Gynecologic Oncology 135 (2014) 100–107

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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno

Src as a novel therapeutic target for endometriosis Kate Lawrenson a, Nathan Lee a, Hugo A.M. Torres b,1, Janet M. Lee a, Doerthe Brueggmann c, P. Nagesh Rao d, Houtan Noushmehr b, Simon A. Gayther a,⁎ a

Department of Preventive Medicine, University of Southern California/Keck School of Medicine, 1450 Biggy Street, Los Angeles, CA 90033, USA Department of Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Núcleo de Apoio à Pesquisa (NAP) denominado Centro de Biologia Sistêmica Integrada (CISBi), São Paulo, Brazil Department of Obstetrics and Gynecology, University of Southern California/Keck School of Medicine, 1450 Biggy Street, Los Angeles, CA 90033, USA d Pathology and Lab Medicine, David Geffen University of California Los Angeles, 22–26 Rehab Cntr, 1000 Veteran Ave, Los Angeles, CA 90024, USA b c

H I G H L I G H T S • EGF plus HC promote anchorage independent growth of endometriosis cells. • Endometriosis cells are sensitive to inhibition of Src and Wnt signaling. • Src is activated in human endometriosis lesions, and in the endometrium of endometriosis patients.

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Article history: Received 4 March 2014 Accepted 16 June 2014 Available online 24 June 2014 Keywords: Endometriosis epithelial cells Endometriosis Three-dimensional in vitro modeling In vivo xenograft Src Clear cell ovarian cancer

a b s t r a c t Background. Endometriosis is a common condition that is associated with an increased risk of developing ovarian carcinoma. Improved in vitro models of this disease are needed to better understand how endometriosis, a benign disease, can undergo neoplastic transformation, and for the development of novel treatment strategies to prevent this progression. Methods. We describe the generation and in vitro characterization of novel TERT immortalized ovarian endometriosis epithelial cell lines (EEC16-TERT). Results. Expression of TERT alone was sufficient to immortalize endometriosis epithelial cells. TERT immortalization induces an epithelial-to-mesenchymal transition and perturbation in the expression of genes involved in the development of ovarian cancer. EEC16-TERT was non-tumorigenic when xenografted into immunocompromised mice but grew in anchorage-independent growth assays in an epidermal growth factor and hydrocortisone dependent manner. Colony formation in agar was abolished by inhibition of Src, and the Src pathway was found to be activated in human endometriosis lesions. Conclusions. This new in vitro model system mimics endometriosis and the early stages of neoplastic transformation in the development of endometriosis associated ovarian cancer. We demonstrate the potential clinical relevance of this model by identifying Src activation as a novel pathway in endometriosis that could be targeted therapeutically, perhaps as a novel strategy to manage endometriosis clinically, or to prevent the development of endometriosis-associated ovarian cancer. © 2014 Elsevier Inc. All rights reserved.

Introduction Endometriosis is a common, benign condition in which proliferative endometrial tissue is found at ectopic locations throughout the body, but most commonly within the peritoneal cavity. Endometriosis affects ⁎ Corresponding author at: Department of Preventive Medicine, University of Southern California/Keck School of Medicine, Harlyne Norris Research Tower, NRT2517G, 1450 Biggy Street, Los Angeles, CA 90033, USA. Fax: +1 323 442 7995. E-mail addresses: [email protected] (K. Lawrenson), [email protected] (N. Lee), [email protected] (J.M. Lee), [email protected] (D. Brueggmann), [email protected] (P.N. Rao), [email protected] (H. Noushmehr), [email protected] (S.A. Gayther). 1 Deceased.

http://dx.doi.org/10.1016/j.ygyno.2014.06.016 0090-8258/© 2014 Elsevier Inc. All rights reserved.

