Oral Oncology xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Molecular mechanisms of HPV induced carcinogenesis in head and neck

Theodoros Rampias a, Clarence Sasaki a, Amanda Psyrri b,⇑
a
Department of Surgery, Section of Otolaryngology, Yale University School of Medicine, New Haven, CT 06511, United States
b
2nd Department of Internal Medicine, Attikon Hospital, National Kapodistrian University of Athens, Athens 12462, Greece

a r t i c l e i n f o a b s t r a c t

Article history: A growing subgroup of oropharyngeal cancers is initiated by infection with high-risk human papillomavi-
Available online xxxx ruses (HPVs). In parallel to mounting epidemiological evidence, solid experimental data support a causal
association between HPV and a subset of oropharyngeal cancers. This Review summarizes the main
Keywords: events of the HPV life cycle, the functions of the viral oncoproteins, and the implications of HPV infection
HPV on their hosts, with an emphasis on carcinogenic mechanisms in oropharyngeal cancer. The demonstra-
Oropharynx cancer tion that HPVs have a role in head and neck carcinogenesis has fuelled the expectation that HPV-targeted
Tumor suppressor pathways
therapeutic strategies may prove curative in these cancers.
Notch
Wnt
Ó 2013 Elsevier Ltd. All rights reserved.

Introduction tobacco-associated counterparts [5]. It is worth noting that

Head and neck squamous cell cancer (HNSCC) represents the
sixth most common cancer diagnosed worldwide and has tradi-
tionally been associated with tobacco and alcohol use [1]. More re-
cently, infection with ‘‘high-risk’’ human papilloma viruses (HPVs)
and especially type 16 has been implicated in the pathogenesis of a
subset of HNSCC, especially those arising from the tonsillar oro-
pharynx. According to the ‘‘Annual Report to the Nation on the Sta-
tus of Cancer 1975–2009 [2], the incidence rates of HPV-associated
oropharyngeal cancers have increased in white men and women.
Based on data from three SEER (Surveillance Epidemiology and
End Results) registries, HPV DNA detection in oropharyngeal tu-
mors increased from 16.3% during the period from 1984 to 1989
to 71.7% during the period from 2000 to 2004 [3]. To the contrary,
the incidence trend of tobacco-associated OSCCs has been declin-
ing due to smoking cessation programs in the US.
In parallel to mounting epidemiological evidence, solid experi-
mental data was added to further support a causal association be-
tween HPV and a subset of oropharyngeal cancer: Rampias et al.
showed that repression of viral oncogene expression in HPV+ oro-
pharyngeal cancer cells induces massive apoptosis and restoration
of p53 and pRb tumor suppressor pathways [4]. This finding fuelled
the expectation that even late stage HPV-associated OSCCs can be
cured by HPV-targeted therapeutic approaches such as therapeutic
vaccines eliciting a cytolytic immune response to cells expressing
these oncoproteins.
HPV-associated OPSCCs represent distinct disease entity in
terms of epidemiology, biology and clinical behavior compared to
⇑ Corresponding author. Tel.: +30 210 5831255; fax: +30 210 5831690.
E-mail addresses: psyrri237@yahoo.com, dp237@email.med.yale.edu (A. Psyrri).
whole-exome sequencing studies conducted, on approximately
100 HNSCC specimens in total, independently by two research
groups [6,7], showed that the mutation rate of HPV-positive tu-
mors was approximately half of that found in HPV-negative HNSCC
(mean of 2.28 mutations/Mb versus 4.83 mutations/Mb; p = 0.004),
consistent with epidemiologic data suggestive of biological differ-
ences between HPV-positive and HPV-negative disease. This re-
view describes the principles of HPV-induced carcinogenesis in
relation to HNSCC.
HPV genomic organization
HPVs are small non-enveloped DNA viruses that infect squa-
mous epithelial cells and give rise to a large spectrum of epithelial
lesions with low malignant potential (mainly warts or papillomas).
A subgroup of HPVs, the ‘‘high-risk’’ HPVs, can cause precancerous
lesions. A small fraction of individuals infected with ‘‘high-risk’’
HPVs will develop cancer which usually occurs many years after
the original infection.
The HPV genome is a small double-stranded circular DNA mol-
ecule of 8000 base pairs (bp) which encodes up to ten proteins.
Only one strand is transcribed into mRNA. The HPV genome con-
tains three regions: a 4000 bp region that encodes proteins in-
volved in viral replication and cell transformation; a 3000 bp
region that encodes the structural proteins of the virus; and a
1000 bp noncoding region that contains the origin of viral DNA
replication and transcriptional regulatory elements. Two clusters
of viral genes, the ‘‘early’’ genes (E1-7) and the ‘‘late’’ genes (L1,
L2), are contained within the HPV genome [8]. The ‘‘early’’ genes
encode two regulatory proteins (‘‘early’’ genes E1 and E2) and three
oncoproteins (‘‘early’’ genes E5, E6 and E7). The ‘‘late’’ genes

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http://dx.doi.org/10.1016/j.oraloncology.2013.07.011

Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
dx.doi.org/10.1016/j.oraloncology.2013.07.011
2 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
Figure 1. Schematic representation of HPV Genome. The HPV genome is a circular
molecule of approximately 7900 base pairs with 8 overlapping reading frames.
encode two structural capsid proteins (L1 and L2) that constitute
the virus particle. The genomic organization is presented in Fig. 1.
The papillomavirus E1 and E2 proteins are required for viral
DNA replication and papilloma formation. E1 is an ATP-dependent
helicase that initiates viral replication in cooperation with the E2
protein. The E2 protein also plays an important role in segregation
of viral DNA plasmids as cells divide [9,10]. By simultaneously
binding to viral genomes and cellular chromatin-associated pro-
teins, during mitosis, the E2 protein ensures proper segregation
of the viral DNA into daughter cells upon cell division [11,12].
The role of the E4 gene is still unknown but its expression is
essential for the productive phase of the papillomavirus life cycle.
The E4 protein is encoded by a gene in the early region that entirely
overlaps with the E2 gene in an alternative translational reading
frame. Nevertheless, the E4 protein is abundantly expressed during
the late stages of the HPV life cycle and may facilitate release of
mature virus from cells [13–15].
The E5 protein is a very short, transmembrane protein encoded
at the 3’ end of the early region of many papillomavirus types. The
E5 protein from bovine papillomavirus type 1 causes tumorigenic
transformation of cultured fibroblasts by inducing ligand-indepen-
dent activation of the platelet-derived growth factor receptor, epi-
dermal growth factor receptor and colony stimulating factor 1
[16,17]. Microarray experiments after HPV16 E5 overexpression
in human epithelial HaCaT cells revealed that E5 plays a major
antiapoptotic role, affects cell-matrix adhesion and interferes with
epithelial differentiation [18]. E5 is considered not to be required
for the late events in HPV-induced carcinogenesis since its coding
sequence is often deleted from the episomal viral DNA during inte-
gration to host genome.
E6 and E7 proteins promote cell-cycle progression and viral
DNA replication in differentiated keratinocytes. The E6 protein of
the high-risk HPV binds and induces the degradation of the p53 tu-
mor suppressor protein via a ubiquitin-mediated process, while
the HPV-E7 protein binds cullin 2 ubiquitin ligase complex and
ubiquitinates the retinoblastoma (Rb) tumor suppressor protein
and related proteins. The late genes L1 and L2 encode viral capsid
proteins utilized in the construction of new viruses. L1 capsid pro-
teins are firstly synthesized in the cytoplasm before being trans-
ported to the nucleus to package viral chromatin. L2 capsid
protein binds specific sites of viral DNA in the nucleus and recruits
L1 for new viral particles to be assembled. The roles of HPV pro-
teins are summarized in Table 1.
