Archives of Medical Research 45 (2014) 525e539

REVIEW ARTICLE
The Role of Signaling Pathways in Cervical Cancer and Molecular
Therapeutic Targets
Joaqu
ın Manzo-Merino,a Adriana Contreras-Paredes,a Elena
e V
azquez-Ulloa,a Leticia Rocha-Zavaleta,b
Alma M. Fuentes-Gonzalez,a and Marcela Lizanoa
a
Unidad de Investigaci
on Biom
edica en C
ancer, Instituto Nacional de Cancerolog
ıa-Instituto de Investigaciones Biom
edicas, Universidad Nacional
Aut
onoma de M
exico, M
exico City, M
exico
b
Departamento de Biolog
ıa Molecular y Biotecnolog
ıa, Instituto de Investigaciones Biom
edicas, Universidad Nacional Aut
onoma de M
exico,
M
exico City, M
exico
Received for publication August 8, 2014; accepted October 29, 2014 (ARCMED-D-14-00436).

Cervical cancer is a public health issue in developing countries. Although the Pap smear
and colposcopy remain the major strategies for detection, most cases are diagnosed in the
late stages. Therefore, a major concern has been to develop early diagnostic approaches
and more effective treatments. Molecular pathways that participate in cervical malignant
transformation have emerged as promising directed therapeutic targets. In this review, we
explore some of the major pathways implicated in cervical cancer development, including
RAF/MEK/ERK, phosphatidylinositol-3 kinase (PI3K/AKT), Wnt/b-catenin, apoptosis
and coupled membrane receptor signaling. We focus on the role of these pathways in
cervical carcinogenesis, their alterations and the consequences of these abnormalities.
In addition, the most recent preclinical and clinical data on the rationally designed
target-based agents that are currently being tested against elements of these pathways
are reviewed. Ó 2014 IMSS. Published by Elsevier Inc.
Key Words: Cervical cancer, Carcinogenesis, Signaling pathways, Inhibitors.

Introduction important cell cycle regulators such as p53 and PDZ domain
containing proteins (4), whereas E7 promotes the inactivation
Cervical cancer is the fourth most common neoplasm in
of pRb (5). In addition, other proteins are overexpressed in
women worldwide. Every year, nearly 529,000 new cases
this neoplasm such as PI3K (phosphatidylinositol 3-kinase),
of cervical cancer are diagnosed. Most cases are reported
EGF-R, b-catenin, and Erk, and anti-apoptotic proteins such
in developing countries where cervical cancer is one of
as the Bcl-2 sub-family (6e9). All of these alterations are part
the major causes of cancer mortality in women (1). Epide-
of the cervical cancer network and present an opportunity for
miological and molecular data indicate that persistent infec-
developing therapies against any of the participating ele-
tion with ‘‘high-risk’’ human papillomavirus (HPV) is a
ments. In this review, we highlight the role of these pathways
prerequisite for cervical cancer development and that it is
in cervical carcinogenesis, their alterations and the conse-
associated with other pathologies, such as head and neck
quences of these abnormalities. In addition, we review the
cancers and anal cancer (2). More than 100 types of HPV
current preclinical and clinical data for designed agents that
have been identified to date; 35 are classified as high-risk
target elements of these pathways.
and another 40 as low-risk based on oncogenic potential (3).
The conventional treatment of cervical cancer consists
HPV overexpression of E6 and E7 oncoproteins is the
of surgery, radiation and chemotherapy alone or in combi-
main oncogenic stimulus for cell transformation in cervical
nation (10). Such treatments remain suboptimal, with resid-
cancer. High-risk E6 proteins induce the degradation of
ual tumors observed in 40e50% of patients with advanced
cancer (11,12). Therefore, there is a need for implementing
Address reprint requests to: Marcela Lizano, Instituto Nacional de
Cancerolog
ıa, Av. San Fernando 22, Col. Secci
on XVI, Tlalpan, Mexico novel therapeutic approaches that, in addition to the stan-
City, D.F., 14080 Mexico; Tel: (þ52) (55) 5628-0400 ext.133; FAX: dard treatments, could improve the overall survival and
(þ52) (55) 5628-0426; E-mail: lizanosoberon@gmail.com quality of life of cervical cancer patients.

