{"paper_id":"7993a4e3-3f7f-482e-b986-1341a349b006","body_text":"UCSF\nUC San Francisco Previously Published Works\nTitle\nInhibition of epidermal growth factor receptor restores decidualization markers in stromal \nfibroblasts from women with endometriosis\nPermalink\nhttps://escholarship.org/uc/item/62x0p6pz\nJournal\nJournal of Endometriosis and Pelvic Pain Disorders, 6(4)\nISSN\n2284-0265\nAuthors\nErikson, David W\nChen, Joseph C\nPiltonen, Terhi T\net al.\nPublication Date\n2014-10-01\nDOI\n10.5301/je.5000198\n \nPeer reviewed\neScholarship.org Powered by the California Digital Library\nUniversity of California\n\nSee\tdiscussions,\tstats,\tand\tauthor\tprofiles\tfor\tthis\tpublication\tat:\t\nhttps://www.researchgate.net/publication/268980925\nInhibition\tof\tepidermal\tgrowth\tfactor\treceptor\nrestores\tdecidualization\tmarkers\tin\tstromal\nfibroblasts\tfrom\twomen...\nArticle\n\t·\tOctober\t2014\nDOI:\t10.5301/je.5000198\nCITATIONS\n2\nREADS\n42\n6\tauthors\n,\tincluding:\nSome\tof\tthe\tauthors\tof\tthis\tpublication\tare\talso\tworking\ton\tthese\trelated\tprojects:\nA\tproject\twith\tprof.\tT\tYanase\tat\tFukuoka\tUniv.\tand\tKyushu\tUniv.\n\t\nView\tproject\nCyclic\tnucleotides\tcompartmentalization\tin\tthe\tcardiovascular\tsystem\n\t\nView\tproject\nDavid\tW\tErikson\nOregon\tHealth\tand\tScience\tUniversity\n48\n\t\nPUBLICATIONS\n\t\t\t\n590\n\t\nCITATIONS\n\t\t\t\nSEE\tPROFILE\nTerhi\tPiltonen\nOulu\tUniversity\tHospital\n46\n\t\nPUBLICATIONS\n\t\t\t\n685\n\t\nCITATIONS\n\t\t\t\nSEE\tPROFILE\nMarco\tConti\nUniversity\tof\tCalifornia,\tSan\tFrancisco\n334\n\t\nPUBLICATIONS\n\t\t\t\n18,873\n\t\nCITATIONS\n\t\t\t\nSEE\tPROFILE\nJuan\tIrwin\nUniversity\tof\tCalifornia,\tSan\tFrancisco\n20\n\t\nPUBLICATIONS\n\t\t\t\n418\n\t\nCITATIONS\n\t\t\t\nSEE\tPROFILE\nAll\tcontent\tfollowing\tthis\tpage\twas\tuploaded\tby\t\nDavid\tW\tErikson\n\ton\t21\tJanuary\t2015.\nThe\tuser\thas\trequested\tenhancement\tof\tthe\tdownloaded\tfile.\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\nJournal of EndometriosisJournal of Endometriosis and Pelvic Pain Disorders (2014; :00) 000-00000 (; :00) 000-00000\n1\nInhibition of epidermal growth factor receptor restores \ndecidualization markers in stromal fibroblasts from \nwomen with endometriosis\nDavid W. Erikson1, Joseph C. Chen1, Terhi T. Piltonen1,2, Marco Conti1, Juan C. Irwin1, Linda C. Giudice1\n1  Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of \nCalifornia – San Francisco, San Francisco, California - USA\n2  Department of Obstetrics and Gynecology and Medical Research Center, University of Oulu and Oulu University Hospital, \nOulu - Finland\nORIGINAL ARTICLE\nDOI: 10.5301/je.5000198\nPurpose: Decidualization comprises specific biochemical and morphological changes in uterine en-\ndometrium essential for establishment of pregnancy. This process is abnormal in women with endo-\nmetriosis, a disorder in which endometrial-like tissue is present outside the uterus. The aim of this \nstudy was to restore cAMP-induced decidualization marker expression in endometrial stromal fibro-\nblasts from women with endometriosis by using chemical inhibitors to PI3K/AKT/mammalian target \nof rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and epidermal growth factor receptor \n(EGFR) signaling pathways in vitro.\nMethods: Endometrial stromal fibroblasts (eSF) from women with (eSF\nendo) and without (eSF non-endo) \nendometriosis were treated with inhibitors to EGFR tyrosine kinase (gefitinib), mTOR (rapamycin)  \nand MAPK kinase 1/2 (MEK1/2) (UO126) during 8-bromoadenosine 3’,5’-cyclic monophosphate  \n(8-br-cAMP)–stimulated decidualization. Decidualization was assessed by evaluating expression \nof insulin growth factor binding protein 1 (IGFBP1), prolactin (PRL) and forkhead box protein O1A \n(FOXO1A) by quantitative real-time PCR.\nResults: Gefitinib restored expression of decidualization markers in eSF\nendo to levels consistent with \nthose in eSFnon-endo. Elevated levels of phosphorylated mTOR in eSF endo were reduced to levels found \nin eSF non-endo, by gefitinib during treatment with 8-br-cAMP. Additional gene expression analyses  \nsuggested dysregulation of EGFR negative feedback regulators in eSFendo.\nConclusions: Results implicate EGFR signaling as an underlying cause for aberrant cAMP-induced \ndecidualization in women with endometriosis, and provide a potential target for management of infertil-\nity associated with the disease. The reduction of p-mTOR levels in eSF\nendo during 8-br-cAMP treatment \nsuggests cooperation between EGR and protein kinase A signaling in the regulation of mTOR in eSF.\nKeywords: Decidualization, Endometriosis, Epidermal growth factor receptor, Fibroblast, Mammalian \ntarget of rapamycin, Mitogen-activated protein kinase\nAccepted: October 28, 2014\nIntRODUCtIOn\nEndometriosis is a chronic estrogen-dependent disorder \naffecting 6%-10% of reproductive aged women (1, 2). It is \ncharacterized by extrauterine endometrial tissue that elic-\nits an inflammatory response mainly in the pelvis (3), con-\ntributing to chronic pelvic pain and associated infertility \ndue to ovulatory dysfunction, poor egg quality, abnormal  \n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n2\nEGFR inhibition restores decidual markers\nwomen with endometriosis (eSF endo) do not express ap-\npreciable amounts of the decidual marker insulin growth \nfactor binding protein 1 (IGFBP1) due to inherent insen-\nsitivity to PKA pathway stimulation (14, 15). t reatment \nof ectopic eSF from ovarian endometriomas with small \n molecule inhibitors of AKt or PI3K during in vitro decidu-\nalization increased expression of IGFBP1 and its upstream \ntranscriptional regulator forkhead box 1 (FOXO1) (16). \nWe previously demonstrated that treatment of stromal \nfibroblasts from eutopic endometrium of women without \n endometriosis (eSFnon-endo) with 8-br-cAMP resulted in up-\nregulation of IGFBP1; whereas eSF endo were refractory  \nto 8-br-cAMP (14). In addition, the inhibition of MAPK \n kinase 1/2 (MEK1/2) with UO126 did not restore 8-br-\ncAMP up-regulation of IGFBP1 in these cells from women \nwith disease (2). thus, the limited use of small molecule \ninhibitors, reported in a few studies, has met with fair \n success in restoring decidualization marker expression in \neSF\nendo.\nHerein, we hypothesized that a combination of overactive \nPI3K/AKt/mtOR, MAPK and EGFR signaling pathways is \ncausative of aberrant 8-br-cAMP-induced decidualization \nmarker expression in eSF from women with endometriosis. \nWe treated eSF with the selective chemical inhibitors ge-\nfitinib (EGFR tyrosine kinase inhibitor), rapamycin (mtOR \ninhibitor) and UO126 concomitantly with 8-br-cAMP , and \nperformed multiplex and quantitative real-time polymerase \nchain reaction (qPCR) analyses to investigate their effects \non decidualization marker expression. Our results indicate \nthat inhibition of EGFR tyrosine kinase activity restored \n expression of specific decidual markers in eSF\nendo to levels \nin eSFnon-endo.