at least 10% of reproductive age women [1] and endometriosis-associated symptoms include pelvic pain, dysmenorrhea and dyspareunia [2]. Endometriosis is a major cause of infertility in reproductive age women. The condition is also associated with a significantly increased risk of developing certain histological subtypes of epithelial ovarian cancer, in particular clear cell ovarian cancer [3–5]. Although commonly treated as a single condition, endometriosis can be stratified into three subtypes: pelvic peritoneal lesions, ovarian cysts (endometriomas) and deep-infiltrating disease occurring on the rectovaginal septum [2,6]. Despite the significant impact of endometriosis on women's health, the underlying biology and origins of the disease are poorly understood. Most evidence suggests that retrograde menstruation is the mechanism by which menstrual blood, containing endometrial cells, is transported

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into the peritoneal cavity via the fallopian tubes and that these endometrial cells can engraft and develop into endometriosis lesions [7–10]. However, retrograde menstruation occurs in 90% of women [11] and it is not known why endometriosis only occurs in a only a subset of these women. While some studies have reported histological and/or molecular differences in the endometrium of endometriosis patients compared to unaffected women [12], reports are often contradictory, and the mechanisms by which endometrial tissues engraft, proliferate and function in the peritoneal organs of endometriosis patients have yet to be elucidated. Despite the commonness of this condition, there is currently no minimally invasive test that can be used to diagnose endometriosis, and there are few treatment options available for patients. One major barrier to endometriosis research aimed at addressing these needs is the relative scarcity of in vitro and in vivo models for studying disease etiology. In vivo models usually comprise freshly isolated human endometrial or endometriosis tissues xenografted into the peritoneum of immunocompromised mice [13–15]. However, because of sample heterogeneity and restricted tissue availability, these models are limited and replication of research findings are challenging. In vitro models of endometriosis are typically human epithelial or stromal cell lines, cultured as primary isolates [16] or ‘immortalized’ using the SV40 large T antigen or overexpression of human hTERT, cyclinD1 and mutant cdk4; but these models are few in number and do not reflect the different subtypes of the disease [17–21]. In this manuscript, we describe establishing and characterizing a novel in vitro model of ovarian endometriosis. This unique model represents a novel research tool for investigating the biology of ovarian endometriosis and neoplastic transformation of endometriosis into clear cell ovarian cancer, and potentially the identification of novel therapies for treating both diseases. Materials & methods Cell culture Endometriosis epithelial cells (EEC16-TERT) were generated from the primary EEC16 epithelial cell line [16], established with informed consent at UCLH, under the approval of the UCLH Ethics Committee. EEC16 was grown in normal ovarian epithelial cell complete medium (NOSECM) [22], which consists of MCDB105:Medium 199 (mixed in a 1:1 ratio) supplemented with 15% fetal bovine serum (FBS, Hyclone), 0.5 mg/ml hydrocortisone, 5 mg/ml insulin (both Sigma), 10 ng/ml epidermal growth factor and 34 mg protein/ml bovine pituitary extract (both Invitrogen) but once immortalized, could be transferred into a basic medium (1:1 MCDB105:Medium 199 plus 15% FBS). OV2008 ovarian cancer cells were maintained in RPMI containing 10% FBS. All cell lines were routinely tested for mycoplasma infection and found to be negative. Viral production and transduction Viral supernatants were produced using standard protocols for cotransfection of HEK293T cells. For immortalization, the pBabe-hygrohTERT, as previously described [23], and pLOX-TERT-iresTK (Addgene, plasmid numbers 1773 and 12245) were used. GFP supernatants were used as a control and the retroviral TERT line selected using 30 U/ml hygromycin. OV2008 and lenti-TERT EEC16 cells were transduced with lentiviral supernatants containing a construct encoding the firefly luciferase gene and a G418 neomycin resistance cassette for mammalian selection (produced by CHLA Vector Core). Luciferase expressing cells were selected using 125 and 300 μg/mL G418 for EEC16 cells and OV2008 cells, respectively. Luciferase activity was confirmed using the Promega Luciferase Assay System (Promega) according to the manufacturer's instructions.