More than 150 human HPV genotypes have been isolated and
there are certainly additional types which have not been identified.
Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
dx.doi.org/10.1016/j.oraloncology.2013.07.011
Some HPVs infect mucosal epithelia whereas other HPVs infect
cutaneous epithelial cells. Mucosal a-HPVs have been studied in
great detail since some of these viruses are the causative agents
for cervical and head and neck cancer. Mucosal a-HPVs are classi-
fied as low and high risk, according to their ability to induce malig-
nant transformation of epithelial cells [19]. HPV type 16 is the
predominant high risk type accounting for 86.7% of all HPV-posi-
tive tumors. Cutaneous b-HPVs are less well studied. They can
cause cutaneous warts and can possibly contribute to non-mela-
noma skin cancers. Cutaneous b-HPVs have intrinsic transforming
activities but do not associate with known cellular target proteins
of high risk mucosal a-HPV E6 proteins, including p53 and cellular
PDZ proteins [20].
In cancers, HPV can be found in episomal (extrachromosomal)
form, integrated form, or a combination of both. Viral integration
into the host-cell genome occurs downstream of the early genes
E6 and E7, often in the E1 or E2 region. This disruption results in
a loss of negative feedback control of oncogene expression by the
viral regulatory E2 protein [21]. Integrant-derived transcripts are
more stable than those derived from episomal viral DNA, and
HPV integration has been associated with a selective growth
advantage for the affected cells [22].
Papillomaviruses life cycle
HPVs are species specific and also display a tropism for squa-
mous epithelia. A large group of HPVs infects cutaneous epithelia,
whereas another sizable group infects mucosal epithelia. Through
wounds or abrasions, HPVs infect basal epithelial cells, which have
the highest proliferation capacity in the epithelial layer and utilize
the host-cell DNA replication machinery to support viral DNA rep-
lication [23–25]. The viral DNA is maintained in the nucleus of in-
fected basal epithelial cells as a low-copy-number and no progeny
virus is produced in these cells. Despite the fact that squamous epi-
thelial cells normally undergo differentiation as they move from
the basement membrane towards the surface epithelium and are
not able to support DNA synthesis, the virus maintains the supra-
basal cells in a state competent for DNA replication and HPV-DNA
replicates to a high copy number in terminally differentiated cells
near the epithelial surface. Similarly, the late viral genes L1 and L2
are expressed only in the highly differentiated cells, where infec-
tious progeny virus is produced and released. The crypt epithelium
of human palatine tonsil is a specialized squamous epithelium that
contains patches of stratified squamous nonkeratinizing epithe-
lium and patches of reticulated sponge-like epithelium. It is also
characterized by the presence of intraepithelial passages that are
filled by non-epithelial cells.
HPV E7 protein and cell cycle progression
The major role of high risk HPV E7 protein is to reprogram ter-
minally differentiating keratinocytes, in order to re-enter the cell
cycle and express proteins that are required for viral genome rep-
lication. Expression of E7 protein in growth arrested cells, indicates
that E7 induces cellular DNA synthesis at the G1 to S transition
[26]. The E7 protein binds and destabilizes the cellular retinoblas-
toma (Rb) tumor suppressor protein and members of the Rb family,
thereby, displacing E2F transcription factor, which then stimulate
expression of E2F-responsive genes, required for cellular (and vir-
al) DNA synthesis and cell cycle progression such as cyclin A and E,
DNA polymerase a, PCNA, hTERT and p21CIP1 which serve to drive
cells into S phase and induce cellular DNA synthesis [27–31]. It has
also been shown that HPV16 E7 hijacks the cullin 2 E3 ubiquitin li-
gase complex to target all three Rb family members for degrada-
tion [27,32].
 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
Table 1
Function of HPV proteins.
HPV Function
protein
Early proteins
E1 Initiation of viral genome replication
E2 Viral DNA replication and transcription. Segregation of viral
genomes
E4 Viral genome packing. Maturation of viral particles
E5 Oncoprotein. Participates in host cell transformation and blocks
apoptosis in late events of HPV carcinogenesis
E6 Major oncoprotein. Inactivates p53 protein. Block apoptosis.
Interacts with many host proteins with PDZ domains
E7 Major oncoprotein. Inactivates pRb protein. Promotes host DNA
synthesis and proliferation. Interacts with many host proteins
Late proteins
L1 Major capsid protein
L2 Minor capsid protein
The E7 protein also inhibits the action of cyclin-dependent ki-
nase inhibitors and interacts with cyclin–cdk complexes such as
cyclin A and cdk2 and other cellular proteins to stimulate cell cycle
progression [33–36]. Bypass of growth arrest by high risk HPV E7
requires both degradation of Rb and abrogation of p21CIP1 inhibi-
tion [37]. It is interesting to note that low risk HPV E7 cannot target
Rb for degradation and also is less efficient in abrogating p21CIP1
activity [33,34].
High risk HPV E7 induces chromatin remodeling and transcrip-
tional activation of specific genes. HPV type 16 E7 interacts with
both histone acetyltransferases (HATs) and histone deacetylases
(HDACs) which results in an increase in the level of histone acety-
lation on E2F regulated promoters [38,39]. In addition, high risk
HPV E7 induces the transcription of histone methyltransferases
(EZH2) and histone demethylases (KDM6A, KDM6B), which play
a crucial role in the epigenetic regulation of gene transcription
[40].
HPV E6 and cellular transformation
Tumor viruses, in order to exert their oncogenic potential, inhi-
bit apoptosis, a host response to infection and unscheduled S phase
entry. As noted in a previous section, in HPV infection, E7 main-
tains an S phase environment within terminally differentiated
keratinocytes, which activate various apoptotic programs. A major
role for E6 protein is to counteract these cellular responses, inhib-
iting the pro-apoptotic functions of p53 protein. The best-studied
function of high risk E6 protein is the binding and targeting of
p53 for ubiquitin-mediated degradation [41]. E6 binds directly to
E6-AP (E6 associated protein) which functions as a specific ubiqui-
tin-ligase for p53 degradation. Low risk HPV E6 protein can also
bind to E6-AP and complex to p53 but the binding does not lead
to p53 degradation. Two different regions of p53 can be bound
by E6, a region in the carboxy terminus that is bound by both
low and high risk HPV E6’s and a core region that correlates with
p53 degradation [42]. Abrogation of the p53 pathway by E6 allows
bypass of p53-mediated checkpoints. In ORSCC, we recently
showed that, repression of HPV type 16 E6/E7 expression using a
retrovirus mediated shRNA expression system in HPV type 16 oro-
pharyngeal cancer cells is associated with a rapid restoration of
p53 and pRb tumor suppressor pathways and massive apoptosis
[4].