0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.arcmed.2014.10.008
526 Manzo-Merino et al./ Archives of Medical Research 45 (2014) 525e539
Signal Transduction Pathways Implicated in Cervical
Cancer
ERK/MAPK Signaling Pathway
The ERK/MAPK pathway, also known as the RAF/MEK
pathway, is a ubiquitous signal transduction pathway that
regulates crucial cellular processes, including proliferation,
differentiation, angiogenesis and survival (13). Impor-
tantly, overexpression or activation of components of this
pathway is believed to contribute to tumorigenesis, tumor
progression and metastatic disease in a variety of solid tu-
mors (14). The ERK/MAPK pathway lies downstream of
various growth factors such as EGF (epidermal growth fac-
tor). Growth factor binding results in receptor phosphory-
lation, which activates an adapter molecule complex
known as GRB2/SHC/SOS. This in turn activates the
RAF/MEK/ERK pathway, which triggers a cascade of spe-
cific phosphorylation events. Within the RAF/MEK/ERK
pathway, the small GTPase RAS and the serine/threonine
kinase RAF are the key molecular signal regulators (15).
Intermediate signaling is regulated by MEK1 and MEK2,
which are responsible for phosphorylating and activating
the final downstream signaling molecules known as
ERK1 and ERK2 (16). ERK1/2 regulates cellular activity
by acting on more than 100 substrates in the cytoplasm
and nucleus, including indirect inducers of gene expres-
sion, transcription factors and cell cycle-related kinases
(17). RAS, another member of the RAF family, also has
a regulatory role in the Erk/MAPK pathway and other
signaling pathways, such as the PI3K/Akt/mTOR, PKC
and the RALGDS pathways, which have been implicated
in carcinogenesis (18,19). Ras is activated in |20% of hu-
man cancers, including cervical cancer, where it has been
linked to the metastatic conversion of tumor cells (20).
Mutations of Ras and Myc amplification are frequently
associated with the development of recurrent cervical can-
cer (21,22).
Therapeutic targets for ERK/MAPK pathway in cervical
cancer. Epidermal growth factor receptor (EGFR) overex-
pression is frequently observed in cervical cancer and is an
independent predictor of poor prognosis in advanced-stage
tumors (23,24). EGFR is a potential target for the treatment
of cancer, and a number of agents targeting EGFR have
been designed (25). There is growing evidence that anti-
EGFR strategies exert their anti-tumor activities in part
by interfering with the MAPK and Akt pathways. Table 1
summarizes the agents used to suppress elements of the
signaling pathways implicated in cervical carcinogenesis
and apoptotic inducers, either in clinical trials and in vitro
models of cervical cancer.
Once the MAPK pathway is activated, the RAF kinase
takes action. Sorafenib is the first oral RAF kinase modu-
lator to receive marketing approval for the treatment of
advanced renal cell carcinoma. This inhibitor interacts with
the ATP-binding cleft of the enzyme and stabilizes the
protein in a conformation that prevents substrate binding
and phosphorylation (26). Sorafenib is being assessed in a
phase I/II clinical trial in combination with radiotherapy
and cisplatin to determine its biological activity in cervical
cancer (www.clinicaltrails.gov; protocol: NCT00510250)
(Table 1 and Figure 1).
Small molecule inhibitors of MEK1/2 are highly specific
protein kinase inhibitors. Since the development of the first
MEK inhibitors PD98059 and U0126 (27), a considerable
number of preclinical studies have investigated various
MEK1/2 inhibitors (28).
PD98059 is a flavonoid and a potent inhibitor of MEK
(29). The suppression of MEK by PD98059 results in resis-
tance to cisplatin, but not to doxorubicin (another common
anti-cancer drug based on DNA intercalation). Both drugs
induce NF-kB activation in SiHa (30). U0126 is a chemi-
cally synthesized organic compound that is a very selective
and potent inhibitor of MEK1 and MEK2. In contrast to
PD098059, which only inhibits inactive MEK, U0126
inhibits both active and inactive MEK-1/2 (31).
CI-1040 is another potent and selective MEK inhibitor
that has displayed preclinical activity in a broad spectrum
of tumor xenografts including those derived from colon
cancer and melanoma (32). CI-1040 treatment produces a
reduction of the pMAPK levels in the cervical tumor cell
line A431.
From the MAPK cascade, the MEK enzymes are the
most attractive molecules for cancer therapy. Fourteen
MEK inhibitors are currently undergoing testing (33), with
two being tested for cervical malignancy, U0126 and trem-
atinib (GSK1120212) or in the treatment of recurrent or
persistent cervical cancer in combination with the Akt
inhibitor GSK214179 (www.clinicaltrials.gov, protocol:
NCT01958112).
The MAPK network has been the subject of intense
research and pharmaceutical scrutiny to identify novel
target-based approaches for cancer treatment. Among these
kinases, RAF and MEK have received substantial attention,
but many more preclinical studies in cervical cancer are
required.
The PI3K/Akt Signaling Pathway
The PI3K signaling pathway is often activated in various
human cancers (34,35). PI3K is a downregulator of the
Ras signaling pathway, and it is a ubiquitous lipid kinase
that is involved in receptor signal transduction (36). This
protein is an intracellular transducer with a catalytic and
a regulatory subunit (37). Activated PI3K converts the
membrane lipid phosphatidylinositol-4,5-bisphosphate
(PtdIns[4,5]P2) to phosphatidylinositol-3,4,5-trisphosphate
(PtdIns[3,4,5]P3). The protein serine-threonine kinases
Akt and phosphoinositide-dependent kinase 1 (PDK1) are
recruited at the PI3K activation sites by directly binding
Table 1. Targeted signal transduction elements in cervical cancer and their mechanism of action

Therapy Signaling pathway Target Mechanism of action Adjuvant treatment tested Clinical protocol References

Sorafenib MAPK Raf kinase Competitive inhibition of Radiotherapy/Cisplatin NCT00510250 Garc
ıa, 2009
the ATP site
PD98059 MAPK Inactive MEK ½ Protein kinase inhibitors Cisplatin and Doxorubicin - Yeh et al. 2002
U0126 MAPK Active and inactive Protein kinase inhibitors - - Favata et al. 1998
MEK ½
Trematinib MAPK MEK ½ Binds and inhibits MEK ½ GSK2141795 NCT01958112 Miller et al. 2014
action
CI-1040 MAPK MEK Induces decrease in pMAPK - - Montagut, 2009
levels
Wortmannin PI3K p110 Irreversible competitor. Radiotherapy/ - Zhang et al. 2009
Competes with the ATP site Chemotherapy/Roscovitine
LY294002 PI3K p110 Reversible competitor. Radiotherapy - Fuhrman et al. 2007
Competes with the ATP site
inducing apoptosis
Quercetin PI3K p110 Competes with ATP - - Huangh et al. 2009
binding De et al. 2000