\nMAtERIAlS AnD MEtHODS\nHuman endometrial samples\nHuman eutopic endometrial tissue samples were obtained in \naccordance with the guidelines of the Declaration of Helsin-\nki, from 24- to 42-year-old women undergoing endometrial  \nbiopsy or hysterectomy for diagnosis or treatment of pelvic \npain, fibroids, prolapse and/or endometriosis (tab. I). no ec-\ntopic lesions or tissues were collected from patients or used \nin the present study. Presence or absence of endometriosis \nwas confirmed by laparoscopic visualization and histologi-\ncal analysis of peritoneal lesions. Staging of endometriosis \nuterine endometrium and/or compromised embryo im-\nplantation (1, 4). Ectopic endometrial lesions (outside the \nuterus) and eutopic endometrium (within the uterus) of \nwomen with this disease are stimulated by estradiol, but \nare resistant to the action of progestins and progesterone \n(P\n4) (5, 6). the latter regulates nociceptive pain thresh-\nolds in innervated endometriosis lesions (7) and, within \nthe uterus, is essential for endometrial epithelial secretory \ntransformation and stromal fibroblast decidualization, crit-\nical for the establishment and maintenance of pregnancy. \nthus, resistance to P\n4 and progestins can have a major \nimpact on pain relief, disease control and fertility success \nof affected women.\nSeveral signaling pathways have been implicated in these \naspects of the pathophysiology of progesterone resistance \nand compromised stromal fibroblast decidualization, in-\ncluding protein kinase A (PKA), mitogen-activated protein \nkinase (MAPK), and phosphatidylinositide-3 kinase/AKt/\nmammalian target of rapamycin (PI3K/AKt/mtOR) (8). Un-\nderstanding involvement and cross-talk among these path-\nways, and whether pathway-specific inhibitors can restore \nendometrial cell function or biomarkers, is of great interest \nas they hold promise to mitigate endometrial cellular dys-\nfunction in the setting of disease.\nConstitutive activation of PI3K/AKt and MAPK pathways \nhas been reported in both eutopic and ectopic endometrial \ntissues in endometriosis (9). We previously found higher \nlevels of phosphorylated (p)-ERK1/2 in stromal fibroblasts \nfrom women with severe endometriosis versus women \nwithout disease in vivo (2). Higher levels of phosphorylated \n(p)-mtOR have been found in ovarian endometrioma tis-\nsues (10), and active AKt1 is elevated in eutopic endo-\nmetrium of women with versus without endometriosis (11). \nIn addition, mRnA expression of epidermal growth factor \nreceptor (EGFR), which is upstream of both PI3K/AKt and \nMAPK pathways, is increased in eutopic endometrium in \nthe early secretory phase in severe versus mild disease \n(12). these data suggest a role for EGFR in increased acti-\nvation of these pathways within the stromal compartment \nof the uterus in women with endometriosis. Consistent \nwith this notion is the finding that members of the EGFR \nfamily HER1, HER2 and HER3 exhibit greater mRnA ex-\npression in whole eutopic endometrium from women with \nversus without disease (13).\nWhen treated with decidualizing stimuli (P\n4, progestin and/\nor 8-bromoadenosine 3’,5’-cyclic monophosphate [8-br-\ncAMP]), eutopic endometrial stromal fibroblasts (eSF) from \n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n3\nErikson et al\nwas defined according to the Revised American Society \nfor Reproductive Medicine classification system (17). no \npatients used hormone treatments within 3 months of sur-\ngery. Samples were obtained through the UCSF nIH Human \nEndometrial tissue and DnA Bank with appropriate institu-\ntional review, approvals and written informed consent from \nall participating subjects, as approved by the University of \nCalifornia San Francisco Committee on Human Research. \nEndometrial samples were processed for cell culture experi-\nments as described below.\neSF isolation and culture\neSF from non-endometriosis (eSF non-endo) (n = 5) and endo-\nmetriosis (eSFendo) (n = 5) subjects were isolated by digest-\ning endometrial tissue samples with collagenase followed \nby filtration as previously described (18, 19). Isolated eSF \nwere cultured in growth medium (phenol red-free medium \nof 3:1 high-glucose phenol red-free DMEM/MCDB-105, \n0.676 mM sodium pyruvate, 10% charcoal-stripped fe-\ntal bovine serum [FBS], 1% penicillin-streptomycin mix, \n50 µg/ml gentamycin, 5 µg/ml insulin), with medium re-\nplaced every 2-3 days for up to 6 passages. All cells used \nin the experiments described herein were from the fourth \npassage. For qPCR and multiplex experiments, confluent \ncells were incubated for 24 hours in low-serum medium \n(3:1 high-glucose phenol red-free DMEM/MCDB-105, \n0.75 mM sodium pyruvate, 50 µg/ml gentamycin, 2% \nFBS) before treatment. For multiplex experiments, cells \nwere incubated in either EGF (20 ng/ml) for 30 minutes \nor 8-br-cAMP (0.5 mM) for 60 minutes with inhibitors to \nmtOR (rapamycin; 10 nM), MEK1/2 (UO126; 10 µM) or \nEGFR t yr1173 t yr992 (gefitinib; 10 µM) alone or in combi-\nnation. these time points were determined by treatment \nof eSF with either EGF or 8-br-cAMP and then observ-\ning the time point at which optimal p-ERK1/2 activation \nTABLE I - HUMAn  PARt ICIPAnt  CHARACt ERISt ICS\nSubject ID Phase Age Procedure Ethnicity Diagnosis Analysis\nS01 PE 28 l aparoscopic \nsupracervical \nhysterectomy\nWhite Dysmenorrhea,  \nmenometrorrhagia,  \novarian cyst\nqPCR\nS02 PE 42 Supracervical \nhysterectomy\nWhite Symptomatic fibroids,  \nadenomyosis\nqPCR,  \nmultiplex\nS03 SE 24 EBx Asian n atural cycle donor qPCR,  \nmultiplex\nS04 PE 37 t ubal ligation, \nEBx\nAsian Undesired fertility qPCR\nS05 PE 39 t ubal ligation, \nEBx\nAsian Undesired fertility qPCR,  \nmultiplex\nS06 SE 37 Operative  \nlaparoscopy, EBx\nWhite Endometriosis, stage IV, pelvic  \nadhesion, adenomyosis\nqPCR,  \nmultiplex\nS07 PE 30 Operative  \nlaparoscopy, EBx\nUnknown Endometriosis, stage IV, pelvic  \npain, uterine bleeding\nqPCR\nS08 PE 32 Operative  \nlaparoscopy, EBx\nWhite Endometriosis, stage IV, pelvic  \npain, uterine bleeding,  \ndysmenorrhea\nqPCR,  \nmultiplex\nS09 PE 42 Operative  \nlaparoscopy, EBx\nWhite Endometriosis, stage IV, pelvic  \npain, pelvic adhesions\nqPCR\nS10 SE 40 Operative  \nlaparoscopy, EBx\nAsian Endometriosis, stage IV, pelvic  \npain, uterine bleeding\nqPCR,  \nmultiplex\nEBx = endometrial biopsy; PE = proliferative endometrium; qPCR = quantitative real-time reverse transcription polymerase chain reaction; SE = secretory  \nendometrium.\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n4\nEGFR inhibition restores decidual markers\n(EGF) or inhibition (8-br-cAMP) occurred (2, 20). All inhibi-\ntors were delivered in dimethyl sulfoxide (DMSO), and all \ntreatments were adjusted to contain 0.1% DMSO. Cells \nwere then harvested for protein analysis as described be-\nlow. For decidualization, cells were treated with 0.5 mM \n8-br-cAMP for 96 hours in low-serum medium, condi-\ntions previously determined optimal for decidualization \n(2, 14), with or without the inhibitors listed above. Media \nwere refreshed after 48 hours. Optimal inhibitor concen-\ntrations were determined using previously published data \non effective doses (2, 21, 22). Samples without treatment \nwere collected at baseline (t = 0) to serve as controls. \nAfter 96 hours of treatment, cells were harvested for RnA \nor protein isolation as described below. All sample treat-\nments were performed in duplicate in  independent ex-\nperiments to ensure reproducibility of  results.