A unique characteristic of the high risk E6 oncoproteins is the
presence of a PDZ binding motif on the extreme C-terminus, a mo-
tif that is absent from all the low risk E6 oncoproteins and thereby
represents a molecular signature for oncogenic potential in the
various HPV types [43,44]. Deletion of this motif in the context
Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
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3
of the whole viral genome results in a virus with reduced replica-
tive capacity and inability to maintain the viral genome in the epi-
somal form [45]. High risk E6 proteins bind to a number of PDZ
domain containing proteins with presumed tumor suppressor
activity that have diverse functions, ranging from control of cell
polarity, control of cell–cell attachment and the regulation of var-
ious signaling pathways [46,47]. Some of these PDZ domain-con-
taining proteins have been confirmed as degradation targets of
E6 protein [48,49]. A recent structural study proposed a model
whereby E6 dimerization is essential for p53 ubiquitination and
degradation [50]. E6 dimerization is also required for degradation
of PDZ domain-containing protein substrates. E6 binding to PDZ
domain-containing proteins may also inhibit their capacity to
interact with other cellular partners, or alternatively, may alter
their correct cellular localization. In addition, the E6 protein
stimulates the expression of telomerase, an RNA dependent DNA
polymerase that maintains the ends of chromosomes in proliferat-
ing somatic cells [51,52].
HPV positive head and neck cancer has a different molecular profile
compared to HPV negative head and neck cancer
Molecular evidence that HPV status determines a separate class
of HNSCC was initially provided by microsatellite analysis studies,
showing that HNSCC with no evidence of HPV infection is charac-
terized by a significant higher incidence of loss of 3p, 9p and/or 17p
chromosomal arms compared to HPV positive HNSCC [53]. LOH at
17p13 is thought to involve TP53, whereas LOH at 9p21 is thought
to involve INK4a, the tumor suppressor gene encoding p16. In HPV
negative HNSCC, inactivation of INK4a, either by chromosomal loss
or by hypermethylation is considered to be the most common way
to disrupt the pRb pathway [54]. On the contrary, HPV positive
HNSCC with E6/E7 viral oncoprotein expression is associated with
low rates of p53 and p16INK4a alterations [53]. In support to these
findings, two recent large scale exome sequencing projects in
HNSCC [6,7] revealed that HPV negative tumors accumulate at
least two times as many mutations. It seems that carcinogenesis
in HPV negative HNSCC is based on acquisition of a large number
of mutations in many different signaling pathways. On the con-
trary carcinogenesis in HPV positive tumors is modulated by the
activities of E6/E7 viral oncoproteins.
Gene expression profile of HPV positive HNSCC: The p16 molecular
phenotype
A number of studies of the gene expression differences between
HPV+ and HPV HNSCCs showed considerable differences in the
gene expression profiles that were specifically associated with
these two subgroups of head and neck cancer, supporting the no-
tion that HPV positive HNSCC has a specific transcription profile
depending partly on E6 and E7 expression. Weinberger et al. [55]
showed that p16 protein status determined by immunohistochem-
istry is reasonable surrogate marker for a biologically and clinically
meaningful HPV infection in OSCC. The authors sought to deter-
mine the incidence and clinical implications of biologically rele-
vant HPV16 infection in a cohort of 107 oropharyngeal squamous
cell cancers treated with primary radiotherapy or surgery followed
by postoperative radiotherapy at Yale University. HPV-16 DNA vir-
al load was determined by Real-Time PCR. In addition, they con-
structed a tissue array composed of these tumors and studied
expression of p53, pRb and p16 proteins using a quantitative
in situ method of protein analysis (AQUA). They hypothesized that
among HPV16 DNA positive cases, p16 expression status would
determine the biologically relevant ones. Their results delineated
3 tumor classes with distinct molecular and clinical features based
on HPV16 DNA presence and p16 expression status: one class of
4 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
HPV16 negative/p16 non-expressing, one class of HPV16 positive/
p16 non-expressing and one class HPV16 positive/p16 expressing
oropharyngeal tumors. Overall survival in class III was 79% com-
pared to the other two classes (20% and 18%, p = 0.0095). Dis-
ease-free survival for the same class was 75% versus 15% and
13% (p = 0.0025). The 5-year local recurrence was 14% in Class III
versus 45% and 74% (p = 0.03). Only patients in class III had signif-
icantly lower p53 and pRb expression (p = 0.017 and 0.001, respec-
tively). Multivariable survival analysis confirmed the prognostic
value of the 3-class model. The authors demonstrated that only
the HPV16 positive/p16 expressing tumors fit the cervical carcino-
genesis model and they are the ones associated with favorable
prognosis.
A comprehensive analysis by Slebos and colleagues, revealed
that CDKN2A (encodes the tumor suppressor p16INK4a protein)
was one of the most significant differentially expressed genes,
since its expression was highly upregulated in HPV+ group [56].
In the same study, other genes identified to have higher expression
in HPV+ group were the cell cycle regulators p18 and CDC7, the
transcription factors TAF7L, RFC4, RPA2, TFDP2 and the cell adhe-
sion molecule TCAM1. Another microarray study between HPV+
and HPV HNSCCs by Schlecht et al. [57] revealed a similar expres-
sion pattern for p18 and TFDP2 expression, that was also found to
be significantly upregulated in HPV+ group. However this study
failed to validate upregulation of p16INK4a in HPV+ group as its
expression levels were low and did not meet the preset twofold
expression threshold. A third microarray analysis by Martinez
et al. [58] succeeded to validate that expression of p16INK4a is
upregulated in HPV+ subgroup and furthermore, showed that the
expression of ZNF238, DNMT1, MCM3, AKR1C3, GRB10, TYK2 and
SART3 were significantly upregulated in HPV+ HNSCC compared
to HPV HNSCC. Unfortunately, the reported gene expression pro-
files show little overlap between studies, which complicates the
data interpretation and the identification of important genes for
tumorigenesis in head and neck epithelia [59]. A recent global
comparative proteomic analysis of HPV+ and HPV HNSCCs using
liquid chromatography-mass spectrometry. revealed that a cluster
of minichromosome maintenance family members (MCM2,6 and
7) are significantly upregulated in HPV+ HNSCC and forms the most
clearly differential set of proteins between these two subgroups
[60]. Minichromosome maintenance proteins function as helicases
for DNA unwinding in early stages of DNA replication during the
cell cycle G1 phase and seems to represent a potential target for
anticancer drug development.
Keratinocyte differentiation and manipulation of cellular pathways by
HPV in head and neck epithelia
Wnt and Notch cell signaling pathways are essential in the reg-
ulation of proliferation and differentiation responses during nor-
mal development. Deregulation of Wnt and Notch signaling is
associated with human cancers. Oncogenic activation of the Wnt/
b-catenin was initially associated with colon cancer where truncat-
ing mutations of APC and mutations of GSK3b phosphorylation
sites of b-catenin are prevalent. Notch is an established tumor sup-
pressor in the skin and in chronic myelomonocytic leukemia
[61,62]. Recent experimental studies also suggest that loss of func-
tion mutations in Notch receptors are associated with head and
neck, lung and cutaneous squamous cell carcinoma [6,7,63]. It is
also well established that Wnt and Notch pathways are attractive
targets for virus interaction and manipulation. Viruses that their
life cycle is associated with the differentiation state of the host cell
and the development of human cancers, such as Epstein-Barr virus
(EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), use
components of these pathways to regulate their own viral gene
Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
dx.doi.org/10.1016/j.oraloncology.2013.07.011
expression and to alter cellular gene expression through mimicry
or manipulation of downstream pathway responses [64].
Papillomaviruses are associated with benign proliferative epi-
thelial lesions in their respective hosts. Since HPV gene expression
and replication is intimately linked to the differentiation state of
the infected keratinocytes, Notch and Wnt pathways are crucial
for the papillomavirus life cycle.
