Biomedicine & Pharmacotherapy 70 (2015) 103–110

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Review

Overview of high-risk HPV’s 16 and 18 infected cervical cancer:

Pathogenesis to prevention
Sabitha Ramakrishnan, Steena Partricia, Ganeshan Mathan *
Bharathidasan University, Tiruchirappalli 620024, Tamilnadu, India

A R T I C L E I N F O A B S T R A C T

Article history: As general, the Human papillomavirus (HPV) causes the most sexually transmitted diseases. Among well
Received 4 December 2014 categorized 80 types, the high-risk types HPV’s 16 and 18 are highly involved in 70% of cervical cancer.
Accepted 30 December 2014 The virulence of HPV is mainly exhibited by E5, E6 and E7 encoded oncoproteins that cause low to high-
grade cervical lesions (CIN-1, 2, 3), leading to form 99.7% of squamous cell and 89% of adenocarcinomas
Keywords: cervical cancer worldwide. This study mainly encircles the major role of E5, E6 and E7 oncoproteins in
HPV 16 HPV 16 and 18. Further discussed the uprising of significant biomarkers and their cellular process in
HPV 18
different stages of cervical cancer with current prevention and treatment regimens. Hence, this
Cervical cancer
Oncoproteins integration will evoke novel markers, potential vaccination and various treatments approaches with
Biomarkers special reference for HPV16 and 18 infected cervical cancers.
Treatments 2015 Elsevier Masson SAS. All rights reserved.
Vaccinations

different recombinant vaccines and adoption of several combina-

1. General information
Globally, HPV causing cervical cancer ranks as the second most
frequent cancer among 15 to 44 years of women that holds deaths
of 265,653 [1]. Among 30 HPV types, known to infect cervix,
vagina, vulva, penis, and anus [2], there are 84.1% of invasive
cervical cancers are attributed mainly by high-risk subtype HPVs
16 or 18 [3,4]. In this, E6 and E7 are notable and the most
responsible oncoproteins for their molecular pathogenesis
[5]. Along with common and specific marker p16INK4A, there
were many significant markers used to identify the different stages
of infection with various cellular processes [6]. It not only detects
disease reliably, but also helps in the choice of multiple therapeutic
alternatives. In addition, the current therapeutics includes
Abbreviations: HPV, Human papilloma virus; VLP, Virus-like particle; CIN, Cervical
intraepithelial neoplasia; EGFR, Epidermal growth factor; COX-2, Cyclooxygenase-
2; CDK, Cyclin-dependent kinase; MDM, Mouse double minute; hTERT, Human
telomerase reverse transcriptase; NSAID, Non-steroidal anti-inflammatory drug;
PI3/Akt, Protein kinase; CTL, Cytotoxic T-lymphocyte; MHC, Major histocompat-
ibility complex; APC, Antigen-presenting cell; HLA, Human leukocyte antigen; DC,
Dentritic cells; Ki-67, Monoclonal antibody; MIB-1, Molecular immunology borstel;
MCM, Minichromosome maintenance.
* Corresponding author. Bharathidasan University, Tiruchirappalli 620024,
Tamilnadu, India. Tel.: +91 994 082 3271; fax: +91 431 240 7045.
E-mail addresses: mathan@bdu.ac.in, mathan_cell@yahoo.com (G. Mathan).
tional drugs for treatment. Hence, the purpose of this review is to
discuss the overview of pathogenicity, usage of markers and varied
preventive and treatment regimens, which specifically target the
E5, E6, and E7 oncoproteins in HPV 16 and 18, infected cervical
cancer.
2. HPV entry and stages
The small non-enveloped icosahedral viruses of HPV contain
double-stranded DNA genome ( 7000–8000 bp), chiefly divided
into three regions. The first two locus control region (LCR) and
early (E) regions encoded proteins for viral gene expression,
replication and survival, whereas a late (L) region is for viral
structural proteins [7]. The early genes (E1 to E4) play a role from
origin of replication to transferring viral genome into the host cells
[8,9]. Excluding other early genes, E5 is the first oncoprotein
encoded by HPV that highly interacts with various transmembrane
proteins [10]. In high-risk HPV, the combination of second
oncoprotein E6 and third oncoprotein E7 aided viral DNA
replication to repress the apoptosis and E7 oncoprotein alone
targeted the cell cycle regulatory pathways to maintain S-phase
state [7]. Finally, the terminal late regions (L1 and L2) encoded viral
capsid proteins during later stages of virion [11].

