Serous ovarian cancer signaling pathways.

REVIEW ARTICLE

Serous Ovarian Cancer Signaling Pathways Ioannis C. Kotsopoulos, MD,* Alexios Papanikolaou, MD, PhD,Þ Alexandros F. Lambropoulos, PhD,Þ Konstantinos T. Papazisis, MD, PhD,þ Dimitrios Tsolakidis, MD, PhD,Þ Panagiota Touplikioti, PhD,§ and Basil C. Tarlatzis, MD, PhDÞ

Abstract: Ovarian cancer is the most lethal malignancy of the female genital tract, mainly due to the failure of early diagnosis and the limitations posed by the conventional chemotherapies. Current research has focused in the study of cascades of various cellular molecular reactions, known as signaling pathways. In this review article, authors try to describe the current knowledge regarding the signaling pathways that influence multiple cellular processes in serous ovarian cancer and especially the pathogenesis. Thorough understanding of the precise role of these pathways can lead to the development of new and more effective targeted therapies as well as novel biomarkers in ovarian cancer. Key Words: Serous ovarian cancer, Signaling pathways, Pathogenesis Abbreviations: BRCA-1, BRCA-2 - breast cancer 1, breast cancer 2, KRAS - Kirsten rat sarcoma viral oncogene homolog, BRAF - v-Raf murine sarcoma viral oncogene homolog B, ERBB2 - v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2, CTNNB1 catenin (cadherin-associated protein), A1, PTEN - phosphatase and tensin homolog, PIK3CA phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit >, ARID1A - AT rich interactive domain 1A (SWI-like), PPP2R1A - protein phosphatase 2 regulatory subunit A>, TP53 - tumor protein p53, CA-125 - cancer antigen 125 or carbohydrate antigen 125 or mucin 16, cell surface associated, E2F3 - E2F transcription factor 3, NICD - intracellular domain of the Notch, Wnt - wingless-type, MMTV integration site family, DSH - dishevelled, GSK-3 glycogen synthase kinase 3, FGF9 - fibroblast growth factor 9, Smo - smoothened, Gli1 - GLI family zinc finger 1, AKT - v-akt murine thymoma viral oncogene homolog, mTOR mechanistic target of rapamycin (serine/threonine kinase), BMP - bone morphogenetic protein, EGF - epidermal growth factor, TGF-> - transforming growth factor >, c-kit - v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog, RTK - receptor tyrosine kinases, LPA - lysophosphatidic acid, VEGF - vascular endothelial growth factor, NF-JB - nuclear factor JB light-chain enhancer of activated B cells, Edg2 - lysophosphatidic acid receptor 1, MAPK mitogen-activated protein kinase, IL-6 - interleukin 6, Jak/STAT3 - Janus kinase/signal transducer and activator of transcription 3, IL-6 - interleukin 6, GRB2 - factor receptor-bound protein 2, GEFs - guanine nucleotide exchange factors, SOS - son of sevenless, GTP guanosine-5¶-triphosphate, ERK - elk-related tyrosine kinase, EMT - epithelial-mesenchymal transition, ET-1 - endothelin 1, c-MET - met proto-oncogene, CSCs - cancer-stem cells

*Obstetrics & Gynecology Department, Medical School, University of Patras, Rion; and †1st Obstetrics & Gynecology Department, Medical School, ‘‘Aristotle’’ University of Thessaloniki; ‡Medical Oncology Department, ‘‘Euromedica’’ General Clinic; and §Cytology Department, Copyright * 2014 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000079

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‘‘Theagenio’’ Cancer Hospital, Thessaloniki, Greece. Address correspondence and reprint requests to Ioannis C. Kotsopoulos, MD, Obstetrics & Gynecology Department, Medical School, University of Patras, Rion, Greece; and Tyrnovou 14, 53100, Florina, Greece. E-mail: [email protected]. No funding was received for this study. The authors declare no conflicts of interest.

International Journal of Gynecological Cancer

& Volume 24, Number 3, March 2014

Copyright © 2014 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.