HPV and Wnt signaling pathway
Wnt ligands control several differentiation processes during
normal tissue homeostasis [65], and they are implicated in patho-
logic conditions such as in certain types of cancer [66]. In the ab-
sence of Wnt ligands, b-catenin forms a ‘‘degradation complex’’
with kinases and scaffold proteins, such as glycogen synthase ki-
nase-3b (GSK3b), casein kinase 1 and 2 (CK1 and CK2), adenoma-
tous polyposis coli (APC), and Axin2. This degradation complex
phosphorylates b-catenin at serine and threonine residues
inducing its ubiquitination and degradation. The activation of
canonical Wnt signaling induces the phosphorylation of the intra-
cellular Dishevelled (Dvl) protein, which eventually interacts with
Axin2 and impedes the formation of the b-catenin degradation
complex; this process leads to the accumulation and nuclear trans-
location of b-catenin. Once it is translocated into the nucleus, b-
catenin binds members of the T-cell factor/lymphoid enhancer fac-
tor (TCF/LEF) family of transcription factors, of which TCF4 is the
best characterized. b-Catenin/TCF4 complexes control the expres-
sion of several target genes that regulate cell polarity, proliferation,
and differentiation including c-jun, c-myc, cyclin D1 [67,68].
The participation of canonical Wnt pathway in cervical cancer is
evident because the nuclear accumulation of b-catenin correlates
with tumor progression in human patients. Nuclear b-catenin is
commonly found in human HPV16-positive invasive carcinoma
biopsies, but it is uncommon in early dysplastic lesions [69,70].
Microarray expression studies conducted on cervical cancer tissues
identified alterations in the expression of regulators of the Wnt sig-
naling pathway [71]. Recent experimental evidence suggests that
Wnt signaling pathway is activated in HPV16-positive HNSCC
and that the Siah-1–dependent ubiquitin/proteasome pathway
plays a significant/pivotal role for b-catenin degradation in
HPV16-positive oropharyngeal cancer cell lines [72]. More specifi-
cally, according to these studies, nuclear b-catenin accumulation
seems to be a direct consequence of E6 and E7 oncoprotein expres-
sion since viral E6/E7 expression is associated with downregula-
tion of the endogenous Seven in absentia homologue (Siah-1) E3
ubiquitin ligase which promotes the proteosomal degradation of
b-catenin through a mechanism independent of GSK3b mediated
phosphorylation. As a consequence, b-catenin is stabilized, translo-
cates into the nucleus and promotes the transcriptional activation
of b-catenin–responsive genes.
Smeets and colleagues showed that immortalization of normal
oral keratinocytes (OKCs) by HPV E6 expression caused an activa-
tion of Wnt pathway which is in consistent with the finding that
nuclear b-catenin protein levels are upregulated in HNSCCs and
HPV16-positive oropharyngeal cancer cell lines [73]. In this study,
the authors demonstrated that Wnt activation is associated with
p53 degradation induced by E6 expression, since the expression
of a dominant negative mutant p53 (R175H) was able to cause a
similar activation of Wnt signaling pathway.
In accordance to these studies, comparative quantitative analysis of
protein expression for 13 different biomarkers in p16/HPV+, p16+/
HPV+, p16+/HPV, p16/HPV subgroups of HNSCC using AQUA fluo-
rescent immunohistochemistry, validated b-catenin as biomarker that
is significantly upregulated in p16+/HPV+ HNSCC tumors [74].
The crucial role of viral E6 expression in activation of Wnt sig-
naling pathway in HPV type 16 positive epithelia has also been
confirmed by in vivo studies. An in vivo model study with a trans-
 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
genic mice expressing the full-length E6 oncoprotein in the basal
layer of stratified epithelia under the control of human keratin
14 promoter (K14E6 mice). This transgenic E6 strain was shown
to display skin hyperplasia and skin cancer at advanced ages and
the expression of the E6 viral oncoprotein was associated with nu-
clear accumulation of b-catenin and the transcriptional activation
of b-catenin–responsive genes in skin epidermis [75]. Conversely,
when the transgenic strain was expressing a truncated E6 oncopro-
tein that lacks the PDZ binding domain (K14E6deltaPDZ mice), nu-
clear b-catenin was not detected and b-catenin–responsive genes
were not transcriptionally activated.
The synergy between Wnt signaling pathway and human papil-
lomavirus was also shown by another in vivo model study using
the double transgenic animal K14-E7/DN87bcat that expresses
the E7 viral oncogene of HPV type 16 along with a constitutively
active amino-terminal truncated b-catenin molecule under the
control of keratin 14 promoter [76]. The K14-DN87bcat transgenic
strain develops benign skin tumors but does not exhibit histopath-
ologic charasteristics of cervical cancer while the K14-E7 strain
displays cervical pathologies after 6 months of estrogen treatment.
The authors analysed the incidence of invasive cervical cancer in
K14-E7, K14-DN87bcat and double transgenic K14-E7/DN87bcat
mice and they observed that invasive cervical cancer developed
in 50%, 11% and 94% of the transgenics at the age of 7 months
old respectively, suggesting that the activation of Wnt pathway
accelerates HPV16-E7 mediated cervical carcinogenesis.
HPV and manipulation of Notch signaling pathway
The Notch receptor and Notch ligands are single-pass trans-
membrane proteins that influence cell fate decisions, proliferation,
and differentiation. In mammals, there are four Notch receptors,
Notch1, 2, 3, and 4, the ligands include Jagged-1and -2 and Del-
ta-like-1, -3, and -4. Ligand binding is followed by two cleavages
of the Notch receptor by ADAM metalloprotease and gamma-
secretase complex, leading to the release of the Notch intracellular
domain (NICD). NICD enters the nucleus and targets the DNA bind-
ing protein CSL (CBF1/Suppressor of Hairless/Lag-1). In the absence
of signaling, DNA-bound CSL is associated with a corepressor com-
plex. Upon Notch signaling, the corepressor complex is displaced
by NotchICD and the coactivator Mastermind, up-regulating target
genes containing CSL binding sites [77].
Figure 2. Manipulation of Wnt and Notch signaling pathway by HPV E6 and E7 proteins.
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5
Recent advances in our understanding of the molecular role of
Notch in HNSCC were provided by whole-exome sequencing stud-
ies [6,7]. The most novel finding to emerge from these sequencing
studies of HNSCC is the discovery of loss of function mutations
within the NOTCH1 gene in 12–15% of cases, and within additional
NOTCH family members in 3%–5%. Analysis of these exome
sequencing data also indicated that Notch ligands (JAG1, JAG2,
DLL1-4), were rarely mutated. The vast majority of Notch 1 muta-
tions in HNSCC, were missense mutations that occurred at or near
identified important domains such as the ligand-binding domain
(EGF repeats 11, 12 and 13) or the ankyrin domains. In addition,
the detected nonsense mutations were predicted to result in trun-
cated Notch1 proteins lacking domains important for transcrip-
tional activation. These data suggest that Notch1 is acting as a
tumor suppressor in head and neck epithelium.
The tumor suppressor role of Notch signaling in SCCs is still
being characterized. However, Notch signaling does promote ter-
minal differentiation of keratinocytes. Papillomaviruses have
evolved to couple the expression of their capsid proteins to termi-
nal differentiation of keratinocytes. Since in the stratified epithelia
Notch signaling has a central role in promoting terminal differen-
tiation [78], one would expect E6 to modulate Notch signaling dur-
ing the viral cycle life in the upper cell layers of the papilloma.
