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

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

1368-8375/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
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

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
Figure 1. Schematic representation of HPV Genome. The HPV genome is a circular a loss of negative feedback control of oncogene expression by the
molecule of approximately 7900 base pairs with 8 overlapping reading frames.
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
encode two structural capsid proteins (L1 and L2) that constitute advantage for the affected cells [22].
the virus particle. The genomic organization is presented in Fig. 1.
The papillomavirus E1 and E2 proteins are required for viral
Papillomaviruses life cycle
DNA replication and papilloma formation. E1 is an ATP-dependent
helicase that initiates viral replication in cooperation with the E2
HPVs are species specific and also display a tropism for squa-
protein. The E2 protein also plays an important role in segregation
mous epithelia. A large group of HPVs infects cutaneous epithelia,
of viral DNA plasmids as cells divide [9,10]. By simultaneously
whereas another sizable group infects mucosal epithelia. Through
binding to viral genomes and cellular chromatin-associated pro-
wounds or abrasions, HPVs infect basal epithelial cells, which have
teins, during mitosis, the E2 protein ensures proper segregation
the highest proliferation capacity in the epithelial layer and utilize
of the viral DNA into daughter cells upon cell division [11,12].
the host-cell DNA replication machinery to support viral DNA rep-
The role of the E4 gene is still unknown but its expression is
lication [23–25]. The viral DNA is maintained in the nucleus of in-
essential for the productive phase of the papillomavirus life cycle.
fected basal epithelial cells as a low-copy-number and no progeny
The E4 protein is encoded by a gene in the early region that entirely
virus is produced in these cells. Despite the fact that squamous epi-
overlaps with the E2 gene in an alternative translational reading
thelial cells normally undergo differentiation as they move from
frame. Nevertheless, the E4 protein is abundantly expressed during
the basement membrane towards the surface epithelium and are
the late stages of the HPV life cycle and may facilitate release of
not able to support DNA synthesis, the virus maintains the supra-
mature virus from cells [13–15].
basal cells in a state competent for DNA replication and HPV-DNA
The E5 protein is a very short, transmembrane protein encoded
replicates to a high copy number in terminally differentiated cells
at the 3’ end of the early region of many papillomavirus types. The
near the epithelial surface. Similarly, the late viral genes L1 and L2
E5 protein from bovine papillomavirus type 1 causes tumorigenic
are expressed only in the highly differentiated cells, where infec-
transformation of cultured fibroblasts by inducing ligand-indepen-
tious progeny virus is produced and released. The crypt epithelium
dent activation of the platelet-derived growth factor receptor, epi-
of human palatine tonsil is a specialized squamous epithelium that
dermal growth factor receptor and colony stimulating factor 1
contains patches of stratified squamous nonkeratinizing epithe-
[16,17]. Microarray experiments after HPV16 E5 overexpression
lium and patches of reticulated sponge-like epithelium. It is also
in human epithelial HaCaT cells revealed that E5 plays a major
characterized by the presence of intraepithelial passages that are
antiapoptotic role, affects cell-matrix adhesion and interferes with
filled by non-epithelial cells.
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- HPV E7 protein and cell cycle progression
gration to host genome.
E6 and E7 proteins promote cell-cycle progression and viral The major role of high risk HPV E7 protein is to reprogram ter-
DNA replication in differentiated keratinocytes. The E6 protein of minally differentiating keratinocytes, in order to re-enter the cell
the high-risk HPV binds and induces the degradation of the p53 tu- cycle and express proteins that are required for viral genome rep-
mor suppressor protein via a ubiquitin-mediated process, while lication. Expression of E7 protein in growth arrested cells, indicates
the HPV-E7 protein binds cullin 2 ubiquitin ligase complex and that E7 induces cellular DNA synthesis at the G1 to S transition
ubiquitinates the retinoblastoma (Rb) tumor suppressor protein [26]. The E7 protein binds and destabilizes the cellular retinoblas-
and related proteins. The late genes L1 and L2 encode viral capsid toma (Rb) tumor suppressor protein and members of the Rb family,
proteins utilized in the construction of new viruses. L1 capsid pro- thereby, displacing E2F transcription factor, which then stimulate
teins are firstly synthesized in the cytoplasm before being trans- expression of E2F-responsive genes, required for cellular (and vir-
ported to the nucleus to package viral chromatin. L2 capsid al) DNA synthesis and cell cycle progression such as cyclin A and E,
protein binds specific sites of viral DNA in the nucleus and recruits DNA polymerase a, PCNA, hTERT and p21CIP1 which serve to drive
L1 for new viral particles to be assembled. The roles of HPV pro- cells into S phase and induce cellular DNA synthesis [27–31]. It has
teins are summarized in Table 1. also been shown that HPV16 E7 hijacks the cullin 2 E3 ubiquitin li-
More than 150 human HPV genotypes have been isolated and gase complex to target all three Rb family members for degrada-
there are certainly additional types which have not been identified. tion [27,32].

