Pigment Epithelium Derived Factor Alleviates Tamoxifen ... · Pigment Epithelium–Derived Factor...

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Cancer Biology and Signal Transduction Pigment EpitheliumDerived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia Keren Goldberg 1 , Hadas Bar-Joseph 1 , Hadas Grossman 1 , Noa Hasky 1 , Shiri Uri-Belapolsky 1 , Salomon M. Stemmer 2 , Dana Chuderland 1 , Ruth Shalgi 1 , and Irit Ben-Aharon 2 Abstract Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptorpositive breast can- cer. Its signicant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro- angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epitheliumderived factor (PEDF) is a potent anti-angio- genic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamox- ifen. Using in vivo mouse models, we demonstrated that tamox- ifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uter- ine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant trans- formation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demon- strated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-a and c-Myc oncoprotein levels were reduced. Our ndings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamox- ifen-induced endometrial pathologies. Mol Cancer Ther; 14(12); 28409. Ó2015 AACR. Introduction Tamoxifen is considered a pivotal component of the hormonal therapy backbone commonly used for treatment of patients with hormone-receptorpositive breast cancer in all settingsadju- vant, metastatic, and as a "primary prevention" risk-reducing strategy for high-risk populations (1, 2). The current American Society of Clinical Oncology guidelines for adjuvant hormonal therapy by tamoxifen have been recently revised; for premeno- pausal patients for 10 years of adjuvant tamoxifen therapy rather than 5 years (3), whereas in postmenopausal patients aromatase inhibitors are considered an alternative, though tamoxifen repre- sents a valid and effective treatment standard. Tamoxifen belongs to a class of selective estrogen receptor modulators (SERM), it binds to estrogen receptor (ER) and elicits estrogen agonistic or antagonistic responses by recruiting diverse sets of corepressors and coactivators, depending on the target tissue (4). Tamoxifen serves as antagonist to ER in breast cancer cells, whereas in the uterus it exerts partial agonistic activity, resulting in a range of endometrial pathologies including hyper- plasia, polyps, carcinomas, and sarcomas (5, 6). Several large- scale studies have reported a 2- to 7-fold increase in the incidence of endometrial cancer in patients treated with tamoxifen (7, 8) compared with control patients. Histopathologic changes in the endometrium were observed after tamoxifen administration mainly in postmenopausal patients (9), and because endometrial hyperplasia is viewed as a possible precursor for malignant transformation, patients with atypical hyperplasia are closely monitored. Tamoxifen is known to exert its effect on the endometrium via activation of estrogen-regulated genes (10); however, recent observations indicate that tamoxifen can also activate different sets of genes that are not regulated by estrogen (11). As demon- strated by gene array proling, these genes are related to cellular processes of DNA replication, cell-cycle progression, and cellular organization (12). Yet, the exact molecular mechanism by which tamoxifen exerts adverse effects on the uterus remains to be elucidated. Angiogenesis, or formation of new blood vessels, is essential for tissue proliferation. Under physiologic conditions, endometrial angiogenesis is tightly regulated by pro- and anti-angiogenic factors (13); estrogen plays a pivotal role in establishing new vascular bed and promoting cellular growth and differentiation 1 Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University,Tel Aviv, Israel. 2 Institute of Oncology, Davidoff Center and Rabin Medical Center, Petah-Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). D. Chuderland, R. Shalgi, and I. Ben-Aharon contributed equally to this article. This work was performed in partial fulllment of the requirements for a PhD degree of K. Goldberg, Sackler Faculty of Medicine, Tel Aviv University, Israel. Corresponding Author: Irit Ben-Aharon, Institute of Oncology, Davidoff Center, Rabin Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Kaplan St., Petach-Tiqva 49100, Israel. E-mail: [email protected] doi: 10.1158/1535-7163.MCT-15-0523 Ó2015 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 14(12) December 2015 2840 on June 3, 2020. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst October 8, 2015; DOI: 10.1158/1535-7163.MCT-15-0523

Transcript of Pigment Epithelium Derived Factor Alleviates Tamoxifen ... · Pigment Epithelium–Derived Factor...

Page 1: Pigment Epithelium Derived Factor Alleviates Tamoxifen ... · Pigment Epithelium–Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia KerenGoldberg1,HadasBar-Joseph1,HadasGrossman1,NoaHasky1,ShiriUri-Belapolsky1,

Cancer Biology and Signal Transduction

Pigment Epithelium–Derived Factor AlleviatesTamoxifen-Induced Endometrial HyperplasiaKeren Goldberg1, Hadas Bar-Joseph1, Hadas Grossman1, Noa Hasky1, Shiri Uri-Belapolsky1,Salomon M. Stemmer2, Dana Chuderland1, Ruth Shalgi1, and Irit Ben-Aharon2