& Volume 24, Number 3, March 2014 Serous Ovarian Cancer Signaling Pathways

Received October 2, 2013, and in revised form November 30, 2013. Accepted for publication December 1, 2013. (Int J Gynecol Cancer 2014;24: 410Y417)

cancer is the most lethal gynecological maligO varian nancy. It accounts for 3% of cancers in women, being

ninth in frequency after cancers of the breast, lung, colon, uterus, thyroid, melanoma, non-Hodgkin lymphoma, and the kidney.1 It also accounts for 31% of the female genital tract malignancies. These tumors consist of a heterogeneous variety of histological types with differences in clinical manifestation, physical history, and biological and genetic background. From the pathological view, serous, mucinous, endometrioid, Brenner, clear cell, squamous, mixed, and undifferentiated are the types of epithelial carcinomas and account for about 90% of the ovarian tumors. The rest 10% of the tumors are classified into germ cell, sex-cord stromal, metastatic, and other extremely rare histological types (eg, sarcomas). Serous carcinoma is the most common epithelial histological type.2 It is well known that about 5% to 10% of ovarian carcinomas result from an inherited predisposition. Mutation in the BRCA-1 and -2 tumor suppressor genes is the most common cause of hereditary ovarian carcinogenesis.3 Apart from this well-studied role of BRCA in hereditary ovarian cancer, there are now clear indications that mutations of these genes are also involved in sporadic cancers. Actually, it seems that epigenetic mechanisms, such as transcriptional silencing of the gene promoter or gene dysfunctions within BRCA-associated pathways, could lead to the pathogenesis of sporadic ovarian cancers. So, almost one third of all ovarian cancers show a ‘‘BRCAness’’ phenotype because of genetic and epigenetic changes.4 For many years, the origin of epithelial ovarian cancer was thought to be the ovarian surface epithelium, also named coelomic epithelium or modified mesothelium.5 However, according to the dualistic model, there are at least 2 different molecular pathways that lead to the ovarian cancer pathogenesis. The so-called type I ovarian cancer includes lowgrade serous, low-grade endometrioid, clear cell, mucinous, and transitional carcinomas. These tumors are generally indolent, usually presented in early stages, and are characterized by specific mutations, including Kirsten rat sarcoma viral oncogene homolog (KRAS); v-Raf murine sarcoma viral oncogene homolog B (BRAF); v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2); catenin (cadherinassociated protein), A1 (CTNNB1); phosphatase and tensin homolog (PTEN); phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit > (PIK3CA); AT-rich interactive domain 1A (SWI-like) (ARID1A); and protein phosphatase 2 regulatory subunit A > (PPP2R1A), but lack of tumor protein p53 (TP53) mutations. They seem to originate from the ovarian epithelium. On the other hand, type II tumors include highgrade serous carcinoma, high-grade endometrioid, undifferentiated carcinoma, and malignant mixed mesodermal tumors

(carcinosarcoma). These tumors exhibit an aggressive behavior, are genetically highly unstable, and are presented in advanced stages. The majority of them (about 80%) express p53 mutations, but rarely harbor the mutations detected in type I tumors. Although it is now widely accepted that endometriosis is the precursor of endometrioid and clear cell carcinomas, serous carcinomas seem to originate from malignant cells of the fallopian tube. These cells originate from ‘‘serous tubal intraepithelial carcinoma.’’ They are implanted after exfoliation on ovarian epithelium deficits, generated after ovulation, giving rise to serous ovarian carcinoma.6,7 In addition, precursor benign lesions called ‘‘serous tubal intraepithelial lesions’’ or ‘‘tubal intraepithelial lesions in transition’’ have also been described.8 Going deeper in this theory, it has been couched that the so called ‘‘tubal carcinogenic pathway’’ implicates p53 and BRCA mutations as well as BRCA dysfunctions, leading to the sequence of serous tubal intraepithelial lesions, serous tubal intraepithelial carcinoma, and finally ovarian cancer or peritoneal carcinoma. On the other hand, in the ‘‘ovarian carcinogenic pathway,’’ the cells of cortical inclusion cysts formulated after ovulations undergo dysplastic proliferation and finally malignant transformation. As a result of the above, our perceptions regarding pelvic surgery alters.8 The role of fallopian tubes and fimbriae, in the management of ovarian cancer and especially in fertility-sparing surgery, should be redefined, as specific group of patient could probably benefit by fimbriectomy.9 Salpingectomy with ovarian conservation, as a preventive method in high-risk women, is a very attractive, reasonable, and practicable model, but requires further studies. However, the superiority of salpingectomy versus tubal ligation, in terms of both effectiveness and cancer prevention, seems to be confirmed.8 The present article focuses mainly to the most common and lethal subtype, the serous ovarian carcinomas. Despite the significant improvements that were achieved in understanding the biology and pathophysiology of ovarian cancer during the past decades, diagnosis still remains a difficult issue. The majority of the patients have intraperitoneal dissemination of the disease at the time of diagnosis.10 The lack of specific clinical symptoms and the absence of an effective screening protocol make early diagnosis difficult, leading to a high mortality rate. CA-125 (cancer antigen 125 or carbohydrate antigen 125 or mucin 16, cell surface associated) and transvaginal ultrasound are characterized by high false-positive results, and routine annual pelvic examinations could not detect early stages of the disease.11 A thorough surgical staging combined with cytoreductive surgery and neoadjuvant or adjuvant chemotherapy consists the criterion standard of treatment management. Despite the advantages in these fields during the past decades, reflecting an overall statistical improvement in survival for

