Rev. Med. Virol. 2015; 25: 24–53.

Published online in Wiley Online Library

(wileyonlinelibrary.com)
Reviews in Medical Virology DOI: 10.1002/rmv.1823

REVIEW
Signaling pathways in HPV-associated cancers
and therapeutic implications
Jiezhong Chen*
School of Biomedical Sciences and Australian Institute for Bioengineering and Nanotechnology, The
University of Queensland, Brisbane, Queensland, Australia

S U M M A RY
Human papillomaviruses (HPVs) are small double-stranded circular DNA viruses with 8 kb genomes. So far, more than
150 HPVs have been identified, and 12 types of HPVs have been conclusively linked to cancer by the International
Agency for Research on Cancer/World Health Organization. Expression of HPV E5, E6 and E7 oncoproteins can alter
multiple signaling pathways to cause cancer. In this review, the signaling pathways activated by these oncoproteins are
summarized, and targeted therapy against key signaling molecules is described. E6 can inactivate tumor protein 53 and
PDZ (post synaptic density protein–drosophila disk large tumor suppressor–zonula occludens-1 proteins) while stim-
ulating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), Wnt and Notch pathways. E7 can inhibit retinoblas-
toma protein and stimulate the PI3K/Akt pathway. Both E6 and E7 can deregulate cellular microRNA expression,
which can alter cellular signaling pathways. E5 can sensitize epidermal growth factor receptor to epidermal growth fac-
tor to increase activation of PI3K/Akt and mitogen-activated protein kinase pathways. E5 can also inhibit the extrinsic
apoptotic pathway. These altered signaling pathways could be critical for the initiation and maintenance of HPV-
associated cancers. Therefore, targeted therapy against the key signaling molecules has therapeutic implications.
Among these, the possibilities of targeting PI3K/Akt, mammalian target of rapamycin, epidermal growth factor recep-
tor and vascular endothelial growth factor have been extensively studied in many cancers. Some inhibitors have been
studied in cervical cancer in both animal models and clinical trials. Although the results are promising, further inves-
tigation is warranted. Copyright © 2015 John Wiley & Sons, Ltd.
Received: 17 May 2014; Revised: 15 October 2014; Accepted: 27 December 2014

*Correspondence to: J. Chen, School of Biomedical Sciences and Australian
Institute for Bioengineering and Nanotechnology, The University of
Queensland, St Lucia, Brisbane, Queensland 4072, Australia.
E-mail: j.chen4@uq.edu.au
Abbreviations used
Akt, protein kinase B; BMI, B cell-specific Moloney murine leukemia
virus integration site 1; CBF-1, C-promoter binding factor 1; CDK2,
cyclin-dependent kinase 2; COX-2, cyclooxygenase-2; CSCs, cancer
stem cells; CSL, CBF-1-Su (H) and LAG-1; CTGF, connective tissue
growth factor; CYR61, cysteine rich 61; DKK-1, dickkopf-related
protein family; EGF, epidermal growth factor; EGFR, epidermal
growth factor receptor; E6AP, E6-associated protein; FZD7, frizzled
family receptor 7; GIPC, GAIP-interacting protein, C terminus;
GSK3b, glycogen synthase kinase 3b; Hes, hairy and enhancer of split;
Hey, Hes-related repressor protein; HPV, human papillomavirus;
Lag-1, lobster agglutinin 1; LIM, Lin11, Isl-1 and Mec-3; MAGI-1,
Membrane-associated guanylate kinase, WW and PDZ domain-
containing protein 1; miRNAs, mircroRNAs; mTOR, mammalian
target of rapamycin; NF-κB, nuclear factor-κB; NFX1, nuclear tran-
scription factor, X-box binding 1; NICD, Notch intracellular domain;
p53, tumour protein 53; PDK1, putative 3-phosphoinositide-dependent
kinase 1; PI3K, phosphoinositide 3-kinase; pRb, retinoblastoma protein;
PTEN, phosphatase and tensin homologue; STAT-1, signal transducer
and activator of transcription 1; TIP-2, Tax-interacting protein-2;
VEGF, vascular endothelial growth factor.
INTRODUCTION
Human papillomaviruses (HPVs) are a family of
small double-circular-stranded DNA viruses with
genomes containing 8 kb DNA sequences [1]. The
sequences of some HPVs, such as HPV16 and
HPV18, which are the most common cancer-related
HPVs, encode two late genes L1 and L2 and six
early genes E1, E2, E4, E5, E6 and E7. Although
E1, E2 and L1 and L2 proteins are essential for all
HPVs, expression of other HPV proteins varies in
different HPVs. For example, HPVs 101, 103 and
108 do not encode E6 [2], while HPV31 expresses
E8 [3]. These HPV proteins maintain replication,
amplification and release of the viruses. Among
them, E5, E6 and E7 are major oncogenes that pro-
mote host cell proliferation to facilitate viral ampli-
fication. E6 and E7 can be integrated into host
genomes in some cases and expressed in high
levels, while E5 can only be expressed from viral
genomes and maintained as episomes in host cells
[4,5]. Infections with some HPVs cause cancers

Copyright © 2015 John Wiley & Sons, Ltd.

