabVision) (1:200); mouse mono-
clonal anti p14ARF(Neomarkers) (1:100); mouse monoclonal anti-p14ARF
(Sigma Aldrich) (1:100); and goat anti-mouse and anti-rabbit secondaries con-
jugated to Alexafluor (488 nm or 594 nm) (Molecular Probes) (1:400). Cervical
intraepithelial neoplasia grade III (CIN3) lesions were described and processed
as previously described (25). Images were taken using an Olympus BH-2 micro-
scope and an Olympus D25 camera and Cell B software (Olympus). For raft
sections, Ki67-positive cells were counted for a minimum of 15 fields of view
(1,000 ?M). For coverslips, a minimum of 15 fields of view and 400 cells were
counted for each slide and expressed as percentages of control numbers. Immu-
nofluorescence images were captured using a Leica AF6000 inverted microscope
and Leica AF imaging software. Exposure times and deconvolution settings were
kept constant within each experiment.
Nucleophosmin is increased at the protein level in E7- and
E6/E7-expressing cells. In an attempt to elucidate data for
novel proteins that are upregulated in the presence of the
HPV-16 E7 oncogene during differentiation, a proteomics ap-
proach was taken. 2-D gel analysis of HFK lysates during 12 h
of methylcellulose-induced differentiation revealed an increase
in the levels of various proteins in cells expressing the E7
oncogene compared to pBabe control cell results (Fig. 1a and
b). Mass spectrometric analysis of the proteins revealed NPM
to be one of the proteins whose level appears to be elevated in
differentiating E7 HFKs. To confirm these findings, HFKs
were transduced with a retrovirus expressing either pBabe con-
trol vector or HPV-16 E7 and cells were resuspended in meth-
ylcellulose for 12 h to induce differentiation. Western blotting
confirmed an increase in NPM in E7-expressing differentiating
cells (Fig. 1c) and proliferating cells (Fig. 1d) compared to
pBabe control cells. Next, we wanted to determine whether
NPM levels are also elevated in E7 cells in a more biologically
relevant model system for keratinocyte differentiation, namely,
an organotypic raft culture system. Sections of organotypic
rafts generated from E7-expressing HFKs showed that NPM
levels were increased compared to the pBabe control raft levels
(Fig. 1e). We also examined NPM levels in E6/E7-expressing
cells, as these proteins are expressed in the context of HPV-16
infection. Western blotting of proteins extracted from organo-
typic rafts and immunofluorescence staining of sections from
organotypic rafts revealed a substantial increase in NPM levels
in cells expressing E7 alone and in E6/E7-expressing cells com-
pared to pBabe control cells (Fig. 1d and e). To determine
whether HPV-16-positive cervical lesions had increased NPM
levels, we used immunohistochemical staining of sections from
matched normal and cervical intraepithelial neoplasia grade
III (CIN3) lesions. Investigation of the CIN3 lesions, all of
which were positive for HPV-16, revealed increased levels of
NPM compared to control epithelia within the same biopsy
specimen (Fig. 1f).
To determine whether the increase in NPM protein levels
observed in E6/E7-expressing cells and rafts was due to in-
creased transcription, RNA from three independent sets of
pBabe- and E6/E7-expressing HFKs was extracted for real-
time PCR. NPM levels were normalized to an RPLP0 control,
and values from the three individual experiments were aver-
aged. Results showed that there was no significant increase in
NPM RNA levels in E6/E7-expressing cells compared to
pBabe control cells, suggesting that the increase in NPM levels
was not due to increased transcription (Fig. 2a).
Increased NPM levels in E6/E7-expressing cells is due to
increased levels of active AKT. Having determined that the
increase in NPM observed in E6/E7-expressing cells was not
transcriptional, we next wanted to identify a possible mecha-
nism to explain the increased protein levels of NPM in these
cells. Previous data from our laboratory show that E7 upregu-
lates AKT activity through deregulation of pRb protein (25). A
recent report (20) has shown that AKT interacts with NPM
and protects it from degradation. Therefore, either AKT was
depleted or the activity was inhibited in pBabe- and E6/E7-
expressing HFKs by small interfering RNAs (siRNA), an AKT
inhibitor (AIV), and a PI3-kinase inhibitor (PI103). Reduced
FIG. 2. Increases in nucleophosmin levels in E6/E7-expressing cells
occur at the protein level and may be due to increased AKT activity.