* 2014 IGCS and ESGO


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all stages (from 29.8% in 1976Y1978 to 49.7% in 1999Y2001), the absolute number of treatment failures still remains high.12 It seems reasonable that the research and development of new sensitive and specific biomarkers are imperative to ensure timely diagnosis of the disease. A number of markers have been indentified and among them human epididymis protein 4 is the relatively most well studied. However, the understanding of the pathogenesis of ovarian cancer at the cellular level cannot be seen without clarification of the molecular pathways involved. Moreover, considering the limitations of the traditional chemotherapies, the focus on therapies based on signaling pathways, also known as targeted therapies, seems to be promising.

MATERIALS AND METHODS We searched in PubMed (US National Library of MedicineVNational Institutes of Health) all articles published until September 2013, focusing on review and original articles regarding mainly serous ovarian carcinoma and especially the pathogenetic processes. We used the combination of the medical heading terms ‘‘serous ovarian cancer’’ and ‘‘signaling pathways.’’ We processed the results, and in some cases we re-search the library using more specific terms on a specific signaling pathway. Articles mainly regarding nonepithelial ovarian cancer were excluded.

MAIN SIGNALING PATHWAYS IN SEROUS OVARIAN CANCER The term signaling pathways refers to molecular paths involved in multiple cellular functions such as proliferation, differentiation, maturation, and apoptosis of the cells. Diversion from their normal function, such as overexpression,


up-regulation or down-regulation, consists the base of multiple theories of tumorigenesis. It is also now well known that ovarian cancer is a genetically complex malignancy that involves alterations in numerous genes. It was found that about 1191 genes are differentially expressed between normal ovarian epithelium and papillary serous ovarian carcinoma.13 The activation (eg, through gene amplification) and the inactivation (through deletion of chromosomal regions, loss of heterozygosity, mutations, and promoter hypermethylation) in a number of these oncogenic or tumor suppressor genes lead to a high genetic instability and cause the genetic polymorphism that characterize ovarian cancer.10 In the present review, an attempt is made to describe both the most important (Fig. 1) and other less well-studied signaling pathways involved in different mechanisms of serous ovarian cancer and especially the pathogenesis. Obviously, isolated molecules implicated in various steps of tumorigenesis are not included at this study.

Notch Notch signaling is a pathway implicated in cellular differentiation, proliferation, and apoptosis. Through this pathway, cells can communicate with neighbor cells, intervening in this way in cellular fate. In human, there are 4 different Notch cellular membrane receptors, called Notch 1Y4. Each receptor is constructed from a large transmembrane protein consisting of an extracellular, a transmembrane, and an intracellular domain. On the other hand, special molecules, called ligands, bind to the extracellular domain, giving rise to 1 of the 3 cleavage events of the Notch protein (the other 2 include the first cleavage during Notch biosynthesis and the third cleavage induced by F-secretase).14 Jagged-1 and -2 and deltalike 1, 3, and 4 are the main ligands in human.15 A second

FIGURE 1. Rough simplified representation of the main signaling pathways implicated in ovarian cancer. Diversion from the normal expression in some points of these paths could lead to various diseases including cancer. (For the references and abbreviations see the text and the lists.)