\nQuantitative RT-PCR\nt otal RnA was isolated from cultured, treated eSF using \nthe nucleoSpin RnA kit (Macherey-nagel, Bethlehem, \nPA, USA) following the manufacturer’s instructions. t otal \nRnA was quantified by UV spectrophotometry (nanodrop, \n Wilmington, DE, USA), and reverse transcription was per -\nformed using random primers and the iScript R t reagents \nfollowing the manufacturer’s protocol (Bio-Rad laborato-\nries, Hercules, CA, USA). mRnA levels were determined \nby qPCR using the Mx 3005 Pro (Stratagene, la Jolla, CA, \nUSA). Primer sets used are listed in Supplemental t able I. \ncDnA (10 ng) was amplified by SYBR Supermix (thermo \nFisher Scientific, Waltham, MA, USA) using the following \nprotocol: (i) DnA polymerase activation at 95°C for 15 min-\nutes; (ii) 40 cycles of denaturation at 95°C for 15 seconds, \nannealing at 60°C for 45 seconds and extension at 72°C \nfor 60 seconds. the absence of primer dimers was deter -\nmined through dissociation curve analysis by performing a \ncycle of denaturation at 95°C for 1 minute followed by 35 \ncycles of increasing temperature for 33 seconds from 65°C \nto 97°C at the end of each analysis. Relative expression \nwas calculated using the relative standard curve method \n(23), where a standard curve is created for each primer set \nusing pooled cDnAs from the entire experimental sample \nset, and efficiency of the cDnA standards is between 90% \nand 110%. Samples were assayed in duplicate, and rela-\ntive gene expression was normalized with beta actin (ACt-\nnB) and t = 0 controls within each group to control for time \nin culture.\nMultiplex assays\nFollowing treatment, cells were washed in phosphate- \nbuffered saline (PBS) and lysed with RIPA buffer contain-\ning a protease inhibitor cocktail (2 mM AEBSF , 1 mM EDtA, \n130 µM Bestatin, E-64 14 µM, 1 µM leupeptin, 0.3 µM \nAprotinin) (Millipore, Bedford, MA, USA). After centrifuga-\ntion to clear cellular debris (10,000 g, 10 minutes, 4°C), \nprotein quantity was evaluated by Bradford assay (Bio-\nRad) using the manufacturer’s instructions. Milliplex MAP \nmultiplex protein assays were performed according to \nthe manufacturer’s protocol (Millipore). Briefly, cell lysates  \n(3 µg protein/well) were diluted 1:1 with Milliplex MAP As-\nsay Buffer 2. the filter plate was pre-wetted with Assay \nBuffer 2, and the buffer removed by vacuum filtration. Mil-\nliplex MAPmates were combined and 25 µl was added per \nwell (Suppl tab. II). After adding 25 µl of diluted cell lysate \nto the appropriate wells, the plate was incubated overnight \nat 4°C with shaking. the lysate was removed by vacuum \nfiltration, washed 3 times with Assay Buffer 2, and 25 µl \nof detection antibody was added for 1 hour at room tem-\nperature. Diluted  streptavidin-phycoerythrin (SAPE) was \nthen added after filtration, the plate was incubated for 15 \nminutes at room temperature, and amplification buffer was \nadded before a final incubation of 15 minutes. the SAPE/\namplification buffer mix was removed by filtration, 150 µl \nof Assay Buffer 2 was added, and the plate was analyzed \non a  BioPlex 200 (Bio-Rad). the ratio of phospho protein \nto glyceraldehyde 3-phosphate dehydrogenase (GAPDH) \nwas calculated for each target.\np-EGFR multiplex assays\nAnalysis of p-EGFR (t yr1173) was performed using a mul-\ntiplex approach similar to that for other phospho protein \ntargets described above. After treatment and total protein \nextraction from cells, lysates were diluted in cell lysis buf-\nfer to a concentration of 10 µg/50 µl and incubated with \nBioPlex Pro magnetic cell signaling beads (Bio-Rad) for \n p-EGFR (tyr1173) and GAPDH overnight at 4°C with shak-\ning. Beads were washed and incubated for 30 minutes with \nantibodies for p-EGFR and GAPDH at room temperature, \nwashed and further incubated with SAPE for 10 minutes at \nroom temperature. Beads were then washed, resuspended \nin Bead Resuspension Buffer and analyzed on a BioPlex \n200 (Bio-Rad). the ratio of phospho protein to GAPDH was \ncalculated for p-EGFR.\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n5\nErikson et al\nData analysis\nAll quantitative data were subjected to analyses of variance \n(AnOVA) using general linear model procedures available \nwith SAS (SAS 2012-2013; SAS Institute, Cary, nC, USA). \nFor transcript comparisons, data were generated from \nfold change of each transcript to ACtnB transcripts using \nindividually determined standard curves. For protein com-\nparisons fold change to GAPDH protein was performed.  \nStatistical models considered variation due to the main \neffect of inhibitor treatment, including no treatment ve-\nhicle control (DMSO). t reatment effects were identified by \nperforming a set of preplanned contrasts to include com-\nparisons of specific groups for each target transcript or \nprotein (24). In all cases, significance was set at a p value \nof <0.05.\nRESUltS\nt o prove/disprove our central hypothesis that overactive \nEGFR signaling in eSF endo is causative of the compro-\nmised decidualization response observed in these cells, \nwe pursued the following: (i) analyzed EGFR pathway ac-\ntivation, including downstream ERK1/2 and mtOR, in eSF \nto determine activation status of these pathways in dis-\nease; (ii) demonstrated compromised eSF decidualization \nresponse on a background of increased EGFR signaling \n(12, 13), and recreated this effect in eSF\nnon-endo by activat-\ning EGFR; and (iii) used small molecule inhibitors to EGFR, \nmtOR and MEK1/2 in an attempt to restore the decidual-\nization response in eSFendo.\neSF from women with endometriosis do not \n decidualize in response to cAMP\nPrevious data have demonstrated that eSF endo exhibit an \naltered response to PKA stimulation (14), which manifests \nin reduced decidualization capacity (2, 15). Our objective \nin this experiment was to demonstrate compromised de-\ncidualization response by eSF on a presumed background \nof increased EGFR signaling (12, 13). After treatment with \ncAMP for 96 hours, we measured expression of 3 key de-\ncidualization genes by PCR, IGFBP1, PRL and FOXO1A, \nand calculated fold changes against expression levels at \nt = 0. IGFBP1 expression increased over 100,000-fold in \neSF\nnon-endo, while there was a minimal increase in eSF endo \n(Fig. 1A). PRL expression increased over 35,000-fold in \neSFnon-endo, with a small increase in eSF endo (Fig. 1B). We \nalso observed a significant increase in FOXO1A expression \nin eSFnon-endo, but little increase in eSF endo (Fig. 1C). these \ndata demonstrate a limited capacity of eSF endo to respond \nto cAMP , and suggest that overactive EGFR found in these \nsubjects could be responsible for aberrant decidualization \nin eSF\nendo.\nComponents of the EGFR signaling pathway are \nactivated in endometriosis\nto better understand the role of signaling in aberrant decidu-\nalization, we analyzed mtOR, EGFR and ERK phosphory-\nlation by 8-br-cAMP at 60 minutes, a time point found to \ndecrease ERK1/2 phosphorylation in eSFnon-endo (2). Contrary \nto our hypothesis, there were no significant differences in p-\nEGFR at baseline (t = 0), and p-EGFR increased in eSFnon-endo \nwhen treated with 8-br-cAMP for 60 minutes, with no con-\ncomitant increase in eSF\nendo (Fig. 2A). p-mtOR was elevated \nin eSFendo at t = 0 and increased in both eSFnon-endo and eS-\nFendo when treated with cAMP , with levels in diseased cells \nremaining significantly higher than in nondiseased cells after \n60 minutes of treatment (Fig. 2B). Finally, p-ERK1/2 was also \nelevated in eSF\nendo at t = 0, and decreased in eSFnon-endo while \nremaining elevated in eSFendo with 60 minutes of cAMP treat-\nment (Fig. 2C). these data indicate differences in response \nof EGFR and mtOR to cAMP and subsequent phosphor -\nylation states that may affect the onset of decidualization  \nin eSF .