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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 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx 3

Table 1 of the whole viral genome results in a virus with reduced replica-
Function of HPV proteins. tive capacity and inability to maintain the viral genome in the epi-
HPV Function somal form [45]. High risk E6 proteins bind to a number of PDZ
protein domain containing proteins with presumed tumor suppressor
Early proteins activity that have diverse functions, ranging from control of cell
E1 Initiation of viral genome replication polarity, control of cell–cell attachment and the regulation of var-
E2 Viral DNA replication and transcription. Segregation of viral ious signaling pathways [46,47]. Some of these PDZ domain-con-
genomes
E4 Viral genome packing. Maturation of viral particles
taining proteins have been confirmed as degradation targets of
E5 Oncoprotein. Participates in host cell transformation and blocks E6 protein [48,49]. A recent structural study proposed a model
apoptosis in late events of HPV carcinogenesis whereby E6 dimerization is essential for p53 ubiquitination and
E6 Major oncoprotein. Inactivates p53 protein. Block apoptosis. degradation [50]. E6 dimerization is also required for degradation
Interacts with many host proteins with PDZ domains
of PDZ domain-containing protein substrates. E6 binding to PDZ
E7 Major oncoprotein. Inactivates pRb protein. Promotes host DNA
synthesis and proliferation. Interacts with many host proteins domain-containing proteins may also inhibit their capacity to
interact with other cellular partners, or alternatively, may alter
Late proteins
L1 Major capsid protein their correct cellular localization. In addition, the E6 protein
L2 Minor capsid protein stimulates the expression of telomerase, an RNA dependent DNA
polymerase that maintains the ends of chromosomes in proliferat-
ing somatic cells [51,52].
The E7 protein also inhibits the action of cyclin-dependent ki-
nase inhibitors and interacts with cyclin–cdk complexes such as HPV positive head and neck cancer has a different molecular profile
cyclin A and cdk2 and other cellular proteins to stimulate cell cycle compared to HPV negative head and neck cancer
progression [33–36]. Bypass of growth arrest by high risk HPV E7
requires both degradation of Rb and abrogation of p21CIP1 inhibi- Molecular evidence that HPV status determines a separate class
tion [37]. It is interesting to note that low risk HPV E7 cannot target of HNSCC was initially provided by microsatellite analysis studies,
Rb for degradation and also is less efficient in abrogating p21CIP1 showing that HNSCC with no evidence of HPV infection is charac-
activity [33,34]. terized by a significant higher incidence of loss of 3p, 9p and/or 17p
High risk HPV E7 induces chromatin remodeling and transcrip- chromosomal arms compared to HPV positive HNSCC [53]. LOH at
tional activation of specific genes. HPV type 16 E7 interacts with 17p13 is thought to involve TP53, whereas LOH at 9p21 is thought
both histone acetyltransferases (HATs) and histone deacetylases to involve INK4a, the tumor suppressor gene encoding p16. In HPV
(HDACs) which results in an increase in the level of histone acety- negative HNSCC, inactivation of INK4a, either by chromosomal loss
lation on E2F regulated promoters [38,39]. In addition, high risk or by hypermethylation is considered to be the most common way
HPV E7 induces the transcription of histone methyltransferases to disrupt the pRb pathway [54]. On the contrary, HPV positive
(EZH2) and histone demethylases (KDM6A, KDM6B), which play HNSCC with E6/E7 viral oncoprotein expression is associated with
a crucial role in the epigenetic regulation of gene transcription low rates of p53 and p16INK4a alterations [53]. In support to these
[40]. 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
HPV E6 and cellular transformation in HPV negative HNSCC is based on acquisition of a large number
of mutations in many different signaling pathways. On the con-
Tumor viruses, in order to exert their oncogenic potential, inhi- trary carcinogenesis in HPV positive tumors is modulated by the
bit apoptosis, a host response to infection and unscheduled S phase activities of E6/E7 viral oncoproteins.