Molecular Targets in Cervical Cancer

Indole-3-carbinol PI3K Akt Inactivates Akt by - NCT00910884 Bell et al. 2000
demethylation of PTEN. Jin et al. 1999
Induces apoptosis Yuan et al. 1999
MK-2206 PI3K Akt Allosteric inhibitor of Akt - - Cherrin et al. 2010
1/2 Tolcher et al. 2009
Genistein PI3K Akt Avoids Akt phosphorylation Radiotherapy NCT00001696 (Completed Yashar et al. 2005
at Ser473 for toxicity) Su-Hyeong et al.
2009
Oridonin PI3K Akt Reduction of Akt - - Hu et al. 2007
phosphorylation
Tripterygium PI3K Akt Reduction of Akt - - Yang et al. 2006
phosphorylation
Temsirolimus PI3K mTOR Forms a complex with the Topotecan NCT01217177 Temkin et al. 2010
FK506 binding protein-12 NCT01026792 (Completed)
Everolimus PI3K mTOR Forms a complex with the Paclitaxel Cisplatin/ NCT01217177 Campone et al. 2009
FK506 binding protein-12 Chemotherapy NCT01026792 (Completed)
Cetuximab EGF EGFR extracellular Avoids the ligand binding Cisplatin/Topotecan (Toxic) NCT00957411 Hertlein et al. 2010
fraction Radiation/Cisplatin NCT00997009 Kurtz et al.Moore
Chemotherapy et al. 2012
Santin et al. 2011
Nimotuzumab EGF EGFR extracellular Avoids the ligand binding Cisplatin/Gemcitabine, NCT02095119, NCT01938105,
fraction chemoradiotherapy, NCT02083211, NCT02039791,
Carboplatin/Paclitaxel, NCT01301612
Brachytherapy
Matuzumab EGF EGFR extracellular fraction Avoids the ligand binding Platinum - Blohmer et al. 2005
Lapatinib EGF EGFR tyrosine kinase Tyrosine kinase inhibitor - NCT00430781 Monk et al. 2010
fraction (Completed)
(continued on next page)

527
 528
Table 1 (continued )
Therapy Signaling pathway Target Mechanism of action Adjuvant treatment tested Clinical protocol References

Bevacizumab VEGF VEGFR-A Avoids the ligand binding Fluorouracil/Capecitabine NCT00025233 (Completed) Wright et al. 2006
Topotecan/Cisplatin NCT00626561 (Terminated) Monk et al. 2009
Radiotherapy/Cisplatin NCT00803062 Zighelboim et al. 2013
Schefner et al. 2011
Pazopanib VEGF VEGF tyrosine kinase Tyrosine kinase inhibitor Lapatinib/Chemotherapy NCT00430781 Monk et al. 2010
fraction
Lapatinib EGF EGFR tyrosine kinase Tyrosine kinase inhibitor - - Monk et al. 2010
fraction
Imatinib PDGF PDGFR kinase fraction Tyrosine kinase inhibitor - - Taja et al. 2006
Gefitinib EGF EGFR tyrosine kinase Tyrosine kinase inhibitor - NCT00049556 Goncalves et al. 2008
fraction (Completed)

Manzo-Merino et al./ Archives of Medical Research 45 (2014) 525e539

Erlotinib EGF EGFR tyrosine kinase Tyrosine kinase inhibitor Cisplatin/Radiotherapy NCT00031993 Nogueira et al. 2008
fraction (Completed) Schilder et al. 2009
3,3-diindolylmethane MAKP Several Downregulates elements of - - Zhu et al. 2012
PI3K the PI3K and MAPK
pathways
Retinoids Apoptosis Fas/FasL EGF, Induces FasL. Deregulates - - Sun et al. 2000
EGF pathway Hembree et al. 1996
N101-2 PI3K/Akt Fas/FasL Arrests cell cycle at sub-G1 - - Kim et al. 2012
Extrinsic apoptosis PI3K/Akt phase. Induces apoptotic
cell death
Silibilin Not yet elucidated Cell cycle-dependent Induces G arrest and - - Zhang et al. 2012
kinases apoptotic cell death
Ganoderic acid derivates Intrinsic apoptosis Not known yet Decrease mitochondrial - - Liu et al. 2012
membrane potential
Recombinant human Apoptosis Death receptor 4/5 Binds the DR directly and Radiation - Maduro et al. 2008
TRAIL acts as an agonist
Luteolin Apotptosis DR5 Induces Bid cleavage and rhTRAIL - Horinaka et al. 2005
caspase-8 activation
Aspirine Intrinsic apoptosis Not specified Suppresses the activation of TRAIL - Im and Jang, 2012
Erk 1/2
Arsenic trioxide Multiple signaling E6 Induces a reduction in E6 NCT00005999 Wen et al. 2012
pathways protein levels with cell (Completed)
cycle arrest in G2-M in
HPV expressing cells
Avenanthramide 2p Wnt/b-catenin b-catenin Induces b-catenin - - Wang et al. 2012
degradation and avoids its
nuclear accumulation
Triptolide Wnt/b-catenin b-catenin Induces b-catenin - - Wang et al. 2012
degradation and avoids its
nuclear accumulation

Different elements designed to block signal transduction through inhibiting certain elements are presented. Current preclinical and clinical evaluations of such compounds are cited.
 Molecular Targets in Cervical Cancer 529

Figure 1. MAPK, PI3K/Akt and Wnt pathways and therapeutic interventions. After ligand binding, the receptors start the signaling cascade downstream the
molecular effectors. In cancer cells signaling pathways are overfunctional because several elements are either overexpressed or overactivated. The figure
shows the main elements in those pathways and the directed therapeutic agents. Most of those agents block tyrosine kinase activity or downstream kinases.
MAbs are intended to block the ligand binding to its receptor and dimerization.

