Central Medical Journal of Obstetrics and Gynecology

Cite this article: Jana S, Ray AK, Chatterjee K, DasMahapatra P, Swarnakar S (2016) ‘Achilles Heel’ for Endometriosis: Pathway to Malignancy? Med J Obstet Gynecol 4(3): 1085.

*Corresponding authorsSnehasikta Swarnakar, Head, Cancer Biology & Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4-Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India, Tel: 91-33-2473-0492 Ext 759; Fax: 91-33-2473-5197; Email:

Submitted: 23 May 2016

Accepted: 20 July 2016

Published: 22 July 2016

ISSN: 2333-6439

Copyright© 2016 Swarnakar et al.

OPEN ACCESS

Review Article

‘Achilles Heel’ for Endometriosis: Pathway to Malignancy?Sayantan Jana1#, Amlan K. Ray2#, Kasturi Chatterjee1, Pramathes DasMahapatra2, Snehasikta Swarnakar1*1Cancer Biology & Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, India2Spectrum Clinic & Endoscopy Research Institute, India#Authors contributed equally

Keywords• Endometriosis• Malignancy• Clears cell carcinoma• Endometrioid carcinoma• Genomic instability• Transition

ABBREVIATIONSEAOC: Endometriosis-Associated Ovarian Cancer; LOH: Loss

of Heterozygosity; FISH: Fluorescence In situ Hybridization; ER: Estrogen Receptor; PR: Progesterone Receptor; IL: Interleukin; VEGF: Vascular Endothelial Growth Factor; IFG: Insulin-Like Growth Factor; TGF: Transforming Growth Factor; PDGF: Platelet Derived Growth Factor; HGF: Hepatocyte Growth Factor; MMP: Matrix Metalloproteinase; SRC: Steroid Receptor Co-Activator; TNF: Tumor Necrosis Factor; EMT: Epithelial to Mesenchymal

Transition; HUMARA: Human Androgen-Receptor; PGK: Phosphoglycerate Kinase; PI3KCA: Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha; KRAS: Kirsten Rat Sarcoma Viral Oncogene Homolog; CGH: Comparative Genomic Hybridization; TP53: Tumor Protein p53; BCL: B Cell Lymphoma; HSD: Hydroxysteroid Dehydrogenase; PGE2: Prostaglandin E2; mTOR: Mechanistic Target of Rapamycin; MAPK: Mitogen Activated Protein Kinase, TGF-β: Transforming Growth Factor; EMI: Early Mitotic Inhibitor; APC: Anaphase Promoting Complex

Abstract

Nearly a century ago, Sampson claimed about an association between endometriosis and ovarian cancer; however, it is in recent few years researchers started to understand the link. Interestingly, the etiology of endometriosis remains a mystery and its pathogenesis is still debated. Endometriosis is believed to be benign disease with existing plausible theories, including retrograde menstruation, coelomic metaplasia, embryonic cell rests, vascular dissemination etc. However, recent notion about endometriosis is more complex and is now believed to be a disease intriguingly associated with neoplastic events. Furthermore, endometriosis and malignancy share various pathological factors, including reduced apoptosis, insensitivity to anti-proliferative signals, sustained angiogenesis, tissue invasion and metastasis. Chromosomal abnormality is another important feature of malignancy and reported to be related with endometriosis and endometriosis-associated ovarian cancer (EAOC). Studies have demonstrated for monoclonality of endometriosis. Genetic abnormalities like loss of heterozygosity (LOH), increased heterozygosity, microsatellite instability, chromosomal loss or gain, genetic polymorphism are known to be involved with endometriosis. Furthermore, different cellular signalling pathways and factors, which are linked with cancer also present in endometriosis. There are two plausible ways for transformation of endometriosis into ovarian cancer: (i) ‘atypical endometriosis’ transition, where typical endometriosis losses benign properties and gains malignant features; (ii) sharing common mechanisms or factors like genetic susceptibility, immune dysregulation with an eventual downstream cascade of events.

This review highlights the key findings in endometriosis research that are linked with malignant transformation. Firstly, we highlight major studies and clinical evidences that support the link between endometriosis and ovarian cancer. Secondly, different phenotypical features that are shared by both endometriosis and cancer are discussed. We also look into on chromosomal abnormalities related to endometriosis and EAOC. Finally, different cellular events and signalling pathways are identified for their possible roles in the transformation of endometriosis into ovarian cancer.