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The entry of HPV particles immediately replicated in squamous
epithelium cells (skin-like cell) of ectocervix through creating a
micro abrasion/wounding at the basement membrane [12]. The L1
capsid protein on the HPV virion surface get interacted with a6b4
integrin’s, caused up regulation of basal epithelial cells. This
allowed incorporation of virus, establishes low episome replication
but the expression of succeeded early gene (E) engendered high
copy numbers that driven terminal differentiation of keratino-
cytes. At last, the expression of late protein (L) constituted virus
assembly, and the viruses were shed from outermost layer of
epithelial cells [13]. Once it got replicates, it began to form CIN,
which has the potential precursor for premalignant dysplastic
changes and it would be graded as mild dysplasia CIN-1, moderate
dysplasia CIN2 and severe dysplasia CIN3 [14]. It further classified
to I–IV stages from preliminary to later stages based on the area
spread out and size of the cancer [15].
3. Molecular mechanism of HPV
The infective mechanisms explained in cervical cancer are more
closely with high-risk HPV 16, 18 types and several oncoproteins
have been expressed during early stage of infection. In specific, E5,
E6 and E7 oncoproteins were major contribution in cervical cancer
development and progression by suppressing or enhancing the
function of various cellular proteins [16,17] (Fig. 1).
3.1. Role of E6 and E7 association
After integration of HPV DNA into the host genome, the
overexpression of two viral genes, E6 and E7 are most virulence for
causing cervical cancer. The E6 prevented the activity of tumor
suppressor p53 whereas the E7 inhibited pRb, which control cell
division by blocking the activity of transcription factors [18–
21]. Since the increased synthesis of E6 and E7, the induction of
disrupted E2 gene regulated both E6 and E7 transcription by
Fig. 1. The major role of E5, E6, and E7 oncoproteins in molecular mechanism of HPV 16 and 18 infected cervical cancer.
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binding and blocked them to acting in the cells [18]. When E6 and
E7 are not bound to E2, the E6 is free to bind with p53 and E7 with
pRb to destroy the tumor suppressing functions. Due to the
absence of p53, the DNA can be kept on accumulating without
repair and removal of pRb that allows cells to undergo cell division.
As a result, it generated the cells with malignant phenotype, which
are several years to develop [13]. Moreover, the genomic instability
has also been raised by E6 and E7 in multiple epithelial cells, yield
various chromosomal abnormalities [7]. These structural changes
could be detected more commonly in chromosomes 1, 3 and 5 with
losses of 3p and 10p for telomerase activation [22]. Thus, the
occurrence of cell cycle progression was not only by external
growth factors, but also by E7 protein, expressed cellular proteins
required for S-phase entry [5].
3.2. Role of E5
Usually, the COX-2 expression associated with cervical cancer
increased lymph node metastasis and radiation therapy resistance
[23–26]. The E5 oncoprotein induced COX-2 expression, performed
numerous changes in cell signalling and gene transcription; alter
angiogenesis and apoptotic process [27,28]. As well as, it activated
EGFR rather inactivating tumor suppressor proteins [21,20].
3.3. Role of E6
Unremarkably, the Notch1 gene had been identified as a novel
target of p53 [29–33]. In cervical keratinocytes, the p53
transactivates Notch1, being as a tumor suppressor to induce the
differentiation process. However, in the presence of E6, the
endogenous Notch1 expression is markedly reduced or absent
when Notch 2 becomes elevated in cervical carcinoma
[30,34]. Another important function of E6 protein is to promote
p53 degradation through E6AP interaction, cellular protein to link
polymorphisms with codon 72 of p53 [35,36]. In addition, E6 also
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interferes with other pro-apoptotic proteins, such as Bak, FADD
and procaspase 8 to prevent apoptosis [37,38]. Mostly, the hTERT
expression in normal cells inhibited telomerase activity to
suppress the senescence process [39]. On the contrary, the E6
induced its activity and thereby formed immortalization of
epithelial cells to maintain telomere length [40,41].
4. Growing of biomarkers
Recent technological advancement apprehended to examine
existing and to explicate new biomarkers for cervical cancer
through comprehensive understanding. This admits broad range of
biochemical entities, such as nucleic acids, proteins, sugars, lipids,
small metabolites, cytogenetic and cytokinetic parameters as well
as whole tumor cells found in the body fluid. However, the growing
of biomarker involved in HPV causing cervical cancer plays a
unique function (Table 1).
4.1. Ki-6 and p16INK4a
This protein expressed during G1, S, G2 and M phase and not in
G0 phase, provided an index for cell growth fraction but it remains
skeptic [42,43]. The expressions of another marker p16INK4a are
tightly controlled in normal cells through inhibiting CDK 4 and
6 and phosphorylation of Rb protein to activate the E2F-driven cell
cycle into S-phase [44–47].
4.2. ProEx C
This protein-based antibody (BD Diagnostics) has to act against
nuclear proteins of MCM 2 and topoisomerase II alpha (TOP2A),
accumulated in HPV-transformed cells. In cervical glandular and
squamous dysplasia, its expression has dramatically elevated due
to increased transcription of S-phase genes (aberrant S-phase
induction) by the action of E7 oncoproteins [48–50]. Whereas, it is
limited in normal cervical epithelium and its notable expression
with six MCMs was seen throughout all phases of the cell cycle
[51–53].
4.3. Cytoactiv HPV-L1
Usually, the L1 capsid protein forms a protective cover for the
viral genetic material with L2 protein, and it acted as a ligand for
host cell surface receptor at the epithelial erosions or mucosal
ulcerations of basal cell layer. The expression of L1 used to be found
in productive phase of HPV infection, but it is progressively lost
during cervical carcinogenesis. Hence, this loss of HPV-L1
expression may indicate the integration of viral DNA into the
human genome. The subsequent work of cytoactive antibody has
to detect the L1 capsid protein, and it may disrupt the L1 gene by
segregating of a viral promoter. And also it may find abnormalities
in transcription factor pathways or in control of L1 protein
translation [54,55].
4.4. Laminin-5 and MIB-1
The previous data indicated that Lam-5 expressions can serve as
a marker for several invasive carcinoma tissues [56–58] The close
correlation of its expression and tumor progression has been found
in different kinds of malignant tumors [59,60]. The high prognostic
value of MIB antibody was particularly present in cervical smears
and there was a significant overexpression of MIB-1 has been