(a) Real-time PCR was carried out on 3 different sets of pBabe- and
E6/E7-expressing HFKs. Data shown in the graph represent averages
of the results of the 3 experiments and show that NPM mRNA levels
are not increased in E6/E7-expressing cells compared to pBabe control
cells (P ? 0.13). (b) Depletion of AKT by siRNA (siAKT) decreases
NPM protein levels in pBabe- and E6/E7-expressing cells. (c and d)
Reductions in AKT activity, but not total protein activity, by the use of
an AKT inhibitor (AIV) and a PI3-kinase inhibitor (PI103) reduce
NPM protein levels.
VOL. 84, 2010HPV16 E6/E7 INCREASES NPM5133
levels of AKT protein correlated with reduced levels of NPM
in both pBabe- and E6/E7-expressing HFKs (Fig. 2b). In ad-
dition, the treatment of cells with AKT or PI-3 inhibitors
resulted in a reduction of AKT activity, as measured by a
reduction in glycogen synthase kinase-3? phosphorylation
(pGSK-3?), and a corresponding decrease in NPM protein
levels (Fig. 2c and d). The half-life of NPM in control and
E6/E7-expressing cells was measured in35S-labeled cells and
was found to be 24 h in control cells, but over the same period
the levels in E6/E7-expressing cells did not change (Fig. 3a). In
line with a role for AKT in NPM stability, when AKT was
depleted and stability determined at 16 h after the35S labeled
amino acids were washed out, the level of NPM in depleted
E6/E7-expressing cells dropped to control cell levels. However,
no change in NPM localization was observed when AKT was
depleted using siRNA (Fig. 3b). Indeed, NPM was localized to
the nucleolus in all cell types examined, including normal HFKs
and pBabe- and E6/E7-expressing cells (data not shown).
Knockdown of NPM reduces proliferation and induces dif-
ferentiation in E6/E7-expressing cells with upregulation of p53
and pRb. Cells expressing E6/E7 have the ability to override
the normal process of cell cycle control and differentiation. To
determine the effect of knockdown of NPM on proliferation
and differentiation, E6/E7-expressing HFKs were infected with
retrovirus expressing scrambled short-hairpin RNAs (shRNA)
expressing either a scrambled shRNA (shScr) or an shRNA
targeting NPM (shNPM) and stable cell lines were generated.
Alternatively, NPM was depleted by RNA interference (RNAi)
molecules (siScr and siNPM) and proliferation was investi-
gated in short-term assays. The cell lines were tested for the
level of proliferation as monolayers and in organotypic rafts by
the use of BrDU incorporation and Ki67 staining, respectively,
FIG. 3. Half-life of NPM in control and E6/E7-expressing keratinocytes. (a) The half-life of NPM is increased in E6/E7-expressing cells
compared to pBabe control cells but is reduced to pBabe levels in E6/E7-expressing cells when AKT is depleted by siRNA. Data in the graph
represent averages of the results of 3 experiments from different time points; densitometry data represent percentages of the value for h 0 for the
control (pBabe). (b) NPM localization is unchanged by AKT depletion in pBabe- and E6/E7-expressing cells. Scrambled control and AKT-depleted
cells were stained for NPM (green), nucleolar marker C23 (red), and DAPI (blue). Images show no change in NPM localization with AKT
knockdown in pBabe- and E6/E7-expressing cells.
5134MCCLOSKEY ET AL. J. VIROL.
while the RNAi-treated cells were studied only as monolayer
cultures. BrDU- and Ki67-positive cells were counted and
quantified against DAPI (4?,6?diamidino-2-phenylindole)-pos-
itive cells, in approximately 15 different fields of view. A 40%
decrease in proliferation of cells in a monolayer (Fig. 4a and b)
and a 50% reduction in proliferation of E6/E7-expressing
HFKs in raft cultures compared to scrambled E6/E7-express-
ing cell results (Fig. 4c and d) were observed. Similar reduc-
tions in proliferation were also observed in pBabe cells when
NPM was depleted (Fig. 4e). Taken together, these results
suggest that E6/E7-expressing cells with reduced NPM levels
are less proliferative than pBabe control cells.
To determine the effects of knockdown of NPM on differ-
entiation, control or NPM-depleted HFKs were differentiated
by two different methods. First, the cells were grown to con-
fluence and then treated with 1.5 mM CaCl2to induce differ-
entiation and harvested at various times thereafter. Alterna-
tively, organotypic rafts were produced with the depleted cells.