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Phenotypic and molecular analysis of EEC16-TERT lines Telomere length was analyzed by real-time PCR according to the methods of Cawthon [24]. To measure telomerase activity the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche) was used according to the manufacturer's instructions. To test p21 expression following irradiation, cells were exposed to 6Gy ionizing radiation from a caesium-137 source and the protein lysates harvested 24 h later. Western blotting was performed using standard protocols; membranes were probed with an anti-p21 antibody (sc-397), p16 (sc-9968), p53 (sc-126), Rb (sc-50, all from Santa Cruz Biotechnology), or β-actin antibody as a loading control (A5060, Sigma). Karyotyping was performed at the Pathology and Lab Medicine, David Geffen University of California Los Angeles. For karyotyping, cultures were seeded onto a 25 cm2 culture flask, fixed and Giemsastained following routine cytogenetic methods. Twenty metaphase cells analyzed using a Zeiss bright field microscope equipped with image analysis hardware and software. Cell line identities were established by typing of short tandem repeats using the Promega PowerPlex 16 HS Assay (Promega). Typing was performed at the University of Arizona Genetics Core and profiles were compared to the ATCC and DSMZ databases, plus published EOC cell line profiles [25]. We were able to confirm that these two lines had identical profiles that did not match any published cell line profiles (Supplementary Table 1). Immunofluorescent cytochemistry, 3D culturing and real-time PCR Immunofluorescent staining was performed using the following antibodies: cytokeratin-7 (clone EPR1619Y, Millipore), pan-cytokeratin (sc-8018, Santa Cruz Biotechnology), vimentin (clone VIM 3B4, Millipore) and Alexa Fluor coupled secondary antibodies. To culture EECs in 3D, cell culture plastics were coated twice with 1.5% polyHEMA (Sigma) dissolved in 95% ethanol (VWR). Coated plates were allowed to dry completely, washed for 5 min with 1X PBS before use, and 1–3 × 106 cells were plated in a final culture volume of 20 ml. Cultures were refed twice per week before processing into paraffin. Spheroids were fixed in neutral buffered formalin (30 min, at room temperature) and transferred into 70% ethanol. At the USC Surgical Pathology Laboratory samples were processed into paraffin, sectioned and stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed at the USC Department of Pathology Immunohistochemistry Laboratory. Gene expression analysis was performed using TaqMan real time PCR probes (ARID1A, Hs00195664_m1; SNAI2, Hs00950344_m1; MLH1, Hs00179866_m1; MSH2, Hs00953527_m1; ACTB, Hs01060665_g1; GAPDH, Hs02758991_g1) and runs on an ABI 7900HT Fast Real-Time PCR system (Applied Biosciences). Data were analyzed using the ΔΔCt relative quantification method. In vivo tumorigenicity assays and live animal imaging In vivo work was performed under the approval and guidance of the University of Southern California Institutional Animal Care and Use Committee. 3 × 106 EEC16-TERT + LUC or OV2008 + LUC cells were injected intra-peritoneally into 6-week old nu/nu mice (Simonsen Laboratories). Animal imaging was performed at the USC Molecular Imaging Core. 1–4% isoflurane was used to anesthetize the animals and 50 μg luciferin administered by tail vein injection. After 1.5 min animals were imaged using the IVIS® Imaging System 200 (Xenogen). Anchorage independent growth assays Anchorage independent growth assays were performed by plating 2 × 104 cells in culture medium containing 0.3% Noble Agar (Sigma) over a base layer of medium containing 0.6% Noble Agar, ovarian cancer cells served as a positive control. After four weeks, cells were fixed and

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stained with 1% p-iodonitrotetrazolium violet (Sigma) in 100% methanol (VWR). Colonies were counted using phase microscopy. Assays were performed using NOSECM or basic medium MCDB105:Medium 199 (1:1) plus 15% fetal bovine serum (FBS, Hyclone), with growth supplements added at the concentrations used in NOSECM. Where stated, inhibitors were added: 10 μM U0126, 10 μM ICG-001, 10 μM LY294002, 1 μM LY450139 (all from Selleck Chemicals) and 50 μM PP2 (Tocris Bioscience). Inhibitors were dissolved in DMSO, control cells received vehicle alone.