Table 1
Various biomarkers used to diagnose the HPV 16 and 18 infected cervical cancer.

S. No Biomarker Cellular process Uses Expression Staining pattern References

1 Ki-67 Increased in epithelial cell Measure cell proliferative Cervical neoplasia Nuclear [94,95]
proliferation of HPV-infected capacity
tissues Used to differentiate benign
versus malignant tumors at
various sites
Predominantly used in
histology applications
2 p16INK4a Increased in irregular cell cycle Serves as a biomarker for Cervical dysplasia Nuclear and [96–100]
inactivation by disrupting the persistent infection in high-risk cytoplasmic
interaction of pRb with HPV
transcription factor E2F in the It facilitates the identification
presence of HPV-E7 oncogene of abnormal cells in cytology
preparations
To interpret the histological
material
3 BD ProEx C Aid to detect the cellular levels It distinguishes true dysplasia Cervical high-grade Nuclear [50–52]
of MCM2 and TOP2A due to from mimics such as reactive/ dysplasia
aberrant transcription of S- reparative changes, immature
phase proteins by the squamous metaplasia, and
interaction of HPV E6 and E7 atrophy
oncoproteins with p53 and Rb
To detect proliferative capacity
4 Cytoactive It found in mild-to-moderate To detect frequently in L1- CIN3 Nuclear [101–104]
HPV-L1 dysplasias, but lost in higher negative intraepithelial lesions
grade intraepithelial neoplasias
5 Laminin-5 It binding to epithelial cells at It seems to be expressed in the Early invasion in the Cytoplasm [105,106,62]
the basement membrane earliest stages, particularly in squamous cell
through the formation of microinvasive lesions carcinoma of the
hemidesmosomes and the uterine cervix
migration of epithelial cells
during wound repair
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6 MIB-1 Helps to detect Ki-67 antigen in To detect proliferative activity Premalignant and Nuclear [63,64]
the G1, S, G2 and M phase, but it in dysplastic lesions malignant lesions of
is absent in the G0 phase the cervix
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observed in normal cervical epithelia to various severities of CINs
[61,62].
5. Treatment strategies
At present, the best possible treatment of cervical cancer
achieved through by combination of cisplatin-based chemother-
apy with radiation. Alongside with the chemotherapeutics/
combinational drug treatments, there are many molecular options
to control cervical cancer by using proteasome inhibitors, NSAIDs
and other combined treatment modalities (Fig. 2).
5.1. Targeting of ERBB protein kinases
The EGFR and many growths receptors impart in ERBB family
formed a tyrosine kinase domain (TKD), regulating transcription,
apoptosis, cell cycle progression, cytoskeletal rearrangement and
differentiation. In general, the EGFR expression is higher in cervical
intraepithelial neoplasia and cervical cancers [63] to activate the
mitogen-activated protein kinase pathway. It also induced sequen-
tial phosphorylation of transcription factors to cause proliferation.
Hence, by miscellaneous using of specific antibodies or small
molecules (gefitinib and erlotinib) found in preclinical studies