As expected, both Western blotting of proteins from CaCl2-
treated cells and immunofluorescence analysis of sections from
organotypic rafts showed low levels of K1 expression in the
E6/E7-expressing scrambled control cells (Fig. 5a and b). In-
terestingly, E6/E7-expressing cells with NPM knockdown
showed increased levels of K1 in both Western blot analysis of
CaCl2-treated cells and immunoflorescence staining of raft
sections (Fig. 5a and b). These data show that when the ele-
vated levels of NPM in E6/E7-expressing cells are reduced,
there is a decrease in proliferation and cells start to differen-
tiate. Therefore, NPM contributes to the inhibition of differ-
entiation observed in E6/E7-expressing cells.
To address the mechanism of decreased proliferation and
increased K1 expression in E6/E7-expressing shNPM cells, we
analyzed the expression of p53 and pRb in these cells. In
E6/E7-expressing HFKs, there were normally low levels of
both p53 and pRb; this was confirmed by Western blotting
(Fig. 5c). However, there was a modest but consistent increase
of both pRb (approximately 28% increase) and p53 (approxi-
mately 24% increase) levels in NPM-depleted E6/E7-express-
ing cells (Fig. 5c).
ARF is relocalized from the nucleolus to nucleoplasm in
E6/E7-expressing cells with the knockdown of NPM. NPM is a
shuttling protein that binds to and influences the localization
of a number of proteins, including p14ARF(ARF). Since ARF
is regulated in part by Rb through E2F and since it stabilizes
p53, we investigated the levels of ARF in E6/E7-expressing
cells compared to control cells. ARF levels were significantly
higher in E6E7 cells (Fig. 6a). Localization of ARF is impor-
tant for biology, so both E6/E7-expressing cells with or without
FIG. 4. Knockdown of NPM decreases proliferation in E6/E7-expressing cells. NPM levels in cells were reduced by the presence of either
short-hairpin molecules (shRNA) or small interfering siRNA. Organotypic rafts and cycling cells were stained with Ki67 and BrDU, positively
staining cells were counted, and percentages compared to DAPI-positive cells were quantified. (a and b) Cycling cells showing a 40% reduction
of BrDU incorporation in siNPM-expressing cells compared to scrambled control cells (siScr). Data represent results from 15 different fields of
view over the course of 3 different experiments. (c and d) Organotypic rafts of cells stably expressing shRNA with respect to NPM (shNPM) showed
a 50% reduction of Ki67 expression in stable shE6/E7-expressing cells compared to scrambled control cells (shScr). Ki67-positive cells were
counted in 15 different fields of view. (e) Proliferation was also decreased by over 40% in pBabe-expressing cells with the knockdown of NPM
siNPM compared to scrambled control cells (siScr). Data in graphs represent averages of the results of 3 experiments.
VOL. 84, 2010 HPV16 E6/E7 INCREASES NPM5135
NPM depletion and control pBabe cells were analyzed for
subcellular localization of ARF. It should be noted that in
control and E6/E7-expressing cells, NPM in HFKs was local-
ized to the nucleolus and colocalized with the nucleolar marker
C23 both in cycling (Fig. 6b) and during differentiation (data
not shown). In pBabe-expressing cells, ARF was localized to
the cytoplasm-nucleoplasm (Fig. 6b), whereas in E6/E7-ex-
pressing shRNA control cells, ARF colocalized with NPM in
the nucleolus (Fig. 6b). In E6/E7-expressing cells with depleted
NPM, ARF was relocalized to the nucleoplasm-cytoplasm
(Fig. 6b). This suggests that in E6/E7-expressing shRNA con-
trol cells, NPM sequesters ARF to the nucleolus and conse-
quently reduces the stabilizing effect of ARF on p53 in these
cells. However, upon depletion of NPM in E6/E7-expressing
cells, ARF relocalizes from the nucleolus to nucleoplasm,
which may result in the stabilization of p53 observed in E6/E7-
expressing HFKs with NPM knockdown (Fig. 5b). This relo-
calization of ARF from the nucleolus to nucleoplasm was also
observed in E6/E7-expressing cells when AKT activity was
inhibited using the AKT inhibitor AIV (Fig. 7a), suggesting
that AKT activity and the ability to stabilize NPM are impor-
tant for E6/E7 to relocalize ARF to the nucleolus.