Signaling pathways in HPV-associated cancers
including cervical cancer, head and neck cancer,
vulvar cancer, vaginal cancer, penile cancer and
anal cancer. So far, more than 150 types of HPVs
have been identified. Among them, 12 types of
HPVs can cause cancer (http://monographs.iarc.
fr/ENG/Monographs/vol90/index.php), and the
list could be expanded with more cancer-related
HPVs identified. HPV16 and HPV18 are responsi-
ble for 50% and 20% of cervical cancer respectively
[5]. Although HPV infections are very common,
only a very small percentage of HPV-infected peo-
ple develop cancer. HPV infections elicit immune
responses, which clear most HPV viruses. Persis-
tent infections greatly increase the risk for carcino-
genesis. Understanding the mechanisms of HPV-
caused cancer could be helpful for the prevention
and treatment of the disease.
Differences in the sequences caused by amino
acids as well as variation in codon usage of E5, E6
and E7 genes have been associated with their
ability to initiate cancer [6–9]. A study has shown
that E7 is sufficient to immortalize cells, while
E6 can increase the effect of E7, that is E6 and E7
can cooperate to immortalize primary human
epithelial cells. The complementary effects of E6
and E7 could be explained by their different down-
stream targets. Inhibition of E6 led to increased
tumor protein 53 (p53) but not retinoblastoma
protein (pRb), while inhibition of E7 resulted in
increased pRb but not p53 [10]. E5 is also an
oncoprotein, which promotes carcinogenic signal-
ing pathways to increase the effects of E6 and E7.
However, E6 and E7 together are not sufficient, re-
quiring introduction of another cancer risk factor
for tumor formation. For example, oncogene Ras
has been shown to cause cancer together with E6
and E7 [11]. Deletion of tumor suppressor gene
RXRα and expression of E6 and E7 together have
also been shown to cause malignant lesions of cer-
vical tissue [12].
Many studies have been performed to elucidate
the mechanisms for E6 and E7 to cause cancer,
showing their interactions with many signaling
molecules. In this review, the effects of these
oncoproteins on intracellular signaling pathways
are summarized in association with the roles of
these signaling pathways in carcinogenesis and
cancer progression. Several microRNAs (miRNAs)
altered by E6 and E7 and associated cell biological
activities are also described. After a discussion of
the changed pathways on cell behaviors including
Copyright © 2015 John Wiley & Sons, Ltd.
25
Figure 1. Signaling molecules altered by E6 protein. Human papil-
lomavirus E6 inhibit tumor protein (p)53, PDZ proteins and some
microRNAs (miRNAs). E6 activates protein kinase B (Akt), Notch
and Wnt pathways. E6 also increases telomerase activity. PDZ, post
synaptic density protein–drosophila disk large tumor suppressor–
zonula occludens-1 protein
proliferation, apoptosis, genomic instability, migra-
tion and drug resistance, the outcomes of targeted
therapy against several altered signaling molecules
are described.
HUMAN PAPILLOMAVIRUS E6 PROTEIN
Human papillomavirus E6 is a small protein
without enzymatic activity. For example, HPV16
E6 consists of 151 amino acids. E6 protein acti-
vates several carcinogenic pathways and inhibits
tumor suppressor protein p53 and post synaptic
density protein–drosophila disk large tumor
suppressor–zonula occludens-1 protein (PDZ)
(Figure 1). The survival pathways activated by
E6 include phosphoinositide 3-kinase (PI3K)/pro-
tein kinase B (Akt), Wnt and Notch. E6 activates
telomerase expression and activity, thereby
inhibiting telomere shortening and increasing cell
immortalization. Several miRNAs have been al-
tered by E6, leading to signaling pathway
changes.
Inactivation of p53 protein
Protein p53, encoded by TP53 gene, is a well-
known tumor suppressor, which prevents cancer
initiation by maintaining genomic stability
[13,14]. It is activated by DNA damage and envi-
ronmental stimuli such as oxidative stress and
osmotic shock as well as viral infections [13,14].
Activated p53 can slow down the cell cycle to
allow damaged DNA to be repaired. Activated
p53 binds to DNA to increase expression of protein
p21, which inhibits cyclin-dependent kinase 2
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DOI: 10.1002/rmv
26
(CDK2) to decrease G1/S transition. Activated p53
can also initiate apoptosis if DNA is severely dam-
aged. Through these processes, p53 prevents accu-
mulation of cells with DNA damage so that it
decreases tumor initiation. In contrast, inactivation
of p53 results in the accumulation of abnormal
cells that have gene mutations, facilitating cancer
initiation.
E6 protein inactivates p53 and thus avoids apo-
ptosis of host cells that have DNA damage. High-
risk type HPV E6 binds to an LXXLL motif (where
L = leucine and X= any amino acid) on cellular E3
ubiquitin ligase E6-associated protein (E6AP) [15–
17]. The formed E6-E6AP complex then recruits
and ubiquitinates p53, which facilitates
proteasome-mediated degradation of p53 (Figure 2)
[18,19]. This E6-induced p53 decrease has been rec-
ognized as a major mechanism for E6 to cause can-
cer. Inactivation of p53 introduced by dominant
negative p53 is sufficient to maintain HPV DNA
with E6 inactivating mutations in human
keratinocytes [20]. In organotypic cultures, an E6
null mutant accumulated high levels of p53, and
Figure 2. E6/p53 pathway. Tumor protein (p)53 family members can
maintain genomic stability through decreasing cell cycle and
increasing apoptosis under stimulation such as ultraviolet (UV),
hypoxia and viral infections. Human papillomavirus E6 can bind
to ubiquitin ligase E6-associated protein (E6AP) or other ligases to
degrade p53. E6 can also act on p63 and p73 directly to destabilize
these proteins
Copyright © 2015 John Wiley & Sons, Ltd.
J. Chen
HPV amplified very poorly [21]. Silencing of p53
or expression of ectopic wild type E6 partially re-
stored amplification, whereas three missense E6
mutations that did not effectively destabilize p53
complemented the null mutant poorly. E6 can de-
grade p53 in E6AP-null mice indicating that other
ubiquitin ligases may also be involved in E6-
mediated p53 degradation [22].
The ability of E6 to degrade p53 has been corre-
lated with HPV’s ability to cause cancer. Mesplede
et al. examined the p53 degradation ability of E6
proteins from 29 types of HPVs [23]. It was found
that variation of the p53 degradation ability was
more than 100 times with high-risk HPVs having
a higher ability to degrade p53. It was also found
that the variants of a type of HPV such as HPV16
and HPV33 had different p53 degradation ability
but the difference was much less than in different
types of HPVs.
While it is certain that degraded p53 by HPV E6
via E6AP plays a critical role in HPV-associated
cancer, other alternatives have also been revealed.
Studies reported that HPV E6 also degraded other
two members of p53 family p63β and p73 [24,25].
These two proteins have similar structures to p53
and also can inhibit cell proliferation and promote
apoptosis [26]. Thus, inactivation of p63β and p73
is important for cancer development [27]. Park
et al. has shown that E6 directly binds to and inac-
tivates p73, but it is not via E3 ubiqitination [25].
Inhibition of post synaptic density protein–
drosophila disk large tumor suppressor–
zonula occludens-1 proteins
The PDZ is a structural domain of 80–90 amino
acids, which is shared by many proteins and
named from the initial letters of three proteins—
post synaptic density protein (PSD95), drosophila
disk large tumor suppressor (Dlg1), and zonula
occludens-1 protein (zo-1) [28]. There are now more
than 250 proteins that have a PDZ domain. PDZ
proteins interact with c-tail amino acids of targeted
proteins to regulate multiple biological processes,
including cell adhesion, tight junction, cell polarity,
cytoskeleton, ion channels and signaling pathways.
Some PDZ proteins are tumor suppressor proteins,
and thus, loss of these proteins facilitates cancer
formation [29]. High-risk types of HPV E6 proteins
have a PDZ-binding motif at their extreme carboxy
termini [30–32]. Binding of E6 to PDZ proteins re-
sults in dysfunction of these proteins.
Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers
Both HPV16 and HPV18 E6 bind to Dlg C-
terminus for transformation and mutation of E6
loses the ability to bind and transform rodent cells
[30,31]. HPV18 E6 protein interacts with TIP-2/
GIPC, leading to polyubiquitination and
proteasome-mediated degradation of the protein
[33]. In HeLa cells, RNAi silencing of E6 decreases
TIP-2/GIPC levels. TIP-2/GIPC can increase ex-
pression of the TGF-beta type-III receptor at the
cell membrane and thus increase the antiprolifera-
tive effect of TGF-beta. Depletion of TIP-2/GIPC
in HeLa cells reduced the antiproliferative effect
of TGF-beta, while silencing of E6 blocked the pro-
liferation of HeLa cells.
HPV E6 acts on PDZ-partitioning defective 3
protein, which regulates tight junctions and cell
polarity [34,35]. E6 caused translocation of PDZ-
partitioning defective 3, leading to the loss of cell
polarity to facilitate tumor formation. Massimi
et al. showed that E6 targeted hDlg, hScrib
(Scribble), MUPP1, membrane-associated
guanylate kinase, WW and PDZ domain-
containing protein 1 (MAGI-1), MAGI-2 and
MAGI-3 through E6AP to increase transformation
[35,36]. HDlg and MAGI-2 interact with phospha-
tase and tensin homologue (PTEN), which regu-
lates the PI3K/Akt pathway [37,38]. E6 has high
affinity for the PDZ domain of MAGI-1 protein,
and mutation of the residue lysine 499 abolished
the effect of E6 on MAGI-1 [39].
Activation of phosphoinositide
3-kinase/protein kinase B
The PI3K/Akt pathway is a major cancer survival
pathway (Figure 3) [40,41]. PI3K regulates Akt and
Rac-1. Akt has a broad range of downstream targets,
which control cell proliferation, cell growth, cell
mobilization, angiogenesis and cell survival [40,41].
The pathway has been associated with increased
cancer initiation, progression, metastasis and drug
resistance. Thus, inhibition of the pathway has been
proposed for the treatment of cancer [42]. Targeted
therapy of PI3K/Akt has been studied extensively
in many cancers such as breast cancer, melanoma,
colon cancer and prostate cancer [40,43,44]. Both
genetic defects and environmental factors are in-
volved in activation of the pathway. Gene defects
have been detected in almost all elements of this
pathway [42]. Obesity has been shown to increase
this pathway, leading to increased cancer inci-
dence [45,46].
Copyright © 2015 John Wiley & Sons, Ltd.
27
Several studies have shown that E6 can activate
this pathway through various mechanisms. E6
inactivates PTEN through PDZ proteins, leading
to increased pAkt as well as increased cell prolif-
eration [37,38,47]. The mammalian target of
rapamycin (mTOR) kinase is a downstream target
of Akt and also activated by blood levels of amino
acids and mitogen-activated protein kinase
(MAPK) pathway. It has been demonstrated that
mTOR is activated by E6 as indicated by increased
ribosomal protein S6 kinase, which is regulated by
mTOR [48,49]. E6-E6AP complex binds and de-
grades the mTOR inhibitor tuberous sclerosis
complex 2 (TSC2). Another study also showed
that HPV16 E6 expression caused an increase in
mTOR complex 1 activity indicated by enhanced
phosphorylation of mTOR as well as its down-
stream targets ribosomal protein S6 kinase and eu-
karyotic initiation factor binding protein 1 under
conditions of nutrient deprivation [50]. However,
a decrease in TSC2 levels in HPV16 E6-expressing
cells was not detected. Instead, upstream kinases
putative 3-phosphoinositide-dependent kinase 1
(PDK1) and mTOR complex 2 were activated,
leading to increased phosphorylation of Akt [50].
In human foreskin keratinocyte (HFK) cells, ex-
pression of HPV16 E6 resulted in sustained activa-
tion of receptor protein tyrosine kinases including
epidermal growth factor receptor (EGFR), insulin re-
ceptor beta and insulin-like growth factor receptor-
beta, which are upstream of the PI3K/Akt pathway
[51]. E6 can also increase signaling adaptor protein
growth factor receptor-bound protein 2 to activate
the PI3K pathway [51].
Activation of Akt can produce a cascade of
changes in downstream targets. Akt can phosphor-
ylate E6 to promote its ability to interact with pro-
tein 14-3-3σ, which is important in carcinogenesis
[52]. HPV has also been associated with increased
expression of c-myc, a downstream protein of Akt
[53–56]. E6 has been reported to act directly on c-
myc, leading to activation of telomerase activity
[10,57]. Controversially, two studies showed that
E6 accelerated c-myc degradation [58,59].
While it is certain that E6 can cause activation
of the PI3K/Akt pathway and its downstream
targets nuclear factor-κB (NF-κB), mTOR, 14-3-3
and c-myc, the effects of E6 on other downstream
targets of Akt are largely not studied. It will
also be interesting to investigate the biological
effects mediated by the E6/PI3K/Akt pathway
Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
28 J. Chen