Abstract

Tamoxifen is a cornerstone component of adjuvant endocrinetherapy for patients with hormone-receptor–positive breast can-cer. Its significant adverse effects include uterine hyperplasia,polyps, and increased risk of endometrial cancer. However, theunderlying molecular mechanism remains unclear. Excessiveangiogenesis, a hallmark of tumorigenesis, is a result of disruptedbalance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown tobe elevated by tamoxifen in the uterus.Pigment epithelium–derived factor (PEDF) is a potent anti-angio-genic factor that suppresses strong pro-angiogenic factors, such asVEGF. Our aim was to investigate whether angiogenic balanceplays a role in tamoxifen-induced uterine pathologies, elucidatethe molecular impairment in that network, and explore potentialintervention to offset the proposed imbalance elicited by tamox-ifen. Using in vivo mouse models, we demonstrated that tamox-ifen induced a dose-dependent shift in endogenous uterine

angiogenic balance favoring VEGF over PEDF. Treatment withrecombinant PEDF (rPEDF) abrogated tamoxifen-induced uter-ine hyperplasia and VEGF elevation, resulting in reduction ofblood vessels density. Exploring the molecular mechanismrevealed that tamoxifen promoted survival and malignant trans-formation pathways, whereas rPEDF treatment prevents thesechanges. Activation of survival pathways was decreased, demon-strated by reduction in AKT phosphorylation concomitant withelevation in JNK phosphorylation. Estrogen receptor-a and c-Myconcoprotein levels were reduced. Our findings provide novelinsight into the molecular mechanisms tamoxifen induces in theuterus, which may become the precursor events of subsequentendometrial hyperplasia and cancer. We demonstrate that rPEDFmay serve as a useful intervention to alleviate the risk of tamox-ifen-induced endometrial pathologies. Mol Cancer Ther; 14(12);2840–9. �2015 AACR.

IntroductionTamoxifen is considered a pivotal component of the hormonal

therapy backbone commonly used for treatment of patients withhormone-receptor–positive breast cancer in all settings—adju-vant, metastatic, and as a "primary prevention" risk-reducingstrategy for high-risk populations (1, 2). The current AmericanSociety of Clinical Oncology guidelines for adjuvant hormonaltherapy by tamoxifen have been recently revised; for premeno-pausal patients for 10 years of adjuvant tamoxifen therapy ratherthan 5 years (3), whereas in postmenopausal patients aromataseinhibitors are considered an alternative, though tamoxifen repre-sents a valid and effective treatment standard.

Tamoxifen belongs to a class of selective estrogen receptormodulators (SERM), it binds to estrogen receptor (ER) and elicitsestrogen agonistic or antagonistic responses by recruiting diversesets of corepressors and coactivators, depending on the targettissue (4). Tamoxifen serves as antagonist to ER in breast cancercells, whereas in the uterus it exerts partial agonistic activity,resulting in a range of endometrial pathologies including hyper-plasia, polyps, carcinomas, and sarcomas (5, 6). Several large-scale studies have reported a 2- to 7-fold increase in the incidenceof endometrial cancer in patients treated with tamoxifen (7, 8)compared with control patients. Histopathologic changes in theendometrium were observed after tamoxifen administrationmainly in postmenopausal patients (9), and because endometrialhyperplasia is viewed as a possible precursor for malignanttransformation, patients with atypical hyperplasia are closelymonitored.

Tamoxifen is known to exert its effect on the endometrium viaactivation of estrogen-regulated genes (10); however, recentobservations indicate that tamoxifen can also activate differentsets of genes that are not regulated by estrogen (11). As demon-strated by gene array profiling, these genes are related to cellularprocesses of DNA replication, cell-cycle progression, and cellularorganization (12). Yet, the exact molecular mechanism by whichtamoxifen exerts adverse effects on the uterus remains to beelucidated.

Angiogenesis, or formation of newblood vessels, is essential fortissue proliferation. Under physiologic conditions, endometrialangiogenesis is tightly regulated by pro- and anti-angiogenicfactors (13); estrogen plays a pivotal role in establishing newvascular bed and promoting cellular growth and differentiation

1Department of Cell and Developmental Biology, Sackler Faculty ofMedicine, Tel Aviv University, Tel Aviv, Israel. 2Institute of Oncology,Davidoff Center and Rabin Medical Center, Petah-Tiqva and SacklerFaculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

D. Chuderland, R. Shalgi, and I. Ben-Aharon contributed equally to this article.

This work was performed in partial fulfillment of the requirements for a PhDdegree of K. Goldberg, Sackler Faculty of Medicine, Tel Aviv University, Israel.

Corresponding Author: Irit Ben-Aharon, Institute of Oncology, Davidoff Center,Rabin Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, KaplanSt., Petach-Tiqva 49100, Israel. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0523

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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within the endometrium at each menstrual cycle (13). In ovari-ectomized mice, tamoxifen stimulates production of uterineVEGF (14), one of the major pro-angiogenic factors in the endo-metrium, known to be upregulated in many pathologies andmalignancies (15).