To determine whether ARF localization to nucleoli in E6/
E7-expressing cells was due to disruption of the p53 and pRb
functions, we next generated HFK cells with knockdown of p53
or of pRb or of both p53 and pRb. NPM levels increased only
when both p53 and pRb were depleted (Fig. 7b), while in cells
with only p53 or pRb depleted, NPM levels are similar to
control cell levels (data not shown). Interestingly, when both
pRb and p53 were depleted, ARF was localized to the nucleoli,
as was the case in E6/E7-expressing cells (Fig. 7c). NPM re-
mains in the nucleoli in these cells (data not shown). These
results suggest that abrogation of the activity of both p53 and
pRb is required for the nucleolar localization of ARF observed
in E6/E7-expressing cells.
The intricate balance between cell proliferation and differ-
entiation is crucial for maintenance of homeostasis and normal
development within the cell, and disruption of either of these
processes may result in oncogenesis. We and others have pre-
viously reported on the ability of the E6 and E7 oncoproteins
to disrupt the normal process of differentiation of HFKs by
targeting key tumor suppressors such as p53 (28) and pRb (17,
37), resulting in increased levels of cell survival proteins such
as AKT (25) and disruption of the cell cycle (24, 31). In this
study, we investigated the possible role of the nucleolar phos-
phoprotein nucleophosmin (NPM) in the differentiation pro-
cess of HFKs. Frequently overexpressed in tumors and highly
proliferating cells, NPM has previously been shown to reduce
the susceptibility of cells to the onset of differentiation and
FIG. 5. Nucleophosmin knockdown induces K1 expression and increases p53 and pRb levels in E6/E7-expressing cells and organotypic
rafts. HFKs were induced to differentiate either in media containing 1.5 mM calcium chloride or in organotypic rafts. (a) Western blot
showing expression of NPM, K1 (an early marker of differentiation), and actin in E6/E7-expressing keratinocytes during induced differen-
tiation at 0, 12, 24, and 36 h after calcium treatment. K1 levels were increased in cells with reduced levels of NPM (E6/E7shNPM) compared
to the results seen with control cells (E6/E7shScr). (b) E6/E7-expressing organotypic rafts, sectioned and stained with H&E (upper panels)
and K1 (lower panels), showing the induction of K1 with knockdown of NPM. (c) Western blots showing the expression of Rb and p53 in
pBabe control and E6/E7-expressing cells with either a scrambled siRNA (siScr) or an siRNA directed to NPM (siNPM).
5136MCCLOSKEY ET AL.J. VIROL.
apoptosis (13, 33). NPM has also been reported to play a
crucial role in sustaining ribosome biogenesis in cancer cells
(12) and is now regarded as important in the development of
various cancers (9, 13, 22), with the first reported NPM inhib-
itor developed as an anticancer agent in 2008 (32). NPM was
identified as one of the proteins that were also upregulated in
a proteomic screening undertaken to characterize proteins al-
tered in expression in differentiating cells expressing E7 and
subsequently shown to be upregulated when both E6 and E7
are present. The function of E6 targeting p53 for degradation,
combined with the ability of E7 to deregulate pRb and family
members as well as AKT, has been extensively characterized
and is known to be required for inhibition of differentiation
and promotion of proliferation. This report describes another
pathway involving NPM that is utilized by cells with E6/E7 to
maintain proliferative capacity and to potentially circumvent
the activity of p53.
Our data show that the upregulation of NPM observed in
E6/E7 was not due to increased transcription. Recent work
(20) has shown that AKT binds to NPM and prevents its
degradation. Previous work had shown that E7 can upregulate
AKT through disruption of the pRb and family member func-
tions (25). Therefore, it was logical to test whether this up-
regulation of AKT was responsible in part for the elevated
levels of NPM detected in E6/E7-expressing cells. Indeed,
when AKT was depleted in E6/E7-expressing HFKs by siRNA
or AKT activity was inhibited by either PI-3 kinase or AKT
inhibitors, there was a marked decrease in NPM levels. Deple-
tion of pRb alone did not cause an increase in NPM levels, and
so it would appear that either another function of E7 is in-
volved or the other pRb family members, p130 and p107, have
a role to play.