entry. As noted in a previous section, in HPV infection, E7 main-
tains an S phase environment within terminally differentiated Gene expression profile of HPV positive HNSCC: The p16 molecular
keratinocytes, which activate various apoptotic programs. A major phenotype
role for E6 protein is to counteract these cellular responses, inhib-
iting the pro-apoptotic functions of p53 protein. The best-studied A number of studies of the gene expression differences between
function of high risk E6 protein is the binding and targeting of HPV+ and HPV HNSCCs showed considerable differences in the
p53 for ubiquitin-mediated degradation [41]. E6 binds directly to gene expression profiles that were specifically associated with
E6-AP (E6 associated protein) which functions as a specific ubiqui- these two subgroups of head and neck cancer, supporting the no-
tin-ligase for p53 degradation. Low risk HPV E6 protein can also tion that HPV positive HNSCC has a specific transcription profile
bind to E6-AP and complex to p53 but the binding does not lead depending partly on E6 and E7 expression. Weinberger et al. [55]
to p53 degradation. Two different regions of p53 can be bound showed that p16 protein status determined by immunohistochem-
by E6, a region in the carboxy terminus that is bound by both istry is reasonable surrogate marker for a biologically and clinically
low and high risk HPV E6’s and a core region that correlates with meaningful HPV infection in OSCC. The authors sought to deter-
p53 degradation [42]. Abrogation of the p53 pathway by E6 allows mine the incidence and clinical implications of biologically rele-
bypass of p53-mediated checkpoints. In ORSCC, we recently vant HPV16 infection in a cohort of 107 oropharyngeal squamous
showed that, repression of HPV type 16 E6/E7 expression using a cell cancers treated with primary radiotherapy or surgery followed
retrovirus mediated shRNA expression system in HPV type 16 oro- by postoperative radiotherapy at Yale University. HPV-16 DNA vir-
pharyngeal cancer cells is associated with a rapid restoration of al load was determined by Real-Time PCR. In addition, they con-
p53 and pRb tumor suppressor pathways and massive apoptosis structed a tissue array composed of these tumors and studied
[4]. expression of p53, pRb and p16 proteins using a quantitative
A unique characteristic of the high risk E6 oncoproteins is the in situ method of protein analysis (AQUA). They hypothesized that
presence of a PDZ binding motif on the extreme C-terminus, a mo- among HPV16 DNA positive cases, p16 expression status would
tif that is absent from all the low risk E6 oncoproteins and thereby determine the biologically relevant ones. Their results delineated
represents a molecular signature for oncogenic potential in the 3 tumor classes with distinct molecular and clinical features based
various HPV types [43,44]. Deletion of this motif in the context on HPV16 DNA presence and p16 expression status: one class of

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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HPV16 negative/p16 non-expressing, one class of HPV16 positive/ expression and to alter cellular gene expression through mimicry
p16 non-expressing and one class HPV16 positive/p16 expressing or manipulation of downstream pathway responses [64].
oropharyngeal tumors. Overall survival in class III was 79% com- Papillomaviruses are associated with benign proliferative epi-
pared to the other two classes (20% and 18%, p = 0.0095). Dis- thelial lesions in their respective hosts. Since HPV gene expression
ease-free survival for the same class was 75% versus 15% and and replication is intimately linked to the differentiation state of
13% (p = 0.0025). The 5-year local recurrence was 14% in Class III the infected keratinocytes, Notch and Wnt pathways are crucial
versus 45% and 74% (p = 0.03). Only patients in class III had signif- for the papillomavirus life cycle.