Figure 3. E6/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. PI3K catalyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into
phosphatidylinositol-3,4,5-trisphosphate (PIP3), which promotes (PDK1) to phosphorylate Akt. Activated Akt can influence cell survival through
mitochondrial Bcl-2 family members, cell proliferation through cyclin D1 and nuclear factor-κB (NF-κB), cell growth through mammalian target
of rapamycin and angiogenesis through vascular endothelial growth factor (VEGF). Akt blocks apoptosis through decreasing tumor protein (p)
53, p21 and p27. Akt also increases genomic instability. E6 can activate PI3K through receptor protein tyrosine kinase or direct interaction with
PI3K. E6 can also block tuberous sclerosis complex 1/2 (TSC1/2) to increase mammalian target of rapamycin complex 1 (mTORC1) activity to in-
crease cell growth and can block pro-apoptotic proteins Bad and Bax to decrease apoptosis. PTEN, phosphatase and tensin homologue; RPTK,
receptor protein tyrosine kinase; HIF, hypoxia-induced factor; S6K, ribosomal protein S6 kinase; GSK, glycogen synthase kinase; Rheb, Ras
homologue enriched in brain; 4E-BP, eukaryotic initiation factor 4E binding protein; MDM2, murine double minute; Foxo1, forkhead box O1;
MAPK, mitogen-activated protein kinase; mTORC2, mammalian target of rapamycin complex 2

in terms of cell proliferation, survival, migration
and drug resistance.
Activation of the Wnt pathway
Wnt ligands and the associated pathway regulate
cellular proliferation and differentiation processes
and thus play critical roles in normal tissue homeo-
stasis [60] and in pathologic conditions such as
cancers [61–63]. Activation of the Wnt pathway
results in accumulation of β-catenin, which in turn
increases transcription of a broad range of genes to
promote cell proliferation. In inactivation status of
the Wnt pathway, β-catenin forms a “degradation
complex” with other proteins including glycogen
synthase kinase-3β (GSK3β), casein kinases, ade-
nomatous polyposis coli and Axin2 and
phosphorylated at serine and threonine residues.
Phosphorylation of β-catenin induces its
ubiquitination by β-TcRP ubiquitin ligase, leading
to degradation. In activation status of canonical
Wnt signaling pathway, intracellular Dishevelled
protein is phosphorylated and interacts with
Axin2, leading to dysfunction of the degradation
complex and accumulation of β-catenin. The accu-
mulated β-catenin is translocated into the nucleus
and binds members of the T-cell factor/lymphoid
enhancer factor family of transcription factors to
regulate target genes including c-jun, c-myc [64],
cyclin D1 [65], multidrug resistance 1[66],
matrilysine [67], Axin2 [68], survivin, vascular en-
dothelial growth factor (VEGF), COX-2 and matrix
metalloproteinases [69]. It also targets positive and