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cleavage event induced by a metalloprotease releases the extracellular domain,16,17 whereas a third cleavage in the transmembrane portion by the enzyme F-secretase releases the intracellular domain of the Notch (NICD).14 Finally, this domain translocates to the nucleus and induces various cellular processes. From the oncological view, Notch behavior is paradoxical, acting like tumor suppressor or oncogene, depending on the cancer type. The first indication of Notch correlation with ovarian cancer began to emerge through a microarray study performed to identify genes that differentially expressed in normal ovarian epithelium and ovarian cancer.14 Among about 44,500 genes, Notch 3 was identified along with other 3 genes (E2F transcription factor 3, GTPase-activating protein, and hematological and neurological expressed 1) to be at least 3-fold up-regulated.18 The Notch-3 protein could be recognized by immunohistochemistry both in the nucleus and in the cytoplasm of malignant cells, and it seems to play an important role in the development and survival of the tumor.19 Moreover, the blockage of the pathway with the use of both F-secretase inhibitors and specific small interfering RNA (siRNA) leads to a decreased cellular proliferation and induces apoptosis in cells that overexpressed Notch-3.19 These findings support the hypothesis that Notch-3 is required for proliferation and survival of the Notch3-overexpressing tumors and that this pathway could be a promising therapeutic target.19 In addition, the Notch-3 overexpression has been correlated to carboplatin resistance and is also implicated in ovarian cancer recurrence leading to a more aggressive phenotype and poorer prognosis.20 Jagged-1 was found to be the main Notch-3 ligand, and the interaction between these 2 proteins promotes ovarian cancer cell proliferation and intraperitoneal dissemination.21 Notch-3 activation enhances a positive regulatory loop inducing Jagged-1 expression, and thus the Notch-3 overexpression causes ligand up-regulation. Similar activity observed in another signaling pathway, the Wnt (wingless-type MMTV integration site family)/A-catenin. These results of Jagged-1 dual regulation suggest that a possibly effective therapy should target both pathways.22 In addition, Jagged-1 silencing through siRNA causes inhibition of endothelial cell migration and tube formation, suggesting a possible role of Notch-3 pathway in tumor angiogenesis.14

Wnt/A-Catenin Wnt signaling pathway plays an important role in cellcell communication and also in multiple cellular procedures during embryonic and adult life, such as differentiation, proliferation, regulation of cell cycle, cellular adhesion, and also cell polarity generation.23 Wnt group of protein includes various secreted lipid-modified signaling proteins.24 Their interaction with serpentine receptors of the Frizzled (Fz) family and members of the low-density lipoproteinYrelated protein family causes initially the activation of a molecules called dishevelled (DSH), then the inhibition of various other molecules such as Axin, glycogen synthase kinase 3 (GSK-3), and the protein adenomatous polyposis coli and finally the stabilization and the nuclear translocation of A-catenin, leading to the expression of specific target genes.25 Two distinguished

pathways could be identified, named canonical and noncanonical Wnt pathway. Each pathway implicates different ligands and related molecules. Abnormal activation of Wnt/A-catenin signaling pathways has been related to multiple diseases such as congenital malformations, cancer, and osteoporosis.26,27 In ovarian carcinoma, this pathway and especially A-catenin mutations are associated with the endometrioid subtype.28,29 Recent studies implicate the overexpression of 2 Wnt target genes, Axin2, and fibroblast growth factor 9 (FGF9), in the development of serous ovarian carcinoma. The messenger ribonucleic acid (mRNA) of these genes is found to be increased in serous papillary ovarian cancers about 55-fold (for Axin2) and 1600-fold (for FGF9).30