The involvement of NPM in the increased proliferation and
disruption of differentiation in different types of cells has been
previously reported (9, 33). E6/E7-expressing HFKs can over-
ride the normal process of differentiation, and in this present
report we provide the first evidence that increased levels of
NPM in HFKs are important for inhibition of differentiation
and proliferation. Stable or transitory knockdown of NPM by
shRNA or siRNA molecules, respectively, in E6/E7-expressing
HFKs led to a decrease in proliferation and an increase in the
levels of the differentiation marker K1. The data demonstrate
FIG. 6. ARF is relocalized from the nucleolus to nucleoplasm in E6/E7-expressing cells with the knockdown of NPM. (a) Western blot showing
an increase in ARF levels in E6/E7-expressing cells. (b) pBabe- and E6/E7-expressing HFKs were treated with either scrambled control or NPM
siRNA. Cells were stained for ARF (green), nucleolar marker C23 (red), and DAPI (blue). ARF was found in the nucleoplasm and cytoplasm in
control cells but in the nucleolus in E6/E7-expressing cells. However, ARF was relocalized in E6/E7-expressing cells from the nucleolus to
nucleoplasm-cytoplasm when NPM was depleted using siRNAs E6 and E7 (E6/E7 siNPM), but no change was observed in the control cells (pBabe
VOL. 84, 2010HPV16 E6/E7 INCREASES NPM5137
a clear role for NPM in suppression of differentiation. NPM is
a nucleolar phosphoprotein that shuttles between the nucleus
and the cytoplasm and takes part in various cellular processes.
It has several interacting partners, some of which can be se-
questered to the nucleolus and rendered inactive (22). One
such target is the tumor suppressor ARF, which has multiple
functions that are dependent on localization within the cell
(16). ARF has been shown to bind to NPM through the same
domain that mediates Mdm2 binding and nucleolar localiza-
tion and as a consequence inhibits the ability of ARF to mod-
ulate p53- and pRb-associated growth arrest functions (18, 38).
Also, NPM has been shown to directly impact levels of p53 (19)
as well as interact with pRb (23, 36). Here we show that
knockdown of NPM in E6/E7-expressing HFKs results in in-
creased levels of p53 and pRb. In support of our results, a
recently developed inhibitor of NPM induced apoptosis, up-
regulated p53, and caused reduced proliferation rates in a
number of cell types (32).
Interestingly, ARF localization is altered in HFKs, where,
upon knockdown of NPM in E6/E7-expressing cells, ARF is
localized in the nucleoplasm and cytoplasm as opposed to
being sequestered in the nucleolus. The underlying mechanism
that allows ARF to be sequestered to the nucleolus by NPM in
E6/E7-expressing cells is the disruption of both p53 and pRb
functions, since knockdown of both of these proteins is re-
quired for the localization of ARF to the nucleoplasm-cyto-
plasm. However, the increased levels of NPM in E7 or E6/E7-
expressing cells do not solely account for the ability of NPM to
sequester ARF to the nucleolus, since E7-expressing cells with
increased NPM levels do not sequester ARF to the nucleolus.
The fact that E7 does not sequester ARF to the nucleolus may
account for the observation of an increase in p53 levels in
E7-expressing cells (6). Therefore, while E7 may upregulate
NPM levels, E6 must carry out another activity that helps
sequester ARF to the nucleolus. In conclusion, our results
provide the first evidence of a role for NPM in HPV-mediated
carcinogenesis and highlight the potential therapeutic use of
an NPM inhibitor in cervical cancer.
This work was supported by grants from the NIH (NIDCR
DE15935) and Wellcome Trust (WT082840AIA).
1. Balsitis, S., F. Dick, N. Dyson, and P. F. Lambert. 2006. Critical roles for
non-pRb targets of human papillomavirus type 16 E7 in cervical carcinogen-
esis. Cancer Res. 66:9393–9400.
2. Band, V., J. A. De Caprio, L. Delmolino, V. Kulesa, and R. Sager. 1991. Loss
of p53 protein in human papillomavirus type 16 E6-immortalized human
mammary epithelial cells. J. Virol. 65:6671–6676.
FIG. 7. ARF is relocalized in HFKs after inhibition of AKT or after depletion of both p53 and pRb. (a) ARF is relocalized from the nucleolus
to nucleoplasm-cytoplasm in E6/E7-expressing cells treated with the AKT inhibitor AIV. (b) Western blot showing HFKs stably expressing shRNA
for both p53 and Rb (shp53/Rb); a reduction in the levels of both proteins and a corresponding increase in NPM protein levels was seen compared
to scrambled (shScr/shScr) control cells. (c) ARF localized to nucleoli in control HFKs with depleted levels of both Rb and p53, and the localization
was the same as that seen in E6/E7-expressing cells.