icantly lower p53 and pRb expression (p = 0.017 and 0.001, respec-
tively). Multivariable survival analysis confirmed the prognostic HPV and Wnt signaling pathway
value of the 3-class model. The authors demonstrated that only Wnt ligands control several differentiation processes during
the HPV16 positive/p16 expressing tumors fit the cervical carcino- normal tissue homeostasis [65], and they are implicated in patho-
genesis model and they are the ones associated with favorable logic conditions such as in certain types of cancer [66]. In the ab-
prognosis. sence of Wnt ligands, b-catenin forms a ‘‘degradation complex’’
A comprehensive analysis by Slebos and colleagues, revealed with kinases and scaffold proteins, such as glycogen synthase ki-
that CDKN2A (encodes the tumor suppressor p16INK4a protein) nase-3b (GSK3b), casein kinase 1 and 2 (CK1 and CK2), adenoma-
was one of the most significant differentially expressed genes, tous polyposis coli (APC), and Axin2. This degradation complex
since its expression was highly upregulated in HPV+ group [56]. phosphorylates b-catenin at serine and threonine residues
In the same study, other genes identified to have higher expression inducing its ubiquitination and degradation. The activation of
in HPV+ group were the cell cycle regulators p18 and CDC7, the canonical Wnt signaling induces the phosphorylation of the intra-
transcription factors TAF7L, RFC4, RPA2, TFDP2 and the cell adhe- cellular Dishevelled (Dvl) protein, which eventually interacts with
sion molecule TCAM1. Another microarray study between HPV+ Axin2 and impedes the formation of the b-catenin degradation
and HPV HNSCCs by Schlecht et al. [57] revealed a similar expres- complex; this process leads to the accumulation and nuclear trans-
sion pattern for p18 and TFDP2 expression, that was also found to location of b-catenin. Once it is translocated into the nucleus, b-
be significantly upregulated in HPV+ group. However this study catenin binds members of the T-cell factor/lymphoid enhancer fac-
failed to validate upregulation of p16INK4a in HPV+ group as its tor (TCF/LEF) family of transcription factors, of which TCF4 is the
expression levels were low and did not meet the preset twofold best characterized. b-Catenin/TCF4 complexes control the expres-
expression threshold. A third microarray analysis by Martinez sion of several target genes that regulate cell polarity, proliferation,
et al. [58] succeeded to validate that expression of p16INK4a is and differentiation including c-jun, c-myc, cyclin D1 [67,68].
upregulated in HPV+ subgroup and furthermore, showed that the The participation of canonical Wnt pathway in cervical cancer is
expression of ZNF238, DNMT1, MCM3, AKR1C3, GRB10, TYK2 and evident because the nuclear accumulation of b-catenin correlates
SART3 were significantly upregulated in HPV+ HNSCC compared with tumor progression in human patients. Nuclear b-catenin is
to HPV HNSCC. Unfortunately, the reported gene expression pro- commonly found in human HPV16-positive invasive carcinoma
files show little overlap between studies, which complicates the biopsies, but it is uncommon in early dysplastic lesions [69,70].
data interpretation and the identification of important genes for Microarray expression studies conducted on cervical cancer tissues
tumorigenesis in head and neck epithelia [59]. A recent global identified alterations in the expression of regulators of the Wnt sig-
comparative proteomic analysis of HPV+ and HPV HNSCCs using naling pathway [71]. Recent experimental evidence suggests that
liquid chromatography-mass spectrometry. revealed that a cluster Wnt signaling pathway is activated in HPV16-positive HNSCC
of minichromosome maintenance family members (MCM2,6 and and that the Siah-1–dependent ubiquitin/proteasome pathway
7) are significantly upregulated in HPV+ HNSCC and forms the most plays a significant/pivotal role for b-catenin degradation in
clearly differential set of proteins between these two subgroups HPV16-positive oropharyngeal cancer cell lines [72]. More specifi-
[60]. Minichromosome maintenance proteins function as helicases cally, according to these studies, nuclear b-catenin accumulation
for DNA unwinding in early stages of DNA replication during the seems to be a direct consequence of E6 and E7 oncoprotein expres-
cell cycle G1 phase and seems to represent a potential target for sion since viral E6/E7 expression is associated with downregula-
anticancer drug development. 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
Keratinocyte differentiation and manipulation of cellular pathways by phosphorylation. As a consequence, b-catenin is stabilized, translo-
HPV in head and neck epithelia cates into the nucleus and promotes the transcriptional activation
of b-catenin–responsive genes.