Analysis of Src activation in human endometriosis datasets Statistical analysis, data collection, and clustering analysis were performed using open source software R (version 3.0.0) and Bioconductor (version 2.12) [26]. GEO datasets were downloaded using bioconductor package GEOquery2 (version 2.26.1) [27]. The Src gene-signature identified by Bild et al. [28] was used to subset the downloaded GEO datasets and the gene annotations were updated using the ‘hgu133plus2.db’ package (version 2.9.0). Samples were classified into two groups (Supplemental Table 2) and intergroup comparisons made by calculating the means of log2-transformed Robust Multi-array Average data values of each group and applying a Welch statistical significance T-test (2-group, two sided) for each probe. The significance was assessed at ≤0.05. A FDR correction was not applied because we tested less than 100 probes. Finally, an endometriosis gene signature was also created using Ingenuity Pathway Analysis (IPA®), imported into NextBio® and compared to gene expression microarray data from functional Src experiments (GSE15161, Mori et al. [43] and GSE17941).

Results Extending the lifespan of endometriosis epithelial cells We have recently described a novel ovarian endometriosis epithelial cell line (EEC16) that is karyotypically normal but exhibits a partially transformed phenotype in vitro [16]. However primary normal cells have a limited in vitro lifespan and so to extend the proliferative potential of EEC16 we overexpressed the catalytic subunit of human telomerase (TERT). The in vitro lifespan of EEC16 was increased following transduction with either lentiviral-TERT (EEC16-TERT-L) or retroviralTERT (EEC16-TERT-R) compared to both control cells transduced with GFP (EEC16-GFP) and primary EEC16 (Fig. 1A). EEC16-GFP and primary cells underwent replicative senescence after 60–70 days while TERTtransduced cells were continuously passaged for over 200 days. There were no significant differences in population doubling times (PDT) between pre-senescent EEC16, EEC16-TERT-L or EEC16-TERT-R lines (PDTs were 28.5 ± 9.8 and 30.7 ± 4.1 days respectively). Telomere lengths in EEC16-TERT-L or EEC16-TERT-R lines (collectively referred to as EEC16-TERT) were significantly increased compared to the primary EEC16 (P b 0.05), but there was no significant difference in telomere length between EEC16-TERT-L and EEC16-TERTR lines (Fig. 1B). Telomerase activity was also detected in EEC16-TERT lines but not in primary cells (Fig. 1C). Immortalized lines retained expression of p16 and Rb, and expression of these proteins did not significantly change following exposure to ionizing radiation in primary and immortalized cells alike. The p53-dependent G1 DNA damage checkpoint remained intact in TERT expressing lines, which we demonstrated by showing upregulated p53 and p21 expression following exposure to ionizing radiation (Fig. 1D). TERT expressing lines also

Fig. 1. Immortalization of EEC16 by viral delivery of telomerase. (A) The EEC16 line was transduced with GFP and TERT viral supernatants. Lentiviral and retroviral delivery of TERT increased the in vitro lifespan of EEC16. Infection with GFP had no effect. (B) Telomere length and (C) telomerase activity is increased in TERT expressing cells. T/S indicates the ratio of telomere length to a standard control gene. (D) TERT immortalized EECs express p16 and pRb and have intact DNA damage repair responses. Expression of p53 and p21 is upregulated following exposure to ionizing radiation (IR). (E) TERT immortalized EECs have normal, female karyotypes. *P N 0.05, two-tailed paired Student's T-test.