5138MCCLOSKEY ET AL.J. VIROL.
3. Cobrinik, D., S. F. Dowdy, P. W. Hinds, S. Mittnacht, and R. A. Weinberg.
1992. The retinoblastoma protein and the regulation of cell cycling. Trends
Biochem. Sci. 17:312–315.
4. Colombo, E., J. C. Marine, D. Danovi, B. Falini, and P. G. Pelicci. 2002.
Nucleophosmin regulates the stability and transcriptional activity of p53.
Nat. Cell Biol. 4:529–533.
5. Dyson, N., P. M. Howley, K. Munger, and E. Harlow. 1989. The human
papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene
product. Science 243:934–937.
6. Eichten, A., M. Westfall, J. A. Pietenpol, and K. Munger. 2002. Stabilization
and functional impairment of the tumor suppressor p53 by the human pap-
illomavirus type 16 E7 oncoprotein. Virology 295:74–85.
7. Genovese, C., D. Trani, M. Caputi, and P. P. Claudio. 2006. Cell cycle
control and beyond: emerging roles for the retinoblastoma gene family.
8. Grisendi, S., R. Bernardi, M. Rossi, K. Cheng, L. Khandker, K. Manova, and
P. P. Pandolfi. 2005. Role of nucleophosmin in embryonic development and
tumorigenesis. Nature 437:147–153.
9. Grisendi, S., C. Mecucci, B. Falini, and P. P. Pandolfi. 2006. Nucleophosmin
and cancer. Nat. Rev. Cancer 6:493–505.
10. Guess, J. C., and D. J. McCance. 2005. Decreased migration of Langerhans
precursor-like cells in response to human keratinocytes expressing human
papillomavirus type 16 E6/E7 is related to reduced macrophage inflamma-
tory protein-3alpha production. J. Virol. 79:14852–14862.
11. Hebner, C. M., and L. A. Laimins. 2006. Human papillomaviruses: basic
mechanisms of pathogenesis and oncogenicity. Rev. Med. Virol. 16:83–97.
12. Herrera, J. E., R. Savkur, and M. O. Olson. 1995. The ribonuclease activity
of nucleolar protein B23. Nucleic Acids Res. 23:3974–3979.
13. Hsu, C. Y., and B. Y. Yung. 2000. Over-expression of nucleophosmin/B23
decreases the susceptibility of human leukemia HL-60 cells to retinoic acid-
induced differentiation and apoptosis. Int. J. Cancer 88:392–400.
14. Huibregtse, J. M., M. Scheffner, and P. M. Howley. 1991. A cellular protein
mediates association of p53 with the E6 oncoprotein of human papilloma-
virus types 16 or 18. EMBO J. 10:4129–4135.
15. Incassati, A., D. Patel, and D. J. McCance. 2006. Induction of tetraploidy
through loss of p53 and upregulation of Plk1 by human papillomavirus
type-16 E6. Oncogene 25:2444–2451.
16. Itahana, K., H. V. Clegg, and Y. Zhang. 2008. ARF in the mitochondria: the
last frontier? Cell Cycle 7:3641–3646.
17. Jewers, R. J., P. Hildebrandt, J. W. Ludlow, B. Kell, and D. J. McCance.
1992. Regions of human papillomavirus type 16 E7 oncoprotein required for
immortalization of human keratinocytes. J. Virol. 66:1329–1335.
18. Korgaonkar, C., J. Hagen, V. Tompkins, A. A. Frazier, C. Allamargot, F. W.
Quelle, and D. E. Quelle. 2005. Nucleophosmin (B23) targets ARF to nu-
cleoli and inhibits its function. Mol. Cell. Biol. 25:1258–1271.
19. Lambert, B., and M. Buckle. 2006. Characterisation of the interface between
nucleophosmin (NPM) and p53: potential role in p53 stabilisation. FEBS
20. Lee, S. B., T. L. Xuan Nguyen, J. W. Choi, K.-H. Lee, S.-W. Cho, Z. Liu, K.
Ye, S. S. Bae, and J.-Y. Ahn. 2008. Nuclear Akt interacts with B23/NPM and
protects it from proteolytic cleavage, enhancing cell survival. Proc. Natl.