Hedgehog The hedgehog signaling is an important pathway in human cells and plays a key role in embryogenesis, involved in stem cell fate, organogenesis, and in proliferation, regeneration, and differentiation of somatic tissues in the adult.31,32 Hedgehog group of proteins contains 3 members named sonic hedgehog, Indian hedgehog, and desert hedgehog. The pathway also includes 2 membrane proteins, the patched-1 receptor that inhibits the second protein called smoothened (Smo). The interaction between the hedgehog proteins that act like ligands and the receptor (patched-1) abrogates its suppressor action on Smo and causes the nuclear translocation of a molecule, the GLI family zinc finger 1 (Gli1). Finally, the latter activates the target genes.33,34 Aberrant activation of the hedgehog signaling pathways seems to be implicated with the epithelial ovarian cancer pathogenesis and tumor cell proliferation, although among studies differences in the expression rate from 20% to 60% were observed.35Y37 Changes in the expression of Gli1, Smo, Ptch1, desert hedgehog, and sonic hedgehog have been described. Overexpression of patched-1 and Gli1 is associated with a poor prognosis.38 Unlike normal tissue, benign and borderline ovarian tumors, sonic hedgehog mRNA is highly expressed in 57.7% of the ovarian malignancies especially in endometrioid and clear cells carcinomas. This percentage is lower (25%) in serous and mucinous carcinomas.38 Moreover, ectopic expression of Gli1 has been associated with cancer cell proliferation, invasiveness, and mobility.38 Regarding the Smo, especially in serous ovarian carcinomas, no clear expression increase was observed, leading to the deduction that hedgehog pathway could be activated without the involvement of Smo.30 Interestingly, patched-1 is down-regulated in ovarian carcinoma, in direct contrast to the overexpression observed in other solid tumors.39 From the therapeutic point, a number of agents have already been tested. Cyclopamine, a hedgehog pathway inhibitor, suppresses the proliferation and clonal growth of ovarian malignant cells in vitro and arrests ovarian tumor growth in vivo.39 Gli1 siRNA could also lead to proliferation arrest.37 Moreover, 2 antagonists of the pathways, named hedgehog interacting protein and growth arrestYspecific 1, are downexpressed in ovarian cancer.40,41 In conclusion, hedgehog signaling pathway is activated in ovarian carcinoma and plays an



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important role in carcinogenesis, and its inhibition could be a promising therapeutic target.

PTENYPI3K/AKT/mTOR Pathway Bone morphogenetic protein (BMP), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor > (TGF->) are the main ligands of the pathway. On the other hand, epidermal growth factor receptor (EGFR), IGFR-1 (insulinlike growth factor [IGF] 1 receptor) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (c-kit) membrane receptors are the receptors. The junction leads to the activation of a number of processes such as amplification of PIK/AKT2 gene, activating mutations in PI3CA gene, signal transmission through receptor tyrosine kinases (RTK) of growth factors, and inactivating mutations in PTEN gene. Finally, these alterations cause cellular proliferation by inactivation of the cell cycle inhibitors and inhibition of the proapoptotic genes.10 Phosphatase and tensin homolog (PTEN) is a dualspecificity phosphatase, with both protein phosphatase and lipid phosphatase activity, encoded by the PTEN gene. It plays a key role in apoptosis, cell cycle arrest, and possibly cell migration. It is one of the known tumor suppressor genes, whose inactivating mutations are associated with the majority of the malignancies in human, in both solid tumors and hematological.42 Phosphatidylinositide 3-kinases (PI3-kinases or PI3Ks) are a family of enzymes of the eukaryotic cell membranes. These types of proteins are involved in multiple cellular processes such as proliferation, survival, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function.43 AKT, or protein kinase B, is a serine/threonine-specific protein kinase and has been shown to be amplified or up-regulated in ovarian cancer and especially in tumorigenesis.44Y46 The PTEN-PI3K/AKT/mTOR signaling pathway seems to play an important role in cell cycle progression, survival, motility, angiogenesis, and immune surveillance.10 It is implicated in pathogenesis of about 70% of clear cell and endometrioid ovarian carcinomas47,48 and also in cell migration and invasion.49Y51 In serous ovarian cancer, multiple components of the pathway are amplified, and the resultant activation leads to cell-cycle progression, decreased apoptosis, and increased metastatic capabilities.52,53

Lysophosphatidic Acid Lysophosphatidic acid (LPA) is a phospholipid derivative that is the ligand of the homonym signaling pathway. LPA1/lysophosphatidic acid receptor 1 (Edg2), LPA2/Edg-4, and LPA3/Edg-7 are the main membrane receptors of the path. The junction between ligand-receptor activates a cascade of events that causes cell proliferation, promotes tumor angiogenesis (through vascular endothelial growth factor [VEGF] and interleukin 8 [IL-8]), up-regulates cyclooxygenase 2 expression, and activates matrix metalloproteinase.10 The main role of the pathway is the regulation of gene expression in both normal and neoplastic cells.54 In ovarian cancer, the pathways seems to be implicated in the pathogenesis of about 90% of the tumors, in almost all the stages of tumorigenesis, from initiation to progression and metastasis.47,55

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Nuclear Factor JB Light-Chain Enhancer of Activated B Cells