\nEGF inhibition of decidualization in eSF from  \nwomen without endometriosis is restored by \ngefitinib\nPrevious experiments have described the inhibition of \ncAMP-induced decidualization by EGF in eSF (25). the \nobjective of this experiment was to inhibit decidualization \nin eSFnon-endo by stimulating EGFR with EGF . Figure 3 shows \nIGFBP1 mRnA expression after 96-hour treatment with \nEGF + 8-br-cAMP and compared with 8-br-cAMP alone. \nResults showed that expression of IGFBP1 in eSF non-endo, \nwhen treated with EGF + 8-br-cAMP was equal to that in \neSFendo and similar to levels found in eSF endo treated with \n8-br-cAMP alone. When the EGFR tyrosine kinase in-\nhibitor gefitinib was added to EGF + 8-br-cAMP , IGFBP1 \n expression in eSFnon-endo was fully restored and allowed par-\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n6\nEGFR inhibition restores decidual markers\nFig. 1 - Endometrial stromal fibroblasts from women with endome -\ntriosis do not decidualize in response to 8-br-cAMP. Endometrial \nstromal fibroblasts from women without endometriosis (Non-Endo) \nor with endometriosis (Endo) were cultured for 96 hours in low-\nserum medium (DMEM-MCDB105 + 2% FBS) with 0.5 mM 8-br-\ncAMP to induce decidualization. Separate cells were collected prior \nto 8-br-cAMP treatment and served as baseline (t = 0 + control). \nAfter 96 hours, cells were harvested for total RNA extraction and \nsubjected to quantitative real-time PCR analysis for A)  insulin-\nlike growth factor binding protein 1 (IGFBP1); B)  prolactin (PRL); \nand C) forkhead box protein 01A (FOXO1A). Gene expression data \nwere calculated using the standard curve method and normalized \nto ACTNB levels in each sample. All data were then normalized to  \nt = 0 to control for time in culture. Data are represented as average \nfold change to t = 0 ± s.e.m. Asterisks indicate significant differ -\nences by ANOVA (p<0.05).\nFig. 2 - 8-br-cAMP regulation of cell signaling activation in en -\ndometrial stromal fibroblasts. Endometrial stromal fibroblasts \nfrom women without endometriosis (Non-Endo) and with endo -\nmetriosis (Endo) were cultured for 60 minutes in low-serum me -\ndium (DMEM-MCDB105 + 2% FBS) containing 0.5 mM 8-br-cAMP. \nCells (n = 3 each Non-Endo and Endo) were harvested for total \nprotein extraction and subjected to multiplex protein analysis for \nA) phosphorylated-epidermal growth factor receptor (p-EGFR); \nB) phosphorylated-mammalian target of rapamycin (p-mTOR); \nand C) phosphorylated-extracellular-signal-regulated kinase 1/2 \n(p-ERK1/2). Phosphorylated-protein values were normalized to \nGAPDH levels for each sample. Data are represented as average \nratios ± s.e.m. Asterisks indicate significant differences by ANOVA \n(p<0.05) where Non-Endo and Endo values were compared within \ntreatment (t = 0 or cAMP).\n\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n7\nErikson et al\ntial restoration of IGFBP1 expression in eSFendo. these re-\nsults indicate that overactivation of EGFR in disease could \nbe a cause for decreased decidualization in response to  \n8-br-cAMP , and that blockade of EGFR tyrosine kinase \nwith gefitinib restores decidualization marker expression in \neSF\nendo.\nEGF increases activation of mTOR in endome-\ntriosis\nWe treated eSF with EGF for 30 minutes alone or with in-\nhibitors to specific signaling pathways, to evaluate EGFR, \nERK and mtOR activation (in a pilot study, 30 minutes was \nfound to be the optimal time for EGF activation of ERK1/2 \nin eSF – data not shown). p-EGFR increased similarly in \neSF\nnon-endo and eSF endo with all treatments, except for gefi-\ntinib + EGF which decreased p-EGFR in eSF independent \nof disease status (Fig. 4A). p-ERK1/2 was also detected \nin similar levels in eSF\nnon-endo and eSF endo, except for t = 0 \nwhere ERK1/2 activation was increased in eSF endo versus \neSFnon-endo. t reatment with both gefitinib and UO126 inhib-\nited ERK activation by EGF (Fig. 4B). mtOR phosphoryla-\ntion was increased in eSFendo versus eSFnon-endo by EGF in all \ntreatments, including treatment with EGF in combination \nwith gefitinib (Fig. 4C). these data demonstrate that EGF \nis able to activate EGFR, ERK and mtOR pathways in eSF , \nindependent of disease state. they also demonstrate that \nwhile gefitinib is able to decrease activation of both EGFR \nand ERK by EGF , the same was not evident with p-mtOR, \nsuggesting an alternate route of activation of mtOR by \nEGF that is EGFR-independent.\nGefitinib restores decidualization marker expres-\nsion in endometriosis\nWe treated eSF with inhibitors to EGFR, mtOR and \nMEK1/2 during cAMP treatment to determine their ef-\nfect on restoring decidualization marker expression, and \n investigated the roles of these signaling pathways on \nthe decidualization process in eSF . IGFBP1 expression \n remained decreased in eSFendo versus eSFnon-endo with 8-br-\ncAMP and rapamycin + 8-br-cAMP , and UO126 + 8-br-\ncAMP . In contrast, when treated with gefitinib, levels of \nIGFBP1 increased in eSF endo to levels equal to that found \nin eSF non-endo (Fig. 5A). Similar results were also found \nwith 2 other decidualization markers, PRL (Fig. 5B) and \nFOXO1A (Fig. 5C). these results indicate that inhibition \nof EGFR, but not downstream mtOR or ERK pathways, \nis able to restore cAMP-induced decidualization marker \nexpression in eSF\nendo.\nFig. 3 - EGF inhibition of decidualiza-\ntion in endometrial stromal fibroblasts \nis restored by gefitinib. Endometrial \nstromal fibroblasts from women with-\nout endometriosis (Non-Endo) or with \nendometriosis (Endo) were cultured \nfor 96 hours in low-serum medium \n(DMEM-MCDB105 + 2% FBS) with \n0.5 mM 8-br-cAMP, 0.5 mM 8-br-\ncAMP + 20 ng/mL EGF or 8-br-cAMP \n+ EGF + 10 µM gefitinib. Inhibitors \nwere delivered in DMSO, and all treat-\nments including cAMP were adjusted \nto contain 0.1% DMSO. Cells were \nharvested for total RNA extraction \nand subjected to quantitative real-\ntime PCR for insulin-like growth factor \nbinding protein 1 (IGFBP1). Relative \ngene expression was calculated using \nthe standard curve method and nor -\nmalized to ACTNB levels in each sam-\nple. Data are represented as average \nfold change to t = 0 ± s.e.m. Different \nletters indicate significant differences \nby 2-way ANOVA (p<0.05).\n\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n8\nEGFR inhibition restores decidual markers\nGefitinib affects mTOR activation in endometriosis\nBecause gefitinib restored decidualization marker expres-\nsion in eSF endo, we investigated signaling pathway activa-\ntion when EGFR tyrosine kinase was inhibited with gefitinib.  \np-EGFR was increased in eSF non-endo versus eSF endo with \n8-br-cAMP treatment, but increased in eSF endo versus  \neSFnon-endo with rapamycin + 8-br-cAMP (Fig. 6A). there \nwere no differences in p-ERK1/2 between eSF non-endo and \neSFendo with any treatment, aside from the t = 0 time point \nand 8-br-cAMP treatment, as observed earlier (Fig. 6B). \nUO126 did not decrease p-ERK1/2 levels in the presence  \nof 8-br-cAMP , as it did when cells were treated with EGF , \nsuggesting the possibility of direct activation of ERK1/2 by \nPKA stimulation in eSF (Fig. 6B). Activation of mtOR was \nhigher at t = 0 in eSF\nendo as described earlier, and remained \nelevated in eSF endo versus eSF non-endo with 8-br-cAMP , ra-\npamycin + 8-br-cAMP , and UO126 + 8-br-cAMP treatment. \nHowever, when cells were treated with gefitinib + 8-br-\ncAMP , the treatment that restored decidualization marker \nlevels in eSF endo, p-mtOR levels were equal in eSF non-endo  \nand eSFendo, although levels in eSF endo with this treatment \nwere lower than those found in eSF endo treated with 8-br-\ncAMP alone (Fig. 6C). thus, these data demonstrate that \ninhibition of the tyrosine kinase domain of EGFR during \ncAMP treatment results in decreased levels of p-mtOR in \nFig. 4 - EGF regulation of cell signaling activation in endometrial stromal fibroblasts. Endometrial stromal fibroblasts from women without \nendometriosis (Non-Endo) and with endometriosis (Endo) were cultured for 30 minutes in low-serum medium (DMEM-MCDB105 + 2% FBS) \ncontaining 20 ng/mL EGF, EGF + 10 µM gefitinib, EGF + 10 nM rapamycin, or EGF + 10 µM UO126. Inhibitors were delivered in DMSO, and all \ntreatments including cAMP were adjusted to contain 0.1% DMSO. Cells were harvested for total protein extraction and subjected to multiplex \nprotein analysis including A) phosphorylated-epidermal growth factor receptor (p-EGFR); B) phosphorylated-extracellular-regulated-kinase \n1/2 (p-ERK1/2); and C) phosphorylated mammalian target of rapamycin (p-mTOR). Phosphorylated-protein values were normalized to GAPDH \nlevels for each sample. Data are represented as average ratios ± s.e.m. Asterisks indicate significant differences between Non-Endo and Endo \nwithin treatment by ANOVA (p<0.05).\n\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n9\nErikson et al\neSFendo to levels equal to eSFnon-endo, suggesting that EGFR-\nmtOR signaling is critical during cAMP-induced decidual-\nization in eSF . When combined with the data above showing \nEGF stimulating mtOR through EGFR-independent means, \nthe data suggest interplay of an EGFR tyrosine kinase and \nPKA signaling in the regulation of mtOR activation in eSF .\nEGF receptor and negative feedback regulator \nexpression is dysregulated in eSFendo\nBecause differences in EGFR activation between eSF non-\nendo and eSF endo were minimal, we evaluated expression of \n several EGF-ligand binding receptors and negative feedback  \nregulators to determine possible alternate regulation of EGFR \nsignaling. Figure 7A shows expression of receptors EGFR  \nand ERBB2, and negative regulators of EGFR,  ERRFI1 and \nPTPRK at t = 0. levels of EGFR, ERBB2 and ERRFI1 mRnA \nwere all higher in eSFendo versus eSFnon-endo; levels of PTPRK \ndid not differ between eSFnon-endo and eSFendo. However, after \n96 hours of 8-br-cAMP treatment, levels of EGFR, ERBB2, \nERRFI1 and PTPRK mRnA all significantly increased in eS-\nF\nnon-endo versus eSFendo (Fig. 7B). these data suggest some \nlevel of involvement of EGFR in decidualization in eSFnon-endo; \nhowever, when stimulated with cAMP , eSFendo do not prop-\nerly regulate activation of EGFR, as suggested by the lower \nlevels of ERRFI1 and PTPRK expression. thus, increased \nFig. 5 - Gefitinib restores decidualization marker expression in endometrial stromal fibroblasts from women with endometriosis. Endometrial \nstromal fibroblasts from women without endometriosis (Non-Endo) and with endometriosis (Endo) were cultured for 96 hours in low-serum \nmedium (DMEM-MCDB105 + 2% FBS) containing 0.5 mM 8-br-cAMP, cAMP + 10 µM gefitinib, cAMP + 10 nM rapamycin, or cAMP + 10 µM \nUO126. Inhibitors were delivered in DMSO and all treatments including cAMP were adjusted to contain 0.1% DMSO. Cells were harvested \nfor total RNA extraction and subjected to quantitative real-time PCR analysis for A)  insulin-like growth factor binding protein 1 (IGFBP1);  \nB) prolactin (PRL); and C)  forkhead box protein 01A (FOXO1A). Gene expression data were calculated using the standard curve method and \nnormalized to ACTNB levels in each sample, and normalized to t = 0 to control for time in culture. Data are represented as average fold change \nto t = 0 ± s.e.m. Asterisks indicate significant differences by ANOVA (p<0.05).\n\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n10\nEGFR inhibition restores decidual markers\nactivation of EGFR may not be causative of aberrant de-\ncidualization in eSF endo, as levels of p-EGFR are actually \nhigher in eSFnon-endo following 8-br-cAMP treatment. Rather, \ndecreased decidualization in response to cAMP may be due \nto sustained unopposed activation of EGFR in eSFendo.\nExpression of select EGFR ligands are increased \nin endometriosis\nIn addition to evaluating EGF family receptors and nega-\ntive regulators, we evaluated mRnA expression of several \nEGFR ligands. Expression of AREG and HBEGF was higher \nin eSFnon-endo than eSFendo at t = 0, while TGFA expression oc-\ncurred at similar levels in eSFnon-endo and eSFendo at t = 0 (Fig. \n8A). However, with 8-br-cAMP treatment, AREG increased \nin eSFendo versus eSFnon-endo and expression was inhibited in \neSF independent of disease with UO126 + 8-br-cAMP (Fig. \n8B). TGFA increased dramatically in  eSF\nendo versus eSFnon-\nendo with 8-br-cAMP , gefitinib + 8-br-cAMP , and UO126 + \n8-br-cAMP; rapamycin + 8-br-cAMP decreased expression \nof TGFA in eSF\nendo (Fig. 8C). Finally, HBEGF expression was \nnot different between eSFnon-endo and eSFendo except with the \ncombination of rapamycin + 8-br-cAMP , where expression \nwas elevated in eSF\nendo versus eSF non-endo (Fig. 8D). these \ndata demonstrate the possibility that autocrine/paracrine \nactivation of EGFR by tGFA or AREG with cAMP treat-\nFig. 6 - 8-br-cAMP regulation of cell signaling pathways in the presence of chemical inhibitors. Endometrial stromal fibroblasts from women \nwithout endometriosis (Non-Endo) and with endometriosis (Endo) were cultured for 60 minutes in low-serum medium (DMEM-MCDB105 + \n2% FBS) containing 0.5 mM 8-br-cAMP, cAMP + 10 µM gefitinib, cAMP + 10 nM rapamycin, or cAMP + 10 µM UO126. Inhibitors were deliv-\nered in DMSO, and all treatments including cAMP were adjusted to contain 0.1% DMSO. Cells were harvested for total protein extraction and \nsubjected to multiplex protein analysis for A)  phosphorylated-epidermal growth factor receptor (p-EGFR); B)  phosphorylated-extracellular-\nregulated-kinase 1/2 (p-ERK1/2); and C) phosphorylated-mammalian target of rapamycin (p-mTOR). Phospho-protein values were normalized \nto GAPDH levels for each sample. Data are represented as average ratios ± s.e.m. Asterisks indicate significant differences between Non-\nEndo and Endo within treatment by ANOVA (p<0.05).\n\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n11\nErikson et al\nFig. 7 - EGF receptor and negative regulator expression in endometrial stromal fibroblasts. Endometrial stromal fibroblasts from women without \nendometriosis (Non-Endo) or with endometriosis (Endo) were cultured for 96 hours in low-serum medium (DMEM-MCDB105 + 2% FBS) with 0.5 \nmM 8-br-cAMP. Cells were harvested for total RNA extraction and subjected to quantitative real-time PCR analysis for epidermal growth factor \nreceptor (EGFR), v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2), ERBB receptor feedback inhibitor 1 (ERRFI1), and \nreceptor-type tyrosine-protein phosphatase kappa (PTPRK) at t = 0 (A) and after 96-hour 8-br-cAMP treatment (B). Gene expression data were \ncalculated using the standard curve method and normalized to ACTNB levels in each sample, and normalized to t = 0 to control for time in culture. \nData are represented as average fold change to t = 0 ± s.e.m. Asterisks indicate significant differences by ANOVA (p<0.05).\nment could result in EGFR activation in eSF endo. the dif-\nferent expression patterns of these ligands depending on \npathway inhibition also provide insight into the complexity of \ntheir regulation in eSF . the similar levels of AREG in eSFnon-\nendo and eSF endo with gefitinib, as well as the higher levels \nof TGFA in eSFendo versus eSFnon-endo with gefitinib suggest \nthat dysregulation of EGFR signaling occurs downstream of  \nligand receptor binding.