Recently, several groups have reported that E6 protein from
HPV5, HPV8 and other cutaneous b-HPVs repress Notch transcrip-
tional activation, and this repression is dependent on a direct inter-
action with the MAML1 [79–81]. MAML1 is a core component of
the transcriptional activation complex that mediates the effects
of the canonical Notch signaling pathway. Binding of cutaneous
b- HPV E6 to MAML1 was found to repress transactivation by
MAML1. The N-terminal basic domain of MAML1 interacts with
the ankyrin repeats of the intracellular domain of Notch receptor
and the DNA-binding factor CSL [77]. Figure 2 presents an over-
view of Wnt and Notch signaling manipulation by HPV. Mapping
the interaction between the paplillomavirus E6 protein and
MAML1 revealed an acidic domain with an LXXLL motif at the C-
terminal fragment of MAML1 that seems to be critical for the
MAML1-HPV E6 protein complex formation. This acidic domain is
part of transcription activation domain 2 (TAD2) which is required
for transcription in vivo and has been proposed to interact with
6 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
additional unknown cellular proteins necessary for Notch tran-
scriptional activation [82].
It is worth noting that in these studies, the expression levels of
endogenous Notch target genes were found repressed by b-HPV E6
proteins. These findings elucidate a mechanism of viral antagonism
of Notch signaling, and suggest that Notch signaling is an impor-
tant epithelial cell pathway target for the human papillomavirus.
However our knowledge about how mucosal a-HPVs manipulate
the Notch signaling is still poor and future studies on the field need
to focus on the mechanism of Notch inhibition by high risk a-HPVs
encoded E6 proteins and the biological implications of inhibition.
HPV and activation of the Phosphatidylinositol 3-Kinase/Akt/mTOR
Pathway in HNSCC
Activation of PI3K pathway is able to drive oncogenesis in mul-
tiple human cancers. When bound to ligand, EGFR activates the
PI3Ks, leading to the phosphorylation of phosphatidylinositol on
the 30 -hydroxyl group [83,84]. The resultant product, phosphati-
dylinositol-3,4,5- trisphosphate, activates AKT kinase [85,86]
which phosphorylates in turn several downstream proteins, regu-
lating cell growth and survival.
Human papillomavirus (HPV) entry into target cells is initiated
by binding to cell surface heparan-sulfonated proteoglycans
(HSPGs). The virus also interacts with secondary receptors, which
are responsible for particle internalization. Upon these early events
of HPV infection, growth factor receptors are often activated trig-
gering the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signal-
ing cascade [87,88]. In a recent study, Surviladze and colleagues
showed that Akt/mTOR signaling pathway is rapidly activated after
exposure of human keratinocytes to HPV type 16 pseudovirions
(PsVs) which deliver a reporter plasmid (pseudogenome) to the
cell, suggesting that HPV infection stimulates the PI3K/Akt/mTOR
pathway potentially via alpha-6 beta-4 integrins [87]. Previous
studies have also shown that high risk HPV E6/E7 protein expres-
sion can activate Akt and mTOR complexes and promote virus sur-
vival [89–91].
Specific components of the EGFR/PI3K pathway have been
found mutated in human cancers, resulting in pathway activation.
Mutations in EGFR, PIK3CA, and PTEN occur commonly in a variety
of cancers, such as lung, breast, prostate cancer, and glioblastoma
[92–95]. Moreover, these mutations cause the constitutive activa-
tion of downstream signaling molecules such as Akt/protein kinase
B (PKB), mammalian target of rapamycin (mTOR) and ribosomal
protein S6 kinase (S6K) Activation of AKT/mTOR pathway which
play a key role in tumorigenesis and survival rate of HNSCC pa-
tients [96].
Previous mutational studies reported that 7–12% of HNSCCs
harbor PI3KCA mutations [97,98], however, in these studies, there
was a lack of mutational data for the downstream effectors of PI3K
pathway. A whole exome sequencing study by Stransky and col-
leagues in a cohort of 74 tumor-normal pairs revealed that 8% of
samples were mutated on PI3KCA gene and 5% of samples were
mutated on PTEN gene [6]. A second whole exome sequencing
study by Agrawal and colleagues in a cohort of 32 primary head
and neck tumors revealed that 6% of samples were mutated on
PI3KCA gene [7]. However, these whole exome sequencing studies
did not revealed a positive association of PI3KCA/PTEN mutations
with the HPV status of samples.
In a recent study, Lui and colleagues, obtained the mutational
profiles of 45 HNSCC tumors by whole exome sequencing using
the Illumina platform. In addition, authors combined all currently
available mutational data from whole-exome sequencing of 151
HNSCC primary tumors and evaluated the mutational events of
genes in MAPK, JAK/STAT, and the PI3K pathways mitogenic path-
ways [99]. Analysis of whole-exome sequencing data revealed that
Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
dx.doi.org/10.1016/j.oraloncology.2013.07.011
the PI3K pathway (PIK3CA, PIK3AP1, PIK3C2A, PIK3C2B, PIK3C2G,
PIK3CB, PIK3CD, PIK3CG, PIK3IP1, PIK3R1/2/3/4/5/6, AKT1/2/3,
mTOR, PTEN, PDK1, TSC1/2, RICTOR and RPTOR) was the most fre-
quently mutated oncogenic pathway (30.5%). Although the number
of HPV-positive HNSCC tumors in their cohort was relatively small
(15 cases), authors were able to identify a subset of HPV positive
HNSCC tumors (3/15, 20%) where PIK3CA or PIK3R1 was the only
mutated cancer gene, suggesting that the tumor progression in this
subset of HPV positive tumors is driven by PI3K pathway muta-
tions alone without any further genomic alterations in critical
oncogenes or tumor suppressor genes.
Conclusions
HPV-associated HNSCC represents a distinct entity from tobac-
co- and alcohol- related HNSCC with different molecular profile.
High risk HPV E6/E7 expression, following viral integration, initiate
a number of malignant transformation processes including bypass
of cell cycle control, DNA synthesis, inhibition of apoptosis and
transcriptional activation of genes that promote proliferation. This
model of tumorigenesis is different from the one that is established
for the tobacco-induced HNSCC which requires the acquisition of a
large number of mutations or deletions in tumor suppressor genes.
However, further investigation into the molecular profiles between
HPV-positive and HPV-negative HNSCC is needed, in order to
understand whether specific gene mutations can enhance the
transforming activities of E6/E7 viral oncoproteins and accelerate
the HPV related tumorigenesis. Human papillomavirus has been
evolved to coexist within epithelial cells and its life cycle is tightly
linked to proliferation and differentiation of the basal epithelial
cells. E6 and E7 viral oncoproteins affect and modulate cellular sig-
naling pathways that regulate the proliferation capacity and the
differentiation state of keratinocytes. Notch, Wnt and PI3K/Akt/
mTOR are cell signaling pathways that are targeted by high risk
HPV in head and neck epithelial cells. The viruses use components
of these pathways to regulate their own viral gene expression and
additionally alter cell gene expression by manipulation of down-
stream pathway responses. Recent experimental studies on HPV-
associated HNSCC, provide evidence that HPV activates PI3K/Akt/
mTOR pathway and promotes b-catenin stabilization and inactiva-
tion of Notch signaling. However further investigation of the inter-
connection between these pathways and the E6/E7 activities is
needed. The understanding of the molecular profile of HPV associ-
ated head and neck cancer will provide opportunities to create new
molecular markers for HPV diagnostics and new therapeutic
approaches.