Pigment epithelium–derived factor (PEDF) is a 50-kDa glyco-protein that belongs to the noninhibitory members of the serineprotease inhibitors (serpin) superfamily. PEDF is one of the mostpotent physiologic inhibitors of angiogenesis (16). The anti-angiogenic effect of PEDF is associated with disruption of endo-thelial proliferation, promotion of endothelial cell apoptosis, anddownregulation of pro-angiogenic factors. Increasing amount ofevidence indicates that under physiologic conditions, an essentialbalance between PEDF and VEGF exists, and PEDF may counter-act the angiogenic potential of VEGF (17). Several studies showedthat PEDF level declines with age, and suggested that this declinemay play a key role in the development of angiogenic-relatedpathologies (18). Moreover, it was shown that decrease in PEDFexpression is one of the mechanisms that promote tumor growth(19), suggesting PEDF as an anticancer therapy (20, 21). We haverecently characterized the expression and regulation of PEDF inhuman and rodent premenopausal endometrium, indicating thatPEDF is dynamically expressed inmice endometrium throughoutthe estrous cycle, in reciprocity to VEGF, and that the balancebetween the two is regulated by estrogen and progestrone (22,23). PEDF is a secreted glycoprotein that promotes a variety ofactivities upon binding to various receptors (20). In our previousstudies, we showed that endometrial cells express Patatin-likephospholipase domain-containing protein 2 (PNPLA2; ref. 22), aPEDF receptor (PEDF-R) thatmediatesmainly prosurvival activity(24). Another key protein that regulates the anti-angiogenicactivity of PEDF is laminin receptor (LR; ref. 25), which has notbeen characterized in the uterus. We have also demonstrated thatstimulation of ECC-1 endometrial cell line with recombinantPEDF (rPEDF) induces a significant decrease in the level of VEGFexpression (22). Finally, we established the role of PEDF as apotential therapeutic agent for endometriosis and ovarian hyper-stimulation syndrome (23, 26).

In the current study, our aim was to explore the mechanismaccountable for tamoxifen-induced endometrial pathologies inpostmenopausal patients, among which these entities representclinical concern. We hypothesized that tamoxifen alters uterineangiogenic balance and that the shift in regulatory angiogenic pro-teins contributes to tamoxifen-induced uterine pathologies. It istherefore suggested that upon this mechanism, a replacement the-rapy using rPEDF may reduce tamoxifen-induced adverse effects.

Materials and MethodsAnimals

Institute for Cancer Research (ICR) female mice (8 weeks old;Harlan Laboratories) were housed in temperature- and humidity-controlled rooms at the animal facilities of the Sackler Faculty ofMedicine, Tel-Aviv University, under artificial illumination for12 hours daily. Food and water were available ad lib. Animal carewas in accordance with institutional guidelines and approved bythe local authorities.

Tamoxifen treatmentTamoxifen tablets (Teva Pharmaceutical Industries) were

pulverized to thin powder, dissolved in ethanol, and diluted

in canola oil. Tamoxifen was administered per os by ablunt gavage needle. Control mice were administered withcanola oil.

OvariectomyMice were anesthetized by an intraperitoneal injection of a

mixture of ketamine hydrochloride (Kepro) and xylazine hydro-chloride (VMD). The dorsal mid-lumbar area was shaved toremove hair and prepared for aseptic surgery. A single skinincision at the dorsal end of the ribcage was made and musclewall was incised, exposing the peri-ovarian fat pad region. Theovary was exteriorized by grasping the peri-ovarian fat with tissueforceps; the oviduct area was ligated and cauterized by extremelyhot forceps to avoid bleeding, thus excising the ovary.The remaining tissue was returned into the peritoneal cavity. Theprocedure was repeated at the other side as well. The muscleincisions were closed with 4-0 absorbable suture (Ethicon) andskin incisions were closed with wound clips (Clay-Adams). Micewere allowed to rest for one week to ensure post-menopausalstate, before experimental onset.

RNA isolation, reverse transcription, and qRT-PCRRNA was extracted using Trizol reagent (Invitrogen), accord-

ing to the manufacturer's instructions, and quantified with theNano-Drop spectrophotometer (ND-1000; Thermo Scientific).Total RNA was reverse transcribed using high capacity cDNAreverse transcription kit (Applied Biosystems; ABI). Changes inthe level of mRNA expression were detected by SYBR greenreagent (SYBR Green PCR Master Mix, Applied Biosystems)using 20-ng cDNA and specific primers, on an ABI Prism 7900Sequence PCR machine (Applied Biosystems). Hypoxanthinephosphoribosyltransferase 1 (HPRT-1) served as an endoge-nous control.

Western blot analysisSamples were subjected to SDS-PAGE and immunoblotted

with the appropriate primary antibodies: anti-PEDF (1:200, sc-25594, Santa Cruz Biotechnology), anti-VEGF (1:200, sc-152,Santa Cruz Biotechnology), anti-phospho-AKT (1:1,000, P-4112, SigmaChemical Company), anti-AKT (1:1,000, #2938, CellSignaling Technology), anti-phospho-JNK (1:1,000, J-4750, Sig-ma), anti-JNK (1:1,000, J-4500, Sigma), anti-ERa (1:1,000, ab2746, Abcam), anti-c-Myc (1:1,000, sc-40, Santa Cruz Biotechnol-ogy), and anti-actin (1:10,000, MAB1501; Millipore). Blots wereincubatedwithhorseradish peroxidase-conjugated secondary anti-bodies (Jackson Immunoresearch) and subjected to enhancedchemiluminescence assay (Thermo Scientific). The intensity of thebands was analyzed by the ImageJ software.