Copyright © 2015 John Wiley & Sons, Ltd. Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers
negative regulators of the Wnt pathway including
Axin2, Wg, FZD7, DKK-1 and sFRP-2 to form auto-
regulation [69].
Accumulated nuclear β-catenin has been com-
monly found in human HPV16-positive invasive
cancer samples but also in early dysplastic lesions,
indicating activation of the Wnt pathway by HPV
oncogenes [70,71]. The nuclear accumulation of β-
catenin correlates with tumor progression in cervi-
cal cancer patients [72]. The accumulated β-catenin
also correlates with HPV infection in cell lines in-
cluding HPV16-positive oropharyngeal cancer cell
lines 147 T and 090, HPV-negative cell line 040 T
and cervical cell lines SiHa (bearing integrated
HPV16) and HeLa (bearing integrated HPV18) [73].
E6 gene silencing in HPV-positive cells reduced nu-
clear β-catenin substantially, suggesting a critical role
of E6 in the activation of the Wnt pathway. The
mechanism has been associated with a protein called
seven in absentia homologue, which increases
proteasomal degradation of β-catenin. E6 can de-
crease seven in absentia homologue mRNA and pro-
tein levels substantially [73]. Lichtig et al. also
showed that HPV16 E6 activated the Wnt/β-catenin
pathway; the mechanism is independent of the abil-
ity of E6 to target p53 for degradation or bind to the
PDZ-containing E6 targets but requires E6AP [74].
In vivo mouse experiments showed that E6 expres-
sion led to accumulation of β-catenin [75]. Full-
length E6 oncoprotein expression in K14E6 mice en-
hanced the nuclear accumulation of β-catenin and
the accumulation of cellular β-catenin-responsive
genes. These effects were not observed when a trun-
cated E6 oncoprotein that lacks the PDZ-binding do-
main was expressed alone (K14E6ΔPDZ mice),
indicating that the effect of E6 was mediated by the
PDZ domain [75]. Although the Wnt pathway may
be a possible mediator for increased β-catenin,
PI3K/Akt is also well known to cause accumulation
of β-catenin through inactivation of GSK3β [76].
Therefore, further studies are needed for a unified
explanation of E6-induced β-catenin accumulation.
Activation of the Notch pathway
Notch signaling plays important roles in both nor-
mal physiological activities such as cell growth, dif-
ferentiation, immune responses and organ
development and various diseases including cancer
[77–81]. Increased expression of the Notch pathway
has been associated with progression of several ma-
lignancies such as prostate cancer, breast cancer,
Copyright © 2015 John Wiley & Sons, Ltd.
29
glioma and head and neck cancers. The pathway
comprises four Notch receptors (Notch 1–4 in
humans), a group of transmembrane proteins, and
their ligands (Jagged1, 2 and delta-like ligand 1, 3
and 4) [82,83]. Binding to their ligands on the sur-
face of neighboring cells leads to the cleavage of
Notch receptor by γ-secretase and subsequent re-
lease of the Notch intracellular domain (NICD)
(Figure 4). As the constitutively active domain of
the Notch receptor, NICD translocates to the nu-
cleus where it binds to and forms a complex with
the transcriptional regulator termed CBF-1-Su (H)
and LAG-1 (CSL) and mastermind-like protein,
leading to the displacement of co-repressors previ-
ously bound to CSL and recruitment of
co-activators. The co-activators then induce expres-
sion of the target genes, such as the hairy and en-
hancer of split (Hes) and Hes-related repressor
protein (Hey) families. Accumulating evidence
indicates that dysregulated NCID can also activate
the PI3K/Akt pathway, which in turn causes a
cascade of target proteins [84–88].
The expression of Notch 1 increases with progres-
sion of cervical cancer; Notch 1 has been detected in
invasive cervical cancer but not low-grade cancer
[89]. Daniel et al. showed that Notch-1 receptor ex-
pression was increased during the progression from
cervical intraepithelial lesions to invasive cervical
carcinoma [90]. Moreover, main cellular localization
of Notch-1 protein changed from cytoplasmic to nu-
clear with the transition from intraepithelial lesions
III to microinvasive carcinoma [90]. The regulation
of the Notch signaling pathway by HPV E6 has been
demonstrated in cervical cancer cell lines. At late
passage but not early W12 cell line, which is an
HPV type-16-positive human cervical low-grade
lesion-derived cell line, jagged1 is upregulated,
while manic fringe is decreased [91]. It was con-
firmed by an increase of Notch-driven report activity
and a decrease of manic fringe promoter-driven re-
port activity in late passage W12. Inhibition of the
Notch pathway by expression of manic fringe,
dominant-negative Jagged1 or RNAi silencing of
Jagged1 inhibits the tumorigenicity of CaSki, an in-
vasive cervical carcinomas-derived cell line. HPV in-
creases the Notch pathway through distinct
mechanisms [92]. HPV16 E6 can interact with cellu-
lar protein NFX1-123 and increase NFX1-123 protein
expression [92]. Overexpression of NFX1-123 in
HPV16 E6-expressing keratinocytes increased
Notch-1 mRNA levels. Weijzen et al. demonstrated
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DOI: 10.1002/rmv
30
Figure 4. E6/Notch pathway. Notch receptor is activated by its ligand Jagged1 from another cell and cleaved by gamma secretase to pro-
duce Notch intracellular domain (NICD), which translocates into the nucleus to form a complex with CBF-1-Su (H) and LAG-1 (CSL) and
mastermind-like protein (maml) to promote transcription of hairy and enhancer of split (Hes) and Hes-related repressor protein (Hey).
NECD, Notch extracellular domain; URR, upstream regulatory region
that presenilin-1 mediated the effect of E6 on the
Notch pathway in mouse and human primary cell
lines transfected HPV16 E6 ([93]).
A controversial opinion about the Notch pathway
in HPV-associated cancer has been proposed. It was
shown that Notch 1 was decreased in HPV-positive
cervical cancer cell lines HeLa, C40I, C4-II, SiHa and
Caski compared with HPV-negative C33a and pri-
mary keratinocytes [94]. However, expression of
Notch 2 in all these cell lines was similar to primary
keratinocytes. Nevertheless, overexpression of
Notch 1 in HPV-positive cells decreased their
growth. This indicates that Notch could be a tumor
suppressor in cervical cancer as in some cancers
such as chronic myelomonocytic leukemia, cutane-
ous and lung cancers [95–97]. It has been demon-
strated that Notch 1 is controlled by p53.
Knockdown of p53 by short interfering RNA
(siRNA) decreased Notch-1 levels, while activation
of p53 by nutlin increased Notch-1 levels in human
keratinocyte cancer cell lines [98]. Suppression of
the Notch signaling pathway together with acti-
vated Ras resulted in cancer cell formation in pri-
mary keratinocytes, providing evidence for
carcinogenic effect of loss of Notch 1[98]. There were
also reports that Notch was lost because of Notch 1
inactivating mutation in 10–15% of patients and the
loss could increase head and neck squamous cell
carcinomas (HNSCC) carcinogenesis [99,100].
Copyright © 2015 John Wiley & Sons, Ltd.
J. Chen
However, a recent study showed that the Notch
pathway was activated in 32% of HNSCC patient
samples and only 9% (4 out of 37) had inactivation
of the pathway, casting doubt whether inactivation
of Notch is required for these cancers [101].
Telomerase activation
Telomerase is a ribonucleoprotein enzyme for main-
taining telomeric structures at the end of chromo-
somes, and its increased activity is important for
carcinogenesis [102–105]. In normal cells, telome-
rase activity is very low so that telomeres shorten
with every round of DNA replication and become
shorter over time, which leads to replicative senes-
cence. Expression of the telomerase catalytic sub-
unit in normal cells increased telomere length,
resulting in indefinite cell proliferation [106]. In can-
cer cells, inhibition of telomerase activity decreased
cell proliferation [107]. Telomerase activation is crit-
ical for the immortalization of primary human
keratinocytes by the high-risk HPV E6 [108].
E6 is able to increase telomerase activity by up-
regulation of telomerase reverse transcriptase
(TERT), which is encoded by the human telomerase
reverse transcriptase (hTERT) gene [108,109]. E6 in-
duces the hTERT promoter via interactions with the
cellular ubiquitin ligase, E6AP. E6 increases hTERT
via NFX1-123. NFX1-123 interacts with hTERT
mRNA and stabilizes it, leading to greater
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Signaling pathways in HPV-associated cancers
telomerase expression [92]. E6 protein also interacts
directly with the hTERT protein [110]. Unlike its ef-
fect on p53, E6 binding to hTERT protein does not
cause protein degradation both in vitro or in vivo
but increases telomerase activation by a post-
transcriptional mechanism [110].
MicroRNAs involved in E6-mediated
signaling pathways
MicroRNAs are small non-coding RNAs contain-
ing 19–24 nucleotides, which regulate gene ex-
pression by targeting transcripts or translation
[111]. In humans, a large number of genes are reg-
ulated by miRNAs, so miRNAs regulate many
biological processes. Studies have shown that
many miRNAs are involved in E6-mediated sig-
naling pathways. Martinez et al. used Ambion
(Ambion Technologies, Austin, TX, USA) arrays
to show that three miRNAs were overexpressed
and 24 underexpressed in cervical cell lines con-
taining integrated HPV-16 DNA [112]. Another
study showed that miRNAs miR-363, miR-33 and
miR-497 were upregulated, whereas miR-155,
miR-181a, miR-181b, miR-29a, miR-218, miR-222,
miR-221 and miR-142-5p were downregulated in
HPV-positive head and neck cancer cells [113].
E6 decreases miR-34a [114,115], which is an im-
portant cell cycle regulator [116,117]. MicroRNA-
34a is a target of p53, and the effect of E6 on
miRNA-34a is mediated by decreased p53
[114,115]. This is consistent with the fact that
miR-34a is reduced in both normal cervical tissue
and cervical lesions with high-risk HPV infection
[118]. Targeting of HPV16 E6 has been shown to
increase miR-34a [119]. One of the targets of miR-
34a is p18Ink4c [120]. Silencing of E6 or overex-
pression of miRNA-34a in HeLa cells decreased
p18Ink4c, while inhibition of miRNA-34a in-
creased p18Ink4c. Protein p18Ink4c is an inhibitor
of CDK4/6, but its role in cervical cancer is not
well studied. Other targets of miR-34a have also
been revealed. MicroRNA-34a is negatively corre-
lated with CDK4, Sirt1 and myeloblastosis protein
[121]. In cell biology, miRNA-34a has been associ-
ated with hypoxia increased epithelial-
mesenchymal transition and docetaxel resistance
in breast cancer [122,123].
Martinez et al. showed that miR-218 was particu-
larly reduced in HPV-positive cervical cancer indi-
cated by microarray, real-time PCR and northern
blot analyses [112]. Expression of HPV16 E6 but
Copyright © 2015 John Wiley & Sons, Ltd.
31
not HPV6 E6 reduced miR-218 expression, while si-
lencing of E6 increased miR-218 expression in cervi-
cal cancer cells. LAMB3, a laminin protein, is
increased in cervical cancer specimens and identi-
fied as a target of miR-218 [113]. Therefore, LAMB3
was increased by E6 [112]. LAMB3 regulates cell dif-
ferentiation, migration, adhesion, proliferation and
survival, so silencing of LAMB3 in cervical cancer
cells reduced cell survival and migration [113]. Con-
sistently, overexpression of miR-218 also reduced
cancer cell migration and invasion in both HPV-
positive and HPV-negative cervical cancer cell lines.
E6 decreases miR-125b [124], which inhibits
cervical cancer growth and promotes apoptosis
via inhibition of the PI3K/Akt pathway [125].
Another study reported that miR-125b was highly
reduced in HPV-positive cells [126]. However, a
recent study showed that miR-125 was increased
in cervical cancer and miR-125b could target the
Bak promoter to reduce its expression and thus
decrease apoptosis [127]. This discrepancy has been
explained by multiple targets of miR-125b with its
roles depending on different expression levels of
target genes [128].
MicroR-375 is decreased in HPV-associated can-
cer, which negatively regulates HPV16 and 18 tran-
scripts [129]. MicroR-375 directly acts on E6AP and
decreases its activity [129]. Thus, transfection of
miR-375 resulted in increased p53, p21 and 14-3-3.
In gastric cancer, miR-375 was shown to inhibit
PDK1 and 14-3-3zeta directly, and overexpression
of miR-375 increased apoptosis via the caspase
pathway [130].
E6 could regulate more miRNAs, so the list
might increase after more studies are performed.
A recent study showed that in organotypic raft cul-
tures of foreskin and vaginal keratinocytes HPV16
and HPV18 changed 13 miRNAs. MicroR-25, miR-
92a and miR-378 were increased, while miR-22,
miR-27a, miR-29a and miR-100 were decreased.
The increased miR-25, miR-92a and miR-378 were
confirmed in cervical patients’ specimens [131].
The significance of these changes in cellular signal-
ing pathways is not elucidated yet.
HUMAN PAPILLOMAVIRUS E7 PROTEIN
E7 is a potent cell cycle regulator with 98 amino
acids containing three domains of E7 called con-
served regions 1–3. It interacts with more than 50
cellular factors [132,133]. The significance of most
of these interactions is unknown. However, several
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DOI: 10.1002/rmv
32
downstream targets have been identified including
its interactions with pRb, the PI3K/Akt pathway
and miRNAs.
Inhibition of retinoblastoma protein
Dysfunction and inactivation of pRb through vari-
ous factors have been associated with initiation of
many cancers [134–136]. E7 is one factor that inacti-
vates pRb [137,138] (Figure 5).
Several mechanisms for E7 to alter pRb signaling
have been elucidated [133,139–141]. Retinoblastoma
protein is a pocket protein, which binds to transcrip-
tion factors E2F1-3 to prevent E2F1-3 functions. When
E7 binds its pocket and inactivates its function to bind
E2F1-3, E2F1-3 is released. E2F1-3 can bind cell cycle
regulators DP-1 or DP-2, leading to increased cell
cycle from G1 to S, decreased apoptosis and increased
genomic instability. HPV16 E7 proteins can destabi-
lize pRb through proteasomal degradation mediated
by cullin 2 ubiquitin ligase complex reprogrammed
by HPV E7 [142]. A difference in an amino acid in
E7 sequence could affect its binding ability and degra-
dation ability, accounting for carcinogenic ability of
high-risk or low-risk HPVs [143,144].
E7 also acts on E2F proteins directly. It binds to
E2F1 and increases its activity [145]. E7 can also
block the transcriptional suppressor activity of
E2F6 [146]. E2F6 is usually upregulated by E2F1
to provide a feedback regulation [146].
In addition, E7 also inactivates other pocket pro-
teins including p130 and p107 [137]. Proteins p130
and p107 regulate E2F4 and E2F5, which also play
key roles in cell cycle regulation. The effects of E7
on three pRb family members may be synergistic
Figure 5. E7/retinoblastoma protein (pRb) pathway. pRb forms a
complex with E2F1 to keep E2F1 in inactivation status. E7 can
bind with pRb, allowing the release of E2F1. E2F1 binds to cell
cycle regulator DP1, causing increasing cell cycle from G1 to S,
decreased apoptosis and increased genomic instability
Copyright © 2015 John Wiley & Sons, Ltd.
J. Chen
[147]. However, knockout of three Rb family mem-
bers in mice is not sufficient to cause cervical carci-
noma although development of high-grade cervical
intraepithelial neoplasia can develop [148].
Activation of phosphoinositide
3-kinase/protein kinase B
Several studies have shown that E7 can activate
the PI3K/Akt pathway. Menges et al. expressed
HPV16 E7 in HFK cells and showed upregulation
of Akt activity in organotypic raft cultures [149].
The mechanism of increased Akt activity has been
regarded as inhibition of pRb by E7 as follows.
First, the ability of E7 to increase Akt activity is
correlated with its ability to bind to and inactivate
Rb. Second, silencing of Rb by short hairpin RNAs
(shRNAs) in differentiated keratinocytes increased
Akt activity. Third, increased Akt activity and loss
of Rb were also correlated in HPV-positive cervical
high-grade squamous intraepithelial lesions.
Protein kinase B upregulation by E7 may be
mediated by protein phosphatase 2A (PP2A)
[150,151]. PP2A is known to inhibit Akt phosphory-
lation. Pim et al. demonstrated that E7 could bind
both the Mr 35 000 catalytic subunit and the Mr
65 000 structural subunit of PP2A to inhibit PP2A
activity [150]. Liu et al. found that PP2A mRNA
and protein levels were detected in 73% and 53% cer-
vical cancer samples respectively but not in normal
cervical tissues [151]. Knockdown of E7 downregu-
lated both mRNA and protein expression of PP2A.
E7 negatively regulates p27 via Akt. HPV16 E7
enhanced both the cytoplasmic retention of p27 in
HFKs, which was reduced by PI3K/Akt inhibition
[152]. A standard wound assay showed that E7 in-
creased the migration of HFKs, which was
Akt/p27 dependent.
Rho family guanosine triphosphatases (GTPases)
play critical roles in cytoskeleton and cell migra-
tion. Under extracellular environment stimulation,
GTPases can transfer inactive guanosine
diphosphate-bound states to the active guanosine
triphosphate-bound form, which in turn changes
cell morphology and promotes cell migration
through Rho family members [153–156]. HPV E7
regulates Rho family members to increase cell mi-
gration through Rac1 and RhoA [152,157]. RhoA
is crucial for efficient cell migration and cell spread-
ing. Todorovic et al. used mass spectrometry identi-
fying p190RhoGTPase-activating protein (p190) as
a binding partner of HPV16 E7 [157]. Protein
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DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers
p190, one of the GTPase-activating protein families
stimulated the intrinsic GTPase activity of the Rho
proteins, leading to Rho inactivation and increased
cell migration.
MicroRNAs altered by E7
E7 decreases miR-203 during keratinocyte differen-
tiation, which is a tumor suppressor and thus in-
creases carcinogenesis [158]. Recently, several
other targets of miR-203 have been identified.
MicroR-203 has been shown to target anti-
apoptotic protein bcl-w to increase cell survival
[159], target survivin to cause G1 cell cycle arrest
[160] and target LIM and SH3 protein 1 to inhibit
migration and invasion [161]. MicroR-203 can also
target p63, which maintains the balance of epithe-
lial cell proliferation and differentiation.
E7 decreases miRNA-205 via pRb [162].
MicroRNA-205 was considered to be a tumor sup-
pressor [163] but has recently been demonstrated to
increase cell proliferation via E2F1 [164]. Lower
miRNA205 has been associated with poor prognosis
of HPV-associated head and neck cancer [165]. Xie
et al. showed that miR-205 expression was frequently
higher in human cervical cancer [166]. In vitro exper-
iments demonstrated that miR-205 promoted cell
proliferation and migration in human cervical cancer
cells. Two miR-205 targets, cysteine rich 61 (CYR61)
and connective tissue growth factor (CTGF), were
identified. Consistently, both CYR61 and CTGF were
downregulated in cervical cancer tissues [166]. Both
CYR61 and CTGF belong to CYR61/CTGF/
nephroblastoma family of growth regulators and
have both carcinogenic or tumor suppressor effect-
dependent tissue types. Overexpression of CYR61
in non-small cell lung cancer cells resulted in
decreased cell proliferation and colony formation
[167]. CTGF reduced cell migration in oral cancer
[168]. However, the effect of CYR61 and CTGF in
cervical cancer still needs to be established.
MicroR-15a, miR-15b and miR-15b have been
shown to be upregulated by E7 through E2F1
and E2F3 [169,170]. The expression of these
miRNAs is increased in cervical cancer samples
[170,171]. These miRs have tumor suppressor ac-
tivity [172,173], decreasing cyclin E1 and leading
to cell cycle arrest [174]. Their upregulation may
inhibit E2F1-promoted cell cycle. MicroR-15 targets
E2F1. Therefore, the regulation of miRs by E7
could play complicated effects in HPV-caused
carcinogenesis.
Copyright © 2015 John Wiley & Sons, Ltd.
33
HUMAN PAPILLOMAVIRUS E5 PROTEIN
E5, a small hydrophobic protein, is also an onco-
gene of HPVs but much less studied compared
with E6 and E7. E5 is not integrated into host ge-
nomes but is expressed by episomes. In an animal
model, E5 alone can also cause cancer. In addition,
E5 can increase the carcinogenic effects of E6 and
E7 [175]. Some signaling pathways activated by
E5 have been revealed (Figure 6). The activation
of signaling pathways by E5 is complementary or
overlapping with E6-mediated and E7-mediated
pathways.
Epidermal growth factor receptor
Epidermal growth factor receptor is a tyrosine
kinase, which consists of an extracellular ligand-
binding domain, a transmembrane region and a
transduction module consisting of a tyrosine
motif and several autophosphorylation sites
[176,177]. EGFR is activated by multiple ligands
such as epidermal growth factor (EGF), epiregulin
and TGFα. EGF/EGFR is a well-known survival
pathway in many cancers. It can increase cell pro-
liferation and decrease cell apoptosis via its
downstream target proteins including Akt, MAPK
and COX-2 [178]. EGFR and Src have feed-
forwarded regulation to activate the activity of
each other through phosphorylation [178]. E5
has been shown to increase sensitivity of EGFR
to the stimulation of EGF [179–181]. Changes of
downstream effectors have also been investigated.
E5 has been shown to increase VEGF expression
via activation of Akt and MAPK [182]. Another
study has shown that it also activates MAPK in-
dependent of EGFR [183]. Activation of Akt can
also block Bax-caused apoptosis [182].
Fas/FasL
E5 has been demonstrated to inhibit the extrinsic
pathway [184]. The pathway is also called cell
death pathway in which specific death receptors
(Drs) are activated by their corresponding ligands,
for examples tumor necrosis factor (TNF) receptor
1 or 2 (TNF R1 and TNF R2) by TNF-alpha, Fas re-
ceptor by Fas ligand, DR1, DR2 by TNF-related
apoptosis-inducing ligand and TNF-like weak in-
ducer of apoptosis by Fas-associated death
domain-like interleukin-1 beta-converting enzyme
[185]. The extrinsic pathway is initiated by FasL,
which binds to Fas receptor, leading to activation
of DRs, causing caspase 8 activation and apoptosis.
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DOI: 10.1002/rmv
34 J. Chen