Conflict of interest statement
None declared.
References
[1] Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of eighteen
major cancers in 1985. Int J Cancer J Int Du Cancer 1993;54:594–606.
[2] Jemal A, Simard EP, Dorell C, Noone AM, Markowitz LE, Kohler B, et al. Annual
report to the nation on the status of cancer, 1975–2009, featuring the burden
and trends in Human Papillomavirus (HPV)-associated cancers and HPV
vaccination coverage levels. J Natl Cancer Inst 2013;105:175–201.
[3] Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, et al.
Human papillomavirus and rising oropharyngeal cancer incidence in the
United States. J Clinical Oncol: Official J Am Soc Clin Oncol 2011;29:4294–301.
[4] Rampias T, Sasaki C, Weinberger P, Psyrri A. E6 and e7 gene silencing and
transformed phenotype of human papillomavirus 16-positive oropharyngeal
cancer cells. J Natl Cancer Inst 2009;101:412–23.
[5] Dayyani F, Etzel CJ, Liu M, Ho CH, Lippman SM, Tsao AS. Meta-analysis of the
impact of human papillomavirus (HPV) on cancer risk and overall survival in
head and neck squamous cell carcinomas (HNSCC). Head Neck Oncol
2010;2:15.
 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
[6] Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, et al.
The mutational landscape of head and neck squamous cell carcinoma. Science
2011;333:1157–60.
[7] Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, et al.
Exome sequencing of head and neck squamous cell carcinoma reveals
inactivating mutations in NOTCH1. Science 2011;333:1154–7.
[8] Engel LW, Heilman CA, Howley PM. Transcriptional organization of bovine
papillomavirus type 1. J Virol 1983;47:516–28.
[9] Ilves I, Kivi S, Ustav M. Long-term episomal maintenance of bovine
papillomavirus type 1 plasmids is determined by attachment to host
chromosomes, which Is mediated by the viral E2 protein and its binding
sites. J Virol 1999;73:4404–12.
[10] Lehman CW, Botchan MR. Segregation of viral plasmids depends on tethering
to chromosomes and is regulated by phosphorylation. Proc Nat Acad Sci USA
1998;95:4338–43.
[11] Bastien N, McBride AA. Interaction of the papillomavirus E2 protein with
mitotic chromosomes. Virology 2000;270:124–34.
[12] Baxter MK, McPhillips MG, Ozato K, McBride AA. The mitotic chromosome
binding activity of the papillomavirus E2 protein correlates with interaction
with the cellular chromosomal protein, Brd4. J Virol 2005;79:4806–18.
[13] Brown DR, Kitchin D, Qadadri B, Neptune N, Batteiger T, Ermel A. The human
papillomavirus type 11 E1–E4 protein is a transglutaminase 3 substrate and
induces abnormalities of the cornified cell envelope. Virology
2006;345:290–8.
[14] Wilson R, Fehrmann F, Laimins LA. Role of the E1–E4 protein in the
differentiation-dependent life cycle of human papillomavirus type 31. J Virol
2005;79:6732–40.
[15] Peh WL, Brandsma JL, Christensen ND, Cladel NM, Wu X, Doorbar J. The viral E4
protein is required for the completion of the cottontail rabbit papillomavirus
productive cycle in vivo. J Virol 2004;78:2142–51.
[16] DiMaio D, Lai CC, Mattoon D. The platelet-derived growth factor beta receptor
as a target of the bovine papillomavirus E5 protein. Cytokine Growth Factor
Rev 2000;11:283–93.
[17] Petti L, Nilson LA, DiMaio D. Activation of the platelet-derived growth factor
receptor by the bovine papillomavirus E5 transforming protein. EMBO J
1991;10:845–55.
[18] Kivi N, Greco D, Auvinen P, Auvinen E. Genes involved in cell adhesion, cell
motility and mitogenic signaling are altered due to HPV 16 E5 protein
expression. Oncogene 2008;27:2532–41.
[19] Klingelhutz AJ, Roman A. Cellular transformation by human papillomaviruses:
lessons learned by comparing high- and low-risk viruses. Virology
2012;424:77–98.
[20] Orth G. Genetics of epidermodysplasia verruciformis: insights into host
defense against papillomaviruses. Semin Immunol 2006;18:362–74.
[21] Woodman CB, Collins SI, Young LS. The natural history of cervical HPV
infection: unresolved issues. Nat Rev Cancer 2007;7:11–22.
[22] Jeon S, Lambert PF. Integration of human papillomavirus type 16 DNA into the
human genome leads to increased stability of E6 and E7 mRNAs: implications
for cervical carcinogenesis. Proc Nat Acad Sci USA 1995;92:1654–8.
[23] Giroglou T, Florin L, Schafer F, Streeck RE, Sapp M. Human papillomavirus
infection requires cell surface heparan sulfate. J Virol 2001;75:1565–70.
[24] Joyce JG, Tung JS, Przysiecki CT, Cook JC, Lehman ED, Sands JA, et al. The L1
major capsid protein of human papillomavirus type 11 recombinant virus-like
particles interacts with heparin and cell-surface glycosaminoglycans on
human keratinocytes. J Biol Chem 1999;274:5810–22.
[25] Shafti-Keramat S, Handisurya A, Kriehuber E, Meneguzzi G, Slupetzky K,
Kirnbauer R. Different heparan sulfate proteoglycans serve as cellular
receptors for human papillomaviruses. J Virol 2003;77:13125–35.
[26] Banks L, Barnett SC, Crook T. HPV-16 E7 functions at the G1 to S phase
transition in the cell cycle. Oncogene 1990;5:833–7.
[27] Boyer SN, Wazer DE, Band V. E7 protein of human papilloma virus-16 induces
degradation of retinoblastoma protein through the ubiquitin-proteasome
pathway. Cancer Res 1996;56:4620–4.
[28] Chellappan S, Kraus VB, Kroger B, Munger K, Howley PM, Phelps WC, et al.
Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7
protein share the capacity to disrupt the interaction between transcription
factor E2F and the retinoblastoma gene product. Proc Nat Acad Sci USA
1992;89:4549–53.
[29] Dyson N, Howley PM, Munger K, Harlow E. The human papilloma virus-16 E7
oncoprotein is able to bind to the retinoblastoma gene product. Science
1989;243:934–7.
[30] Gonzalez SL, Stremlau M, He X, Basile JR, Munger K. Degradation of the
retinoblastoma tumor suppressor by the human papillomavirus type 16 E7
oncoprotein is important for functional inactivation and is separable from
proteasomal degradation of E7. J Virol 2001;75:7583–91.
[31] Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM. Complex
formation of human papillomavirus E7 proteins with the retinoblastoma
tumor suppressor gene product. EMBO J 1989;8:4099–105.
[32] Huh K, Zhou X, Hayakawa H, Cho JY, Libermann TA, Jin J, et al. Human
papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin
ligase complex, which contributes to degradation of the retinoblastoma tumor
suppressor. J Virol 2007;81:9737–47.
[33] Funk JO, Waga S, Harry JB, Espling E, Stillman B, Galloway DA. Inhibition of
CDK activity and PCNA-dependent DNA replication by p21 is blocked by
interaction with the HPV-16 E7 oncoprotein. Genes Dev 1997;11:2090–100.
Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
dx.doi.org/10.1016/j.oraloncology.2013.07.011
7
[34] Jones DL, Alani RM, Munger K. The human papillomavirus E7 oncoprotein can
uncouple cellular differentiation and proliferation in human keratinocytes by
abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev 1997;11:2101–11.