Histologic evaluationUteri were excised, fixed in 4% paraformaldehyde in PBS,

dehydrated by passage through an ascending series of alcohols,embedded in paraffin, sectioned (6 mm), and placed on slides(Propper). Slides for histologic evaluation were stained withhematoxylin and eosin (Bio-Optica).

IHCSections were deparaffinized by running the slides through

xylene (15 minutes) and a descending series of alcohols (5minutes each) to a total re-hydration in distilled water. The

PEDF Alleviates Tamoxifen-Induced Endometrial Hyperplasia

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sections were placed into preheated antigen unmasking solution(H-3300; Vector Laboratories Inc.), cooled on ice to 50�C, re-heated for three more minutes and cooled on ice to roomtemperature. The slides were then rinsed in PBS and incubatedfor 1 hour with PBSTg (0.2% Tween-20, 0.2% gelatin in PBS), re-rinsed in PBS, blocked for 10minutes in blocking solution (927B;Cell Marque), and incubated overnight at 4�C with anti-CD34(1:200, CL8927AP; Cedarlane Laboratories) primary antibody.Before and after applying the secondary goat anti-rat AlexaFlour555-conjugated antibody (1:400, Cell Signaling Technolo-gy) and a nuclear marker (Hoechst 3342; Sigma), sections wererinsed in PBSTg (�3), in PBS (�3), and mounted with mowiol(Sigma) before being scanned by a laser confocal microscope(Leica TCS SP5).

rPEDF productionHis-tagged human rPEDF (NM_002615.4) was expressed in

Escherichia coli BL21. Bacteria were allowed to grow at 30�C toOD600 nm of 0.5 to 0.6, induced for 4 to 5 hours by isopropyl-L-thio-b-D-galactopyranoside (0.5 mmol/L; Sigma), centrifuged,and their pellets lysed. Recombinant protein was purified by ionmetal affinity chromatography with Ni-NTA His-Bind resin(Merck KGaA) according to the manufacturer's protocol. Proteinsof the eluted fractions were resolved by SDS-PAGE, dyed byGelCode (Blue Stain Reagent, Thermo scientific), or Westernblotted with a specific anti-PEDF antibody (�57 kDa). Eluateswith 90% purity were dialyzed against Tris buffer pH 10.

Statistical analysisData were expressed as mean � SEM. A Student t test (equal

variance, one-tailed) was used to statistically evaluate the differ-ences between control and experimental results. Differences wereconsidered statistically significant when P < 0.05.

ResultsShort-term tamoxifen treatmentmodulates uterine VEGF/PEDFbalance in vivo

We have established an in vivo mouse model for examiningthe effect of tamoxifen on postmenopausal uterine tissue (seeSupplementary Fig. S1 for the experimental design). To study thesubacute effect we used the short-term treatment model, whichdemonstrated a dose-dependent increase of uterine weight(Fig. 1A), as described in the literature (27). The histologicappearance (Supplementary Fig. S2) showed that tamoxifencaused an increase of uterine glands and epithelial height as wellas enlargement of the stromal compartment (27). We found thatthe level of VEGF protein in the uterus was upregulated in a dose-dependentmanner (Fig. 1B andE), correlatedwithuterineweight.Thereafter, we evaluated the effect of tamoxifen on the expressionof PEDF protein, and found that PEDF levels were downregulated(Fig. 1C and E) as tamoxifen doses were increased, yielding areciprocal manner of VEGF and PEDF expression (Fig. 1D),indicating impaired uterine angiogenic balance after tamoxifenadministration.

Figure 1.Short-term tamoxifen treatmentmodulates uterineVEGF/PEDFbalance in vivo. Various concentrations of tamoxifenwere administered for 7days toovariectomizedmice. Uteri were excised and lysed. Proteins were resolved by SDS-PAGE and immunoblotted with anti-VEGF or anti-PEDF antibodies. mRNA was extractedusing TRIZOL reagent and subjected to RT-PCR. A, uterine weight. B, VEGF protein expression. C, PEDF protein expression. D, VEGF/PEDF protein ratio.E, representative Western blot of VEGF, PEDF, and actin as a loading control. Protein expression was quantified by ImageJ and calibrated to actin as a loadingcontrol. F, PEDF-R mRNA calibrated with HPRT-1. n ¼ 3–4 mice/group. The ratio between each treatment and control is plotted as mean � SEM (� , P < 0.05;�� , P < 0.01), statistically significantly different from control value, using t test.