\nno significant differences in p-AKt , p-PtEn, p-P70S6K, \ncleaved PARP or active caspase 3 between eSF\nnon-endo and \neSFendo were observed regardless of treatment time or inhibi-\ntors present (Suppl Figs. 1 and 2). Simultaneous treatment \nwith any 2-inhibitor combination did not have an additive \neffect on decidual marker gene expression (Suppl Fig. 3).\nDISCUSSIOn\nHerein, we used selective chemical inhibitors to block the \nEGFR, mtOR and MAPK signaling pathways in an effort \nto induce decidualization marker expression in eSF from \nwomen with endometriosis. Using gefitinib, an inhibitor of \nthe EGFR tyrosine kinase domain (t yr1173 and t yr992), \nwe were able to restore gene expression of 3 key mark-\ners of decidualization: IGFBP1, PRL and FOXO1A. t o our \nknowledge, this is also the first study providing evidence of \nthe dysregulation of protein tyrosine phosphatase recep-\ntor type kappa (PtPRK), a negative feedback regulator of \nEGFR, during 8-br-cAMP-induced decidualization in eSF . \nthese observations are crucial to understanding the de-\ncidualization process in eSF and its aberrant regulation in \nendometriosis.\nDecidualization\nDecidualization is a complex process that involves a \nnumber of autocrine/paracrine loops involving cytokines \nand growth factors and other secreted molecules includ-\ning interleukin-11 (Il-11), PRl, IGFBP1 and HBEGF (26). \nPreviously, studies have demonstrated a blunted expres-\nsion of specific decidual markers in response to activa-\ntion of the cAMP/PKA pathway in eSF from women with \nendometriosis versus those without disease (2, 14, 15). \nWe also previously determined that there is no defect in \ncAMP hydrolysis in eSF\nendo, indicating that the diminished \nresponse of these cells to cAMP/PKA stimulation may be \ndue to insensitivity of certain genes to PKA stimulation \nor a difference in the regulation of cell signaling path-\nways in eSF endo (14). In the present study, IGFBP1, PRL \nand FOXO1A expression increased in eSF endo to levels \nconsistent with those found in eSF non-endo, by treatment \nwith the EGFR tyrosine kinase domain inhibitor gefitinib  \n(Fig. 5), suggesting dysregulation of EGFR signaling in \neSFendo during cAMP-induced decidualization.\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n12\nEGFR inhibition restores decidual markers\nWe detected elevated levels of p-ERK1/2 and p-mtOR in \neSFendo when compared with normal counterparts prior to \ntreatment with 8-br-cAMP , both of which are downstream \nof EGFR in their respective pathways. We previously found \nan increase in EGFR gene expression and gene expression \nof selected EGFR ligands in eSF from women with severe \nendometriosis versus women with mild disease (12), while \nanother study showed decreased expression of EGFR in \neSF from women with disease (27). A decreased response \nto EGF by eSF\nendo (28) and an inhibitory effect of EGF on \n8-br-cAMP-induced decidualization (25) have also been \nreported. these data collectively suggest it is possible that \nseveral mechanisms are operating with regard to EGFR \nand other ERBB receptors in eSF\nendo, which may be affect-\ned by increased copy number or higher levels of tyrosine \nkinase activity. It is also possible that negative regulators \nof EGFR, including ERRFI1 and PtPRK, do not respond \nto stimuli in eSF\nendo as they do in eSF non-endo, thus affect-\ning down-regulation of EGFR tyrosine kinase. Evidence for \ndysregulation at different levels within the EGFR system is \napparent in the current study from the impaired regulation \nof EGFR, ERBB2, ERRFI1 and PTPRK expression by 8-br-\ncAMP in eSF\nendo, which remained essentially nonrespon-\nsive to 8-br-cAMP (Fig. 7). Collectively, these data coupled \nFig. 8 - Expression of select EGFR ligands in endometrial stromal fibroblasts. Endometrial stromal fibroblasts from women without endome -\ntriosis (Non-Endo) and with endometriosis (Endo) were cultured for 96 hours in low-serum medium (DMEM-MCDB105 + 2% FBS) containing \n0.5 mM 8-br-cAMP, cAMP + 10 µM gefitinib, cAMP + 10 nM rapamycin, or cAMP + 10 µM UO126. Inhibitors were delivered in DMSO, and all \ntreatments including cAMP were adjusted to contain 0.1% DMSO. Cells were harvested for total RNA extraction and subjected to quantita -\ntive real-time PCR analysis for amphiregulin (AREG), heparin-binding epidermal growth factor-like growth factor (HBEGF), and transforming \ngrowth factor alpha (TGFA) at t = 0 (A)  or after 96-hour treatment with 8-br-cAMP (B-D) . Gene expression data were calculated using the \nstandard curve method and normalized to ACTNB levels in each sample, and normalized to t = 0 to control for time in culture. Summary of \n8-br-cAMP-induced pathways in decidualization of human endometrial stromal fibroblasts in women without endometriosis (Normal) and \nwith disease (Endometriosis). A)  Relative expression data calculated at t = 0. B-D)  Average fold change to t = 0 ± s.e.m. Asterisks indicate \nsignificant differences by ANOVA (p<0.05).\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n13\nErikson et al\nsion of a number of cancers, presumably by stimulating \ntumor cell proliferation (34). this raises the possibility that \nendogenous tGFA acts as an autocrine/paracrine ligand \nstimulating EGFR signaling that inhibits decidualization. \nHowever, changes in TGFA or AREG expression did not \ncorrelate with decidualization marker changes consistently \nacross treatments. Addition of rapamycin in combination \nwith 8-br-cAMP reduced TGFA expression, but this was \nnot correlated with increased expression of decidualization \nmarkers in eSF\nendo. Conversely, 8-br-cAMP in combination \nwith gefitinib had no effect on TGFA expression, but in-\ncreased expression of decidualization markers in eSF\nendo. \ntherefore, increased availability of EGFR ligand alone does \nnot seem to account for the inhibition of decidualization \nmarker expression in eSF endo, suggesting a potential dys-\nregulation of the EGFR signaling pathway residing down-\nstream from ligand-receptor binding.\nmTOR and decidualization markers\nAnother observation made in this study is the relationship \nbetween mtOR activation and expression of decidualization \nmarker genes. levels of p-mtOR were constitutively elevat-\ned in eSFendo versus eSFnon-endo and in all cases when treated \nwith 8-br-cAMP (Fig. 6). the one exception occurred when \neSF were treated with gefitinib in addition to 8-br-cAMP , \nwhich decreased p-mtOR in eSF\nendo to levels equal to those \nin eSFnon-endo. Addition of gefitinib to 8-br-cAMP treatment \nwas also when we observed equivalent levels of expression \nof IGFBP1, PRL and FOXO1A in eSFendo and eSFnon-endo (Fig. \n5). this would suggest that  inhibition of EGF signaling in eS-\nFendo causes a sufficient decrease in the elevated levels of \np-mtOR for decidualization to occur. cAMP or its analogues \nare not known to directly activate the mtOR signaling path-\nway. A study previously performed in thyroid cells demon-\nstrated that cAMP , in combination with tSH, could activate \nmtORC1, which then led to downstream activation (35). this \nactivation of mtOR by the cAMP/tSH combination occurred \nindependent of AKt phosphorylation, suggesting direct ac-\ntivation of mtOR by cAMP/tSH. this observation, coupled \nwith our own data, suggests that cAMP may stimulate mtOR \nsignaling through nonclassic yet still unclear mechanisms. \nthus, it is possible that the dysregulation of EGFR signal-\ning we propose is occurring downstream of ligand-receptor \nbinding results in hyperactivation of mtOR in eSF\nendo that is \nnormalized to that of eSFnon-endo with inhibition of the EGFR \ntyrosine kinase domain.