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retained the normal 46,XX karyotype of the primary cells (Fig. 1E). Finally, we evaluated the phenotype of EEC16 in vivo after intraperitoneal injection of luciferase labeled EEC16-TERT-L/R cells (EEC16-TERT-LUC) into immunocompromised mice, with luciferase expressing ovarian cancer cells (OV2008-LUC) used as a positive control. Within 32 days, mice injected with OV2008-LUC cells showed widespread disease throughout the peritoneal cavity, but there was no evidence of cell growth in animals injected with EEC16-TERT-LUC or with vehicle (Supplementary Fig. 1). We monitored EEC16-TERT-LUC animals for 7 months without observing any bioluminescent signal; neither were there any external signs of endometriosis, suggesting that the EEC16TERT-LUC models are non-tumorigenic in immunocompromised mice (Supplementary Fig. 1).

Epithelial-to-mesenchymal transition of TERT-immortalized EEC cells Primary and TERT-expressing EEC16 share similar morphologies, but differentially express epithelial and mesenchymal markers: primary EEC16 expresses keratin (epithelial features) and vimentin but lacks E-cadherin expression (both mesenchymal features) [16]. By contrast, EEC16-TERT cultures showed neither cytokeratin expression nor expression of other epithelial markers (BerEP4), but maintained expression of vimentin (Fig. 2A & B). This suggested a commitment to a mesenchymal phenotype occurred when the EEC16 primary cell isolate was immortalized through expression of TERT. In support of this, we observed a 4- to 10-fold increase in SNAI2 (also known as Slug) gene expression when EEC16-TERT lines were compared to the primary cultures; this transcription factor is known to be involved in epithelialto-mesenchymal transition (EMT) (Fig. 2C i). EEC16-TERT cultures also exhibited significantly lower expression of tumor suppressor

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genes associated with clear cell ovarian cancer (ARID1A, MLH1 and MSH2) compared to primary EEC16 (Fig. 2C ii–iv). Influence of growth factors on the phenotype of EEC16-TERT cells EEC16-TERT lines form colonies in anchorage independent growth assays, at efficiencies similar to ovarian cancer cell lines. This phenotype was dependent on growth media composition. These cell lines were established in a basic medium supplemented with serum, epidermal growth factor (EGF), hydrocortisone (HC), insulin and bovine pituitary extract (BPE). Removing all supplements except serum inhibits anchorage independent colony formation (Fig. 3, see NOSECM versus Basic Medium). By testing the effects of withdrawing each supplement individually, we established that removal of either EGF or HC in serum-containing medium significantly inhibited colony formation (P N 0.015, two-tailed paired Student's T-test, Fig. 3A–C). Conversely, by supplementing the basic medium with growth factors individually and in combination, we were able to demonstrate that the combination of EGF plus HC was responsible for anchorage independent growth phenotype we observed (Fig. 3D). Src activation in endometriosis models and primary tissues Anchorage-independent growth is an EMT-associated phenotype. To identify the key pathways active in EEC16-TERT-L/R-LUC lines we inhibited several pathways implicated in EMT-ERK, PI3K, Src, Wnt and Notch using specific inhibitors, and then evaluated anchorage independent growth. Inhibition of Src signaling completely abolished colony formation versus vehicle control (P = 0.002) and inhibition of Wnt signaling reduced colony formation by 85% (P = 0.002 versus vehicle control). Inhibition of ERK, PI3K and Notch signaling had no significant

Fig. 2. Immortalized EEC16 cells undergo an epithelial to mesenchymal transition and differentially express EOC genes. (A) Immunofluorescent staining of primary and TERT-expressing EEC16 cells. Primary cells stain positive for cytokeratin 7, pan-cytokeratin and vimentin. Expression of TERT is associated with a loss of cytokeratin expression but maintenance of vimentin expression. EEC16-TERT-L is shown, EEC16-TERT-R cells showed the same effect. Flourescence microscopy, 40 × magnification. (B) 3D models of EEC16-TERT do not express pan-cytokeratin (an epithelial marker) and do express vimentin. Primary EEC16 3D cultures express both markers. Brown color denotes positive staining, samples are counterstained with eosin (blue), light microscopy. (C) Immortalized EEC16 lines upregulate SNAI2 and downregulate tumor suppressor genes that have been implicated in clear cell and endometrioid ovarian cancer development.