Figure 6. E5-induced pathways. E5 can sensitize epidermal growth factor receptor (EGFR) to epidermal growth factor stimulation, leading
to increased activation of EGFR downstream pathways phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated
protein kinase (MAPK)/extracellular signal-regulated kinases (ERK). Akt can upregulate vascular endothelial growth factor (VEGF) to in-
crease angiogenesis and block Bax to decrease apoptosis. E5 can also block the Bap31-induced Fas/FasL apoptotic pathway

E5 can inhibit this pathway via various mechanisms.
In HaCaT cells, E5 expression reduced FasL-
stimulated Fas by twofold and subsequently inhibited
caspase 8 activation [186,187]. TNF-related apoptosis-
inducing ligand-induced cell death was also inhibited
by E5. The mechanism could be the binding of E5 to
Bap31 protein, which is supported as follows: (1) E5
and Bap31 can be coimmunoprecipitated; (2) they
are colocalized in the endoplasmic reticulum; and (3)
deletion of C terminus of E5 results in the loss of the
interaction [188]. When Bap31 is cleaved into
p20Bap31, it promotes cell apoptosis through the DR
pathway, and binding of E5 could prevent such
cleavage [189]. A recent report demonstrated that E5
could activate A4, a small transmembrane lipopro-
tein, which in turn increased cell proliferation. A4 is
known to interact with Bap31 [190].
THE CONSEQUENCES OF SIGNALING
ALTERATIONS IN HUMAN
PAPILLOMAVIRUS-INFECTED CELLS
Cell proliferation
Cancer is defined as abnormal cell growth charac-
terized by increased cell proliferation and de-
creased apoptosis. Abnormally increased cell
proliferation is one of the main characteristics of
cancer [191]. Although not all hyperproliferations
cause cancer, increased cell proliferation could
facilitate accumulation of mutated genes necessary
for carcinogenesis. Cell proliferation is caused by
cell growth and division controlled by cell cycle.
Cell cycle proceeds in four phases as indicated in
Figure 7 including G1, S, G2 and M phases [192].
Each phase of a cell cycle is regulated by a family
of serine/threonine protein kinases, which are het-
erodimers consisting of a CDK (catalytic) and a cy-
clin (regulatory) [192]. E6 and E7 can affect these
cyclins via various pathways to promote cell prolif-
eration. Among them, cyclin D is most studied. E6
can activate the Akt/myc pathway and decrease
p16 to increase cyclin D [53]. Increased cyclin B1
by E6 has also been reported, which promotes G2
phase [193–195]. HPV 16 E7 can increase cyclin A
by direct interaction [196,197]. Silencing of E6/E7
by siRNA reduced cervical cancer cell proliferation
in both in vitro and in vivo [198,199].
Debnath et al. studied co-operative effects of acti-
vation of Akt and expression of E7 or cyclin D1 in
cultured cancer cells [200]. It was found that Akt
activation alone or E7 expression alone was insuffi-
cient to maintain cell proliferation without growth
factors. However, the combination of activation of
Akt and expression of E7 markedly increased cell
proliferation as indicated by cell proliferation
marker Ki-67Akt downstream target mTOR that
has been shown to play a key role as rapamycin-
inhibited cell growth in E6/E7 transduced