to PtdIns(3,4,5)P3 (38). The proteins recruited to
PtdIns(3,4,5)P3 facilitate the phosphorylation of Akt by
PDK1 (39). This phosphorylation stimulates the catalytic
activity of Akt, resulting in the phosphorylation of other
proteins that affect cell cycle entry, cell proliferation, and
anti-apoptosis.
The termination of PI3K signaling by the degradation of
PtdIns(3,4,5)P3 can be mediated by at least two different
types of phosphatases. One is Src-homology 2-containing
phosphatase (SHIP), which dephosphorylates the 5 position
of the inositol ring to produce PtdIns(3,4)P2. The other is
PTEN (phosphatase and tensin homologue deleted on chro-
mosome (10), which dephosphorylates the 3 position of
PtdIns(3,4,5)P3 to produce PtdIns(4,5)P2. In many human
cancer types, deleted PTEN and upregulated PI3K lead to
the enhanced cellular synthesis of PtdIns(3,4,5)P3. Thus,
PI3K and PTEN are deeply involved in cell survival
pathways, which include the regulation of gene expression,
vesicular trafficking, cellular metabolism and cytoskeletal
rearrangements (40e42). In fact, growing evidence sug-
gests PTEN as a promising biological marker because
PTEN expression correlates with tumor incidence, size
and progression, making this protein an excellent candidate
for early diagnosis and an opportunity for therapeutic inter-
vention (43).
The constitutive activation of the PI3K/Akt signaling
pathway has been firmly established as a major determinant
of tumor cell growth and survival in a number of solid tu-
mors (44). AKT1 gene alterations have been reported in a
genomic analysis identifying an amplification of the region
14q32.33 (45). These results were validated by expression
profiles in cervical cancer specimens, supported by the
observation that the PI3K catalytic subunit is amplified in
cervical cancers (46).
HCCR oncogenes, which are related to the tumorigen-
esis process through their interaction with p53, regulate
PI3K/Akt signaling in cervical cancer (47).
Therapeutic targets for PI3K/Akt pathway in cervical
cancer. The first relatively specific PI3K inhibitors
described were Wortmannin (Wm), a naturally occurring
metabolite of Penicillium funiculosum, and LY294002
(2-(4-morpholinyl)-8-phenyl-chromone), derived from the
flavonoid quercetin (48). Both pharmacologic agents target
the p110 catalytic subunit of PI3K (49) by competing with
ATP binding (50).
LY294002 is a reversible PI3K inhibitor that has a very
short half-life, is insoluble in aqueous solutions, and has
off-target activity and high toxicity. These characteristics
have limited its clinical application (49,51). However, this
metabolite has been shown to cause apoptosis in various hu-
man cervical cancer derived cells in vitro, in addition to cell
growth inhibition (52), through the up-regulation of the
FOXO1 nuclear activity by reducing PI3K mediated phos-
phorylation (53).
LY294002 has also been reported to cause a potent
radiosensitization via double-strand DNA break repair
inhibition in HeLa cells (54). Conversely, Wm is a potent
irreversible inhibitor of PI3K (55). It enhances apoptosis
in several cell lines associated with the inhibition of
Akt activation. Wm radiosensitizes cancer cells (49) by
decreasing the activities of both the ATM protein and
530 Manzo-Merino et al./ Archives of Medical Research 45 (2014) 525e539
DNA-PK (DNA-dependent protein kinase catalytic subunit)
(56). In addition, Wm promotes a chemosensitizer effect in
HeLa cells when challenged with the experimental drug
roscovitine (57).
Quercetin is a naturally occurring flavonoid that can
inhibit a broad range of protein kinases. In fact, it is the pro-
totypal compound from which a variety of derivatives,
including LY294002, were designed (50). Huangh et al.
showed that quercetin inhibits HeLa cell proliferation and
induces cell death via the Ca2þ-dependent mitochondrial
apoptosis pathway (58). In addition, quercetin inhibits the
adhesion, migration and invasion of HeLa cells (52).
Finally, testing of quercetin in mouse models showed
that this agent is able to arrest or reverse the progression
of cervical neoplasia (59). However, quercetin has not been
investigated in clinical trials to date.
The phytochemical indole-3-carbinol (I3C) is an
anti-carcinogenic and anti-estrogenic compound found
abundantly in cruciferous vegetables such as broccoli and
brussels sprouts. It inhibits the growth of benign tumors
of laryngeal tissue caused by HPV-16 in a mouse model
and was shown to be effective in the treatment and preven-
tion of laryngeal papillomas caused by HPVs (60). It is
clear that I3C inactivates Akt in tumor cells by upregulating
the tumor suppressor protein PTEN. The mechanism by
which I3C upregulates PTEN is unknown, but the demethy-
lation of PTEN by I3C (61) and the upregulation of the
transcription factor Egr-1, a regulator of PTEN expression,
are potential mechanisms (62).
Bell and co-workers carried out a clinical trial using oral
I3C to treat women with cervical intraepithelial neoplasia
(CIN) (63). The authors reported a statistically significant
regression of CIN in patients treated with I3C, compared
with placebo. Moreover, indole-3-carbinol has been used
as adjuvant treatment for recurrent respiratory papillomato-
sis (RRP) with promising outcomes (64).
Once the PI3K/Akt pathway is activated the number of
outputs increases as the signal moves down the pathway. In-
hibiting PI3K should provide the maximum inhibition of all
outputs, whereas inhibiting downstream targets, such as Akt,
and even further downstream targets, such as mammalian
target of rapamycin (mTOR), will give progressively more
selective output inhibition (65). Although Akt is the central
player in this important pro-survival pathway, few direct
Akt inhibitors have been developed that may be directly
translated to the treatment of humans (66). A series of naph-
thyridine and naphthyridinone allosteric dual inhibitors of
Akt1 and 2 have been developed (67). MK-2206 is a com-
pound from this class of Akt inhibitors. Treatment of the cer-
vical cell line C33A with MK-2206 resulted in the
redistribution of Akt from the membrane to the cytoplasm
(68). Phase I studies in solid tumors suggested that MK-
2206 is an effective therapeutic drug (69).
Banerjee et al. found that genistein (40 ,5,7-
trihydroxyisoflavone) treatment reduced the level of the
phosphorylated Akt protein at Ser473 compared to control
cells, resulting in a dose-dependent induction of apoptosis in
cells that display constitutively active Akt (70). Genistein is
a small molecule found in high abundance in natural soy prod-
ucts (71). Genistein significantly suppressed the cell growth of
HeLa and CaSki cells, perhaps due to Akt inhibition (72). It
also functions as a radiosensitizer for cervical cancer cells
such as CaSki and ME180 (73). This drug has been tested
in numerous clinical trials of prostate, breast and pancreatic
cancer. However, genistein has not been tested in patients with
cervical neoplasia until recently (clinicaltrials.gov).
Two more natural products have shown Akt inhibitory