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Fig. 3. Immortalized EEC16 form colonies in anchorage independent growth assays in an EGF and hydrocortisone dependent manner. Anchorage-independent growth assays performed on (A) EEC16-TERT-L and (B) EEC16-TERT-R cultures. Cells were grown in Basic Medium, supplemented with 15% serum and glutamine only, or growth factor rich NOSE-complete medium (NOSECM). NOSECM is basic medium that is supplemented with EGF, hydrocortisone (HC), insulin (INS) and bovine pituitary extract (BPE). Agar assays were performed using Basic Medium, NOSECM, or NOSECM with a supplement removed. Withdrawal of EGF and HC significantly impaired colony formation. (C) Examples of colonies formed by EEC16 lines grown in NOSECM, basic medium and NOSECM without supplements. EEC16 data shown, EEC16-TERT-L and EEC16-TERT-R colonies were highly similar in appearance. Arrows indicate examples of colonies; arrowheads point to single cells. (D) Addition of EGF, HC, BPE or INS to Basic Medium does not induce agar colony formation by EEC16-TERT-L cultures, but the combination of EGF plus HC induces colony formation at rates that are not significantly different to NOSECM. P N 0.05, relative to NOSECM control. Two-tailed paired Student's T-test. n.s., not significant.

effect (Fig. 4). Src has not previously been implicated in endometriosis and so, using two independent publicly available datasets (GSE7305 and GSE7307) we looked for evidence of Src pathway activation in human endometriosis tissues compared to normal eutopic endometrium. In both datasets we identified an overlap between a Src signature from Bild et al. [28] and endometriosis gene expression profiles (Fig. 5 and Supplementary Fig. 2). Using the Src signature gene-set, we identified 37 differentially expressed probes (14 up- and 23 down-regulated) from GSE7305, 30 differentially expressed probes (5 up- and 25 downregulated) from GSE7307. We integrated this result with the Src signature expression directionality as reported by Bild et al. [28] and identified 32 concordant genes when both datasets were merged. We performed a Fishers Exact test for each data set compared to the Bild et al. (2006) set (using a 2 × 2 table), and confirmed that the association is statistically significant (P-value b 0.03). We also found a core set of 11 Src-regulated genes that were differentially expressed in the endometrial tissues of endometriosis patients compared to unaffected controls (Fig. 5, GSE6364, P b 0.02, Fisher's Exact test), suggesting differential Src signaling can be detected in eutopic endometrium of affected women compared to controls. Finally, when we created a 321 gene endometriosis signature using Ingenuity Pathway Analysis (Supplementary Table 2), and compared this to data from 3 different functional Src experiments, we also found a significant overlap, with gene expression changes tending to correlate between the endometriosis signature and the functional experiments (Supplementary Fig. 3). These data show that Src activation occurs in our endometriosis model as well as in human endometriosis tissues.

Discussion Endometriosis is a common chronic, benign gynecological condition in women. Endometriosis causes pain and infertility and is associated with an increased risk of developing a particularly lethal subtype of epithelial ovarian cancer [5]. There is a clinical imperative to identify novel targets that can be used for the treatment of endometriosis, since more efficacious therapies for this disease could be a realistic way to treat infertility and chronic pain, and to prevent deaths from endometriosisassociated ovarian cancer. However, the types of experimental models that have successfully been used in translational cancer research have not yet been developed for endometriosis. The goal of this study was to establish an in vitro model of ovarian endometriosis that could be used for the discovery and testing of novel molecular targets for the disease. We created TERT-immortalized EEC16 lines because detailed cell biology studies in in vitro models usually require cells to be extensively passaged beyond the capacity of primary cells. It is now well established that many human cell types can be successfully immortalized using TERT alone, without abrogating the p53 and Rb pathways through the introduction of the SV40 large T-antigen [29–31]. Immortalized EEC16 lines maintained a normal diploid female karyotype, and the p53 DNA damage response pathway remained intact. There were some limitations of our model, particularly the absence of progesterone receptor (PRA) and estrogen receptor-alpha (ERα) expression. Other researchers have also reported reduction in ERα expression after culture and immortalization of EECs, which can be circumvented by overexpressing