Nuclear factor J light-chain enhancer of activated B cells (NF-JB) is a key protein in immune response regulation. Proinflammatory cytokines such as IL-1 and tumor necrosis factor > as well as various growth factors bind to endothelial growth factor receptors directly activating the signaling pathway. However, the activation of the path could also be achieved indirectly though other pathways such as mitogenactivated protein kinase kinase kinase 3/mitogen-activated protein kinase (MAPK). The final downstream effects include decreased apoptosis and increase of proliferation, angiogenesis, inflammation, and activation of antioxidant proteins.10 Cell survival, angiogenesis, and metastasis through gene regulation are the main actions of the pathway. In ovarian cancer, it is implicated in the pathogenesis of more than 50% of the tumors.10,47,56,57

IL-6/Janus KinaseYSignal Transducer and Activator of Transcription Among various tumorigenic theories in ovarian cancer, immunopathogenesis plays a key role. According to this hypothesis, immune deficiencies presented in the ovarian tumor environment contribute to the development of ovarian carcinoma. These deficiencies include the presence of regulatory T cells, the inhibition of natural killer cytotoxic response, the accumulation of myeloid suppressor cells in the neoplasm, the abnormalities on interferon signaling, the expression of membrane molecules, and finally the secretion of cytokines that promote tumor growth.58 One of the most important aforementioned molecules is IL-6. Interleukin 6 is the main ligand of a signaling pathway that implicates the activation of LPA and NF-JB as well as the Ras signaling pathways.10 The interaction between ligand (IL-6) and receptor (IL-6R> and a signal transducer named gp130) induces receptor dimerization, activating downstream molecules such as Jak and STAT3 (Janus kinaseYsignal transducer and activator of transcription). The pathway regulates multiple cellular functions such as survival, proliferation, and differentiation. The activated Jaks could also induce the cascade of various signaling pathways such as MAPK and PI3K/AKT.59 In ovarian cancer, the pathway is implicated in neovascularization by inhibition of endothelial cell proliferation.10 There is evidence for involvement in the tumorigenesis of 70% of ovarian carcinomas.47,60

Mitogen-Activated Protein Kinase Epidermal growth factor and Trk A/B are the main ligands of the path. The ligand binds to the corresponding receptor named EGFR.10 Downstream molecules include growth factor receptorYbound protein 2 and GEFs (guanine nucleotide exchange factors) SOS (Son of Sevenless). The activation of the latter, promote the removal of guanosine diphosphate from the K-ras molecule. The junction of the Ras with guanosine5¶-triphosphate activates the Ras.61,62 The activated Ras, subsequently activates various intracellular molecules such as the protein kinase activity of RAF kinase, MEK, and * 2014 IGCS and ESGO




finally MAPK.63 Mitogen-activated protein kinase could activate through phosphorylation and various transcription factors. This pathway, otherwise known as Ras-Raf-MEK-ERK (elk-related tyrosine kinase) pathway, is implicated in cell growth, division, differentiation, survival, and death.10,64 In ovarian cancer, MAPK causes the up-regulation of CCND1 (the gene encoding G1/S-specific cyclin-D1), COBRA 1, and transglutaminase 2 that provoke uncontrolled proliferation.10 The pathway is implicated in the tumorigenesis of less than 50% of ovarian cancer and mainly of low-grade micropapillary serous and serous borderline tumors.47,65 In high-grade serous carcinomas, the pathway is less active, and its activation serves as a good prognostic factor in patients with advancedstage high-grade serous cancer.66 In tumors with K-ras and B-raf mutation, inactivation of MAPK path could be a potential target-based therapy.67

Transforming Growth Factor A

Transforming growth factor A signaling pathway displays the paradox of the dual role, both as promoter and tumor suppressor. Thus, in early stages, it stimulates cell death, whereas in advanced stages, it promotes oncogenesis.68 The path is implicated in proliferation, migration, and apoptosis as well as intervenes in the modification of cell shape.69 Transforming growth factor A acts like ligand and binds with the corresponding cellular membrane receptors and activates both SMAD and non-SMAD pathways. Both TGF-A and its receptor are usually expressed in low levels in normal cells, but are up-regulated in ovarian cancer.70 Especially the TGF-A1 has been associated with the proliferation and progression of epithelial ovarian cancer. In serous and endometrioid carcinomas, TGF-A1 mRNA is less expressed compared with clear cell and mucinous tumors. It is suggested that TGF-A1 mRNA expression could identify the tumor’s aggressiveness and predict the prognosis of patients with ovarian cancer.71 Moreover, it seems that it could be used as a marker or mediator of chemoresistance in advanced serous ovarian cancer.72