potential: oridonin, which is isolated from Rabdosia rubes-
cens (74), and triptolide, the main active component of the
traditional Chinese herbal medicine Tripterygium wilfordii
Hook (75). Both products were tested in HeLa cells where
they were effective apoptotic agents with concomitant
reduction of Akt phosphorylation.
Downstream in the PI3K/Akt pathway there is a distal
component known as mTOR. Rapamycin, the prototypic
mTOR inhibitor, was discovered in 1975 on the island of
Rapa Nui as a potent anti-fungicide that is produced natu-
rally by Streptomyces hygroscopicus (76). More recently,
Rapamycin analogues such as CCI-779 and RAD-001 have
been explicitly designed for development as anti-cancer
drugs (50). These drugs are small molecule inhibitors that
function intracellularly, forming a complex with the
FK506 binding protein-12 (FKBP-12), which is then recog-
nized by mTOR (77).
CCI-779, known as temsirolimus, is an intravenously
administered agent approved by the U.S. Food and Drug
Administration (FDA) and the European Medicines Agency
(EMEA) for the treatment of advanced renal cell carcinoma
(77,78). CCI-779 has anti-tumor activity in a variety of other
human cancer models (79). In fact, O100 clinical trials for
this drug are registered at the clinical trial website. A phase
I trial undertaken by Temkin et al. tested this drug in combi-
nation with topotecan in a variety of gynecological malig-
nancies and showed that the regimen may be safe for
women who have not previously received radiation (80).
Another rapamycin analog, RAD-001, known as
everolimus, is an oral agent that has recently obtained
U.S. FDA and EMEA approval for the treatment of
advanced renal carcinoma (77). Everolimus displays
antiproliferative effects on cancer cells, yields anti-
angiogenic activity in established tumors, and has
synergistic activity with paclitaxel in preclinical models
(81). Some clinical trials are now recruiting patients
with cervical tumors (www.clinical trails.gov; protocol:
NCT01217177) (Table 1 and Figure 1).
The EGFR/VEGFR Signaling Pathway
Epidermal grow factor receptor (EGFR) and its family
members Her-2/Erb-2, Her-3/Erb-3 and Her4/Erb-4
 Molecular Targets in Cervical Cancer
belong to a tyrosine kinase receptor family. Together with
their ligands, these receptors are involved in O70% of all
cancers (82). EGFR is associated not only with the prolif-
eration of tumor cells but also with enhanced tumor cell
survival, angiogenesis and metastatic spread (83). At least
six ligands are known to directly activate EGFR:
epidermal growth factor (EGF), transforming growth
factor alpha (TGF-a), heparin binding EGF-like growth
factor (HB-EGF), amphiregulin, betacellulin, and epiregu-
lin. These proteins are synthesized as transmembrane
precursors and are proteolytically cleaved by metallopro-
teases to release the mature growth factor in an autocrine
or paracrine manner (84). Extracellular ligand binding
causes dimerization of the EGFR, which becomes auto-
phosphorylated at distinct tyrosine residues by the recep-
tor’s intrinsic kinase activity and, depending on the
ligand, intracellular pathways can be activated (85). The
best-known pathways are the ERK1/2 and PI3 kinases as
previously described.
Many studies have shown that the overexpression of
EGFR is linked to the development of cervical cancer
(8,24,86,87) and that its expression is correlated with poor
prognosis and metastasis (23,88e90). Because EGFR pro-
teins are cell surface receptors, they can be directly targeted.
The relationship between EGFR expression and cervical can-
cer has led to the development of many therapeutic agents
targeting this receptor (91), with the monoclonal antibodies
(mAb) the most promising in a series of clinical trials.
Another method of targeting EGF is the use of small-
molecule, adenosine triphosphate-competitive inhibitors of
the tyrosine kinase receptors (92), as reviewed here.
Angiogenesis is a fundamental step in the formation of
metastasis. EGF-activated signals may induce the synthesis
of vascular endothelial growth factor (VEGF), promoting
angiogenesis, tumor growth cell survival, and metastasis
(93). In cervical cancer, the angiogenic process is related
to VEGF expression (94), which correlates with severity
in precursor lesions and invasive disease (95). The VEGF
family includes VEGF-A to F, with VEGF-A as the most
important member. Cooper et al. evaluated intra-tumoral
microvessel density (IMD) in patients treated with pelvic
irradiation and reported that a high vascularity was associ-
ated with poor survival rates in which IMD acted as a prog-
nostic marker (96).
Platelet-derived growth factor. Platelet-derived growth fac-
tor (PDGF) is a very strong stimulator of blood vessel forma-
tion and has also been associated with the pathogenesis of
cervical cancer. This ligand and its receptors are frequently
expressed in cervical cancer-derived cell lines (97).
Therapeutic targets for EGFR and VEGFR in cervical can-
cer. Cetuximab is a human/murine chimeric immunoglob-
ulin G2 mAb that targets EGFR. Preclinical studies showed
that cetuximab binds to EGFR with high affinity, inducing
531
receptor dimerization, internalization and downregulation,
which in turn inhibits receptor activation and signaling
(98). Cetuximab has been approved for the treatment of
squamous cell carcinoma of the head and neck in combina-
tion with radiotherapy (99). In addition, cetuximab
combined with irinotecan increased response rates and
progression-free survival in patients with colorectal cancer
(100). However, a recent report by Hertlein et al. found no
advantage for cetuximab monotherapy in patients with
advanced cervical cancer (101), whereas Kurtz et al.
demonstrated that the combination of cisplatin-topotecan-
cetuximab induces a high rate of serious adverse and/or
fatal events at the standard dose and schedule (102). In
another study, the feasibility of cetuximab combined with
whole pelvic radiation and cisplatin compared with cetuxi-
mab combined with extensive field radiation and cisplatin
was investigated. The latter combination was highly toxic
and not feasible for the treatment of patients with cervical
cancer (103). Santin et al. demonstrated that cetuximab is
well tolerated as a mono-drug with limited activity in the
study population, with specific activity and limited effect
on patients with squamous cell histology (104).
Nimotuzumab, another EGFR inhibitor, inhibits the
EGF-related signaling and ligand-independent signaling,
but at higher concentrations than cetuximab (105,106),
suggesting its potential use as a therapy in tumors of epithe-
lial origin (107). Because EGFR is overexpressed in
a considerable number of tumors of the cervix, it is
currently undergoing testing in a variety of clinical
trials alone or in combination (www.clinicaltrails.gov;
protocols: NCT02095119, NCT01938105, NCT02083211,
NCT02039791, NCT01301612). Two studies are testing
the efficacy and tolerability of cetuximab in cervical cancer
patients (www.clinicaltrials.gov; protocols: NCT00957411
and NCT00997009).