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Fig. 4. Src activation in models of endometriosis. (A) Anchorage independent growth assays performed in medium containing EGF + HC and LYS450139, PP2, ICG-001, U0126 and LYS249002 to inhibit Notch, Src, Wnt, MAPK and PI3K signaling respectively. Control cells received vehicle alone. Colonies formed in the presence of a Wnt inhibitor were visibly smaller than control colonies. (B) Phase contrast microscopy shows loss of anchorage independent growth results from Src and Wnt inhibition.

this gene [21]. We find that the EEC16-TERT-L model can be readily transduced in the laboratory, and is therefore amenable to studies incorporating overexpression/knockdown of genes including PRA and ERα. Immortalization of EEC16 was associated with changes in the expression of markers that indicated that an epithelial-to-mesenchymal transition (EMT) had occurred. These changes were independent from the transduction method (retroviral or lentiviral). In our experience, TERT-immortalized OSECs do not undergo EMT [23,31] suggesting this phenomenon is a feature of EEC16 and/or other endometriosis derived cell lines. The mechanisms underlying accentuated EMT after immortalization are not fully clear as TERT is not known to induce EMT. We postulate that the prolonged time in culture in the presence of epidermal growth factor and hydrocortisone may promote EMT as a similar effect has been reported for primary OSECs [32]; but unlike in OSECs, inhibition of MAPK and PI3K signaling did not have a significant impact on the EMT phenotype of EEC16-TERT. EMT is a key process in the pathogenesis of human endometriosis [33] and also in neoplastic transformation [34]. In support of this, we observed a reduction in expression of key tumor suppressor genes implicated in the development of endometriosis-associated ovarian cancers (EAOC) in EEC16-TERT lines, including ARID1A. This suggests that the EEC16-TERT models mimic the very earliest stages in the development of EAOC and that, by introducing additional genetic alterations, these cell lines will be particularly useful for modeling neoplastic transformation in EAOC. A number of genes have now been implicated in the development of EAOC, including PTEN, ARID1A, HNF1B, WNT4 and CDKN2BAS1 [35–40]. Perturbing expression of these genes in these endometriosis models could be used to ascertain the functional role of these genes during EAOC tumorigenesis and to identify protein biomarkers, expressed by

Fig. 5. Src activation occurs in human endometriosis tissues. Gene expression changes in endometriosis tissues more frequently go in the same direction as in the Bild et al. (2006) Src signature. GSE7305 and GSE7307 are endometriosis expression datasets from the Human Body Index study and GSE6364, is a dataset of endometrial tissues of women with endometriosis compared to unaffected controls (mid-secretory phase endometrium) from Burney et al., 2007. Each row is labeled with official gene symbols and the corresponding Affymetrix U133 v2.0 probe identification separated by a dot. Each column shows the expression directionality in each dataset; Red: upregulated in endometriosis compared to eutopic endometrium, green: downregulated in endometriosis, white: no statistically significant change in expression.

partially transformed endometriosis cells, which could be assayed immunohistochemically to identify those women most at risk of developing cancer. Transformation models could also lead to the discovery of blood-borne biomarkers of early-stage disease that could be used to detect EAOC when disease is still confined to the ovary. Both strategies could save many women's lives by reducing rates of EAOC-associated mortality. EEC16-TERT cells showed evidence of neoplastic characteristics in anchorage independent growth assays, which was dependent on