SECONDARY SIGNALING PATHWAYS IN SEROUS OVARIAN CANCER The term ‘‘secondary’’ is used in this study in order to include signaling pathways that are either less important in various processes of ovarian cancer and especially in oncogenesis or, mainly, are less well studied. Thus, the known data regarding these paths are limited. Much of these are implicated to the epithelial-mesenchymal transition (EMT), which is correlated to ovarian carcinogenesis.10 Through this process, epithelial cells undergo a series of cellular events that lead to the loss of their epithelial characteristics. The cells assume de novo a mesenchymal cell phenotype characterized by an increase in migration abilities, invasion, and apoptosis resistance.10,73

Hepatocyte Growth Factor A specific tyrosine-kinase, named met proto-oncogene (c-MET), is the receptor of the plasminogen-like protein HGF.10,74 The pathway that is mediated from HGF is not autonomous, but activates other paths such as MAPK, PI3K,

and AKT and thus causes the effects promoted from these pathways (see above).68

Endothelin 1 The compound of the ligand (ET-1) with the receptor (ETA or ETB) activates the pathway, which then acts through various other paths such as MAPK, PI3K/Akt, and integrinlinked kinase.75,76 In ovarian cancer, the activation of the path down-regulates E-cadherin; increases levels of A-catenin, Snail, and other mesenchymal markers; and promotes EMT.76

Estrogen Pathway

17A-Estradiol binding to the estrogen receptor > promotes the inhibition of E-cadherin and the overexpression of Snail and Slug, enhancing metastatic potential of ovarian malignant cells.77,78 Thus, the pathway can potentiate tumor progression by EMT induction.77 On the other hand, ERA acts as antagonist.10

Bone Morphogenic Protein 4 Bone morphogenic proteins are the larger member of the TGF-A protein superfamily. This group includes about 30 cytokines that signal through transmembrane serine/ threonine kinase receptors.79 Bone morphogenic protein 4 signaling pathway is implicated in various cellular processes such as self-renewal, proliferation, differentiation, apoptosis, and quiescence.80 Metabolic diseases such as obesity, diabetes, vascular calcification, hereditary hemorrhagic telangiectasia, and pulmonary hypertension as well tumorigenesis are related to dysregulation of BMP pathways.81 In primary ovarian cancer, BMP-4 signaling pathway is related to the EMT through the activation of Snail and Slug and to a more aggressive phenotype.82 Cells undergo morphological alterations such as increased cellular adhesion, motility, and invasion.82 However, BMP-4 overexpression before chemotherapy is an independent prognostic factor of longer progression-free time and overall survival.83

Insulinlike Growth Factor The union of IGF with its receptor (IGFR) activates various downstream pathways such as PI3K/AKT/mTOR, RAF, and MAP. The path is overexpressed in low-grade serous ovarian cancer and has been related to the pathogenesis of these tumors. Immunostaining of endometrioid carcinomas with IGF-1 also reveals strong expression, whereas highgrade serous carcinomas and clear cell cancers show less intense staining. In mucinous carcinomas, there is no expression of IGF-1 receptor.84

DISCUSSION Signaling pathways implicated in various processes of ovarian cancer are numerous and often complicated and redundant. Focusing on serous carcinomas (without taking grade into consideration), it can be said that Notch-3 pathway is essential for the proliferation and survivor of the tumors. Moreover, overexpression of 2 target genes of the Wnt path, namely, Axin2 and FGF9, may promote the development of these carcinomas. On the other hand, hedgehog pathway is active without the involvement of Smo membrane protein,