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DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers
Figure 7. Human papillomaviruses, cell cycle and cell proliferation. A cell undergoes a cycle for proliferation with four phases, G1, S, G2
and M. Cell cycle is sophisticatedly regulated by cyclins and cyclin-dependent kinases (CDKs). E7 can block Rb/E2F to increase cyclin-E
activity and thus accelerate cell cycle. E7 can also promote cyclin-A activity. E6 increases the activities of cyclins D and B
epithelial cells [201]. PDZ protein MAGI-1 has been
shown to mediate the E6-induced cancer cell prolif-
eration [39], and expression of MAGI-1 through
disrupting its interaction with E6 resulted in de-
creased proliferation.
In patients with HPV-associated cancers, cell pro-
liferation in tumor tissues is increased. It has been
shown that cell cycle proteins p16, Ki-67, cyclin
D1, p53 and ProEx C are altered in 144 cervical tis-
sue samples, showing increased proliferation [202].
Molecules related to cell proliferation have a signif-
icant increase from low-grade to high-grade lesions
and cancer in HPV-associated cancers [203].
Apoptosis
Another characteristic of cancer cells is decreased
cell apoptosis [191]. There are two pathways in-
volved in cell apoptosis: extrinsic, also called the
death receptor pathway, and intrinsic, also called
the mitochondrial pathway. E5/E6/E7 can de-
crease apoptosis through both apoptotic pathways
via altering multiple signaling pathways. Targeting
HPV E6 with peptide aptamers caused cancer cell
apoptosis [204]. Qi et al. showed that simultaneous
silencing of HPV18 E6 and E7 induced apoptosis
in HeLa cells, indicating an important role for
these oncoproteins in maintaining cancer cell sur-
vival [205].
E6 inhibition of p53 can greatly reduce intrinsic
apoptosis to reduce DNA damage-initiated apopto-
sis. Smeets et al. showed that inactivation of p53
Copyright © 2015 John Wiley & Sons, Ltd.
35
and pRb was sufficient to cause oral keratocyte im-
mortalization [206]. Activation of PI3K/Akt by
E5/E6/E7 can increase cell survival by reducing
the intrinsic apoptotic pathway [207]. PDZ protein
MAGI-1 has been shown to mediate the E6-
decreased cancer cell apoptosis [39]. Overexpres-
sion of MAGI-1 through disruption of its interac-
tion with E6 resulted in increased apoptosis. E5
can decrease both extrinsic and intrinsic pathways
(Figure 6) [182,184]. Therefore, HPVs use multiple
oncoproteins, which alter multiple signaling path-
ways to decrease host cell apoptosis.
Genomic instability
Genomic instability plays a key role in carcinogen-
esis. It promotes gene mutations, which in turn af-
fect cell proliferation and apoptosis. HPVE5, E6
and E7 could increase genomic instability via sev-
eral mechanisms. Akt activation can cause genomic
instability [208]. Alteration of the pRb/E2F1 path-
way by E7 also leads to genomic instability [209]. In-
activation of p53 decreases DNA damage-induced
cell death and increases tolerance to genomic insta-
bility. HPV-caused genomic instability is initiated
by abnormal amplification of centrosomes [5]. Cen-
trosomes are microtubule-organizing centers,
consisting of a pair of centrioles and pericentriolar
proteins [210]. Centrosomes are important regula-
tors of cell division to ensure a cell is divided into
two [210]. In normal conditions, centrosome dupli-
cates once prior to mitosis to form two spindle
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36
poles. The duplication begins in late M phase/early
G1 phase. HPV E7 causes rapid induction of centro-
somes with more than two copies, leading to abnor-
mal cell division and genomic instability [211,212].
The pathway is mediated by CDK2, which is acti-
vated by Akt [213,214]. Increased CDK2 activity in
turn promotes polo-like kinase 4, a rate-limiting en-
zyme in centriole replication [215]. It is known that
deletion of polo-like kinase 4 causes defects in cen-
triole replication, while overexpression results in
multiple centrioles [215].
Genomic instability can cause accumulation of
gene mutations. In cervical cancer, many gene mu-
tations have been reported with several mutations
involved in the PI3K/Akt pathway including
PIK3CA, PTEN and EGFR and Ras [216–220]. These
mutations can further activate the PI3K/Akt path-
way in cervical cancer. A report showed that
PIK3CA mutated at 31.3%, Kirsten rat sarcoma viral
oncogene homologue at 8.8% and EGFR at 3.8% of
cervical cancers [218]. The list of gene mutations in
HPV-associated cervical cancer has been expanded
in a recent study including recurrent E322K substi-
tutions in the MAPK1 gene (8%), inactivating muta-
tions in the HLA-B gene (9%) and mutations in
EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53
(5%) and ERBB2 (6%) [221]. This study also con-
firmed previously reported mutations such as
PIK3CA (14%), PTEN (6%) and STK11 (5%). There-
fore, HPV oncogenes initiate signaling pathway al-
terations, which cause genomic instability, leading
to accumulation of gene mutations, which accelerate
changes of signaling pathways necessary for
carcinogenesis.
Drug resistance
Drug resistance to anticancer therapeutic agents is
a major reason responsible for treatment failure
and high rate of cancer-related deaths [222–224].
Patients may not respond to initial therapy (inher-
ent drug resistance) or develop drug resistance sub-
sequently after effective initiation (acquired
resistance) [222]. Multiple mechanisms for drug re-
sistance have been elucidated including drug target
mutations, increased drug detoxification system to
reduce intracellular drug concentrations, increased
resistance to apoptosis and abnormal activation of
signaling pathways [225–232].
PI3K/Akt is well known to cause drug resistance
in cancer. Activation of the pathway by insulin,
IGF-1 or mutations has been associated with drug
Copyright © 2015 John Wiley & Sons, Ltd.
J. Chen
resistance to chemotherapeutic drug 5-fluorouracil
(5-FU) and oxaliplatin [229,230]. In cervical cancer,
activation of PI3K/Akt causes resistance to radio-
therapy, and inhibition of the pathway increased
efficacy of radiation [233]. A recent study
demonstrated that activation of the PI3K/Akt
pathway by E6 caused drug resistance to cisplatin
in HPV-associated lung cancer mediated by the
downstream target of the PI3K/Akt pathway
inhibitors of anti-apoptotic protein [234]. Overex-
pression of hTERT increased 5-FU effect on HeLa
cells to cause apoptosis [235].
Targeting these signaling pathways has been an
effective strategy to overcome drug resistance in
cancer treatment [236,237]. Many approaches, par-
ticularly, small molecule inhibitors, have been de-
veloped for the treatment. The combination
therapy of targeted therapy with other treatment
regime is detailed in the late section—therapeutic
implications.
Cell mobility and metastasis
Cell migration plays a key role in cancer metas-
tasis. To be able to migrate to a new place, a can-
cer cell must detach from the original site,
migrate to the new place, attach and grow in a
new environment. Signaling pathways play key
roles in the process of cancer metastasis [238].
They can control cell morphological change, cell
polarity and cell mobility to meet the require-
ments of migration. Many signaling changes
caused by HPV E6 and E7 are associated with
cell morphology and migration such as the
PI3K/Akt and RhoA pathways [152,157].
MicroRNAs have also been associated with cell
morphology control. Therefore, HPV E6 and E7
can also facilitate cancer metastasis. Loss of
PTEN has been associated with increased metas-
tasis in patients [239]. Activation of MAPK in-
creased metastasis of cervical cancer [240].
Antiviral treatment has been shown to have
anti-metastatic effect in HPV-positive cells [241].
Cancer stem cells
It has been realized that cancer cells in a tumor or in
a cell line are heterogeneous, which are composed
of many different phenotypic cancer cells
[242,243]. One small population of cancer cells is
called cancer stem cells (CSCs). These cells have
the property of renewal and can differentiate to
other types of cancer cells. CSCs are considered to
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DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers
be only cells within a tumor that can proliferate ex-
tensively and drive tumorigenic growth [242,243].
The other types of cancer cells are terminal differen-
tiated cells, which are not renewed. CSCs have also
characteristics of normal stem cells, that is slow cy-
cling, altered DNA repair machinery and high ex-
pression levels of anti-apoptotic genes and ATP-
binding cassette (ABC) transporters [244]. These
characteristics render CSCs drug resistant, for ex-
ample CSCs are less sensitive to cisplatin than
non-CSCs in HNSCC [245].
Cancer stem cells have been identified in acute
myeloid leukemia and later found in other cancer
types as well, for example breast cancer, brain can-
cer, colon cancer, neck and head cancer, pancreas
cancer, liver cancer and melanoma. In cervical can-
cer, CSCs have been isolated by various biomarkers
including cell surface markers, Hoechst staining
and formation of spheres. Qi et al. isolated sphere
cells from cervical cancer cell line HeLa [246]. These
stem cells account for only about 1% of total popu-
lation. They are small, round cells with increased
biomarkers Oct3/4, CD133 and breast cancer resis-
tance protein. However, aldehyde dehydrogenase
is not changed. Stem cells isolated have been shown
to have increased drug resistance to chemothera-
peutic agent Trichostatin and radiation treatment.
CSCs have also been isolated from two other cervi-
cal cell lines SiHa and CaLo by their increased abil-
ity to efflux Hoechst 33342 [247]. These cells had
increased ABCG2 and ABCB1.
Not many studies have been performed for the
role of HPV in CSCs. It has been compared with
HPV-positive HNSCC cancers with HPV-negative
ones for the amount of CSCs contained. Zhang
et al. found that HPV16-positive HNSCC had a
greater intrinsic CSC pool than HPV-negative
HNSCC [248], while Tang et al. showed that the
proportion of CSC was not significantly different
in HPV-positive or HPV-negative HNSCC cell lines
[245]. These experiments may not be able to pro-
vide valuable evidence for the role of HPV in CSCs
because other cancers can be caused by risk fac-
tors, which also affect CSC abundance. Transduc-
tion of HPV negative cancer cells with HPV
E6/E7 can reflect the role of E6/E7 in CSCs. It
has been shown to increase colony formation that
was observed in both CSCs and non-CSCs. How-
ever, its effect on stemness is not well studied. This
is an important area, where detailed studies are
warranted.
Copyright © 2015 John Wiley & Sons, Ltd.
37
THERAPEUTIC IMPLICATIONS
HPV-associated cancers that cannot be removed by
surgery can be treated by multiple approaches such
as chemotherapeutic agents, antiviral therapy, im-
munotherapy, radiotherapy, phytochemicals and
targeted therapy (Figure 8).
Chemotherapy
A number of chemotherapeutic agents have been
used in cervical cancer. Cisplatin has been shown
to be most effective among all drugs tested includ-
ing cisplatin, carboplatin, 5-FU, cyclophosphamide,
chlorambucil, melphalan, methotrexate, vincristine,
bleomtcin, adriamtcin, mitomycin C, ifosfamimde,
pclitaxel, irinotecan, gemcitabine, vinorelbine, do-
cetaxel, doxorubicin and mitolactol. However, cis-
platin only has 10–20% of responses [249]. An
increase of dosage of cisplatin resulted in severe re-
nal toxicity. Carboplatin is a derivative of cisplatin
but with reduced renal toxicity. Overall, single che-
motherapeutic agents can only produce low effi-
cacy, which can only extend patients’ lives to
12 months and thus is regarded as a palliative ther-
apy [250]. As single agents are not efficient, various
combination therapies have been applied. Chemo-
therapeutic agents have been used for double or
triplet combination therapy to increase therapeutic
effectiveness. For example, cisplatin has been used
together with 5-FU, and the combination therapy
showed various responses from 22% to 58% [251–
253]. Cisplatin and capecitabine together produced
responses of 30–50% [254,255]. However, the me-
dian survival was not improved.
The use of chemotherapeutic agents usually
results in drug resistance caused by activation of
signaling pathways. Combination of chemothera-
peutic agents and targeted therapy could be more
effective, as activation of signaling pathways is
one of the major reasons for drug resistance of che-
motherapeutic agents. For example, 5-FU can stim-
ulate the Src and PI3K/Akt pathways, resulting in
drug resistance and inhibition of Src that increased
the efficacy of 5-FU markedly [256].
Antiviral and anti-oncogene therapies
Because expression of HPV oncogenes through epi-
somes or integrated E6 and E7 genes is important
for the maintenance of cancer cells, elimination or
inhibition of HPVs, HPV oncogenes has been
studied for the treatment of HPV-associated can-
cers. Several antiviral agents have been tested.
Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
38 J. Chen