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We evaluated the expression of PEDF-Rs in the uterus andfound that short-term tamoxifen treatment upregulated theexpression level of both PNPLA2 and LR (Fig. 1F).

rPEDFdownregulates both tamoxifen-induced hyperplasia andVEGF elevation, and modifies signaling pathways

We chose the dose of 2.5 mg/mouse tamoxifen as an optimaldose and added subcutaneous injections of rPEDF (10 mg/kg,every third day). We administered rPEDF to mice according totwo regimens (see Supplementary Fig. S1 for experimentaldesign): (1) starting on day 1 of tamoxifen administration("rPEDF prevention"), (2) starting on day 4 of tamoxifen

administration ("rPEDF treatment"). When rPEDF was addedon days 4 to 7 of tamoxifen administration ("rPEDF treatment"regimen), significant decreases in uterine weight (Fig. 2A) andin uterine VEGF level (Fig. 2B and C) were observed, comparedwith the values obtained in mice treated with tamoxifen alone;suggesting that rPEDF had a significant favorable effect ontamoxifen-induced uterine hyperplasia. This effect may beattributed, at least in part, to PEDF's ability to resist tamoxi-fen-induced VEGF elevation. No effects on uterine weight oruterine VEGF level were observed in mice treated according tothe "rPEDF prevention" regimen (data shown in Supplemen-tary Fig. S3).

Figure 2.rPEDF downregulates both tamoxifen-induced hyperplasia and VEGF elevation, and modifies signaling pathways. Ovariectomized mice were administered for7 days with tamoxifen (2.5 mg/mouse) or with a combination of tamoxifen and rPEDF (10 mg/kg; days 4–7). Uteri were excised, lysed, proteins resolvedby SDS-PAGE, and immunoblotted with anti-VEGF antibody (B and C), anti-p-AKT (D and E), or with anti-p-JNK antibodies (F and G). A, uterine weight.B, VEGF protein expression. C, representative Western blot analyses of VEGF and actin as a loading control. Protein expression was quantified by ImageJ andcalibrated to actin as a loading control. Each value is normalized to that of control (tamoxifen-only) level. Tamoxifen, n ¼ 23; tamoxifenþrPEDF, n ¼ 16.D, p-AKT protein expression. E, representative Western blot of p-AKT. Protein expression was quantified by ImageJ and calibrated to gen-AKT as a loadingcontrol. F, p-JNK protein expression. G, representative Western blot of p-JNK. Protein expression was quantified by ImageJ and calibrated to gen-JNK asa loading control. Each value is normalized to that of control (tamoxifen-only) level (n ¼ 4 mice/treatment group). Data are plotted as mean � SEM (� , P < 0.05;�� , P < 0.01), statistically significantly different from control value, using t test.

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Examination of signaling pathways known to mediate cellulargrowth and survival (PI3K/AKT/mTOR pathway) as well as apo-ptosis [c-Jun N-terminal kinases (JNK)] revealed a significantdecrease in phosphorylation of AKT (Fig. 2D and E) and anincrease in phosphorylation of JNK (Fig. 2F andG) inmice treatedwith the "rPEDF treatment" regimen compared with micetreated with tamoxifen alone. Mice of the "rPEDF prevention"cohort showed an increase in phosphorylation of JNK and nosignificant change in phosphorylation of AKT compared withmice treatedwith tamoxifen alone (data shown in SupplementaryFig. S3).

Prolonged tamoxifen treatment modulates uterine VEGF/PEDFbalance in vivo

We established a prolonged-treatment model in which tamox-ifen was administered to mice continuously for a period of 1month. The effect of prolonged tamoxifen treatment was similarto that of the short-term treatment; namely, an increase in uterineweight (Fig. 3A), upregulation of VEGF protein (Fig. 3B and E),and downregulation of PEDF protein (Fig. 3C). VEGF/PEDF ratiowas significantly increased (Fig. 3D), suggesting a long-lastingeffect of tamoxifen.

Evaluation of the expression of PEDF-Rs after 1 month oftamoxifen treatment (Fig. 3F) revealed that PNPLA2 level wassignificantly increased, whereas that of LR did not change signif-icantly. This suggests that following tamoxifen treatment endog-

enous PEDF will more likely bind to PNPLA2 and induce cellularsurvival than promote anti-angiogenic activity through LR.

A prolonged combined treatment of tamoxifen and rPEDFdownregulates tamoxifen-induced hyperplasia, VEGFelevation, and blood vessels density

We examined the long-term effectiveness of rPEDF treatmentadministered in vivo according to the prolonged tamoxifen(2.5 mg/mouse) treatment model and added rPEDF injections(2 mg/kg, every third day). Based on our finding that "rPEDFtreatment" regimen was more effective than "rPEDF prevention"regimen, we started rPEDF injections on day 8 of tamoxifentreatment (see Supplementary Fig. S1 for experimental design).Uterine weight did not differ in the group treated with thecombination compared with tamoxifen-only (Fig. 4A; P ¼ 0.2).The expression level of uterine VEGF protein was significantlydecreased in mice of the combined "tamoxifenþrPEDF" micecompared with that of the "tamoxifen-only" group (Fig. 4B andC); suggesting a durable, long-lasting effect of rPEDFon the uterusafter 1 month of treatment.

Changes in tissue vasculature were examined by IHC stainingwith CD34. Tamoxifen caused a remarkable increase in thedensity of uterine blood vessels (Fig. 4E and H) compared withcontrol (Fig. 4D and G), whereas addition of rPEDF (tamoxi-fenþrPEDF treatment) caused a decrease of CD34 staining (Fig.4F and I) compared with tamoxifen-only.