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INTRODUCTION

Overview on endometriosis and associated ovarian cancers

Epithelial ovarian cancer, with or without endometriosis, consists of five major histological subgroups: clear-cell, endometrioid, mucinous, high-grade serous and low-grade serous carcinomas, with distinct clinical, pathological and molecular features [1-6]. Large scale population study on ovarian cancer and endometriosis suggested that some subtypes of ovarian cancer, such as clear-cell and endometrioid adenocarcinomas show higher association with pre-existent endometriosis as compared to the other subtypes of ovarian cancer [7,8]. Among other tumors, Mullerian-type borderline tumors are also believed to be associated with endometriosis. These are known as endometriosis-associated ovarian cancers (EAOC). Clear cell carcinoma is further subdivided into cystic, adenofibromatous and indeterminate clear cell carcinoma. Histopathologically, clear cell carcinoma consists of clear, hobnail cells and flattened epithelial cells with abundant clear cytoplasm lining tubules and cysts, and grows in a solid or tubular or glandular pattern. Clear cell carcinomas represent approximately 5-10% of epithelial ovarian cancers and are associated with endometriosis in the pelvic cavity [9]. Endometrioid adenocarcinomas show histological features similar to endometrium with overt gland formation, sometimes accompanied by squamous differentiation. Mucinous carcinomas also exhibit overt gland formation; however, the tumor cell cytoplasm is significantly mucin-rich in comparison to endometrioid carcinoma. For serous carcinomas, papillary or solid growth with slit-like spaces and nuclear atypia are the typical histological feature. Müllerian tumors are mixed carcinomas with presence of sarcomatous differentiation rather than sarcomas [10].

Researchers have often identified presence of atypical endometriosis during ovarian cancer development from pre-existent endometriosis. Atypical endometriosis arises from ovarian typical endometriosis with distinct histological features. Atypical endometriosis is characterized by increased nuclear to cytoplasmic ratio, large hyperchromatic or pale nuclei with pleomorphism, cellular crowding, stratification and tufting etc [11]. In a study by Fukunaga et al., atypical endometriosis was reported in ~54% of clear cell carcinomas and in 42% of ovarian endometrioid cancinomas [12]. In a study with 37 cases of EAOC, 22 cases showed transition from typical endometriosis to atypical endometriosis, and transition of atypical endometriosis into ovarian cancers were found in23 cases [13].

In a collective study by Hepas et al., ovary is the primary site for 78.7% of endometriosis, while extragonadal sites represent ~21.3% of endometriosis cases [14]. Patients with ovarian endometriosis were accounted 69% for endometrioid adenocarcinoma, 13.5% for clear cell carcinoma, 11.6% for sarcoma and 6% for other rare cell type cancers. In contrast, extraovarian site endometriosis exhibited clear cell carcinoma and adenocarcinoma as the most prevalent cancers [14]. Kobayashi et al., followed-up a cohort of 6,398 women with a clinically documented ovarian endometrioma in Shizuoka (between 1985 and 1995) up to 2002 and reported 46 ovarian cancer incidences,

resulting the standardized incidence ratio (SIR) value of 8.95. The study further reported about increased incidence ratio value of 13.2 for women over 50 years of age [15]. In another study, accumulated reports from 1966 to 2011 (collected from pubmed) showed endometriosis-associated malignancies for 483 reported cases, among which 89% of cases had epithelial malignancy and 11% had mesenchymal malignancy [16].

