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 Cervical cancer is the fourth most common neoplasm in women worldwide. Every year, nearly 529,000 new cases of cervical cancer are diagnosed. Most cases are reported in developing countries where cervical cancer is one of the major causes of cancer mortality in women (1). Epidemiological and molecular data indicate that persistent infection with ‘‘high-risk’’ human papillomavirus (HPV) is a prerequisite for cervical cancer development and that it is associated with other pathologies, such as head and neck cancers and anal cancer (2). More than 100 types of HPV have been identified to date; 35 are classified as high-risk and another 40 as low-risk based on oncogenic potential (3). HPV overexpression of E6 and E7 oncoproteins is the main oncogenic stimulus for cell transformation in cervical cancer. High-risk E6 proteins induce the degradation of Address reprint requests to: Marcela Lizano, Instituto Nacional de Cancerolog
ıa, Av. San Fernando 22, Col. Secci
on XVI, Tlalpan, Mexico City, D.F., 14080 Mexico; Tel: (þ52) (55) 5628-0400 ext.133; FAX: (þ52) (55) 5628-0426; E-mail: [email protected]

important cell cycle regulators such as p53 and PDZ domain containing proteins (4), whereas E7 promotes the inactivation of pRb (5). In addition, other proteins are overexpressed in this neoplasm such as PI3K (phosphatidylinositol 3-kinase), EGF-R, b-catenin, and Erk, and anti-apoptotic proteins such as the Bcl-2 sub-family (6e9). All of these alterations are part of the cervical cancer network and present an opportunity for developing therapies against any of the participating elements. In this review, we highlight the role of these pathways in cervical carcinogenesis, their alterations and the consequences of these abnormalities. In addition, we review the current preclinical and clinical data for designed agents that target elements of these pathways. The conventional treatment of cervical cancer consists of surgery, radiation and chemotherapy alone or in combination (10). Such treatments remain suboptimal, with residual tumors observed in 40e50% of patients with advanced cancer (11,12). Therefore, there is a need for implementing novel therapeutic approaches that, in addition to the standard treatments, could improve the overall survival and 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

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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). Importantly, overexpression or activation of components of this pathway is believed to contribute to tumorigenesis, tumor progression and metastatic disease in a variety of solid tumors (14). The ERK/MAPK pathway lies downstream of various growth factors such as EGF (epidermal growth factor). Growth factor binding results in receptor phosphorylation, 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 specific 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 expression, 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 human 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 cancer (21,22). Therapeutic targets for ERK/MAPK pathway in cervical cancer. Epidermal growth factor receptor (EGFR) overexpression 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 antiEGFR 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 modulator 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 resistance 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 chemically 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 trematinib (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 Signaling pathway

Target

Mechanism of action

Sorafenib

MAPK

Raf kinase

PD98059 U0126

MAPK MAPK

Trematinib

MAPK

Inactive MEK ½ Active and inactive MEK ½ MEK ½

CI-1040

MAPK

MEK

Wortmannin

PI3K

p110

LY294002

PI3K

p110

Quercetin

PI3K

p110

Indole-3-carbinol

PI3K

Akt

MK-2206

PI3K

Akt

Genistein

PI3K

Akt

Oridonin

PI3K

Akt

Tripterygium

PI3K

Akt

Temsirolimus

PI3K

mTOR

Everolimus

PI3K

mTOR

Cetuximab

EGF

EGFR extracellular fraction

Nimotuzumab

EGF

EGFR extracellular fraction

Avoids the ligand binding

Matuzumab Lapatinib

EGF EGF

EGFR extracellular fraction EGFR tyrosine kinase fraction

Avoids the ligand binding Tyrosine kinase inhibitor

Competitive inhibition of the ATP site Protein kinase inhibitors Protein kinase inhibitors Binds and inhibits MEK ½ action Induces decrease in pMAPK levels Irreversible competitor. Competes with the ATP site Reversible competitor. Competes with the ATP site inducing apoptosis Competes with ATP binding Inactivates Akt by demethylation of PTEN. Induces apoptosis Allosteric inhibitor of Akt 1/2 Avoids Akt phosphorylation at Ser473 Reduction of Akt phosphorylation Reduction of Akt phosphorylation Forms a complex with the FK506 binding protein-12 Forms a complex with the FK506 binding protein-12 Avoids the ligand binding