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although sonic hedgehog mRNA is only moderately (25% of the tumors) highly expressed in serous ovarian malignant tumors. The activation of PTEN-PI3K/AKT/mTOR pathway is implicated to cell cycle progression, decreased apoptosis, and increased metastatic potential. In addition, NF-JB pathway is related to the carcinogenesis of more than 50% of serous tumors. Mitogen-activated protein kinase signaling is implicated in the pathogenesis of less than 50% of low-grade as well as borderline serous tumors, whereas in grade 3 carcinomas, it is less active. High-grade ovarian carcinomas consist the majority of ovarian malignant tumors, and despite the advantages in both diagnosis and treatment, achieved during the past decades, it still remains a lethal disease. The high rate of mortality is mainly due to the lack of an efficient screening, the inability of early diagnosis, and the limitations posed by the conventional chemotherapies. Despite the fact that initial treatment approach is effective in almost 70% of the patients, recurrences are frequent. This has been attributed in part both to the surgical failure to remove the microscopic foci of the disease and the limitations of the classic anticancer agents. These limitations seem to play a key role in a subpopulation of cells, inside the heterogeneous group of cells that form tumors, called cancerstem cells (CSCs).85 Cancer-stem cells exhibit normal stem cells characteristics such as unlimited asymmetrical division (the capability to create both themselves and differentiated [nontumorigenic] daughter cells). More interestingly, sufficient evidence supports that CSCs are implicated in tumor formation and could escape the action of conventional chemotherapies, causing cancer relapse. According to our knowledge, CSCs are present in ovarian cancer, showing heterogeneity of multiple subpopulations, reflecting the cellular complexity of these tumors.85 In addition, other mechanisms related to CSCs, such as EMT, seem to play a key role in the resistance to conventional platinum-based chemotherapies. Some of the described signaling pathways such as TGF-A pathway could activate EMT.86 Extrapolating the above knowledge in the present study could be easily done making clear that CSCs consists one of the main objectives of targeted therapies. Independently on whether CSCs are the only subpopulation that could give rise to cancerous cells (also known as CSCs model) or contribute with other subpopulation in the creation of the tumor (known as clonal evolution model), their role in both pathogenesis and recurrence seems to be established.85 The majority of the previously mentioned main signaling pathways have been found to be related to CSCs.87 Blocking the silenced apoptotic signaling pathways or the unregulated and overexpressed paths that drive to survival, growth, proliferation, metastasis, and angiogenesis in CSCs could be a very promising theory. Theoretically, a ‘‘dysfunctional’’ signaling pathway could be targeted in multiple points, from the ligand to the mRNA of the ‘‘affected’’ gene. So, targeted agents such as monoclonal antibodies could direct toward ligands or cellular surface receptors, blocking the flow of the path. Bevacizumab, a VEGF receptor inhibitor that targets angiogenesis, is the most well known humanized monoclonal antibody already

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used in clinical practice, both as single agent and in combination therapy for primary and recurrent disease.88 Ramucirumab and cediranib are other VEGF receptor inhibitors. Sunitinib, a receptor tyrosine kinase inhibitor, is implicated in multiple targets such as VEGF receptor and EGFR. Pazopanib could inhibit all VEGF receptors. Another promising molecule with tyrosine kinase inhibition action is vatalanib, whereas Intedanib is a multikinase inhibitor acting in multiple growth factor receptors. Moreover, thalidomide, a famous agent used in the past during pregnancy, is nowadays known for its interaction with FGF receptor 2, acting like an antiangiogenic molecule in recurrent ovarian cancer. In addition, the PTEN/PI3K-AKTmTOR pathways could also be targeted. Temsirolimus is an mTOR inhibitor. Perifosine inhibits AKT, whereas XL147 and enzastaurin block PI3K. Gefitinib, erlotinib, cetuximab, pertuzumab, matuzumab, and lapatinib are implicated in EGF pathway. Other molecules such as sorafenib, vandetanib, imatinib, dasatinib, and cabozantinib are considered multipathway agents. The Notch signaling pathway could be targeted with F-secretase inhibitors. Finally, small specific RNA molecules, called siRNAs, could target a specific mRNA, causing the silencing of the ‘‘affected’’ gene.88 As it became clear in the present review, ovarian carcinogenesis implicates multiple and often tortuous signaling pathways. The discovery of new paths and the detailed understanding of both the mechanism and the role of every known path are critical steps in the way of new and more effective treatment approaches. It also seems the study of possible interaction between the pathways is very important. The currently known targeted agents are promising, but their exact efficacy needs more studies. The need to find new and potentially more effective agents becomes imperative. Collectively, it can be said that epithelial and especially serous ovarian cancer signaling pathways offer numerous application possibilities of targeted therapies, which will focus on the inhibition of various intrapathway molecules.

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