A population based cohort study by Brinton et al., on hospital discharges among 20,686 women with endometriosis (from 1969 to 1983) in Swedish population found that the risk of ovarian cancer was particularly elevated among subjects with a long-standing history of ovarian endometriosis (Relative Risk (RR) 4.2, 95%CI 2.0-7.7) [17]. A further follow up study of Swedish population was conducted with discharged patients of 64,490 from year 1969-2000 to evaluate the consistency in risk factor. The study found no further increased overall risk of cancer (SIR 1.04, 95% CI 1.00-1.07) with follow up, and the previously increased risk of ovarian cancer was maintained (SIR 1.43, 95% CI 1.19-1.71). Women with early diagnosed and long-standing endometriosis showed a higher risk of ovarian cancer, with SIR of 2.01 and 2.23, respectively [18]. In another cohort study of 21,646 infertile patients carried out from 1982 to 2002 in Australia found that chances of ovarian cancer was higher (Hazard Ratio (HR) 2.33, 95% CI 1.02-5.35) among women with endometriosis, which further increased in nulliparous women(HR 3.11, 95% CI 1.13-8.57) as compared to parous women(HR 1.52, 95% CI 0.34-6.75) [19]. Recently, Pearce et al., conducted a worldwide study among 7911 women with invasive ovarian cancer to assess the association between endometriosis and histological subtypes of ovarian cancer [8]. The study reported that the endometriosis is associated with a significantly increased risk of clear-cell carcinoma (SIR 3·05, 95% CI 2·43–3·84, p<0·0001), low-grade serous (SIR 2·11, 95% CI 1·39–3·20, p<0·0001) and endometrioid invasive ovarian cancers (SIR 2·04, 95% CI 1·67–2·48, p<0·0001). The study also showed no association between endometriosis and risk of mucinous (SIR 1·02, 95% CI 0·69–1·50, p=0·93) or high-grade serous invasive ovarian cancer (SIR 1·13, 95% CI 0·97–1·32, p=0·13), or borderline tumours of serous (SIR 1·20, 95% CI 0·95–1·52, p=0·12), and mucinous subgroups(SIR 1·12, 95% CI 0·84–1·48, p=0·45) [8].

Recent meta-analysis using published literature on endometriosis from 1990 to 2012 accumulated a total 444255 patients from 1625 studies [20]. The study found that progression-free survival was no different from EAOC to non-EAOC, while endometrioid and clear cell carcinomas were more common in EAOC (RRs, 1.759 and 2.606; 95% CIs, 1.551–1.995 and 2.225–3.053 respectively). The serous carcinoma was less frequent in EAOC than in non-EAOC (RR, 0.733; 95% CI, 0.617–0.871), however no difference was found for mucinous carcinoma between these two groups (RR, 0.805; 95% CI, 0.584–1.109) [20].

Endometriosis and malignancy: Common cellular features and pathological responses

Endometriosis manifests certain cellular features and pathological responses that are common to cancer [3]. Both of the diseases share increased proliferative responses along

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with decreased apoptosis and insensitivity to anti-proliferative signals. Similar to endometriosis, certain types of ovarian cancers are estrogen dependent. Self-sufficiency of growth factors and sustained pathological angiogenesis are also common in both the diseases. Apart from cancer, endometriosis is probably the only disease with metastatic nature. The phenotypes of cancer cells include limitless replicative properties; fortunately, no reports claim limitless replicative properties in endometriosis. It is safe to assume that endometriosis lacks the phenotype.

Self-sufficiency in growth factors

Endometriosis is principally regulated by estrogen-induced signalling; however, EAOC can be both estrogen receptor (ER) positive (endometrioma) and negative (clear cell carcinoma). In addition, different endometriotic lesions exhibit differential ER expressions. For example, active red lesion endometriosis showed significantly elevated ER-α expressions than inactive black lesion [21]. Moreover, recent study found that ER-β play specific and more active role in endometriosis progression than ER-α [22]. Endometriosis is also associated with inherited ER and PR (progesterone receptor) as well as cytochrome P450 polymorphisms [3]. Furthermore, endometriosis exhibits increased levels of growth factors in systemic fluids and endometriotic lesions, which include vascular endothelial growth factor (VEGF), insulin-like growth factor (IFG), transforming growth factor (TGF)-β, platelet derived growth factor (PDGF), hepatocyte growth factor (HGF) etc [3,23].

Reduced apoptosis

Endometriosis show decreased apoptotic responses in clinical samples and experimental animal studies [24]. In endometriosis patients, sloughed endometrial cells contain reduced percentage of apoptotic cells in comparison to control women [25]. Furthermore, apoptotic index was significantly lowered in the glandular epithelium of endometriosis patients than control women [26]. Decreased apoptotic responses in endometriosis can result from different factors: (a) increased anti-apoptotic Bcl-2 expression and reduced pro-apoptotic Bax expressions in ectopic tissues [27]. (b) Increased soluble FasL in serum and peritoneal fluid in women with higher stages of endometriosis [28]. (c) Increased levels of pro-inflammatory cytokines (e.g. IL-8), survivin and metalloproteinase activities that modulate apoptotic responses. For example, elevated matrix metalloproteinase (MMP)-9 generate a ~70 kDa steroid receptor coactivator (SRC)-1 fragment in endometriosis that prevent tumor necrosis factor (TNF)-α mediated apoptosis of human epithelial cells [29]. (d) Elevated ER-β responses reduce apoptotic responses and modulate inflammasome to drive endometriosis [22]

