Role of GPR30 in Endometrial Pathology After Tamoxifen for Breast Cancer

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Research www.AJOG.orgnistic effect in breast cancer, it acts as anestrogen agonist in the uterus.9 This ago-nistic effect of tamoxifen can stimulate cellgrowth and proliferation in endometrialtissue, enhancing the risk of endometrialabnormalities,includingendometrialcarci-noma.10,11 Interestingly, the classic ER an-tagonists tamoxifen and fulvestrant act asagonists for GPR3012 and stimulate cell

cohort. The role of GPR30 for tamoxifen-induced endometrial abnormalities wasconfirmed in vitro, using endometrial car-cinoma cell line.

MATERIALS AND METHODSMaterialsAll chemicals and antibiotics for a cell

czuk), Lublin Medical University,blin, Poland.

ceived March 26, 2010; revised June 19,0; accepted July 21, 2010.

prints not available from the authors.

s study was supported in part by Grant931 from Deutsche Krebshilfe.

2-9378/$36.00010 Published by Mosby, Inc.and function as hormone-induciblenscription factors. In addition to thisassic or genomic effect of estrogen,

nongenomic effects obeen demonstrated tates the proliferativein breast, endometriacer cells (review6).

Breast cancer is thecer in women and is pgen-dependent.7 The eof choice for breast canpatients remains the seceptor modulator (SERthough tamoxifen has

m the Department of Obstetrics andnecology (Drs Ignatov, Eggemann,choff, Costa, Ignatov, and Mr Smith),to-von-Guericke University, Magdeburg,rmany; Department of Pathology (Drsessner and Kalinski), Otto-von-Guerickeiversity, Magdeburg, Germany; and theD Department of Gynecology (Drproliferation and growth: 10.1016/j.ajog.2010.07.034

Dstrogen. It hast GPR30 medi-ects of estrogennd ovarian can-

st common can-ominantly estro-ocrine treatmentpremenopausal

tive estrogen-re-) tamoxifen.8 Al-estrogen antago-

the tamoxifen stimulatory effects in endo-metrial carcinoma cells in vitro14,16 and ishighly expressed in endometrial carci-noma with poor prognosis.17

In this regard, the activation of GPR30may be a possible mechanism leading toendometrial abnormalities in breast can-cer patients after tamoxifen treatment. Weare the first to describe the distribution ofGPR30 in endometrial tissue of patientstreated with tamoxifen for breast cancerare mediated by 2 estrogen receptorss), ER and ER, which belong tosteroid hormone receptor superfam-

protein-coupled receptor, GPR30, wasclaimed to be a new membrane-boundestrogen receptor involved in the rapid

that GPR30/EGFR crosstalk is an impor-tant component of tamoxifen agonistic ac-tivity in breast cancer cells.15 Moreover, itSIC SCIENCE: GYNECOL

ole of GPR30 in endofter tamoxifen for brnja Ignatov, MD; Holm Eggemann, MD;ert Roessner, MD; Serban D. Costa, MD

JECTIVE: This study was undertaken to evaluarotein-coupled estrogen receptor in endometd with tamoxifen treatment of breast cancer pa

UDY DESIGN: We investigated whether G-proteptor plays a role in mediating proliferating effeetrial carcinoma cells. These results wererotein-coupled estrogen receptor expressionue from a cohort of 95 breast cancer patients,or another adjuvant therapy.

SULTS: In vitro tamoxifen significantly stimulaed protein kinase phosphorylation and cell pro

this article as: Ignatov T, Eggemann H, Semczuk0;203:595.e9-16.

strogens are key regulators of differ-ent cellular processes involved in the

ysiology of female reproductive tractd mammary gland.1 The physiologicGY

etrial pathologyst cancer

drzej Semczuk, MD; Bobbie Smith; JoachhD; Thomas Kalinski, MD, PhD; Atanas

he potential role ofpathology associ-nts.

-coupled estrogenof tamoxifen in en-mpared with theern in endometrialo received tamox-

the mitogen-acti-ration of endome-

trial cell lines via G-prowas a significant correlaceptor expression and t(P .006). Moreover, Gwas predictive of an earing or suspect endome(P .019).