Wnt and Notch cell signaling pathways are essential in the reg- Smeets and colleagues showed that immortalization of normal
ulation of proliferation and differentiation responses during nor- oral keratinocytes (OKCs) by HPV E6 expression caused an activa-
mal development. Deregulation of Wnt and Notch signaling is tion of Wnt pathway which is in consistent with the finding that
associated with human cancers. Oncogenic activation of the Wnt/ nuclear b-catenin protein levels are upregulated in HNSCCs and
b-catenin was initially associated with colon cancer where truncat- HPV16-positive oropharyngeal cancer cell lines [73]. In this study,
ing mutations of APC and mutations of GSK3b phosphorylation the authors demonstrated that Wnt activation is associated with
sites of b-catenin are prevalent. Notch is an established tumor sup- p53 degradation induced by E6 expression, since the expression
pressor in the skin and in chronic myelomonocytic leukemia of a dominant negative mutant p53 (R175H) was able to cause a
[61,62]. Recent experimental studies also suggest that loss of func- similar activation of Wnt signaling pathway.
tion mutations in Notch receptors are associated with head and In accordance to these studies, comparative quantitative analysis of
neck, lung and cutaneous squamous cell carcinoma [6,7,63]. It is protein expression for 13 different biomarkers in p16/HPV+, p16+/
also well established that Wnt and Notch pathways are attractive HPV+, p16+/HPV, p16/HPV subgroups of HNSCC using AQUA fluo-
targets for virus interaction and manipulation. Viruses that their rescent immunohistochemistry, validated b-catenin as biomarker that
life cycle is associated with the differentiation state of the host cell is significantly upregulated in p16+/HPV+ HNSCC tumors [74].
and the development of human cancers, such as Epstein-Barr virus The crucial role of viral E6 expression in activation of Wnt sig-
(EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), use naling pathway in HPV type 16 positive epithelia has also been
components of these pathways to regulate their own viral gene confirmed by in vivo studies. An in vivo model study with a trans-

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genic mice expressing the full-length E6 oncoprotein in the basal Recent advances in our understanding of the molecular role of
layer of stratified epithelia under the control of human keratin Notch in HNSCC were provided by whole-exome sequencing stud-
14 promoter (K14E6 mice). This transgenic E6 strain was shown ies [6,7]. The most novel finding to emerge from these sequencing
to display skin hyperplasia and skin cancer at advanced ages and studies of HNSCC is the discovery of loss of function mutations
the expression of the E6 viral oncoprotein was associated with nu- within the NOTCH1 gene in 12–15% of cases, and within additional
clear accumulation of b-catenin and the transcriptional activation NOTCH family members in 3%–5%. Analysis of these exome
of b-catenin–responsive genes in skin epidermis [75]. Conversely, sequencing data also indicated that Notch ligands (JAG1, JAG2,
when the transgenic strain was expressing a truncated E6 oncopro- DLL1-4), were rarely mutated. The vast majority of Notch 1 muta-
tein that lacks the PDZ binding domain (K14E6deltaPDZ mice), nu- tions in HNSCC, were missense mutations that occurred at or near
clear b-catenin was not detected and b-catenin–responsive genes identified important domains such as the ligand-binding domain
were not transcriptionally activated. (EGF repeats 11, 12 and 13) or the ankyrin domains. In addition,
The synergy between Wnt signaling pathway and human papil- the detected nonsense mutations were predicted to result in trun-
lomavirus was also shown by another in vivo model study using cated Notch1 proteins lacking domains important for transcrip-
the double transgenic animal K14-E7/DN87bcat that expresses tional activation. These data suggest that Notch1 is acting as a
the E7 viral oncogene of HPV type 16 along with a constitutively tumor suppressor in head and neck epithelium.