Insensitivity to anti-proliferative signals

Limited studies were available regarding the mechanisms for insensitivity to anti-proliferative signal in endometriosis. However, it is believed that progesterone receptor (PR) isoforms play the critical role. Endometriosis prevalently expresses the PR-A isoform, instead of the stimulatory PR-B isoform, which might result for insensitivity to anti-proliferative signal [30]. Moreover, endometriosis showed differential expressions for

p27Kip1 in active red and inactive black lesions of peritoneal endometriosis [31].

Sustained angiogenesis

Similar to cancer, endometriosis is also associated with pathological angiogenesis. Endometriosis patients show elevated levels of VEGF in systemic fluid and ectopic lesions. Elevated VEGF receptor (VEGFR) expression is present with advancement of the disease [32]. The pathological angiogenesis in endometriosis and cancer governs through similar inflammatory molecules where pool of growth factors guide angiogenic tip cells to invade into the implant. Apart from VEGF, several other pro-angiogenic factors elevate in endometriosis including HGF, IL-8 , IL-15, macrophage migration inhibitory factor, neutrophil-activating factor, TNF-α, erythropoietin and angiogenin [33]. In addition, MMPs are also involved in angiogenesis and cellular remodelling [34].

Tissue invasion and metastasis

Ectopic growth of endometriosis requires tissue remodelling followed by invasion through basement membrane. Endometriotic niche consists of several proteases, including MMPs and ADAMs, which cleave different extracellular matrix components [35]. Moreover, MMPs can cleave different cellular adherent molecules, including E-cadherin that promote release of cells from its basement membranes [36]. Similar to cancer, endometriotic cells possess metastatic properties, thus can spread through systemic fluid to dock at other sites [37]. The disease affects various organs including, ovary, bladder, rectal walls, gastrointestinal tracts, and even lungs. N-cadherin-positive and E-cadherin negative endometriotic cells possess increased invasive character, which are similar to invasive cancer cells [38]. Moreover, recent studies have identified epithelial to mesenchymal-like transition (EMT) in endometriosis suggesting its direct involvement in invasion and metastasis [39].

GENOMIC INSTABILITY AND ENDOMETRIOSISMost neoplasms are monoclonal in origin, and clonal

outgrowth is thought to be a fundamental feature of human neoplasm. Several studies documented the association of endometriosis with genetic abnormalities like loss of heterozygosity (LOH), increased heterozygosity at chromosome 17 anuploidy, microsatellite instability, chromosomal loss or gain [4,5].

Clonality of endometriotic lesions

Clonal selection and outgrowth is a fundamental feature for neoplasm. Although neoplastic tumor is often heterogeneous, the origin usually depends on single population of transformed cells. The parent cell population contain a special growth advantage over others and accumulates further genetic changes in daughter cells that provide additional advantage for cancer development. In female, clonality can be detected by assessing two X chromosomes and the status of inactivation of one X chromosome due to methylation. Genes like hypoxanthine phosphoribosyl transferase harbour polymorphism and restriction site for methylation-sensitive endonucleases, are being used for accessing clonality status [5].

Limited studies were reported for clonality status in

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endometriosis. Among 8 studies mentioned here, 4 used HUMARA (human androgen-receptor) gene as an X-linked polymorphic gene, 3 used phosphoglycerate kinase-1 (PGK-1) gene and one used both HUMARA and PGK-1. Five out of 8 studies found a total of 97 specimens of endometrial glands and cyst having 100% monoclonality [40-44]. Nabeshima et al., found 100% monoclonality (n=22) for individual glands, however different glands within same lesion have polyclonal origin. Three other studies had reported 60% (3/5), 82% (14/17) and 6% (2/32) of monoclonal results [45-47]. Mayr et al., collected 32 samples from various sites of 13 endometriosis patients and found that only 2 have monoclonal origin, thus only 15.4% of patients bear monoclonal tissue [45]. However, the process for studying the clonality has a major drawback, as precise capture of epithelial cells is required and any contamination of stromal cells (which are polyclonal in origin) may cause erroneous results.