CONCLUSION: G-proteinrole in tamoxifen-induce

Key words: estrogen, G

t al. Role of GPR30 in endometrial pathology after

re are some rapid or nonclassicnaling effects, including stimulationphospholipase C, cAMP, calcium,sitol phosphate, and ERK upreg-. Particularly in cu

ECEMBER 2010 AmericanBischoff, MD;atov, MD

-coupled estrogen receptor. In vivo, therebetween G-protein-coupled estrogen re-

tamoxifen-induced endometrial pathologyrotein-coupled estrogen receptor positivitydevelopment of symptoms, such as bleed-thickness, induced by tamoxifen therapy

upled estrogen receptor plays an importantdometrial abnormalities.

30, tamoxifen

oxifen for breast cancer. Am J Obstet Gynecol

ast and endometrial cancer cells,R30 activates the human epidermalwth factor receptor (EGFR) via releaseheparin-bound growth factor (HB-lture, 4-OH-tamoxifen and 17--es-

Journal of Obstetrics& Gynecology 595.e9

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Research Basic Science: Gynecology www.AJOG.org

59diol (E2), were obtained from Sigma-rich (Steinheim, Germany). GPR30nist G1 was obtained from Calbio-

em (Darmstadt, Germany). GPR30tibody was purchased from Antibod--online (Aachen, Germany). The an-odies against total and phosphory-ed MAPK and -actin were from Cellnaling (Frankfurt am Main, Germany).dium was obtained from Sigma-Al-ch. GPR30 antisense oligonucleotidesre purchased from MWG-Medical (Mi-, Italy). Lipofectamin 2000 transfectiongent was obtained from Invitrogenarlsruhe, Germany).

ll cultureman endometrial carcinoma cellsEC-1A) were obtained from Ameri-

Type Culture Collection (Manassas,). They were routinely cultured inEM/F12 containing 5% fetal-calf se-

(FCS), 100 U/mL penicillin and0g/mL streptomycin. To perform

experiments, the cells were seeded inell plates at a density of 60,000 cells

r well. To test the effect of estradiol,oxifen and GPR30 agonist G1, the

ls were treated with phenol red-freedium supplemented with charcoal-ipped FCS 48 hours before the assay.en, cells were incubated for 5 daysth 10 nM E2, 1 nM G1, and 100 nM. At

end of the treatment, the cells weresed twice with saline solution andnted using a Coulter counter as de-

ibed.18 After treatment of HEC-1Als for 10 minutes with 100 nM tamox-n, the phospho-MAPK (pMAPK) andal-MAPK (tMAPK) were detected bymunoblotting. Furthermore, HEC-1Als were treated with 100 nM tamoxifener transfection with GPR30 antisensegonucleotides (GPR30/AS) or with con-l scrambled oligonucleotides (Con/AS).

periments with GPR30tisense oligonucleotidesr these experiments, the cells wereshed twice in phosphate-buffered sa-e, then transfected with 0.2 MR30 antisense oligonucleotide (5=-GGGAAGTCACATCCAT-3=) or withM random control (5=-GATCTCAG-CGGCAAAT-3=). The transfection was

formed with Lipofectamine 2000 trans- ifen

5.e10 American Journal of Obstetrics& Gynecologtion reagent as previously described.19

e experiments were performed 24 hourser transfection. The efficacy of GPR30ocking down was detected by Westerntting.

stern blottingtein cell lysis and Western blottingcedures were performed as previ-

sly described.15 Briefly, 25 L of theples were separated by SDS-PAGE

d blotted onto nitrocellulose mem-nes. The membranes were blocked

th 3% dry milk in TBS/Tween and in-bated for 2 hours at room temperatureT) with antibodies against GPR30, di-ed 1:1000, total or phosphorylatedPK diluted 1: 2000, and -actin di-

ed 1:10000. A peroxidase-conjugatedtirabbit antibody, diluted 1:10,000,s used for a second incubation (30nutes). West Pico Supersignal sub-ate (Perbio, Bonn, Germany) was leftthe membrane until distinct bands

d developed. A MagicMark standardvitrogen) was used to identify the mo-ular weights. The enhanced chemilumi-cence membrane images were quanti-using the GeneGnome and GeneTools

age scanning and analysis package (Syn-e, Cambridge, UK).