active amino-terminal truncated b-catenin molecule under the The tumor suppressor role of Notch signaling in SCCs is still
control of keratin 14 promoter [76]. The K14-DN87bcat transgenic being characterized. However, Notch signaling does promote ter-
strain develops benign skin tumors but does not exhibit histopath- minal differentiation of keratinocytes. Papillomaviruses have
ologic charasteristics of cervical cancer while the K14-E7 strain evolved to couple the expression of their capsid proteins to termi-
displays cervical pathologies after 6 months of estrogen treatment. nal differentiation of keratinocytes. Since in the stratified epithelia
The authors analysed the incidence of invasive cervical cancer in Notch signaling has a central role in promoting terminal differen-
K14-E7, K14-DN87bcat and double transgenic K14-E7/DN87bcat tiation [78], one would expect E6 to modulate Notch signaling dur-
mice and they observed that invasive cervical cancer developed ing the viral cycle life in the upper cell layers of the papilloma.
in 50%, 11% and 94% of the transgenics at the age of 7 months Recently, several groups have reported that E6 protein from
old respectively, suggesting that the activation of Wnt pathway HPV5, HPV8 and other cutaneous b-HPVs repress Notch transcrip-
accelerates HPV16-E7 mediated cervical carcinogenesis. tional activation, and this repression is dependent on a direct inter-
action with the MAML1 [79–81]. MAML1 is a core component of
HPV and manipulation of Notch signaling pathway the transcriptional activation complex that mediates the effects
The Notch receptor and Notch ligands are single-pass trans- of the canonical Notch signaling pathway. Binding of cutaneous
membrane proteins that influence cell fate decisions, proliferation, b- HPV E6 to MAML1 was found to repress transactivation by
and differentiation. In mammals, there are four Notch receptors, MAML1. The N-terminal basic domain of MAML1 interacts with
Notch1, 2, 3, and 4, the ligands include Jagged-1and -2 and Del- the ankyrin repeats of the intracellular domain of Notch receptor
ta-like-1, -3, and -4. Ligand binding is followed by two cleavages and the DNA-binding factor CSL [77]. Figure 2 presents an over-
of the Notch receptor by ADAM metalloprotease and gamma- view of Wnt and Notch signaling manipulation by HPV. Mapping
secretase complex, leading to the release of the Notch intracellular the interaction between the paplillomavirus E6 protein and
domain (NICD). NICD enters the nucleus and targets the DNA bind- MAML1 revealed an acidic domain with an LXXLL motif at the C-
ing protein CSL (CBF1/Suppressor of Hairless/Lag-1). In the absence terminal fragment of MAML1 that seems to be critical for the
of signaling, DNA-bound CSL is associated with a corepressor com- MAML1-HPV E6 protein complex formation. This acidic domain is
plex. Upon Notch signaling, the corepressor complex is displaced part of transcription activation domain 2 (TAD2) which is required
by NotchICD and the coactivator Mastermind, up-regulating target for transcription in vivo and has been proposed to interact with
genes containing CSL binding sites [77].

Figure 2. Manipulation of Wnt and Notch signaling pathway by HPV E6 and E7 proteins.

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
6 T. Rampias et al. / Oral Oncology xxx (2013) xxx–xxx

additional unknown cellular proteins necessary for Notch tran- the PI3K pathway (PIK3CA, PIK3AP1, PIK3C2A, PIK3C2B, PIK3C2G,
scriptional activation [82]. PIK3CB, PIK3CD, PIK3CG, PIK3IP1, PIK3R1/2/3/4/5/6, AKT1/2/3,
It is worth noting that in these studies, the expression levels of mTOR, PTEN, PDK1, TSC1/2, RICTOR and RPTOR) was the most fre-
endogenous Notch target genes were found repressed by b-HPV E6 quently mutated oncogenic pathway (30.5%). Although the number
proteins. These findings elucidate a mechanism of viral antagonism of HPV-positive HNSCC tumors in their cohort was relatively small
of Notch signaling, and suggest that Notch signaling is an impor- (15 cases), authors were able to identify a subset of HPV positive
tant epithelial cell pathway target for the human papillomavirus. HNSCC tumors (3/15, 20%) where PIK3CA or PIK3R1 was the only
However our knowledge about how mucosal a-HPVs manipulate mutated cancer gene, suggesting that the tumor progression in this
the Notch signaling is still poor and future studies on the field need subset of HPV positive tumors is driven by PI3K pathway muta-
to focus on the mechanism of Notch inhibition by high risk a-HPVs tions alone without any further genomic alterations in critical
encoded E6 proteins and the biological implications of inhibition. oncogenes or tumor suppressor genes.