Figure 8. Therapy strategy against human papillomavirus (HPV)-associated cancers. HPV oncogenes E5, E6 and E7 activates cellular signaling
pathways to promote cancer cell survival. Treatment strategy can target the process in different levels including decreasing HPVs and their
oncogene expression by short hairpin RNAs (shRNAs) or small molecule inhibitors, targeting cellular signaling pathways by inhibitors or
phytochemicals and killing cancer cells by chemotherapy or radiotherapy or immunotherapy

Cidofovir has been shown to restore p53 and in-
crease radiosensitivity in HPV-associated cancers
[257]. RNA interference has been used to inhibit ex-
pression of E6 and E7 [258]. Transduction of
lentiviral vectors delivered shRNA against E6/E7
into HeLa cells caused apoptosis [259–262]. Combi-
nation of shRNA against both E6 and VEGF was
more effective for the treatment of cervical cancer
than single shRNA or combination therapy with
chemotherapeutic drugs [262,263]. It has been
shown that siRNA against E6 increased the sensi-
tivity of SiHa cells to cisplatin [264]. In a xenograft
model of cervical cancer, injection of shRNA
against E6/E7 reduced tumor growth [265]. A
range of small molecule inhibitors have been devel-
oped to inhibit E6 and E7 or their binding to part-
ner proteins [266].
Targeted therapy
As E6/E7 affects cancer via signaling pathways,
modulation of these signaling pathways has thera-
peutic implications. As multiple signaling path-
ways have been altered by E6 and E7, it is
possible to use multiple small chemicals to reverse
these pathways such as reactivation of p53, inhibi-
tion of the PI3K/Akt pathway, inhibition of the
Notch pathway and supplementation of decreased
miRNAs. Among them, targeted therapies against
the EGFR, VEGF, Src, PI3K/Akt and Erk path-
ways have been the best studied to date (Figure 9).
Anti-phosphoinositide 3-kinase/protein
kinase B
Activation of the PI3K/Akt pathway has been as-
sociated with increased cancer cell proliferation,
decreased apoptosis, increased cell migration and
decreased drug sensitivity in many cancers in-
cluding cervical cancer [41,45,229,267,268].
Targeted therapy against PI3K/Akt has been used
in many cancers [236,269]. Noh et al. showed that
activation of Akt by HPV 16 E7 was responsible
for cancer cells to escape immune responses
[270]. Therefore, targeting PI3K/Akt may be an
effective approach for the treatment of HPV-
associated cancer.
Several studies have tested the effect of
PI3K/Akt pathway inhibitors on cervical cell lines.
Rashmi et al. used two allosteric Akt inhibitors

Copyright © 2015 John Wiley & Sons, Ltd. Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers 39

(SC-66 and MK-2206) in combination with the glu-
cose analog 2-deoxyglucose in C33A cells, which
have activating PIK3CA (E545K, E542K) and
inactivating PTEN (R233*) mutations [271]. Re-
sults showed that inhibition of the PI3K/Akt by
these inhibitors decreased cell viability through a
non-apoptotic mechanism. 2-Deoxyglucose further
increased cell viability. SC-66 also inhibited cell
migration. Xia et al. tested the effect of
PI3K/Akt/COX-2 pathway inhibitors Ly294002
and celecoxib on the sensitivity of radiotherapy
in HeLa cells and found that the combination of
Ly294002, celecoxib and radiation produced treat-
ment effects [233].
The combination of inhibition of PI3K/Akt and
radiotherapy has also been tested in an animal
model of cervical cancer [272,273]. In BALB/C
nude mice with xenografted HeLa cells, LY294002
decreased cell survival and xenograft tumor
growth. However, the combination of LY294002
and radiation resulted in synergistic reduction of
tumor growth [273].
Anti-mammalian target of rapamycin
Inhibitors of PI3K/Akt downstream signaling mol-
ecule mTOR have also been developed. These in-
hibitors have been tested in cervical cancer cell
lines and animal models. Among them,
temsirolimus has been used in a phase-II clinical
trial. It increased cervical cancer patient survival
time, that is 3% partial response, and 57.6% had
stable disease [274]. Coppock et al. established a
mouse cancer cell line by transducing both E6/E7
oncogenes and mutated H-Rasv12 gene into mouse
oropharyngeal epithelial cells for the test of the ef-
fect of rapamycin [201]. The cells can grow into tu-
mor in a xenograft model. Rapamycin has been
used to treat the mice in combination with cis-
platin, decreasing cancer cell proliferation and
prolonging long-term survival rate of mice with
the xenograft tumors.
Anti-vascular endothelial growth factor
Human papillomavirus oncogenes cause upregula-
tion of VEGF, which can increase cancer cell sur-
vival and angiogenesis. In cervical cancer, VEGF
is overexpressed. Therefore, anti-VEGF has been
used in clinical trial for the treatment of cervical
cancer. Bevacizumab, a monoantibody against
VEGF, is a most-studied agent for anti-VEGF ther-
apy. A recent study showed that addition of
bevazumab to chemotherapeutic cisplatin–
paclitaxel or topotecan–paclitaxel regime increased
treatment efficacy, increasing response rate from
36% to 48% and overall survival time from 13.3
months to 17.0 months [275]. It can also increase

Figure 9. Targeted therapy against key signaling pathways in human papillomavirus-associated cancers. Many small molecule inhibitors
have been developed to target key signaling molecules in human papillomavirus-associated cancers including epidermal growth factor
receptor (EGFR), extracellular signal-regulated kinases (ERK), vascular endothelial growth factor (VEGF), Src and mammalian target of
rapamycin (mTOR). Dual inhibitors against both phosphoinositide 3-kinase (PI3K) and mTOR have also been developed. Akt, protein ki-
nase B; GSK, glycogen synthase kinase