\nwith restoration of decidual marker expression in eSF endo \nfollowing treatment with gefitinib, demonstrate that the \nEGFR signaling system plays a key role in the aberrant ex-\npression of decidualization markers seen in endometriosis.\nEGFR inhibitors\nOur study showed lower levels of ERRFI1 expression in \n8-br-cAMP-treated eSF endo compared with their normal \ncounterparts, consistent with its known altered expres-\nsion in endometrium from women with endometriosis (26,  \n29-31). ERRFI1 has also previously been shown to be in-\nvolved in regulation of uterine homeostasis in response \nto E2 in mice (31), and knockdown of ERRFI1 in human \nbronchiolar epithelial leads to an increase in both p-EGFR \nand p-AKt (32), resulting in increased cell proliferation. the \ncurrent study also revealed the dysregulation of another \nnegative receptor of EGF signaling in eSF\nendo, receptor-\ntype protein tyrosine phosphatase kappa (PtPRK), to our \nknowledge not previously reported. PtPRK acts as a regu-\nlator of EGFR activity by dephosphorylating the receptor at \ntyrosines 1068 and 1173 (33), and its silencing increases \nbasal and EGF-stimulated EGFR tyrosine kinase phos-\nphorylation and downstream ERK activation. In the cur -\nrent study, 8-br-cAMP-stimulated levels of both ERRFI1 \nand PTPRK were markedly increased by gefitinib, and \nshowed less prominent changes with the other inhibitors, \nbut invariably with a persistent blunted response in eSF\nendo \ncompared with controls. Alternatively, it is possible that the \n effect of gefitinib on dephosphorylation of p-EGFR tyr1173 \nresulted in decreased expression of PTPRK in eSF\nendo. the \nsame effect was not seen in eSF non-endo, however, indicat-\ning differences in how eSF respond to these stimuli in the \ncontext of disease. Combined, these results indicate that, \nregardless of EGFR expression or activation, eSF endo may \ndemonstrate a compromised capacity to regulate EGFR \nactivity during 8-br-cAMP decidualization.\nEGFR ligands\nthe expression of several EGFR ligands was evaluated to \ninvestigate the potential for autocrine or paracrine activa-\ntion of EGFR signaling in eSF\nendo. In the present study, 8-br-\ncAMP increased TGFA and AREG expression selectively in \neSF\nendo. While similar observations in endometriosis have \nnot been reported, the interaction of tGFA with EGFR in \nan autocrine manner has been implicated in the progres-\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n14\nEGFR inhibition restores decidual markers\nand aromatase P450 inhibitors to block ovarian estradiol \nsynthesis and/or action on endometrial tissue, prevent \nretrograde menstruation and provide a less optimal envi-\nronment for lesion survival and growth (36). Most of these \ncurrent medical therapies affect ovulation and endometrial \ncyclicity, thus having no positive effect of fertility in women \nwith disease, and in fact delay fertility potential. Further -\nmore, no drug that has been used to date has proven to be \neffective at restoring infertility in subfertile endometriosis \npatients versus placebo (36). the data presented herein \ndemonstrating that the use of gefitinib restores 8-br-cAMP-\ninduced decidualization marker expression in women with \nendometriosis indicate that chemical inhibitors of EGFR \ntyrosine kinase activation warrant further exploration as \npotential treatments for endometriosis-related infertility. \nthe use of an EGFR inhibitor could be one alternative, as \nit may not have negative effects on reproductive functions, \nalthough this remains undetermined.\nSummary and conclusions\nHerein, we have demonstrated the restoration of the de-\ncidualization markers IGFBP1, PRl and FOXO1A in eSF \nfrom women with endometriosis by inhibiting EGFR tyrosine \nkinase activity with gefitinib. these experiments have also \nprovided insight into the roles of EGFR, mtOR and MAPK \nsignaling in 8-br-cAMP-induced decidualization. Future \nstudies are warranted to investigate the involvement of these \npathways in the complex process of decidualization, and \nevaluate the potential for, and safety of, EGFR inhibitors in \nthe clinical management of endometriosis-related infertility.\nFinancial Support: this project was supported by the Eunice  \nKennedy Shriver national Institute of Child Health and Human  \nDevelopment/national Institutes of Health (nICHD/nIH) through  \ncooperative agreement U54HD 055764-06 as part of the Special-\nized Cooperative Centers Program in Reproduction and Infertility \nResearch (l.C.G.) and F32HD074423 (J.C.C.).\nConflict of Interest: none of the authors has any financial interest \nrelated to this study to disclose.\nAddress for correspondence:\nlinda C. Giudice, MD, PhD, MSc\nDistinguished Professor and Chair\nDepartment of Obstetrics, Gynecology  \nand Reproductive Sciences\nUniversity of California – San Francisco\n505 Parnassus Avenue M1496\nSan Francisco, CA 94143-0132, USA\nlinda.Giudice@ucsf.edu\nSummary of observations\nEGF is known to signal via multiple pathways including \nERK1/2 and mtOR via AKt . It is evident that in eSF non-endo \n8-br-cAMP activates mtOR, but not ERK1/2 in our model. \nIn eSF\nendo 8-br-cAMP has no effect on constitutively high \nlevels of p-ERK1/2, but activates mtOR such that it re-\nmains higher than in eSF\nnon-endo. this differential activation of \nmtOR correlates with the differential expression of decidu-\nalization markers in 8-br-cAMP-treated eSF\nnon-endo/eSFendo, \nand further, parallels changes in eSF non-endo in response to \ninhibitor treatments. the involvement of EGFR is inferred \nfrom the effects of gefitinib, which normalizes decidualiza-\ntion marker expression in eSFendo and at the same time re-\nduces mtOR activation to normal eSF non-endo levels, while \nnonselectively activating p-ERK1/2 in both eSF non-endo and \neSFendo. this is consistent with the known involvement of \nmtOR signaling in protein synthesis versus MAPK path-\nway in proliferation. Despite these parallel observations, \nthere are inconsistencies in the model. Activation of mtOR \nby 8-br-cAMP is associated with  decidualization marker \nexpression in eSF\nnon-endo, but rapamycin has no effect on \ndecidualization marker expression. In addition, overactive \nmtOR correlates with no  expression of decidualization \nmarkers. this suggests that cAMP signaling in eSF non-endo \ndoes not depend on mtOR to induce decidualization mark-\ner expression. While it is not entirely clear from these data \nhow constitutively active EGFR may prevent decidualiza-\ntion marker expression in eSF\nendo, inhibition of EGFR tyro-\nsine kinase with gefitinib restores expression of 3 classic \ndecidualization markers to similar levels to those found in \neSFnon-endo. this posits overactive EGFR as key in the ab-\nerrant decidualization response of eSF endo to 8-br-cAMP \nthrough dysregulation of downstream pathways.