HPV and activation of the Phosphatidylinositol 3-Kinase/Akt/mTOR Conclusions

Pathway in HNSCC
HPV-associated HNSCC represents a distinct entity from tobac-
Activation of PI3K pathway is able to drive oncogenesis in mul- co- and alcohol- related HNSCC with different molecular profile.
tiple human cancers. When bound to ligand, EGFR activates the High risk HPV E6/E7 expression, following viral integration, initiate
PI3Ks, leading to the phosphorylation of phosphatidylinositol on a number of malignant transformation processes including bypass
the 30 -hydroxyl group [83,84]. The resultant product, phosphati- of cell cycle control, DNA synthesis, inhibition of apoptosis and
dylinositol-3,4,5- trisphosphate, activates AKT kinase [85,86] transcriptional activation of genes that promote proliferation. This
which phosphorylates in turn several downstream proteins, regu- model of tumorigenesis is different from the one that is established
lating cell growth and survival. for the tobacco-induced HNSCC which requires the acquisition of a
Human papillomavirus (HPV) entry into target cells is initiated large number of mutations or deletions in tumor suppressor genes.
by binding to cell surface heparan-sulfonated proteoglycans However, further investigation into the molecular profiles between
(HSPGs). The virus also interacts with secondary receptors, which HPV-positive and HPV-negative HNSCC is needed, in order to
are responsible for particle internalization. Upon these early events understand whether specific gene mutations can enhance the
of HPV infection, growth factor receptors are often activated trig- transforming activities of E6/E7 viral oncoproteins and accelerate
gering the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signal- the HPV related tumorigenesis. Human papillomavirus has been
ing cascade [87,88]. In a recent study, Surviladze and colleagues evolved to coexist within epithelial cells and its life cycle is tightly
showed that Akt/mTOR signaling pathway is rapidly activated after linked to proliferation and differentiation of the basal epithelial
exposure of human keratinocytes to HPV type 16 pseudovirions cells. E6 and E7 viral oncoproteins affect and modulate cellular sig-
(PsVs) which deliver a reporter plasmid (pseudogenome) to the naling pathways that regulate the proliferation capacity and the
cell, suggesting that HPV infection stimulates the PI3K/Akt/mTOR differentiation state of keratinocytes. Notch, Wnt and PI3K/Akt/
pathway potentially via alpha-6 beta-4 integrins [87]. Previous mTOR are cell signaling pathways that are targeted by high risk
studies have also shown that high risk HPV E6/E7 protein expres- HPV in head and neck epithelial cells. The viruses use components
sion can activate Akt and mTOR complexes and promote virus sur- of these pathways to regulate their own viral gene expression and
vival [89–91]. additionally alter cell gene expression by manipulation of down-
Specific components of the EGFR/PI3K pathway have been stream pathway responses. Recent experimental studies on HPV-
found mutated in human cancers, resulting in pathway activation. associated HNSCC, provide evidence that HPV activates PI3K/Akt/
Mutations in EGFR, PIK3CA, and PTEN occur commonly in a variety mTOR pathway and promotes b-catenin stabilization and inactiva-
of cancers, such as lung, breast, prostate cancer, and glioblastoma tion of Notch signaling. However further investigation of the inter-
[92–95]. Moreover, these mutations cause the constitutive activa- connection between these pathways and the E6/E7 activities is
tion of downstream signaling molecules such as Akt/protein kinase needed. The understanding of the molecular profile of HPV associ-
B (PKB), mammalian target of rapamycin (mTOR) and ribosomal ated head and neck cancer will provide opportunities to create new
protein S6 kinase (S6K) Activation of AKT/mTOR pathway which molecular markers for HPV diagnostics and new therapeutic
play a key role in tumorigenesis and survival rate of HNSCC pa- approaches.
tients [96].
Previous mutational studies reported that 7–12% of HNSCCs
harbor PI3KCA mutations [97,98], however, in these studies, there Conflict of interest statement
was a lack of mutational data for the downstream effectors of PI3K
pathway. A whole exome sequencing study by Stransky and col- None declared.
leagues in a cohort of 74 tumor-normal pairs revealed that 8% of
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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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