Matuzumab, a humanized immunoglobulin G1 mono-
clonal anti-EGFR antibody, was effective in patients with
cervical cancer that progressed after treatment with
platinum-based chemotherapy. In the 38 patients enrolled
in the study, the antibody was well tolerated, with two cases
of partial response and nine cases with stable disease (108).
Bevacizumab is a mAb directed against VEGF-A. It was
the first anti-angiogenic agent that was successfully used in
colorectal cancer (109). Bevacizumab delayed the time to
progression by 4 months and increased the median survival
in patients. Bevacizumab has also been tested in cervical
cancer patients in whom prior therapy failed. Studies of
bevacizumab plus fluorouracil or capecitabine found clin-
ical benefit in 67% of patients. Therapy was well tolerated;
thus, bevacizumab showed anti-tumor activity in pretreated
cervical cancer (110). Subsequent studies demonstrated a
favorable toxicity profile and similar results for bevacizu-
mab (111). Another study showed that the combination of
bevacizumab with topotecan and cisplatin induces an
improvement in both the overall survival and progression-
532 Manzo-Merino et al./ Archives of Medical Research 45 (2014) 525e539
free survival of patients with recurrent or persistent cervical
cancer (112). Another study of bevacizumab in combina-
tion with radiotherapy and cisplatin showed a very safe pro-
file with an improvement of overall survival and without
adverse events (113). An independent study is currently
testing the efficacy of paclitaxel and cisplatin or topotecan
in combination with or without bevacizumab (www.
clinicaltrails.gov; protocol: NCT00803062).
The tyrosine kinase inhibitors (TKIs) pazopanib and la-
patinib, which target VEGF and EGF, respectively, have
demonstrated antiangiogenic activity in a phase II clinical
trial performed by Monk and co-workers (2010) in women
with recurrent or advanced cervical cancer (114). This trial
compared antiangiogenesis therapy (pazopanib) with
EGF-based therapy (lapatinib). The authors suggest the su-
periority of pazopanib due to its favorable toxicity profile,
easier administration (oral), high antiangiogenic activity
and higher overall survival (11.6 weeks longer than the
lapatinib arm). Erlotinib, another TKI, has been evaluated
as monotherapy (115), as has gefitinib (116). These were
well tolerated but without an objective response. The
authors declared that monotherapy with those agents had
no effect in patients with advanced or recurrent cervical
cancer. However, another phase I trial showed that the
use of erlotinib is feasible, presenting an acceptable toxicity
profile in patients undergoing erlotinib in combination with
cisplatin and radiotherapy (117). Finally, the potential use
of another TKI, imatinib, has been tested in an in vitro pilot
study using cervical cancer-derived cells. The authors
showed that imatinib inhibited cell growth by hampering
the phosphorylation of PDGFR, suggesting that it may be
a good target for the development of a therapy for cervical
cancer (97) (Table 1 and Figure 1).
The Apoptotic Signaling Pathways
The life and death of cells must be balanced to maintain
tissue homeostasis. Disruption of this balance can lead to
a severe disturbance that may result in cancer (118). Cells
have an intrinsic mechanism to control tissue homeostasis
linked to apoptosis. Defects in the apoptosis-inducing path-
ways can eventually lead to the expansion of a population
of neoplastic cells.
In principle, there are two alternative pathways that
initiate apoptosis. One pathway, occasionally referred to
as the extrinsic pathway, is mediated by death receptors
on the cell surface, which are activated when the death-
inducing ligand binds to the death receptors (DR). Upon
binding, caspases (cysteine aspartyl-specific proteases)
8 and 10 become activated which, in turn, activates cas-
pases 3, 6 and 7. The other apoptotic pathway, the intrinsic
pathway, is mediated by the mitochondria. Fas and TRAIL
receptors are the main regulatory elements in the extrinsic
pathway and are expressed by a broad panel of normal
epithelial cells (119).
The intrinsic apoptotic pathway involves procaspase-9,
which is activated downstream of mitochondrial pro-
apoptotic events at the so-called apoptosome, a cytosolic
death signaling protein complex formed upon release of
cytochrome c from the mitochondria. In this case, it is
the dimerization of procaspase-9 molecules at the Apaf-1
scaffold that is responsible for caspase-9 activation (120).
Once the initiator caspases have been activated, they can
proteolytically activate the effector procaspases 3, 6, and
7, which subsequently cleave a specific set of protein sub-
strates, including the procaspases themselves (120).
Various proteins regulate the apoptotic process at
different levels. FLIPs (FADD-like interleukin-1b
converting enzyme-like protease) and FLICE/caspase 8
(FADD-like ICE) inhibitory proteins interfere with the initi-
ation of apoptosis directly at the level of death receptors.
The IAPs (inhibitor of apoptosis proteins) constitute
another class of regulatory proteins. IAPs bind to and
inhibit caspases. They might also function as ubiquitin li-
gases, promoting the degradation of the caspases that they
bind (121).
A very important feature in cancer establishment is the
modulation of apoptosis mediated by either viruses or onco-
genes supporting proliferation, immortalization, transfor-
mation and finally cancer (122). Cervical cancer is not an
exception, with a disruption in the apoptotic pathway pri-
marily due to the continued expression of E6 protein. The
E6 protein binds to p53 with the aid of E6AP and prevents
p53 from inducing apoptosis by targeting it for degradation
via the ubiquitin-proteasome pathway (123). In malignant
cells, these physiological apoptotic pathways are often
disturbed, which allows for uncontrolled cell proliferation
(124).
Downregulation of Fas expression is a common abnor-
mality in gynecological cancers (125). One study focused
on the pattern of expression of apoptotic-related proteins
showed that Fas is lost as long as the lesion progresses.
The FasL, TRAIL and death receptors (DR) 4 and 5 are
frequently expressed in the cytoplasm of tumor cells in cer-
vical cancer patients. Because DR4, DR5, and TRAIL are
frequently observed, the absence of TRAIL expression
was associated with a higher pathological complete
response rate to radiotherapy (126,127). Conversely, HPV
E5 protein impairs the TRAIL- and FasL-mediated
apoptosis by downregulating the Fas receptor and inter-
fering with the DISC formation upon the TRAIL signal
in raft cultures of E5 expressing keratinocytes, which were
completely protected from FasL- and TRAIL-induced cell
death (128). Similarly, when UV radiation is used to induce
stress, E5 expressing human keratinocytes are protected
from apoptosis (129).
Therapeutic targets for apoptotic signaling pathways in
cervical cancer. The induction of apoptosis is most likely
the underlying mechanism of therapeutic strategies that
 Molecular Targets in Cervical Cancer 533