Adjuvant treatment tested Radiotherapy/Cisplatin

Clinical protocol NCT00510250

Cisplatin and Doxorubicin GSK2141795

References Garc
ıa, 2009

NCT01958112

Yeh et al. 2002 Favata et al. 1998 Miller et al. 2014

-

-

Montagut, 2009

Radiotherapy/ Chemotherapy/Roscovitine Radiotherapy

-

Zhang et al. 2009

-

Fuhrman et al. 2007

-

-

-

-

Huangh et al. 2009 De et al. 2000 Bell et al. 2000 Jin et al. 1999 Yuan et al. 1999 Cherrin et al. 2010 Tolcher et al. 2009 Yashar et al. 2005 Su-Hyeong et al. 2009 Hu et al. 2007

-

-

Yang et al. 2006

-

NCT00910884

Radiotherapy

NCT00001696 (Completed for toxicity)

Topotecan Paclitaxel Cisplatin/ Chemotherapy Cisplatin/Topotecan (Toxic) Radiation/Cisplatin Chemotherapy Cisplatin/Gemcitabine, chemoradiotherapy, Carboplatin/Paclitaxel, Brachytherapy Platinum -

NCT01217177 NCT01026792 (Completed) NCT01217177 NCT01026792 (Completed) NCT00957411 NCT00997009

Temkin et al. 2010

Molecular Targets in Cervical Cancer

Therapy

Campone et al. 2009 Hertlein et al. 2010 Kurtz et al.Moore et al. 2012 Santin et al. 2011

NCT02095119, NCT01938105, NCT02083211, NCT02039791, NCT01301612 NCT00430781 (Completed)

Blohmer et al. 2005 Monk et al. 2010 (continued on next page) 527

528

Table 1 (continued ) Signaling pathway

Target

Mechanism of action

Adjuvant treatment tested

Bevacizumab

VEGF

VEGFR-A

Avoids the ligand binding

Fluorouracil/Capecitabine Topotecan/Cisplatin Radiotherapy/Cisplatin

NCT00025233 (Completed) NCT00626561 (Terminated) NCT00803062

Pazopanib

VEGF

Tyrosine kinase inhibitor

Lapatinib/Chemotherapy

NCT00430781

Lapatinib

EGF

Imatinib Gefitinib

PDGF EGF

Erlotinib

EGF

3,3-diindolylmethane

MAKP PI3K

VEGF tyrosine kinase fraction EGFR tyrosine kinase fraction PDGFR kinase fraction EGFR tyrosine kinase fraction EGFR tyrosine kinase fraction Several

Retinoids

Apoptosis

Fas/FasL EGF,

N101-2

PI3K/Akt Extrinsic apoptosis

Fas/FasL PI3K/Akt

Silibilin

Not yet elucidated

Ganoderic acid derivates

Intrinsic apoptosis

Cell cycle-dependent kinases Not known yet

Recombinant human TRAIL Luteolin

Apoptosis

Death receptor 4/5

Apotptosis

DR5

Aspirine

Intrinsic apoptosis

Not specified

Arsenic trioxide

Multiple signaling pathways

E6

Avenanthramide 2p

Wnt/b-catenin

b-catenin

Triptolide

Wnt/b-catenin

b-catenin

Tyrosine kinase inhibitor

-

Tyrosine kinase inhibitor Tyrosine kinase inhibitor

-

Tyrosine kinase inhibitor Downregulates elements of the PI3K and MAPK pathways Induces FasL. Deregulates EGF pathway Arrests cell cycle at sub-G1 phase. Induces apoptotic cell death Induces G arrest and apoptotic cell death Decrease mitochondrial membrane potential Binds the DR directly and acts as an agonist Induces Bid cleavage and caspase-8 activation Suppresses the activation of Erk 1/2 Induces a reduction in E6 protein levels with cell cycle arrest in G2-M in HPV expressing cells Induces b-catenin degradation and avoids its nuclear accumulation Induces b-catenin degradation and avoids its nuclear accumulation

Cisplatin/Radiotherapy

Clinical protocol

References Wright et al. 2006 Monk et al. 2009 Zighelboim et al. 2013 Schefner et al. 2011 Monk et al. 2010