tients and study designobtained ethical approval from Otto-

n-Guericke University Ethic Commit-. The study included 95 patients withmary breast cancer diagnosed between9 and 2004 in the Department of Ob-

trics and Gynecology, University Schooledicine, Magdeburg, Germany, and in

IInd Department of Gynecology, Lub-Medical University, Lublin, Poland.

ey were classified into a tamoxifenup and 2 control groups. The tamox-

group (group I) consisted of 48 pa-nts with ER-positive breast cancer whoeived daily tamoxifen after breast sur-y and in whom vaginal bleeding and/orpicious endometrial thickness was ob-ved at the periodic checkup. All theseients had undergone endometrial bi-sy. A biopsy was performed if endome-l thickness of5 mm was determined.e first control group (group II) includedpatients who also receive daily tamox-

, but did not have symptoms or endo- the

y DECEMBER 2010trial abnormalities. The daily dose of ta-xifen for both groups was 20 mg.

oup III consisted of 25 patients who didt receive adjuvant therapy with tamox-. The time to symptoms was calculated

m the beginning of tamoxifen treat-nt to the first time the patients reportedptoms or suspicious endometrial

cknesswasobserved.Allpatientsunder-nt a periodic checkup with gynecologicmination, followed by ultrasound. Theients in the control groups underwent a

sterectomy because of prolapses, ovar-,pelvic,orotherpathologynot linkedtooxifen therapy. Endometrial tissueeloped from the curettages and hyster-

omieswasfixedinformalinandembed-in paraffin. The histology was classi-as follows: normal/atrophic (inactive)ometrium, endometrial hyperplasia

d/or endometrial polyp, and endome-l carcinoma.linicopathologic data regarding di-osis and treatment, epidemiologic

tors, as well as follow-up data, weretained retrospectively from the clini-records or the local physicians.

munohistochemistrymunohistochemistry was performedalready described.20 Briefly, sectionsformalin-fixed and paraffin-embed-

d breast cancer specimens (3.0 mck) were mounted on SuperFrost Plusss slides (Menzel, Braunschweig, Ger-ny) and dried overnight. A Bench-rk XT (Ventana, Unterhaching, Ger-ny) conducted the immunostaining.e slides were incubated with affinity-rified rabbit antibody against GPR30uted 1:500 for 32 minutes at 37C, af-

antigen retrieval with Protease Ientana) for 10 minutes. The reactionsre visualized by 5,5==-di-aminobenzi-e (DAB) detection. The slides werenterstained with hematoxylin anderslipped after being embedded inunting medium.

aluation of GPR30munoreactivityR30 immunoreactivity was detectedthe cytoplasm and in the nucleus of

cells. Some endometrial tissueswed clear GPR30 staining pattern in

cytoplasm and weak expression in

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www.AJOG.org Basic Science: Gynecology Researchnucleus (Figure 1), whereas othersmonstrated higher expression in thecleus. In contrast, the expression ofclassical ER was detected exclusively

the nucleus. No detectable cytoplas-c or plasma membrane staining wasserved for ER (Figure 1). The GPR30ression was classified in accordance

th the dominant immunostaining pat-n. GPR30 immunoreactivity was clas-ed as already described15,21 using thelowing grading system: staining ex-sity was categorized as 1 (10% pos-e cells), 2 (10-50% positive cells), or 350% positive cells), and staining in-sity was categorized as 0 (negative), 1eak), 2 (moderate), or 3 (strong). Theividual categories were multiplied toe a total immunoreactive score (IRS).S scores ranged between 0 and 9.

IGURE 1mmunohistochemistry for GPR30 an

nd B, Example of moderate nuclear staining of Gunoreactivity is moderately detected in both cytegative expression of GPR30 in the nucleus comgnification 100; B, D, F, H, J, L: original maestrogen receptors; GPR30, G-protein-coupled receptor.

tov. GPR30 and tamoxifen-induced endometrial pathology.R30 expression was classified as fol- sens: negative (IRS between 0 and 2), lowde (IRS 35), and high grade (IRS6-9). GPR30 protein expression wasided into 3 categories: negative, when0% of the cells showed negative orderate staining intensity; low grade,en 10-50% of the cells demonstratedderate staining; high grade, when0% showed strongly positive cells.

tistical analysise statistical calculations were performedng SPSS version 13.0 (SPSS, Chicago,. Association between GPR30 expres-n and the tamoxifen-induced endome-l pathologies was evaluated usinghers exact test or the 2 test. The inter-between the start of tamoxifen treat-nt and the diagnosis of bleeding and/orpicious endometrial thickness was cho-

R in endometrial tissue

0 with negative cytoplasmic staining and C and Dsm and nucleus with G and H, moderate immund with moderate cytoplasmic staining and K andcation 400.