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epidermal growth factors (EGF) and hydrocortisone (HC). We found that this was dependent on active Src and Wnt signaling. A central role for Src signaling in endometriosis has not previously been reported and so we evaluated Src activation in primary human endometriosis tissues to validate this finding. We found a significant correlation between a Src gene signature [28] and gene expression profiles of human endometriosis tissues compared to biopsies of normal endometrium. We also found evidence for Src activation in the endometrium of endometriosis patients. This has particular clinical relevance as currently there is no diagnostic test that can accurately detect endometriosis in endometrial biopsy specimens. Patients with severe symptoms can undergo laparoscopic surgery to obtain a histological diagnosis of endometriosis, but the majority of endometriosis cases in the US do not require invasive surgery and so are managed based on symptoms alone. A molecular clinical test for endometriosis based, for example, on the 10 core Src-regulated genes we identified, would avoid cases of misdiagnosis in women with non-specific pelvic symptoms and could improve treatment of these patients. Activated Src signaling commonly occurs in cancer, and this study raises the interesting hypothesis that Src activation could be a driver of endometriosis-associated ovarian cancer. Moreover, if the Src signature is differentially activated in the eutopic endometrium of women at highest risk of developing endometriosisassociated ovarian cancer, endometrial biopsy may represent a noninvasive screening tool to detect early-stage endometriosis-associated ovarian cancer. Anchorage independent growth assays can be considered to model seeding of endometrial/endometriosis cells throughout the peritoneal cavity. Single endometrial cells found within the peritoneal cavity following retrograde menstruation [41] are mimicked by suspending single cell endometriosis cell suspensions in semi-solid medium. It is proposed that the near universal process of retrograde menstruation is the mechanism by which endometrial cells reach the peritoneal cavity, although it is not currently known why endometriosis only develops in ~10% of women. The results observed with Src inhibition led us to postulate that Src activation may promote the survival of ectopic endometrial cells in the peritoneal cavity following retrograde menstruation and that Src activation in endometrial tissues of women may therefore perhaps predispose to endometriosis. Larger studies of Src signaling in primary endometriosis lesions and endometrial tissues will be required to confirm this hypothesis. Src inhibitors are currently being evaluated in clinical trials as novel anti-cancer treatments and could perhaps be used in the future for treatment of endometriosis particularly if novel Src inhibitors are developed that have less severe side effects at active doses [42]. Treatment with Src inhibitors would be predicted to impede the development of new endometriosis lesions. In addition, Src inhibitors have also been shown to block angiogenesis, proliferation, invasion and migration of tumor cells and therefore are likely to exert similar pleiotropic effects on endometriosis cells [42]. In conclusion, we have established novel TERT-immortalized models of ovarian endometriosis. Many fundamental questions in endometriosis biology remain unanswered and so given the paucity of robust experimental models for endometriosis, it is likely that these new models will have many diverse applications for understanding the underlying biology and etiology of endometriosis. Perhaps more significantly, these models can be used as tools for identifying novel therapeutic targets for the disease. We demonstrated the potential power of these models to identify the Src pathway as a novel, putative therapeutic target for endometriosis. Validation of this finding in more detailed follow-up studies, combined with the fact that Src kinase inhibitors have already been developed, could lead to rapid clinical applications emerging from the development of these models. Research support This work was supported by departmental funds from the Departments of Preventive Medicine and Obstetrics and Gynecology at

the USC Keck School of Medicine. KL is funded by the National Institutes of Health grant number 5 U19 CA148112-02 and an Ann Schreiber Program of Excellence award from the Ovarian Cancer Research Fund (POE/USC/01.12). The work was performed within the USC Norris Comprehensive Cancer Center which is supported by a Cancer Center Support Grant (award number P30 CA014089) from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. Funding support for this project was in part supported by CNPq (12.1.17598.1.3) to H.N. Author contributions K.L. and S.A.G. designed and lead the study. K.L. and N.L. performed most of the experiments. J.M.L. performed the Western blotting and D.B. assisted with a preliminary xenograft assay. P.N.R. performed the karyotyping and H.A.M.T. and H.S. performed the Src signature analysis in primary tumors. All authors were involved in writing the paper and had final approval of the submitted and published versions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2014.06.016. Conflict of interest statement None to declare.

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