[35] Noya F, Chien WM, Broker TR, Chow LT. P21cip1 Degradation in differentiated
keratinocytes is abrogated by costabilization with cyclin E induced by human
papillomavirus E7. J Virol 2001;75:6121–34.
[36] Tommasino M, Adamczewski JP, Carlotti F, Barth CF, Manetti R, Contorni M,
et al. HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin
A. Oncogene 1993;8:195–202.
[37] Helt AM, Funk JO, Galloway DA. Inactivation of both the retinoblastoma tumor
suppressor and p21 by the human papillomavirus type 16 E7 oncoprotein is
necessary to inhibit cell cycle arrest in human epithelial cells. J Virol
2002;76:10559–68.
[38] Avvakumov N, Torchia J, Mymryk JS. Interaction of the HPV E7 proteins with
the pCAF acetyltransferase. Oncogene 2003;22:3833–41.
[39] Baldwin A, Huh KW, Munger K. Human papillomavirus E7 oncoprotein
dysregulates steroid receptor coactivator 1 localization and function. J Virol
2006;80:6669–77.
[40] Holland D, Hoppe-Seyler K, Schuller B, Lohrey C, Maroldt J, Durst M, et al.
Activation of the enhancer of zeste homologue 2 gene by the human
papillomavirus E7 oncoprotein. Cancer Res 2008;68:9964–72.
[41] Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. The E6
oncoprotein encoded by human papillomavirus types 16 and 18 promotes the
degradation of p53. Cell 1990;63:1129–36.
[42] Li X, Coffino P. High-risk human papillomavirus E6 protein has two distinct
binding sites within p53, of which only one determines degradation. J Virol
1996;70:4509–16.
[43] Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, et al. Recognition
of unique carboxyl-terminal motifs by distinct PDZ domains. Science
1997;275:73–7.
[44] Kiyono T, Hiraiwa A, Fujita M, Hayashi Y, Akiyama T, Ishibashi M. Binding of
high-risk human papillomavirus E6 oncoproteins to the human homologue of
the Drosophila discs large tumor suppressor protein. Proc Nat Acad Sci USA
1997;94:11612–6.
[45] Lee C, Laimins LA. Role of the PDZ domain-binding motif of the oncoprotein E6
in the pathogenesis of human papillomavirus type 31. J Virol
2004;78:12366–77.
[46] Gardiol D, Kuhne C, Glaunsinger B, Lee SS, Javier R, Banks L. Oncogenic human
papillomavirus E6 proteins target the discs large tumour suppressor for
proteasome-mediated degradation. Oncogene 1999;18:5487–96.
[47] Nguyen ML, Nguyen MM, Lee D, Griep AE, Lambert PF. The PDZ ligand domain
of the human papillomavirus type 16 E6 protein is required for E6’s induction
of epithelial hyperplasia in vivo. J Virol 2003;77:6957–64.
[48] Roberts S, Delury C, Marsh E. The PDZ protein discs-large (DLG): the ‘Jekyll and
Hyde’ of the epithelial polarity proteins. FEBS J 2012;279:3549–58.
[49] Facciuto F, Cavatorta AL, Valdano MB, Marziali F, Gardiol D. Differential
expression of PDZ domain-containing proteins in human diseases –
challenging topics and novel issues. FEBS J 2012;279:3538–48.
[50] Zanier K, Ould M’hamed ould Sidi A, Boulade-Ladame C, Rybin V, Chappelle A,
Atkinson A, et al. Solution structure analysis of the HPV16 E6 oncoprotein
reveals a self-association mechanism required for E6-mediated degradation of
p53. Structure 2012;20:604–17.
[51] Gewin L, Galloway DA. E box-dependent activation of telomerase by human
papillomavirus type 16 E6 does not require induction of c-myc. J Virol
2001;75:7198–201.
[52] Veldman T, Horikawa I, Barrett JC, Schlegel R. Transcriptional activation of the
telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein. J
Virol 2001;75:4467–72.
[53] Braakhuis BJ, Snijders PJ, Keune WJ, Meijer CJ, Ruijter-Schippers HJ, Leemans
CR, et al. Genetic patterns in head and neck cancers that contain or lack
transcriptionally active human papillomavirus. J Natl Cancer Inst
2004;96:998–1006.
[54] Shintani S, Nakahara Y, Mihara M, Ueyama Y, Matsumura T. Inactivation of the
p14(ARF), p15(INK4B) and p16(INK4A) genes is a frequent event in human oral
squamous cell carcinomas. Oral Oncol 2001;37:498–504.
[55] Weinberger PM, Yu Z, Haffty BG, Kowalski D, Harigopal M, Brandsma J, et al.
Molecular classification identifies a subset of human papillomavirus–
associated oropharyngeal cancers with favorable prognosis. J Clinical Oncol:
Official J Am Soc Clinical Oncol 2006;24:736–47.
[56] Slebos RJ, Yi Y, Ely K, Carter J, Evjen A, Zhang X, et al. Gene expression
differences associated with human papillomavirus status in head and neck
squamous cell carcinoma. Clin Cancer Res: Official J Am Assoc Cancer Res
2006;12:701–9.
[57] Schlecht NF, Burk RD, Adrien L, Dunne A, Kawachi N, Sarta C, et al. Gene
expression profiles in HPV-infected head and neck cancer. J Pathol
2007;213:283–93.
[58] Martinez I, Wang J, Hobson KF, Ferris RL, Khan SA. Identification of
differentially expressed genes in HPV-positive and HPV-negative
oropharyngeal squamous cell carcinomas. Eur J Cancer 2007;43:415–32.
[59] Braakhuis BJ, Brakenhoff RH, Leemans CR. Gene expression profiling in head
and neck squamous cell carcinoma. Curr Opin Otolaryngology Head Neck
Surgery 2010;18:67–71.
[60] Slebos RJ, Jehmlich N, Brown B, Yin Z, Chung CH, Yarbrough WG, et al.
Proteomic analysis of oropharyngeal carcinomas reveals novel HPV-associated
biological pathways. Int J Cancer J Int du Cancer 2013;132:568–79.
8 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx
[61] Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, van Noort M, et al.
Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 2003;33:
416–21.
[62] Klinakis A, Lobry C, Abdel-Wahab O, Oh P, Haeno H, Buonamici S, et al. A novel
tumour-suppressor function for the Notch pathway in myeloid leukaemia.
Nature 2011;473:230–3.
[63] Wang NJ, Sanborn Z, Arnett KL, Bayston LJ, Liao W, Proby CM, et al. Loss-of-
function mutations in Notch receptors in cutaneous and lung squamous cell
carcinoma. Proc Nat Acad Sci USA 2011;108:17761–6.
[64] Hayward SD, Liu J, Fujimuro M. Notch, Wnt signaling: mimicry and
manipulation by gamma herpesviruses. Sci STKE: Signal Transduction
Knowledge Environ 2006;2006:re4.
[65] Cadigan KM, Nusse R. Wnt signaling: a common theme in animal
development. Genes Dev 1997;11:3286–305.
[66] Polakis P. Wnt signaling and cancer. Genes Dev 2000;14:1837–51.
[67] He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, et al.
Identification of c-MYC as a target of the APC pathway. Science 1998;281:
1509–12.
[68] Tetsu O, McCormick F. Beta-catenin regulates expression of cyclin D1 in colon
carcinoma cells. Nature 1999;398:422–6.