-

Monk et al. 2010

-

Taja et al. 2006 Goncalves et al. 2008

NCT00049556 (Completed) NCT00031993 (Completed)

Nogueira et al. 2008 Schilder et al. 2009 Zhu et al. 2012

-

-

-

-

-

-

Sun et al. 2000 Hembree et al. 1996 Kim et al. 2012

-

-

Zhang et al. 2012

-

-

Liu et al. 2012

Radiation

-

Maduro et al. 2008

rhTRAIL

-

Horinaka et al. 2005

TRAIL

-

Im and Jang, 2012

NCT00005999 (Completed)

Wen et al. 2012

-

-

Wang et al. 2012

-

-

Wang et al. 2012

Different elements designed to block signal transduction through inhibiting certain elements are presented. Current preclinical and clinical evaluations of such compounds are cited.

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

Therapy

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 chromosome (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 suggests 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 intervention (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 tumors (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 tumorigenesis 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 human 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 phosphorylation (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

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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 prototypal 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 prevention 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 demethylation 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 papillomatosis (RRP) with promising outcomes (64). Once the PI3K/Akt pathway is activated the number of outputs increases as the signal moves down the pathway. Inhibiting 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 naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed (67). MK-2206 is a compound from this class of Akt inhibitors. Treatment of the cervical 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 MK2206 is an effective therapeutic drug (69). Banerjee et al. found that genistein (40 ,5,7trihydroxyisoflavone) 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 products (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 rubescens (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 naturally 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 recognized 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 combination with topotecan in a variety of gynecological malignancies 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 antiangiogenic 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 proliferation 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 epiregulin. These proteins are synthesized as transmembrane precursors and are proteolytically cleaved by metalloproteases to release the mature growth factor in an autocrine or paracrine manner (84). Extracellular ligand binding causes dimerization of the EGFR, which becomes autophosphorylated at distinct tyrosine residues by the receptor’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 proteins are cell surface receptors, they can be directly targeted. The relationship between EGFR expression and cervical cancer 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 smallmolecule, 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 associated with poor survival rates in which IMD acted as a prognostic marker (96). Platelet-derived growth factor. Platelet-derived growth factor (PDGF) is a very strong stimulator of blood vessel formation 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 cancer. Cetuximab is a human/murine chimeric immunoglobulin 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 combination 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-topotecancetuximab 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 cetuximab 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 epithelial 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 monoclonal 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 clinical 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 bevacizumab (111). Another study showed that the combination of bevacizumab with topotecan and cisplatin induces an improvement in both the overall survival and progression-

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free survival of patients with recurrent or persistent cervical cancer (112). Another study of bevacizumab in combination with radiotherapy and cisplatin showed a very safe profile 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 lapatinib, 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 superiority 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 pathways 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 deathinducing ligand binds to the death receptors (DR). Upon binding, caspases (cysteine aspartyl-specific proteases) 8 and 10 become activated which, in turn, activates caspases 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 proapoptotic 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 substrates, 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 initiation 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 ligases, 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 oncogenes supporting proliferation, immortalization, transformation and finally cancer (122). Cervical cancer is not an exception, with a disruption in the apoptotic pathway primarily 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 abnormality 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 cervical 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 interfering 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

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have been or are being tested. Several approaches have successfully 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 radiotherapy 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

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and IGF signaling pathways (137), induction of Fas and increase 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 cancer 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 caspase 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 receptors (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.