Figure 3.Prolonged tamoxifen treatment modulates uterine VEGF/PEDF balance in vivo. Tamoxifen (2.5 mg/mouse) was administered for 1 month (5 days/week) toovariectomized mice. Uteri were excised and lysed. Proteins were resolved by SDS-PAGE and immunoblotted with anti-VEGF or anti-PEDF antibodies.mRNA was extracted using TRIZOL reagent and subjected to RT-PCR. A, uterine weight. B, VEGF protein expression. C, PEDF protein expression. D, VEGF/PEDFprotein ratio. E, representative Western blot of VEGF and actin as a loading control. F, PEDF-R mRNA calibrated with HPRT-1. n ¼ 6–7 mice/treatment group.Protein expression was quantified by ImageJ and calibrated to actin as a loading control. The ratio between each treatment and control is plotted asmean � SEM (�, P < 0.05; �� , P < 0.01), statistically significantly different from control value, using t test.

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Combined treatment of tamoxifen and rPEDF modulates ERaand c-Myc expression patterns

We used RT-PCR to examine the expression pattern of hor-monal receptors in the uteri ofmice treated according to the short-term and prolonged in vivomodels; assessing the mRNA levels ofERa and ERb and progesterone receptor (PR). Short-term tamox-ifen treatment caused an upregulation of ERa mRNA level, buthad no significant effect on that of ERb and PR (Fig. 5A). rPEDFimpaired the tamoxifen-induced upregulation of ERamRNA. Thelevels of ERa protein along the various treatments resembled

those of ERa mRNA, though with no statistical significance (Fig.5B, P ¼ 0.1).

Prolonged tamoxifen treatment upregulated mRNA levels ofboth ERa and ERb, but had no significant effect on PR expression(Fig. 5D). The combined treatment of tamoxifenþrPEDF signif-icantly reduced ERa mRNA level compared with tamoxifen. Thelevel of ERa protein expression at the various treatments corre-lated with that of ERa mRNA (Fig. 5E).

Finally, we tested the effect of rPEDF on c-myc protein. Short-term tamoxifen treatment increased the level of c-Myc protein,

Figure 4.Prolonged combined treatment of tamoxifen and rPEDF downregulates tamoxifen-induced hyperplasia, VEGF elevation, and blood vessels density. Ovariectomizedmice were treated for 1 month with tamoxifen (2.5 mg/mouse) or with a combination of tamoxifen and rPEDF (2 mg/kg), every third day. Uteri were excised,lysed, proteins resolved by SDS-PAGE, and immunoblotted with anti-VEGF antibody. A, uterine weights. B, VEGF protein expression. C, representativeWestern blot of VEGF and actin as a loading control. D–F, representative CD34 IHC staining (red) of uteri of control (D), tamoxifen (E), and tamoxifenþrPEDF (F)treated mice. G–I, magnification of selected areas (marked by white boxes in D–F). DNA labeling by Hoechst 32242 (blue), n ¼ 6 mice/group. Protein expressionwas quantified by ImageJ and calibrated to actin as a loading control. Each value was normalized to control (tamoxifen-only) value. Data are presentedas mean � SEM, � , P < 0.05, statistically significantly different from control value, using t test.

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whereas rPEDF causedno significant change (Fig. 5C). The level ofc-Myc was significantly increased after a prolonged tamoxifentreatment as well, and significantly decreased after a combinedtamoxifenþrPEDF treatment (Fig. 5F).

DiscussionTamoxifen-induced uterine pathologies present a major clin-

ical challenge. To date, the mechanism that accounts for thespectrum of tamoxifen-induced endometrial hyperplasia-meta-plasia remains unclear. It is postulated that it involves alterationsin uterine angiogenesis, though only paucity of supporting evi-dence exists. In the current study, we hypothesized that tamoxifenexerts its effects on the uterus by modulating uterine angiogenicbalance; we studied the mechanisms using an in vivo mousemodel. Because tamoxifen is prescribed in the clinical setting ona daily basis for an extended period, we established two modelsthat represent the subacute and the long-term effects. Our resultswere in accordance with previous reports and showed that tamox-ifen upregulates the level of uterine VEGF.We showed, for the firsttime, that the level of uterine PEDF is downregulated aftertamoxifen treatment, resulting in an impaired balance of VEGFand PEDF, and demonstrated that tamoxifen-induced increase in

uterine weight correlates with VEGF increase and PEDF decrease.PEDF was shown to be a potent effector of angiogenesis and wasproposed as a potential therapeutic intervention for other angio-genesis-related pathologies. We suggested using rPEDF as areplacement therapy for reducing tamoxifen-induced adverseeffects, and investigated its effect on key proteins involved inregulation of cells proliferation, survival, and apoptosis.