Chromosomal aberration and endometriosis

The emergence of cancer from precursor cells requires stepwise accumulation of genetic changes (namely, the ‘multi hit’ model). Thus to develop ovarian cancer from endometriosis may follow two possible ways: (i) the initial cells of reflux menstrual fluid may contain special variants that have higher survival properties including cellular attachment, resistant to apoptosis, proliferation, escape from immune cells etc. Further, somatic mutations in oncogenes or tumor suppressor genes might ignite malignant properties in these cells. (ii) The endometriosis and ovarian carcinoma share co-existence with similar risk factors e.g. genetic predisposition and immune dysregulation etc, however the diseases represent differential events and originate from two distinct causative factors.

Although classical karyotypic analysis have not found any chromosomal abnormalities in pelvic endometriosis implants, FISH (fluorescence in situ hybridization) analysis revealed increased anuploidy frequency at chromosome 17 in endometriosis specimens [48]. Similar to ovarian cancers, increased heterogeneity at chromosome 17 aneuploidy were found in endometriosis samples.

LOH was reported in various studies using different microsatellite markers, and it was found that the LOH frequency increases with the progression of endometriosis to endometrioid ovarian tumour [49]. Jing et al., found LOH in 27.5% cases with one or more loci on chromosome 9p(18%), 11q(18%), 22q(22%) out of 40 endometriotic specimens [46]. In a subsequent study by the same group ~64% (9/14) cases showed LOH at one or more loci. Nine out of 11 cases of ovarian endometriosis adjacent to ovarian carcinoma showed common LOH at different chromosomal loci, including 9p21 (31%), 11q23 (20%), 22q13 (31%), 4q (8%), 5q13-q14 (25%) and 6q14-q15 (27%) [50]. Sato et al., reported LOH in 56.5% of 23 cases of endometriotic cysts at the chromosomal position of 10q23.3 [51]. Goumenou et al., studied 22 endometriotic samples and LOH was reported at loci 9p21 (27.3%), 1q21 (4.5%) and 17q13.1 (4.5%), corresponded for p16INK4, APOA2 (apolipoprotein A2) and TP53 respectively [52]. In 4 late stage endometriosis cases (out of 15) Bischoff et al., found LOH at 17q13,where tumor suppressor gene BRCA1 (breast cancer 1) is present [53]. In contrast, Prowse et al., investigated 12 polymorphic satellite markers correspondent to

p16INK4, APOA2, PTEN, TP53 etc in 17 ovarian endometriotic samples and found no LOH, except one at 8p22 loci [54].

Comparative genomic hybridization (CGH) allows the analysis of entire genome and identification of chromosomal gain or loss. CGH analysis of 18 endometriosis samples showed 83% cases of recurrent gene copy number alteration. Loss of genetic material was observed in chromosome 1p (50%), 22q (50%), 5p (33%), 6q (27%), 7p (22%), 9q (22%), 17q and gain of genetic material was observed in 1q, 6q, 7q, 17q [55]. Although different outcomes of chromosomal aberration in endometriosis stand far apart from being conclusive, especially for 17p loci, it certainly indicates increased genetic aberration for endometriosis followed by EAOC development.

Oncogenes and tumor suppressor genes

PTEN (Phosphatase and tensin homolog), located at chromosomal arm 10q23.3, is a tumor suppressor gene and due to its phosphatase activity acts as a negative regulator of PI3K. Sato et al., found LOH at 10q23.3 in 13 out of 23 ovarian cysts and found missense mutations, deletions of PTEN in 7 out of 34 ovarian endometriotic cysts [51]. Although no further studies have reported any mutation at PTEN, two other reports have found LOH at chromosome 10q23.3. Using genetically modified mouse with conditional deletion of PTEN, endometriosis is reported to have higher possibility to develop ovarian tumor. Furthermore, with combined mutation of K-ras (Kirsten rat sarcoma viral oncogene homolog) and PTEN, tumor originates from ovarian epithelium within only 7 weeks in mouse model of endometriosis [56].