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Wnt Signaling Pathway The Wnt signaling pathway is critical in embryonic development and is a large family of secreted growth factors that control cell fate, proliferation, migration, tissue architecture, and organogenesis (142,143). Wnt also plays an important role in adult organisms because it regulates the homeostasis of hematopoiesis (144), angiogenesis (145) and adipogenesis (146). Deregulation of the Wnt pathway has been implicated in cancer (147). It is believed that three different pathways can be activated upon Wnt receptor activation: the canonical Wnt/b-catenin cascade, the noncanonical planar cell polarity (PCP) pathway, and the Wnt/Ca2þ pathway. The canonical pathway is the best understood. Wnt receptors include the Frizzled family (Fzd) and co-receptors such as the low density lipoprotein receptor-related proteins-5/6 (LRP). Once bound by their cognate ligands, the Fzd/LRP co-receptor complex activates the canonical pathway, which is characterized by the accumulation of b-catenin in the cytoplasm and nucleus. Cytoplasmic b-catenin protein levels are controlled by ubiquitination and degradation, which are induced by phosphorylation by active GSK3b (148). Activation of the Wnt signaling pathway inhibits GSK3b and causes the accumulation of b-catenin in the cytoplasm. The excess of b-catenin promotes its entrance to the nucleus where it forms an active transcriptional complex with Tcell factor (TCF), which then activates the transcription of target genes including c-myc (149) and cyclin D (150,151). In order for GSK3b to phosphorylate b-catenin, the presence of adenomatous polyposis coli (APC gene product) and axin are required (152). PP2A is also necessary because it inhibits Wnt signaling through its direct interactions with APC and axin (153,154). Several mutations in several components of the Wnt/b-catenin pathway have been studied and identified in a variety of cancers. Wnt signaling may contribute to the development of cervical carcinoma because the activation of the canonical Wnt pathway is necessary to induce the transformation of HPV immortalized cells (155). Some authors have found that b-catenin expression is increased in a high proportion of cervical cancer specimens as assessed by cytoplasmic and nuclear positive staining, whereas mutations were found in no more than 20% of the samples (7,156). It has been proposed that b-catenin may be activated by the inactivation of negative regulators such as APC and axin. It is possible that negative regulators of the Wnt/b-catenin pathway could be inactivated by methylation because the axin and APC genes have enriched CpG islands in their promoters, which are hypermethylated in cervical cancer (157). Another mechanism involved in the upstream activation of the Wnt/b-catenin pathway is the over-expression of

activators such as Wnt ligands, frizzled receptors and disheveled (Dvl). In cervical cancer cell lines, the overexpression of Wnt10B-14 (158,159), Fzd-10 (160), and Dvl1 (161) has been reported. In a genome expression analysis conducted in HPV-16associated cervical cancer cases, a significant increase in Wnt-4, -8a, Fzd2, GDK3b and b-catenin compared with normal cervical epithelia was observed (162). Continuous turnover of epithelia is ensured by the selfrenewal capacity of tissue-specific stem cells (163). In the same way, cancer stem cells maintain epithelial tumors and b-catenin signaling is essential in sustaining the phenotype of cancer stem cells. In chemically derived mouse skin tumors, ablation of the b-catenin gene results in the loss of cancer stem cells and complete tumor regression (164). Therefore, the b-catenin gene or other elements involved in the Wnt/b-catenin pathway may be targeted to eliminate cancer stem cells with the goal of eradicating squamous cell carcinoma. Multiple therapeutic interventions have been proposed for the Wnt pathway. Related to cervical cancer, phytochemicals with known antioxidant, anti-inflammatory and chemopreventive properties have been tested in the cervical cancer-derived cell line, HeLa (165). Naturally occurring polyphenols have antiproliferative effects through the attenuation of the Wnt pathway by inducing cellular b-catenin degradation and a reduction in the nuclear abundance of b-catenin. Some compounds have been tested in clinical assays for solid tumors, trying to reach distinct Wnt regulatory levels with the use of small molecules, antibodies or peptides (166) directed to different targets such as Wnt ligands, Frizzled receptors or b-catenin. It is clear that due to the complexity of Wnt signaling, this pathway cannot be targeted using a single strategy. Additional studies are required to identify possible signaling nodes as targets for Wnt for successful therapeutic interventions in cervical cancer cells without affecting normal cells (Table 1 and Figure 1).

Conclusions and Future Directions Cervical cancer remains a public health issue among women, primarily in developing countries, despite the use of the Papanicolaou smear and colposcopy programs. Although the use of screening tests could prevent cervical cancer development, 80% of cases are diagnosed in the terminal stages. Surgery, radiation and chemotherapy alone or in combination are the conventional treatments for such neoplasms, but some of the cases will persist or recur. Effective treatment for advanced and/or recurrent cervical cancer is needed as well as reliable markers for prognosis related to treatment. Thus, signal transduction elements are promising targets for new drugs. Taken together,

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preclinical and clinical data suggest that combinatorial therapies 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 Ciencia y Tecnolog
ıa CB-166808 and CB-151493.

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