Copyright © 2015 John Wiley & Sons, Ltd. Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
40
treatment efficacy of radiation and cisplatin alone
[276]. Many small molecule inhibitors of VEGF
have also been developed and tested in clinical tri-
als with benefit but limited improvement of sur-
vival time.
Anti-epidermal growth factor receptor
Epidermal growth factor receptor gene amplifica-
tion has been associated with poorer outcome of
cervical cancer, and thus, anti-EGFR has been em-
phasized in the treatment of cervical cancer [277].
Both antibody against EGFR (cetuximab) and small
molecule inhibitors of EGFR (gefitinib and erloti-
nib) have been tested. Cetuximab produced syner-
gistic effects with antiviral agent cidofovir in
HeLa and Me180 cells in both in vitro cell cultures
and in vivo xenograft model [278]. Cetuximab has
also been tested in a clinical trial with platinum
and 5-FU in 121 head and neck cancer patients
and extended overall survival time to 11 months
[279]. Another phase-II clinical trial with combina-
tion of cetuximab and cisplatin showed that the re-
gime was well tolerated but cetuximab had no
further benefit than cisplatin alone [280]. Gefitinib
also resulted in stable disease in 20% of patients
of recurrent cervical cancer in a clinical trial [281].
Clinical trials of erlotinib showed safety [282] but
limited benefit even in combination with topotecan
[283] and cisplatin [284].
Applications of phytochemicals
Phytochemicals could be used for the treatment of
cancer because of their ability to inhibit multiple
signaling pathways. Some phytochemicals have
been well studied in cervical cancer such as epi-
gallocatechin gallate (EGCG), resveratrol and
curcumin and quercetin. EGCG is a major green
tea component, which decreases cell proliferation
and increases drug sensitivity in cervical cancer
[285,286]. The mechanisms are inhibition of several
key molecules of signaling pathways. In Caski,
EGCG induced G1 arrest and apoptosis [287,288].
Treatment of HeLa cells with EGCG induced
dose-dependent and time-dependent inhibitions
of proliferation and increased apoptosis [289].
EGCG activated the mitochondrial apoptotic path-
way indicated by increases of reactive oxygen spe-
cies, Bax/Bcl-2 ratio, cytochrome-c release, cleavage
of procaspase-3 and -9 and poly(ADP-ribose)-poly-
merase. EGCG also inhibited the activity of Akt
and NF-κB and reduced expression levels of cyclin
Copyright © 2015 John Wiley & Sons, Ltd.
J. Chen
D1. EGCG has been demonstrated to increase the
sensitivity to cisplatin [285].
Curcumin has also been shown to suppress HPV
oncogene expression and thus increase p53 and
pRb [290]. Singh et al. showed that curcumin de-
creased estrodiol-induced cervical cancer cell pro-
liferation and caused apoptosis [291] Curcumin
can decrease the levels of E7, proliferating cell nu-
clear antigen and cyclin D1 elevated by estrogen
but not E6, telomerase and p16. Curcumin over-
came drug resistance to cisplatin in SiHa cells by
inhibiting metalloprotease 1, P-glycoprotein, cyclin
D1 and upregulation of p53, peripheral benzodiaz-
epine receptor, p21 and p27 [292]. Nanoparticle-
packed curcumin increased the sensitivity of cervi-
cal cancer to chemotherapeutic agent paclitaxel
through inhibition of the Akt pathway [293]. In a
xenograft model using CaSki cells, curcumin re-
duced tumor size by decreasing VEGF, COX-2
and EGFR [294]. A cream containing curcumin
eliminated HPV+ cells in mice [295].
Quercetin has also caused HeLa apoptosis and
cell cycle arrest in G2/M phase [296,297]. It was
shown to increase expression of p53 and p21 and
decrease cyclin D1. The mitochondrial apoptotic
pathway was activated with upregulation of pro-
apoptotic bcl-2 family members and cytochrome
c and Apaf-1 and caspases and downregulation
of bcl-2 and survivin. Quercetin also inhibited
NF-κB [296].
Immunotherapy
An effective immune system is necessary for the
eradication of cancer cells, which have been weak-
ened by chemotherapy or targeted therapy. Al-
though infection of HPV can elicit immune
responses against virus and can clear virus in most
cases, HPV oncogenes E5, E6 and E7 can inhibit im-
mune responses to escape the clearance. E5 reduces
HLA-1 expression by interacting with HLA-1
heavy chain [298]. E7 can reduce STAT-1 and de-
crease transporter associated with antigen
processing/interferon regulatory factor and thus
reduce HLA class 1 [299]. Therefore, HPV antigen
presentation is reduced, and immune responses
against HPV-positive cells are decreased.
Various immunotherapies have been tried in cer-
vical cancer and vulval intraepithelial neoplasia
(VIN) to strengthen the immune responses to elim-
inate virus or against cancer cells. Imiquimod, a
toll-like receptor agonist, has been tested to activate
Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
Signaling pathways in HPV-associated cancers
innate immune cells such as dendritic cells, mono-
cytes and macrophages in HPV-associated cancers.
Imiquimod increased HPV clearance and reduced
tumor size in a clinical trial with 59 cervical cancer
patients [300]. The mechanism was shown to be the
normalization of immune cell counts [301] and in-
creased CD8+ cell recognition to E7 immunization
[302]. Topical application of 5% imiquimod cream
in 62 VIN patients resulted in 47 complete re-
sponses and 19 partial responses [303]. The effect
of imiquimod was time-dependent, and patients
had complete regression rate 32% (6 out of 19) at
week 10, 58% (11 out of 19) at week 20 and 63%
(12 out of 19) at week 52 [304]. E6 and E7 proteins
have been used to elicit specific immune response
to HPV-positive cells. The test in 20 patients with
VIN showed partial efficacy with increased CD4
and CD8 T-cell responses [305]. E7 has been carried
by a naked DNA vector for its expression in cells to
increase MHC processing, resulting in increased
immunogenicity of E7 and overcoming immune
tolerance [306]. Imiquimod increased the effect of
this DNA vaccine [307]. Cytokines have also been
used to activate immune system in treating cervical
cancer. The most commonly used cytokines are IL-2,
IL-12, granulocyte-macrophage-CSF and IFN-alpha.
Systemic application of cytokines is highly toxic, but
local application is well tolerated [308].
Antiestrogen therapy
Estrogen plays a key role in several cancers such as
endometrial, prostate, colon and breast cancers. Ep-
idemiological evidence has shown that estrogen is
also important in cervical cancer [309]. In an animal
model, estrogen has been shown to have
cooperating effect with HPV 16 oncogenes E6 and
E7 [310–312]. There are two types of estrogen re-
ceptors—estrogen receptor-alpha (ERα) and estro-
gen receptor-beta (ERβ). The activation of these
receptors has opposite effects in some cancers. In
colon cancer, ERα increases carcinogenesis via acti-
vation of signaling pathways Akt and MAPK,
while ER-beta inhibits these pathways. Both ERα
and ERβ are expressed in cervical cancer [313].
ERα is decreased, while ERβ is maintained in cervi-
cal cancer [313,314]. Interestingly, ERα expression
in stromal cells is increased [314], and ERα in these
cells is necessary for the development of cervical
cancer [315]. The signaling pathways altered by es-
trogen in cervical cancer are not well studied. A re-
cent study using microassay showed that it
Copyright © 2015 John Wiley & Sons, Ltd.
41
increased activator protein 1, NF-κB and MAPK
[316]. More detailed studies are needed to elucidate
the role of estrogen in the carcinogenesis of cervical
cancer. Nevertheless, ERα has been demonstrated
to play an important role in cervical cancer in ani-
mal experiment, and selective estrogen receptor
modulators raloxifene can decrease tumor growth
[317,318].
In addition to inhibiting ERα, activation of ERβ
has also been tested for the treatment of cervical
cancer. Genistein, a phytoestrogen, which
stimulates ERβ, induced apoptosis of HeLa cells
[319] and sensitized chemotherapeutic agents cis-
platin and camptothecins [320,321]. Geistein was
shown to inhibit the PI3K/Akt, NF-κB, MAPK
and MMP-9 pathways and activate apoptotic path-
ways [321–324].
Antiestrogen phytochemical indole-3-carbinol
was shown to prevent incidence of cervical cancer
in a transgenic model [325]; 19 out of 25 mice devel-
oped cervical cancer in control group, while only 2
out of 24 had cancer in indole-3-carbinol supple-
mented group. In cervical cancer cell lines, indole-
3-carbinol was shown to cause apoptosis through
the intrinsic pathway indicated by the reduction
of Bcl-2 protein [326]. Indole-3-carbinol also
prevented PTEN loss [327] and decreased cyclin-E
levels [328].
Targeting cancer stem cells
It has been recognized that CSCs play key roles
in cancer treatments. Conventional therapy can
result in decreased differentiated cancer cells
while CSCs are resistant to the therapy. The ther-
apy could eliminate differentiated cancer cells,
leading to initial shrinkage of tumor. However,
remained CSCs will continue to produce differen-
tiated cancer cells, causing relapse of cancer.
Therefore, elimination of CSCs could lead to cur-
able cancer.
Signaling pathways are important in maintaining
CSCs, such as the PI3K/Akt and MAPK pathways.
Akt activation has been associated with stemness
in various cancers such as pancreatic cancer, ovarian
cancer, breast cancer and glioma [329–332]. As stem
cells are drug resistant, increased stemness may
account for increased drug resistance. Inhibition of
PI3K/Akt pathway caused a decreased sphere
formation in cancer cell populations in breast cancer,
glioma and prostate cancer [333–335]. Studies have
also shown that IGF-1 is associated with colon
Rev. Med. Virol. 2015; 25: 24–53.
DOI: 10.1002/rmv
42 J. Chen

CSCs [336]. Activation of the PI3K/Akt pathway
decreases the sensitivity of CSCs to chemothera-
peutic drugs [335,337]. The downstream of the Akt
pathway such as GSK/β-catenin has been demon-
strated to be of importance in CSCs [330]. Thus,
targeting signaling pathways may eradicate CSCs.
This has been demonstrated by a recent break-
through by inhibiting signaling molecule BMI-1 to
reduce colon CSCs’ renewal ability [338]. The ap-
proach may be applicable in HPV-associated cancer.
A recent study showed that CD200, a membrane
protein, was overexpressed in HPV-positive
HNSCC cell lines and the overexpression of CD200
was associated with increased BMI-1 [339]. In vivo,
CD200 increased resistance to chemoradiation, indi-
cating the importance of BMI-1 in HPV-associated
cancers, and inhibition of BMI-1 may have similar
effect as in colon cancer.
In summary, various approaches have been
used for the treatment of HPV-associated cancers,
especially cervical cancer. However, the single
treatment approach has very limited effect. Com-
bination therapy may be promising, particularly
the combination of targeted therapy with other
approaches. Single inhibition of a signaling mole-
cule in cervical cancer could be not effective. This
may be due to the activation of multiple signaling
pathways by HPV oncogenes E5, E6 and E7.
Simultaneous inhibition of two or more key sig-
naling molecules may produce much better ef-
fects. At present, the inhibition of multiple
molecules in HPV-associated cancers has not been
optimized.
CONCLUSIONS
HPV oncogenes E5, E6 and E7 play key roles in
HPV-associated cancer by activating multiple can-
cer survival signaling pathways and inhibiting can-
cer suppressor proteins. Altered signaling
pathways in turn promote cell proliferation, de-
crease cell apoptosis and increase cell migration
and drug resistance. The effects of E5, E6 and E7
could be synergistic. Targeting E5-mediated, E6-
mediated and E7-mediated signaling pathways is
of therapeutic value. Among them, the PI3K/Akt
pathway activated by E5, E6 and E7 as well as mu-
tations of genes that encode elements of the path-
way could be inhibited, which may provide an
effective approach for the treatment of HPV-
associated cancer. Although anti-EGFR and anti-
VEGF have been used in clinical trials in cervical
cancer, outcomes are not satisfactory. As multiple
signaling pathways are activated by HPV onco-
genes, it may be necessary to inhibit multiple sig-
naling molecules for the treatment of HPV-
associated cancers.

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