TP53 (tumor protein p53) is located at the short arm of chromosome 17 and mutation of p53 gene is related with different cancers. Still now, limited studies on endometriosis have found LOH at chromosome 17 [53], almost no reports with endometriosis have found any p53 mutation. There is exclusively one case of endometriosis adjacent to endometriod ovarian carcinoma, where TP53 mutation (Tyr-to-Cys at codon 220) was found, however the adjacent cancer was reported not to harbour the mutation [57]. Although no p53 mutation is linked to endometriosis, over expression of p53 is reported in endometriotic lesions. Saintz et al., found a distinct upregulation of p53 with transition of typical endometriosis to atypical endometriosis to cancer [58].

BCL (B cell lymphoma)-2 is a proto-oncogene located at chromosomal arm 18q21.3 and bcl-2 protein acts as anti-apoptotic molecule. In endometriotic tissues, no chromosomal aberration was reported for BCL-2 gene, however reports confer upregulation of Bcl-2 in endometriosis both in clinical and experimental models [24, 27]. Nezhat et al., found positive association of Bcl-2 in benign and malignant ovarian endometriotic cysts with 23% positive for endometriotic cysts and 67%, 74% for endometrioid and clear cell carcinoma respectively [59]. ARID1A (AT-rich interactive domain-containing protein 1A) is a tumor suppressor gene, located at chromosome 1p, and mutation of ARID1A is common to endometrioid and clear cell ovarian carcinoma (~46 and 30% of cases). In endometriosis-associated clear cell carcinoma, mutation of ARID1A was reported and speculated to be present only during the pre-neoplastic event

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[60]. From prevalence data of ARID1A in non-atypical and atypical endometriosis, Yamamoto et al., found that loss of ARID1A occurs only during early event of cancer transformation from endometriosis [61].

Other factors

Multiple genetic alterations were reported in the pathogenesis of endometriosis-associated neoplasm. Molecular events, such as PIK3CA (phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha) activation mutation, and K-ras activation mutation have been indentified in EAOC [62,63]. Serous borderline tumours were reported with B-raf and ERBB2 mutation, whereas K-ras mutation was found in Mullarian-mucinous borderline tumours [64-66]. Furthermore, abnormal methylations for hMLH1 (human mutL homologue) and p16 were reported (~8.6 and 2.1% of cases respectively) in malignant transformed endometriosis [67]. These factors, either cumulative or individual, might direct endometriosis into malignant transition.

PATHWAYS TO CARCINOGENESIS

Iron load and oxidative stress

Endometriosis represents repetitive haemorrhage, iron accumulation and subsequent oxidative stress at the ectopic sites [68]. Endometriotic cysts contain significantly increased levels of free iron as compared to non-endometriotic cysts and might responsible for carcinogenesis through persistent oxidative load [69]. Sustained oxidative stress results into inflammatory responses with the progression of disease. The possible mechanism for carcinogenesis lies in the reduction of ferric to ferrous iron by superoxide and hydrogen peroxide, which in turn catalyses the formation of hydroxyl radicals and promotes lipid peroxidation, protein oxidation, DNA damage and mutagenesis [70]. Iron induced carcinogenesis is well reported in animal models and iron-catalyzed oxidative damage targets specific genes including p15 and p16 tumor suppressor genes [71].

Estrogen effect

Endometriosis, being an estrogen-dependent disease, possibly associated with hyperestrogenism for EAOC development [2]. Aromatase, usually absent in eutopic endometrium, present in high levels in endometriosis and constitutively catalyze the production of estrone and estradiol from andostenedione and testosterone [72]. In addition, endometriotic tissues contain 17β-HSD (hydroxysteroid dehydrogenase) type-1, instead of the 17β-HSD type-2, which is more potent in converting estrone to estradiol and thus locally support the proliferative microenvironment of endometriotic cells [73]. Furthermore, estrogen stimulates the production of prostaglandin E2 (PGE2) through positive feedback mechanism,thus establishes a microenvironment of self-sufficient estrogenic milieu. Along with the estrogenic effects, any additive features of genetic aberration, like methylations or mutation, may trigger malignant pathways. However, estrogenic effects may follow separate pathways for development of different EAOCs, as both estrogen-dependent (endometriod) and independent (clear cell) carcinomas are prevalent with endometriosis.