Our research is the first to demonstrate the significant impact oftamoxifen on the expression of uterine blood vessels, revealed byenhanced staining of the endothelial marker CD34. We suggestthat the increase in blood vessels density may be attributed to theimpaired balance between pro- and anti-angiogenic factors,induced by tamoxifen. Moreover, treatment with rPEDF restoredthe angiogenic balance by resisting tamoxifen-induced uterineVEGF elevation, as seen by the decrease of both uterineweight andCD34-marked blood vessels. Former evidence characterizing thechanges in uterine vasculature after tamoxifen treatment in post-menopausal patients was not persistent, possibly due to limitedsample size and increased heterogeneity (28, 29).

Endometrial hyperplasia, especially atypical hyperplasia, isgenerally viewed as a possible precursor for malignant transfor-mations (30). Expression of VEGF and vascular density, twoimportant markers of angiogenesis, were found to be increased

Figure 5.Combined treatment of tamoxifen and rPEDF modulates ERa and c-Myc expression patterns. Ovariectomized mice were treated for 1 month with tamoxifen(2.5 mg/mouse, 5 days/week) or with a combination of tamoxifen and rPEDF (2 mg/kg). Uteri were excised and lysed. mRNA was extracted using TRIZOLreagent and subjected to RT-PCR. Proteins were resolved by SDS-PAGE and immunoblotted with anti-ERa or anti-c-Myc antibodies. A–C, short-termtreatment, 7 days; D–F, prolonged treatment, 1 month. RT-PCR was performed with specific primers and calibrated with HPRT-1. Protein expression wasquantified by ImageJ and calibrated to actin as a loading control. Data were normalized to control (tamoxifen-only) level (n ¼ 4 mice/group in short-termexperiments; n ¼ 6 mice/group in prolonged experiments). Data are presented as mean � SEM (� , P < 0.05; ��, P < 0.01), statistically significantly differentfrom control value, using t test.

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inmanypremalignant lesions (31) and are known to play a role inthe spectrum of tissue malignant transformation. Our in vivomodels verified that tamoxifen enhanced both hyperplasia andangiogenic activation in treated mice. Furthermore, administra-tion of rPEDF reduced the levels of both parameters (vasculardensity and VEGF(, implying a reduced risk of endometrialtumorigenesis following tamoxifen treatment.

Based on the ability of PEDF to elicit non-angiogenic cellularpathways as well, we examined its effect on signaling pathwaysmediating cell proliferation, survival, and apoptosis. Activation ofthe PI3K/AKT/mTOR pathway, a signaling pathway that plays animportant role in cellular growth and survival, has recently beenimplicated in endometrial cancer (32). Activated AKT initiates acascade of downstream signaling events in endometrial cancercells that promote cellular growth, metabolism, proliferation,survival, migration, apoptosis, and angiogenesis. JNKs, key med-iators of stress and inflammation, were implicated in apoptoticresponses to DNA damage and to cytotoxic agents (33, 34).Activation of JNK correlates with induction of apoptosis inendometrial cancer cells (35). PEDF was previously shown toinduce apoptosis bydownregulationofphosphorylated-AKT (36)and upregulation of phosphorylated-JNK (37). CombinedrPEDF–tamoxifen treatment causes a significant increase in thephosphorylation of the pro-apoptotic signaling molecule JNK,and a decrease in the phosphorylation of the prosurvival signalingmolecule AKT, when compared with treatment with tamoxifen

alone.Our data indicated that rPEDF treatment reduces activationof proliferation-associated signaling pathways.

Hormone receptors (ER and PR) expression is a fundamentalprinciple in tamoxifen treatment for breast cancer. We hypothe-sized that due to its SERM traits, tamoxifen-induced effects on theendometrium may involve endometrial ER. The effect of tamox-ifen on the ER may entail recruitment of processes as tissueproliferation and VEGF regulation, two major pathways relevantto increased hyperplasia and possibly to malignant transforma-tions. Moreover, a study conducted in Ishikawa endometrial cellline suggests that tamoxifen-induced upregulation of VEGF ismediated mainly through ERa, and not through ERb (38). ERbwas shown to modulate, and possibly suppress, ERa-mediateduterine gene transcription (39); the activity of tamoxifen maytherefore be determined by the relative expression of ERa and ERbin the tissue (40). Our results showed that rPEDF treatmentinduces a specific decrease in the expression of uterine ERa,without modifying the expression of ERb and PR, implying ashift toward higher activity of ERb over ERa. The decrease in ERaexpression confers dual implication with regard to tamoxifen-treated uterus: First, the ER-related signaling, known to mediatehyperplastic response, is diminished, and, second, the uterineresponse to tamoxifen is reduced because there are less tamoxifen-binding receptors per cell. c-Myc protein, known to induce pro-liferation and to play a role in the development of endometrialcarcinoma (41), is upregulated by tamoxifen (42, 43). In ourmice

Figure 6.Suggested mechanism of action for tamoxifen and rPEDF—schematic representation. Tamoxifen treatment (top) causes increased vascularization by (i)downregulation of PEDF, (ii) upregulation of VEGF, and (iii) downregulation of laminin receptor (LR). In addition, tamoxifen causes increased hyperplasiaby (i) upregulation of ERa, (ii) upregulation of c-Myc, and (iii) upregulation of Patatin-like phospholipase domain-containing protein 2 (PNPLA2). Thesecombined processes are suggested to account for the increased risk of endometrialmalignant transformation under tamoxifen treatment. PEDF treatment (bottom)causes decreased vascularization by downregulation of VEGF, and decreased hyperplasia by (i) downregulation of ERa, (ii) downregulation of c-Myc, (iii)downregulation of phosphorylated-AKT, and (iv) upregulation of phosphorylated-JNK (iii and iv, during the short-term treatment). PEDF treatment restoredthe uterine natural angiogenic balance, and decreased pathologic hyperplasia, thus possibly reducing the risk of endometrial malignant transformation.