Acad. Sci. U. S. A. 105:16584–16589.
21. Li, J., X. Zhang, D. P. Sejas, G. C. Bagby, and Q. Pang. 2004. Hypoxia-
induced nucleophosmin protects cell death through inhibition of p53. J. Biol.
22. Lim, M. J., and X. W. Wang. 2006. Nucleophosmin and human cancer.
Cancer Detect. Prev. 30:481–490.
23. Liu, X., Z. Liu, S. W. Jang, Z. Ma, K. Shinmura, S. Kang, S. Dong, J. Chen,
K. Fukasawa, and K. Ye. 2007. Sumoylation of nucleophosmin/B23 regulates
its subcellular localization, mediating cell proliferation and survival. Proc.
Natl. Acad. Sci. U. S. A. 104:9679–9684.
24. Longworth, M. S., and L. A. Laimins. 2004. Pathogenesis of human papil-
lomaviruses in differentiating epithelia. Microbiol. Mol. Biol. Rev. 68:362–
25. Menges, C. W., L. A. Baglia, R. Lapoint, and D. J. McCance. 2006. Human
papillomavirus type 16 E7 up-regulates AKT activity through the retinoblas-
toma protein. Cancer Res. 66:5555–5559.
26. Mu ¨nger, K., J. R. Basile, S. Duensing, A. Eichten, S. L. Gonzalez, M. Grace,
and V. L. Zacny. 2001. Biological activities and molecular targets of the
human papillomavirus E7 oncoprotein. Oncogene 20:7888–7898.
27. Mu ¨nger, K., and P. M. Howley. 2002. Human papillomavirus immortaliza-
tion and transformation functions. Virus Res. 89:213–228.
28. Mu ¨nger, K., W. C. Phelps, V. Bubb, P. M. Howley, and R. Schlegel. 1989. The
E6 and E7 genes of the human papillomavirus type 16 together are necessary
and sufficient for transformation of primary human keratinocytes. J. Virol.
29. Nguyen, D. X., L. A. Baglia, S. M. Huang, C. M. Baker, and D. J. McCance.
2004. Acetylation regulates the differentiation-specific functions of the reti-
noblastoma protein. EMBO J. 23:1609–1618.
30. Patel, D., S. M. Huang, L. A. Baglia, and D. J. McCance. 1999. The E6
protein of human papillomavirus type 16 binds to and inhibits co-activation
by CBP and p300. EMBO J. 18:5061–5072.
31. Patel, D., A. Incassati, N. Wang, and D. J. McCance. 2004. Human papillo-
mavirus type 16 E6 and E7 cause polyploidy in human keratinocytes and
up-regulation of G2-M-phase proteins. Cancer Res. 64:1299–1306.
32. Qi, W., K. Shakalya, A. Stejskal, A. Goldman, S. Beeck, L. Cooke, and D.
Mahadevan. 2008. NSC348884, a nucleophosmin inhibitor disrupts oligomer
formation and induces apoptosis in human cancer cells. Oncogene 27:4210–
33. Qing, Y., G. Yingmao, B. Lujun, and L. Shaoling. 2008. Role of Npm1 in
proliferation, apoptosis and differentiation of neural stem cells. J. Neurol.
34. Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M.
Howley. 1990. The E6 oncoprotein encoded by human papillomavirus types
16 and 18 promotes the degradation of p53. Cell 63:1129–1136.
35. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric
sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68:
36. Takemura, M., F. Ohoka, M. Perpelescu, M. Ogawa, H. Matsushita, T.
Takaba, T. Akiyama, H. Umekawa, Y. Furuichi, P. R. Cook, and S. Yoshida.
2002. Phosphorylation-dependent migration of retinoblastoma protein into
the nucleolus triggered by binding to nucleophosmin/B23. Exp. Cell Res.
37. Werness, B. A., K. Munger, and P. M. Howley. 1991. Role of the human
papillomavirus oncoproteins in transformation and carcinogenic progres-
sion. Important Adv. Oncol. 1991:3–18.
38. Zhang, Y. 2004. The ARF-B23 connection: implications for growth control
and cancer treatment. Cell Cycle 3:259–262.
VOL. 84, 2010HPV16 E6/E7 INCREASES NPM5139