Fig. 2. The current treatment modalities by using different kinds of drugs for the treatment of HPV 16 and 18 infected cervical cancer.
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exerted anti-EGFR properties and also showed improvement when
combined with radiotherapy [64–67].
5.2. Other treatment modalities
At present, the combination of anti-EGFR agents with NSAIDs
and recombinant human TRAIL has been proven to be an effective
treatment for cervical cancer. On the other hand, the evoking RNA
technique introduced short interfering RNA (Si RNA) for homo-
logous mRNA degradation and their coded protein for selective
silencing of E6 or E7 expression [68]. Although in current practice,
the cisplatin has turned to be the most active single agent to treat
metastatic and recurrent squamous cervix cancer. Nevertheless,
there were a number of experimental strategies, such as
introduction of the bax and functional p53 gene, inhibition of
the JNK pathway and treatment of tumors with aldose reductase
inhibitors to overcome cisplatin resistance. This includes cisplatin
accumulation, faster repair of cisplatin adducts, modulating
apoptotic pathways, upregulation of transcription factors and
the loss of p53, but it still remain under preclinical or clinical level.
Beyond all, it made efficient when combine with other platinum
drugs, radio- and emerging gene therapy regimens [69]. The recent
investigation also includes the active component of emodin (1,3,8-
trihydroxy-6-methylanthraquinone) isolated from herb, Polygo-
num cuspidatum are traditionally used in China [70], exerted anti-
proliferative effects in human cervical cancer [71–75].
6. Vaccination types
HPV vaccine adopted as the most effective option to prevent
cervical cancer, routinely recommended for 11 and of 12 years of
girls, since, there was no clear evidence in contraceptive methods
to confer protection. The current emergence of two licensed
vaccines, quadrivalent vaccine (Gardasil) and bivalent vaccine
(Cervarix) has formulated non-infectious VLPs most specific for
HPV 16 and 18, attained through recombinant DNA technology,
acquired protection and substantially reduced the incidence of
cervical cancer [76]. From technical perspective, this vaccination
has been categorized based on their own properties (Table 2).
capsid proteins. Hence, this vaccine type produced by viral L1
capsid protein, self-assemble to form virus-like particles (VLPs) to
induce high levels of neutralizing antibodies when expressed in a
recombinant system [77]. In phase I/II clinical trials, these
intramuscular vaccination of VLPs found to induce significant
antibody titres in HPV 16 infected cervical secretions [78,79]. Also,
the recent investigation shown the potentials of other HPV type
vaccines other than 16 and 18 [80].
6.2. Therapeutic vaccines
It mainly targeted already established HPV infections in the
lower genital tract [81]. In contrast to the prophylactic, this
antigenic determinant was derived from early HPV proteins (e.g.,
E2, E6, and E7) rather than late proteins. Since these foreign viral
proteins, E6 and E7 had complete antigenic peptides/epitopes than
a mutant cellular protein. Accordingly, it is being good targets to
develop antigen-specific vaccines for HPV 16 and apart from E6, E7
has expressed abundant immunological characterization
[82,83]. This vaccine has essentially classified according to the
following vectors.
6.2.1. Viral and bacterial vector vaccines
In phase II clinical trial, the live recombinant Vaccinia virus
encoded E6 and E7 from HPV 16, 18 was conjugated with MHC
class I molecules by using Vaccinia vectors. This has been
administered at early stage of cervical cancer patients to generate
strong CTL activity [84]. By using attenuated bacteria (e.g., Listeria
monocytogenes, Escherichia coli), serve as a carriers to deliver
plasmids encoding genes or proteins of interest to antigen-
presenting cells (APCs). After phagocytosis, the production of
listeriolysin O from L. monocytogenes moved into cytoplasm and
facilitated the delivery of antigens to both MHC-I and MHC-II
pathways. The attenuated Salmonella and Bacillus Calmette–
Guerin (Mycobacterium bovis) are called to be safe bacterial
vaccine vectors, encoded HPV 16-L1 and E7 proteins to induce E7-
specific antibody and cytotoxic immune responses [85]. As like
viral vaccines, it also had pre-existing immunity that inhibited
repeat immunization limit.