have been or are being tested. Several approaches have suc-
cessfully induced apoptosis in cervical cancer-derived cell
lines by using chemical compounds such as luteolin (130)
and aspirin (131), either alone or in combination with radio-
therapy or by using soluble apoptotic inducers such as
TRAIL (132).
Recently, a number of pro-apoptotic agents have been
tested in cervical cancer cells. The N101-2 compound, a
flavonoid derivate, showed clear apoptotic effects in CaSki
and SiHa cells by arresting the cell cycle at the G1 phase,
activating intrinsic pathways, and inhibiting the PI3K/Akt
signaling pathway (133). Similarly, silibilin induced a
dose-dependent apoptotic death in HeLa cells by activating
both the mitochondrial and death receptor-mediated
pathway (134). Meanwhile, ganoderic acid derivatives
demonstrated therapeutic potential in HeLa cells, inducing
apoptosis by decreasing mitochondrial membrane potential
in a very specific manner (135).
Retinoids, a class of compounds related to vitamin
A, may play a role as chemopreventive agents in
HPV-associated CIN and cervical cancer. In vitro treatment
of HPV-immortalized keratinocytes and cervical carcinoma
cell lines with retinoids downregulated E6/E7 transcription
and upregulated wild type p53 expression (136), with
several consequences including the deregulation of EGF
and IGF signaling pathways (137), induction of Fas and in-
crease in Fas/Fas ligand-mediated apoptosis (138). I3C has
exhibited chemopreventive effects in preclinical studies.
This agent has anti-estrogenic activities and induces
apoptosis (139). Transgenic mice harboring tumors were
fed a diet supplemented with I3C, and the incidence of can-
cer in these animals was dramatically reduced (60).
Conversely, 3,3-diindolylmethane (DIM), an important
polymer derived from I3C, exerts antiproliferative and
pro-apoptotic effects in a cellular model by affecting the
MAPK and PI3K pathways when used as a mono-drug
(140).
A promising preclinical report from Wen et al. found
that arsenic trioxide induces apoptosis by specifically
targeting HPV-expressing cells, decreasing cell viability
48e60% compared with control cells, which decreased
16% (141). This effect is due to downregulation of E6
expression and the subsequent rescue of p53 and cas-
pase 3 levels that are conducive to cell death. Certainly,
the end point for cervical cancer therapy is to induce cell
death within the tumor, and most of the registered
protocols at www.clinicaltrails.gov aim to improve
overall survival and progression-free survival with a
high apoptotic rate and low toxic profile (Table 1 and
Figure 2).