[69] Shinohara A, Yokoyama Y, Wan X, Takahashi Y, Mori Y, Takami T, et al.
Cytoplasmic/nuclear expression without mutation of exon 3 of the beta-
catenin gene is frequent in the development of the neoplasm of the uterine
cervix. Gynecol Oncol 2001;82:450–5.
[70] Uren A, Fallen S, Yuan H, Usubutun A, Kucukali T, Schlegel R, et al. Activation of
the canonical Wnt pathway during genital keratinocyte transformation: a
model for cervical cancer progression. Cancer Res 2005;65:6199–206.
[71] Perez-Plasencia C, Vazquez-Ortiz G, Lopez-Romero R, Pina-Sanchez P, Moreno
J, Salcedo M. Genome wide expression analysis in HPV16 cervical cancer:
identification of altered metabolic pathways. Infectious Agents Cancer 2007;2:
16.
[72] Rampias T, Boutati E, Pectasides E, Sasaki C, Kountourakis P, Weinberger P,
et al. Activation of Wnt signaling pathway by human papillomavirus E6 and E7
oncogenes in HPV16-positive oropharyngeal squamous carcinoma cells. Mol
Cancer Res: MCR 2010;8:433–43.
[73] Smeets SJ, van der Plas M, Schaaij-Visser TB, van Veen EA, van Meerloo J,
Braakhuis BJ, et al. Immortalization of oral keratinocytes by functional
inactivation of the p53 and pRb pathways. Int J Cancer J Int du Cancer
2011;128:1596–605.
[74] Rampias T, Pectasides E, Prasad M, Sasaki C, Gouveris P, Dimou A, et al.
Molecular profile of head and neck squamous cell carcinomas bearing p16
high phenotype. Annals Oncol: Official J Eur Soc Med Oncol/ESMO 2013.
[75] Bonilla-Delgado J, Bulut G, Liu X, Cortes-Malagon EM, Schlegel R, Flores-
Maldonado C, et al. The E6 oncoprotein from HPV16 enhances the canonical
Wnt/beta-catenin pathway in skin epidermis in vivo. Mol Cancer Res: MCR
2012;10:250–8.
[76] Bulut G, Fallen S, Beauchamp EM, Drebing LE, Sun J, Berry DL, et al. Beta-
catenin accelerates human papilloma virus type-16 mediated cervical
carcinogenesis in transgenic mice. PLoS ONE 2011;6:e27243.
[77] Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the
activation mechanism. Cell 2009;137:216–33.
[78] Egloff AM, Grandis JR. Molecular pathways: context-dependent approaches to
Notch targeting as cancer therapy. Clin Cancer Res: Official J Am Assoc Cancer
Res 2012;18:5188–95.
[79] Meyers JM, Spangle JM, Munger K. The HPV8 E6 protein interferes with NOTCH
activation during keratinocyte differentiation. J Virol 2013.
[80] Brimer N, Lyons C, Wallberg AE. Vande Pol SB. Cutaneous papillomavirus E6
oncoproteins associate with MAML1 to repress transactivation and NOTCH
signaling. Oncogene 2012;31:4639–46.
Please cite this article in press as: Rampias T et al. Molecular mechanisms of HPV induced carcinogenesis in head and neck. Oral Oncol (2013), http://
dx.doi.org/10.1016/j.oraloncology.2013.07.011
[81] Tan MJ, White EA, Sowa ME, Harper JW, Aster JC, Howley PM. Cutaneous beta-
human papillomavirus E6 proteins bind Mastermind-like coactivators and
repress Notch signaling. Proc Nat Acad Sci USA 2012;109:E1473–80.
[82] McElhinny AS, Li JL, Wu L. Mastermind-like transcriptional co-activators:
emerging roles in regulating cross talk among multiple signaling pathways.
Oncogene 2008;27:5138–47.
[83] Kurosu H, Maehama T, Okada T, Yamamoto T, Hoshino S, Fukui Y, et al.
Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110beta is
synergistically activated by the betagamma subunits of G proteins and
phosphotyrosyl peptide. J Biol Chem 1997;272:24252–6.
[84] Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular
function of phosphoinositide 3-kinases: implications for development,
homeostasis, and cancer. Annu Rev Cell Dev Biol 2001;17:615–75.
[85] Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-
oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 1997;
275:665–8.
[86] Klippel A, Kavanaugh WM, Pot D, Williams LT. A specific product of
phosphatidylinositol 3-kinase directly activates the protein kinase Akt
through its pleckstrin homology domain. Mol Cell Biol 1997;17:338–44.
[87] Surviladze Z, Sterk RT, DeHaro SA, Ozbun MA. Cellular entry of human
papillomavirus type 16 involves activation of the phosphatidylinositol 3-
kinase/Akt/mTOR pathway and inhibition of autophagy. J Virol 2013;87:
2508–17.
[88] Marsh M, Helenius A. Virus entry: open sesame. Cell 2006;124:729–40.
[89] Menges CW, Baglia LA, Lapoint R, McCance DJ. Human papillomavirus type 16
E7 up-regulates AKT activity through the retinoblastoma protein. Cancer Res
2006;66:5555–9.
[90] Pim D, Massimi P, Dilworth SM, Banks L. Activation of the protein kinase B
pathway by the HPV-16 E7 oncoprotein occurs through a mechanism
involving interaction with PP2A. Oncogene 2005;24:7830–8.
[91] Spangle JM, Munger K. The human papillomavirus type 16 E6 oncoprotein
activates mTORC1 signaling and increases protein synthesis. J Virol 2010;84:
9398–407.
[92] Arteaga CL. EGF receptor mutations in lung cancer: from humans to mice and
maybe back to humans. Cancer Cell 2006;9:421–3.
[93] Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr Opin Oncol
2006;18:77–82.
[94] Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, et al. PTEN, a putative
protein tyrosine phosphatase gene mutated in human brain, breast, and
prostate cancer. Science 1997;275:1943–7.
[95] Albanell J, Codony-Servat J, Rojo F, Del Campo JM, Sauleda S, Anido J, et al.
Activated extracellular signal-regulated kinases: association with epidermal
growth factor receptor/transforming growth factor alpha expression in head
and neck squamous carcinoma and inhibition by anti-epidermal growth factor
receptor treatments. Cancer Res 2001;61:6500–10.
[96] Nathan CO, Amirghahari N, Abreo F, Rong X, Caldito G, Jones ML, et al.
Overexpressed eIF4E is functionally active in surgical margins of head and
neck cancer patients via activation of the Akt/mammalian target of
rapamycin pathway. Clin Cancer Res: Official J Am Assoc Cancer Res 2004;
10:5820–7.
[97] Kozaki K, Imoto I, Pimkhaokham A, Hasegawa S, Tsuda H, Omura K, et al.
PIK3CA mutation is an oncogenic aberration at advanced stages of oral
squamous cell carcinoma. Cancer Sci 2006;97:1351–8.
[98] Cohen Y, Goldenberg-Cohen N, Shalmon B, Shani T, Oren S, Amariglio N, et al.
Mutational analysis of PTEN/PIK3CA/AKT pathway in oral squamous cell
carcinoma. Oral Oncol 2011;47:946–50.
[99] Lui VW, Hedberg ML, Li H, Vangara BS, Pendleton K, Zeng Y, et al. Frequent
mutation of the PI3K pathway in head and neck cancer defines predictive
biomarkers. Cancer Discovery 2013.