6.1. Prophylactic vaccines 6.2.2. Protein, dendritic and DNA vaccines

The production of HPV 16 encoded E7 peptide-based vaccines
The L1 encoded protein is highly conserved among different may be further enhanced by using of adjuvants, fusion proteins or
Papilloma virus species; therefore, it has been used to identify HPV anchor-modified peptide epitopes [86]. The growing of DNA

Table 2
Various types of vaccination against HPV 16 & 18 infected cervical cancer.

S. No Vaccines Types Activity Remarks References

1 Prophylactic Bivalent Protective against HPV 16 and 18 Mild side effects include pain where [91]
vaccines (cervarix) the shot was given and fever, dizziness, nausea
Quadrivalent Protective against HPV 16, 18, 6, 11 Mild side effects include pain where the [92]
(Garadasil) shot was given and fever, dizziness, nausea
Pentavalent (VLPs) Protective against HPV 16, 18, 45, 31, and 33, Not found [93]
prevent 83% of all cervical carcinomas
Heptavalent (VLPs) Protective against HPV 52 and 58, Not found [93]
prevent 87% of the cervical cancer
2 Therapeutic Vector-based-viral/ Protective against HPV 16 Highly immunogenic [93]
vaccines bacterial Safety concerns
Peptide-based Protective against HPV 16 Safety, easy to produce, weakly immunogenic, [90]
MHC restriction
Protein-based Protective against HPV 16 Safety, less HLA restriction. Weak activator [90]
of cell-mediated immunity
DNA-based Protective against HPV 16 and 18 They are easy to produce, store, weakly [90]
immunogenic and generate sustained antigenic
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expression
Dendritic cell-based Protective against HPV 16 and 18 They are highly immunogenic, difficult to [90]
produce and biodeliver
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vaccines allowed sustained antigen expression on MHC–peptide
complexes. This bypassed the peptide-based vaccines by directly
transduced the DNA coding for antigen at APCs and the synthesized
peptides could be presented at the patient’s own HLA molecules
[87]. The incorporations of DCs are the primary mediators of DNA
vaccine, enhanced the weak intrinsic potency of naked DNA
vaccines. It also induced immune response by modifying
intracellular or intercellular movement of antigen to improve
DNA vaccine potency. Since DCs had limited life span, the co-
administration of E7-containing DNA with anti-apoptotic proteins
(Bcl-xl, Bcl-2) enhanced DC survival and E7-specific immune
responses for tumor treatment [88]. Another strategy to improve
delivery and antigenicity of HPV DNA vaccines is the use of
encapsulation [89], demonstrated the recombinant, full-length,
E7-pulsed, autologous DCs, could elicit a specific CD8+ CTL
response against HPV 16 or 18 infected cervical cancer patients.
Conclusively, it faces more challenges when compared to
prophylactic vaccines in stimulating the immune system and
immunocompromised state of cancer patients [90].
7. Conclusion
Although, there were numerous reports discussed in the causes
and treatment of HPV-infected cervical cancer, this review
exclusively conferred the complete pathogenesis of E6 and E7
oncoproteins and their treatment diversification solely against
HPVs 16 and 18. Altogether, this may bring root for further
modification of prophylactic vaccines and combinational therapies
to create novel treatments. And also for an effective diagnostics, it
enlisted the importance of immunocytochemical detection along
with traditional Pap screening. Ultimately, it will be an anchor for
further research in the area of treatment and prevention and this
overall aspect would pave the way to understand the unclear
concepts of HPV causing cervical cancer and also the pros and cons
on recent therapeutics, which more concern to HPVs 16 and 18.
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