Figure 2. Apoptotic signaling pathway and current agents inducing apoptosis. The apoptotic pathway starts when the death signal is received by death re-
ceptors (DR) allowing the intracellular activation of caspase 8. The signal proceeds through the effector caspase cleavage with cell death as the final end.
Caspase 8 induces Bid activation which, in turn, allows cytochrome C release from mitochondria, with subsequent caspase 9 activation enhancing the
apoptotic signal. The yellow arrows show the targeted elements that have been tested either in clinical and preclinical cervical cancer models studies by
different agents (bold) with the final aim of activating the apoptotic pathway via extrinsic or intrinsic mechanisms.
534 Manzo-Merino et al./ Archives of Medical Research 45 (2014) 525e539

Wnt Signaling Pathway activators such as Wnt ligands, frizzled receptors and
disheveled (Dvl). In cervical cancer cell lines, the overex-
The Wnt signaling pathway is critical in embryonic devel-
pression of Wnt10B-14 (158,159), Fzd-10 (160), and Dvl-
opment and is a large family of secreted growth factors that
1 (161) has been reported.
control cell fate, proliferation, migration, tissue architec-
In a genome expression analysis conducted in HPV-16-
ture, and organogenesis (142,143). Wnt also plays an
associated cervical cancer cases, a significant increase in
important role in adult organisms because it regulates the
Wnt-4, -8a, Fzd2, GDK3b and b-catenin compared with
homeostasis of hematopoiesis (144), angiogenesis (145)
normal cervical epithelia was observed (162).
and adipogenesis (146). Deregulation of the Wnt pathway
Continuous turnover of epithelia is ensured by the self-
has been implicated in cancer (147).
renewal capacity of tissue-specific stem cells (163). In the
It is believed that three different pathways can be
same way, cancer stem cells maintain epithelial tumors
activated upon Wnt receptor activation: the canonical
and b-catenin signaling is essential in sustaining the pheno-
Wnt/b-catenin cascade, the noncanonical planar cell polar-
type of cancer stem cells. In chemically derived mouse skin
ity (PCP) pathway, and the Wnt/Ca2þ pathway. The
tumors, ablation of the b-catenin gene results in the loss of
canonical pathway is the best understood.
cancer stem cells and complete tumor regression (164).
Wnt receptors include the Frizzled family (Fzd) and
Therefore, the b-catenin gene or other elements involved
co-receptors such as the low density lipoprotein
in the Wnt/b-catenin pathway may be targeted to eliminate
receptor-related proteins-5/6 (LRP). Once bound by their
cancer stem cells with the goal of eradicating squamous cell
cognate ligands, the Fzd/LRP co-receptor complex acti-
carcinoma.
vates the canonical pathway, which is characterized by
Multiple therapeutic interventions have been proposed
the accumulation of b-catenin in the cytoplasm and nu-
for the Wnt pathway. Related to cervical cancer, phyto-
cleus. Cytoplasmic b-catenin protein levels are controlled
chemicals with known antioxidant, anti-inflammatory and
by ubiquitination and degradation, which are induced by
chemopreventive properties have been tested in the cervical
phosphorylation by active GSK3b (148). Activation of
cancer-derived cell line, HeLa (165). Naturally occurring
the Wnt signaling pathway inhibits GSK3b and causes
polyphenols have antiproliferative effects through the atten-
the accumulation of b-catenin in the cytoplasm. The
uation of the Wnt pathway by inducing cellular b-catenin
excess of b-catenin promotes its entrance to the nucleus
degradation and a reduction in the nuclear abundance of
where it forms an active transcriptional complex with T-
b-catenin.
cell factor (TCF), which then activates the transcription
Some compounds have been tested in clinical assays for
of target genes including c-myc (149) and cyclin D
solid tumors, trying to reach distinct Wnt regulatory levels
(150,151).
with the use of small molecules, antibodies or peptides
In order for GSK3b to phosphorylate b-catenin, the pres-
(166) directed to different targets such as Wnt ligands, Friz-
ence of adenomatous polyposis coli (APC gene product)
zled receptors or b-catenin. It is clear that due to the
and axin are required (152). PP2A is also necessary because
complexity of Wnt signaling, this pathway cannot be tar-
it inhibits Wnt signaling through its direct interactions with
geted using a single strategy. Additional studies are
APC and axin (153,154). Several mutations in several com-
required to identify possible signaling nodes as targets for
ponents of the Wnt/b-catenin pathway have been studied
Wnt for successful therapeutic interventions in cervical
and identified in a variety of cancers. Wnt signaling may
cancer cells without affecting normal cells (Table 1 and
contribute to the development of cervical carcinoma
Figure 1).
because the activation of the canonical Wnt pathway is
necessary to induce the transformation of HPV immortal-
ized cells (155). Some authors have found that b-catenin
Conclusions and Future Directions
expression is increased in a high proportion of cervical
cancer specimens as assessed by cytoplasmic and nuclear Cervical cancer remains a public health issue among
positive staining, whereas mutations were found in no more women, primarily in developing countries, despite the use
than 20% of the samples (7,156). of the Papanicolaou smear and colposcopy programs.
It has been proposed that b-catenin may be activated by Although the use of screening tests could prevent cervical
the inactivation of negative regulators such as APC and ax- cancer development, 80% of cases are diagnosed in the ter-
in. It is possible that negative regulators of the Wnt/b-cat- minal stages. Surgery, radiation and chemotherapy alone or
enin pathway could be inactivated by methylation in combination are the conventional treatments for such
because the axin and APC genes have enriched CpG islands neoplasms, but some of the cases will persist or recur.
in their promoters, which are hypermethylated in cervical Effective treatment for advanced and/or recurrent cervical
cancer (157). cancer is needed as well as reliable markers for prognosis
Another mechanism involved in the upstream activation related to treatment. Thus, signal transduction elements
of the Wnt/b-catenin pathway is the over-expression of are promising targets for new drugs. Taken together,
 Molecular Targets in Cervical Cancer
preclinical and clinical data suggest that combinatorial ther-
apies will improve outcomes in those patients, and that
progress on this issue will help in managing those patients
in the future. Although some new molecular targeting drugs
have shown to be successful, prevention will always be the
best therapy.
Acknowledgments
This work was partially supported by Consejo Nacional de Cien-
cia y Tecnolog
ıa CB-166808 and CB-151493.
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