PEDF Alleviates Tamoxifen-Induced Endometrial Hyperplasia

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model tamoxifen treatment causes an increase in the level ofc-Myc protein, compared with its level in control mice. Admin-istration of rPEDF prevents c-Myc elevation, suggesting adecreased risk of malignant development. Our results also indi-cate that tamoxifen treatment changes the expression patterns ofreceptors known to mediate PEDF activity. By upregulating thelevel of PNPLA2, induction of prosurvival pathway is preferredover that of anti-angiogenic activity via the LR.

Two therapeutic options, Levonorgestrel and Metformin, wereinvestigated as potential candidates to reduce the risk of devel-oping endometrial cancer following tamoxifen treatment.Though Levonorgestrel-releasing intrauterine system (LNG-IUS)was indicated as a suppressor of endometriumproliferation, thereis not enough evidence regarding its efficacy in reducing endo-metrial hyperplasia and cancer (44). Moreover, the use of LNG-IUS in the setting of hormonal therapy for breast cancer, isquestionable due to its possible interaction with the antiestro-genic therapy (45). When considering the safety of PEDF treat-ment to patients with breast cancer—PEDF was already shown toinhibit the growth of breast cancer cells in vitro, to exhibit anti-tumor activity in vivo, and to reduce the resistance of tumors totamoxifen (46). Thus, PEDF treatment may have dual efficacy—treating breast cancer and minimizing the risk of endometrialpathogenesis. The other candidate, the antidiabetic drug Metfor-min, exerts an antiproliferative effect on the endometrium oftamoxifen-treated mice via inhibition of mTOR (47). Interesting-ly, Metformin increases PEDF levels in patients with type IIdiabetes (48), and our data demonstrated that rPEDF decreasedthe activity of PI3K/AKT/mTOR pathway. However, inhibition ofPEDF by Metformin was described (49), thus further research isneeded to fully comprehend the connection among these obser-vations. The beneficiary effect of rPEDF in protecting the uterusand the fact that it does not compromise the safety of patientswithbreast cancer, imply that rPEDF therapy could minimize gyneco-logic follow-up of tamoxifen-treated women and reduce medicalinterventions caused by tamoxifen.

Our study suggests that angiogenesis-related changes contrib-ute to tamoxifen-induced uterine hyperplasia, and therefore inhi-bition of VEGF can improve tamoxifen's effect on the uterus both

short- and long-term treatment models. Tamoxifen-induced vas-cular proliferation clearly inhibited in mice of the prolongedrPEDF treatment model. Prolonged rPEDF treatment downregu-lates VEGF and modulates expression patterns of ERa, thusaltering uterine response to tamoxifen treatment, diminishingestrogen-related signal activation, and reducing susceptibility totamoxifen-induced adverse effects. Moreover, the expression of c-Myc oncoprotein is downregulated after rPEDF treatment, possi-bly reducing the risk of endometrial malignant transformation(proposed mechanism of action is depicted in Fig. 6). By admin-istering PEDF, we restored the altered angiogenic balance ofuterine tissue to its natural state, and reduced the cascade ofevents leading to pathologic hyperplasia.

Disclosure of Potential Conflicts of InterestK. Goldberg, H. Bar-Joseph, D. Chuderland, S.M. Stemmer, R. Shalgi and

I. Ben-Aharon have ownership interest in U.S. Provisional Application no.61/972,325. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: K. Goldberg, D. Chuderland, R. Shalgi, I. Ben-AharonDevelopment of methodology: K. Goldberg, D. Chuderland, I. Ben-AharonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Goldberg, H. Bar-Joseph, H. Grossman, N. Hasky,S. Uri-BelapolskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Goldberg, S.M. Stemmer, D. Chuderland,R. Shalgi, I. Ben-AharonWriting, review, and/or revision of the manuscript: K. Goldberg, S.M. Stem-mer, D. Chuderland, R. Shalgi, I. Ben-AharonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I. Ben-AharonStudy supervision: D. Chuderland, R. Shalgi, I. Ben-Aharon

The costs of publication of this articlewere defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 22, 2015; revised September 8, 2015; accepted September 21,2015; published OnlineFirst October 8, 2015.

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2015;14:2840-2849. Published OnlineFirst October 8, 2015.Mol Cancer Ther   Keren Goldberg, Hadas Bar-Joseph, Hadas Grossman, et al.   Endometrial Hyperplasia

Derived Factor Alleviates Tamoxifen-Induced−Pigment Epithelium

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