Epithelial to mesenchymal transition

Endometriotic cells show increased cellular invasiveness and reported to be metastatic in nature. However, until now the mechanism was not well understood. Recent studies identified epithelial to mesenchymal transition (EMT) in endometriosis [39]. Although the proportion of EMT was found higher in pelvic endometriosis, ovarian endometriosis was also reported to have the phenotype. Furthermore, the transition for cellular phenotypes were involved with N-cadherin positive, E-cadherin negative endometriotic cells [74]. Increased MMP activities found in late endometriosis play important roles in EMT. Apart from increased cellular invasiveness and metastatic properties, development of cellular transition can be an important event in clonal selection and may trigger monoclonality over benign endometriosis to develop EAOC.

Signalling pathways for Neoplasm

Several signalling pathways were documented in endometriosis that were common with cancer; however, factors only constraint to potential trigger for neoplasm are mentioned here. AKT signalling responses were involved with endometriosis progression and its pathways include PI3K, mTOR (mechanistic target of rapamycin), and MAPK (mitogen activated protein kinase) responses [75]. However, AKT is negatively regulated by PTEN, which often found to be mutated in different cancer [76]. Deletion of PTEN in experimental endometriosis showed accelerated development of ovarian cancer through upregulated AKT-mTOR phosphorylation and p70 S6 kinases [56]. Moreover, tumors were positive for FKHR (also known as FOXO1) and MAPK expressions which were downstream effectors for K-ras and AKT signalling pathways indicating its potential involvement in EAOC development [56].

The occurrence of EMT in endometriosis may indicate different signalling possibility in endometriosis and associated transition to ovarian cancer. Transforming growth factor (TGF-β) was reported in ectopic endometrial lesions and involved with disease progression [77]. Over expression of TGF-β resulted in induction of EMT in different in vivo and in vitro cancer models and might play an important role in endometriosis associated EMT-like changes [78]. MMPs were also associated with endometriosis and inflammation, and reported to result direct EMT-like processes through Rac1B mediated signalling pathways [79].

Polo like kinases were recently reported to be present in ectopic endometriosis [80] and involved in phosphorylation of Emi 1(Early mitotic inhibitor 1). Emi1 regulates cell cycle progression through interacting with anaphase promoting complex(APC) [81]. Overexpression of Emi1 causes mitotic abnormality and genomic instability and tumorigenesis [82]. Thus, Emi/APC signalling in endometriosis and clear cell carcinoma can be an important clue to uncover the mechanisms for EAOC development.

Although mutation of p53 is not still reported in endometriosis, further studies are warranted for role of p53 in EAOC development from endometriosis. Abundant expressions of p53 were already reported in endometriosis [53]. Wnt and Notch signalling pathways were recently found to be involved in the

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pathogenesis of endometriosis, and might play important roles in cellular transition [83,84]. Ras/Raf/MAP Kinase is another important signalling cascade that is involved with different cellular events and pathogenesis of endometriosis.

CONCLUSION AND FUTURE DIRECTIONSMolecular pathogenesis of endometriosis and associated

neoplastic processes are challenging areas for researchers. Oxidative stress, hyperestrogenism, genetic aberration and cellular events seem to be inextricably intertwined in neoplastic transformation of endometriosis and are frequently found in association with clear cell and endometrioid carcinomas, suggesting direct correlation of ovarian malignancies with endometriotic deposits. However, why the specified two subtypes, but not any other subtypes of ovarian cancers are associated with ovarian endometriosis, is still unclear. Recent findings on the roles of ARID1A in endometriosis are interesting, and might be helpful to shed light on the topic. In addition, the possibility of early organogenic defects cannot be ruled out for endometriosis-EMT-EAOC development. Identification of EMT-like processes can highlight over the origin of endometriosis; although ‘Sampson’s hypotheses’ may also explain the possibility of EMT through MMP or TGF-β mediated responses. Moreover, K-ras mutated, PTEN deleted murine model for ‘endometriosis to malignancy’ study provided a new insight to endometriosis research [56]. It is evident that genetically modified murine models are necessary to further explore pathogenesis of endometriosis and associated malignancies. However, explanation for EAOC development from benign endometriotic lesions is still incomplete. The other research wings related to immunology and environmental toxicants on endometriosis development should be given importance. Furthermore, studies with global genomic and proteomic profiling will be helpful in future to understand the disease development better and in management of clinical conditions.

ACKNOWLEDGEMENT This work is supported by the Council of Scientific and

Industrial Research, India [grant number BSC0111-INDEPTH & BSC0119-HUM].

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