\nClinical translation\nEndometriosis-related infertility is presumably due, in part, \nto endometrium that is nonreceptive to embryo implanta-\ntion, as well as poor egg quality and reduced ovarian re-\nserve due to intrapelvic inflammation. Current therapies \nhave focused on a variety of fertility medications, surgery \nor assisted reproductive technologies to achieve preg-\nnancy, which are costly and can result in treatment-related \nside effects (1). t reatment of endometriosis-related pain \nhas largely relied on the use of drugs such as combined \ncontraceptive steroids, progestogens, GnRH agonists \n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n15\nErikson et al\n15. Klemmt PA, Carver JG, Kennedy SH, Koninckx PR,  \nMardon HJ. Stromal cells from endometriotic lesions and \nendometrium from women with endometriosis have re-\nduced decidualization capacity. Fertil Steril. 2006;85(3):  \n564-572.\n16. Yin X, Pavone ME, lu Z, Wei J, Kim JJ. Increased activation \nof the PI3K/AKt pathway compromises decidualization of \nstromal cells from endometriosis. J Clin Endocrinol Metab. \n2012;97(1):E35-E43.\n17. American Society for Reproductive. Revised American Soci-\nety for Reproductive Medicine classification of endometriosis: \n1996. Fertil Steril. 1997;67(5):817-821.\n18. t ulac S, Overgaard Mt , Hamilton AE, Jumbe nl, Suchanek \nE, Giudice lC. Dickkopf-1, an inhibitor of Wnt signaling, is \nregulated by progesterone in human endometrial stromal \ncells. J Clin Endocrinol Metab. 2006;91(4):1453-1461.\n19. Aghajanova l, Velarde MC, Giudice lC. the progesterone re-\nceptor coactivator Hic-5 is involved in the pathophysiology of \nendometriosis. Endocrinology. 2009;150(8):3863- 3870.\n20. Yamamoto t , Cui XM, Shuler CF . Role of ERK1/2 signaling \nduring EGF-induced inhibition of palatal fusion. Dev Biol. \n2003;260(2):512-521.\n21. Gong HC, Wang S, Mayer G, et al. Signatures of drug \nsensitivity in nonsmall cell lung cancer. Int J Proteomics. \n2011;2011:215496.\n22. Eum KH, lee M. t argeting the autophagy pathway using ec-\ntopic expression of Beclin 1 in combination with rapamycin \nin drug-resistant v-Ha-ras-transformed nIH 3t3 cells. Mol \nCells. 2011;31(3):231-238.\n23. larionov A, Krause A, Miller W. A standard curve based \nmethod for relative real time PCR data processing. BMC \nBioinformatics. 2005;6(1):62.\n24. Chen JC, Frankshun Al, Wiley AA, et al. Milk-borne lacto-\ncrine-acting factors affect gene expression patterns in the \ndeveloping neonatal porcine uterus. Reproduction. 2011; \n141(5):675-683.\n25. Sakamoto t , t anaka t , Umesaki n, Ogita S. Epidermal \ngrowth factor inhibits 8-Br-cAMP-induced decidualization \nof human endometrial stromal cells. Horm Res. 2000;53(6): \n294-299.\n26. Aghajanova l, t atsumi K, Horcajadas JA, et al. Unique tran-\nscriptome, pathways, and networks in the human endome-\ntrial fibroblast response to progesterone in endometriosis. \nBiol Reprod. 2011;84(4):801-815.\n27. Huang JC, Yeh J. Quantitative analysis of epidermal growth \nfactor receptor gene expression in endometriosis. J Clin En-\ndocrinol Metab. 1994;79(4):1097-1101.\n28. Mellor SJ, thomas EJ. the actions of estradiol and epider -\nmal growth factor in endometrial and endometriotic stroma \nin vitro. Fertil Steril. 1994;62(3):507-513.\n29. Burney RO, t albi S, Hamilton AE, et al. Gene expression \nanalysis of endometrium reveals progesterone resistance \nand candidate susceptibility genes in women with endome-\ntriosis. Endocrinology. 2007;148(8):3814-3826.\nREFEREnCES\n1. Giudice lC. Clinical practice. Endometriosis. n Engl J Med. \n2010;362(25):2389-2398.\n2. Velarde MC, Aghajanova l, nezhat CR, Giudice lC. In-\ncreased mitogen-activated protein kinase kinase/extra-\ncellularly regulated kinase activity in human endometrial \nstromal fibroblasts of women with endometriosis reduces \n3′,5′-cyclic adenosine 5′ -monophosphate inhibition of cy-\nclin D1. Endocrinology. 2009;150(10):4701-4712.\n3. Gerbie AB, Merrill JA. Pathology of endometriosis. Clin Ob-\nstet Gynecol. 1988;31(4):779-786.\n4. Papaleo E, Ottolina J, Viganò P , et al Deep pelvic endome-\ntriosis negatively affects ovarian reserve and the number of \noocytes retrieved for in vitro fertilization. Acta Obstet Gyne-\ncol Scand. 2011;90(8):878-884.\n5. Kitawaki J, Kado n, Ishihara H, Koshiba H, Kitaoka Y ,  \nHonjo H. Endometriosis: the pathophysiology as an estrogen- \ndependent disease. J Steroid Biochem Mol Biol. 2002;83   \n(1-5): 149-155.\n6. Aghajanova l, Velarde MC, Giudice lC. Altered gene expres-\nsion profiling in endometrium: evidence for progesterone re-\nsistance. Semin Reprod Med. 2010;28(1):51-58.\n7. Alvarez P , Chen X, Hendrich J, et al. Ectopic uterine tissue as \na chronic pain generator. neuroscience. 2012;225:269-282.\n8. Kim JJ, Yin X. Signaling pathways in endometriosis (eutopic/\nectopic). In: Endometriosis: science and practice. Giudice \nlC,  Evers JlH, Healy Dl, eds. Blackwell, Oxford, UK. 2012: \n164-172.\n9. Honda H, Barrueto FF , Gogusev J, Im DD, Morin PJ. Serial \nanalysis of gene expression reveals differential expression \nbetween endometriosis and normal endometrium. Possible \nroles for AXl and SHC1 in the pathogenesis of endometrio-\nsis. Reprod Biol Endocrinol. 2008;6(1):59.\n10. Yagyu t, tsuji Y , Haruta S, et al. Activation of mammalian tar-\nget of rapamycin in postmenopausal ovarian endometriosis. \nInt J Gynecol Cancer. 2006;16(4):1545-1551.\n11. laudanski P , Szamatowicz J, Kowalczuk O, Kuz ´micki M, \nGrabowicz M, Chyczewski l. Expression of selected tumor \nsuppressor and oncogenes in endometrium of women with \nendometriosis. Hum Reprod. 2009;24(8):1880-1890.\n12. Aghajanova l, Giudice lC. Molecular evidence for differ -\nences in endometrium in severe versus mild endometriosis. \nReprod Sci. 2011;18(3):229-251.\n13. Ejskjaer K, Sorensen BS, Poulsen SS, Mogensen O, Forman \nA, nexo E. Expression of the epidermal growth factor sys-\ntem in eutopic endometrium from women with endometrio-\nsis differs from that in endometrium from healthy women. \nGynecol Obstet Invest. 2009;67(2):118-126.\n14. Aghajanova l, Horcajadas JA, Weeks Jl, et al. the protein \nkinase A pathway-regulated transcriptome of endometrial \nstromal fibroblasts reveals compromised differentiation and \npersistent proliferative potential in endometriosis. Endocri-\nnology. 2010;151(3):1341-1355.\n\n© 2014 Wichtig Publishing - ISSN 2035-9969\n16\nEGFR inhibition restores decidual markers\nlates epidermal growth factor receptor function. J Biol Chem. \n2005; 280(52):42694-42700.\n34. Ciardiello F , tortora G. Interactions between the epidermal \ngrowth factor receptor and type I protein kinase A: biological \nsignificance and therapeutic implications. Clin Cancer Res. \n1998;4(4):821-828.\n35. Blancquaert S, Wang l, Paternot S, et al. cAMP-dependent \nactivation of mammalian target of rapamycin (mtOR) in thy-\nroid cells. Implication in mitogenesis and activation of CDK4. \nMol Endocrinol. 2010;24(7):1453-1468.\n36. Evers JlH. Medical therapy of endometriosis: subfertility. \nIn: Endometriosis: science and practice. Giudice lC,Evers  \nJlH, Healy Dl, eds. Blackwell, Oxford, UK. 2013:449-454.\n30. t albi S, Hamilton AE, Vo KC, et al. Molecular phenotyp-\ning of human endometrium distinguishes menstrual cycle \nphases and underlying biological processes in normo- \novulatory women. Endocrinology. 2006;147(3):1097-1121.\n31. Jeong JW, lee HS, lee KY , et al. Mig-6 modulates uterine \nsteroid hormone responsiveness and exhibits altered ex-\npression in endometrial disease. Proc natl Acad Sci USA. \n2009;106(21):8677-8682.\n32. Jin n, Cho Sn, Raso MG, et al. Mig-6 is required for appro-\npriate lung development and to ensure normal adult lung \nhomeostasis. Development. 2009;136(19):3347-3356.\n33. Xu Y , t an lJ, Grachtchouk V, Voorhees JJ, Fisher GJ.  \nReceptor-type protein-tyrosine phosphatase-kappa regu-\nView publication statsView publication stats","source_license":"CC0","license_restricted":false}