J Obstet Gynecol 2010.as endpoint of the study. The associa- ou

DECEMBER 2010 American Jn between GPR30 expression and theerval was calculated by the Kaplan-ier survival method, and the equality ofvival curves was tested by the log rankt. For paired observations, statisticalalysis was carried out using Student tt. Curve fittings were performed withsm program (GraphPad Software, Sanego, CA). The results are expressed asans of 6 determinations standard de-tion (SD). Results were considered sta-ically significant if thePvalue was .05.

SULTS, G1, and tamoxifen stimulate cellwth and MAPK phosphorylationendometrial cellse first examined the effects of E2,R30 agonist G1, and the selective ERdulator tamoxifen on cell growth. In

clear immunostaining of ER. E and F, GPR30activity of ER in the nucleus. I and J, Exampleuclear ER expression. A, C, E, G, I, K: originallowgradiv1mowhmo5

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59nces significantly stimulated cellwth in HEC-1A (Figure 2, A). The

ongest proliferation effect was ob-ved with E2. However, the fact that

IGURE 2ell growth effects of E2, G1 and tam

HEC-1A cells were treated for 5 days with 10 nM-free medium containing 5% charcoal-strippedlter counter. The experiments were repeatedarate measurements SD. B, HEC-1A cells wespho-MAPK (pMAPK) and total-MAPK (tMAPK)ibodies. Total MAPK was used as loading controtov. GPR30 and tamoxifen-induced endometrial pathology., as a highly specific agonist for in

5.e12 American Journal of Obstetrics& GynecologR30, increased cell growth suggestedPR30-mediated cell proliferation.hen we examined the effects of these

ubstances on MAPK phosphorylation

ifen in endometrial cancer cells

, 1 nM G1, and 100 nM tamoxifen in phenoll calf serum. The cells were counted using aeast 3 times. Each data point is mean of 6eated for 10 minutes with 100 nM tamoxifen.e detected by immunoblotting using specificnormalize the pMAPK expression.J Obstet Gynecol 2010.HEC-1A cells. The cells were treated inh

y DECEMBER 201010 minutes with 10 nM E2, 1 nM G1,d 100 nM tamoxifen or with mediuma control. There was a significant in-ase in MAPK phosphorylation afterubation with all three substances

pared with the negative controlgure 2, B), whereas the level of totalPK remained unchanged.

moxifen-induced cell growth isersed by knocking down GPR30investigate the role of GPR30 in ta-xifen-induced endometrial cell pro-ration in more detail, HEC-1A cellsre transfected with GPR30-specifictisense oligonucleotides. Scrambledgonucleotides were used as a control.e transfection of HEC-1A cells withR30 antisense oligonucleotide abro-ed their response to tamoxifen (Fig-3, A), suggesting that GPR30 takes a

nificant part in tamoxifen-induceddometrial cell proliferation. GPR30ockdown and the specificity of GPR30tibody were confirmed by Westernt analysis (Figure 3, B). Taken to-her, all these data suggest an involve-nt of GPR30 signaling pathway in thecess of tamoxifen-induced endome-

al proliferation in vitro.

dometrial pathologythe study groupe wanted to further analyze the contri-tion of GPR30 to the development of

oxifen-induced endometrial pathol-y. For this purpose, we used tissue

ples of patients who were symptom-c during tamoxifen treatment forast cancer. As negative control, wed samples from women who received

other adjuvant therapy or tamoxifent never developed symptoms. Thean age of the whole study group was

.02 14.1 years, 54.94 12.8 years forup I, 71.73 10.9 years for group II,

d 67.08 11.6 years for group III. Thedian follow up of the study was 42nths (range, 577 months). All 95 pa-

nts received primary breast surgeryth or without radiotherapy. The pa-nts in group I and II received tamox-n with or without chemotherapy asjuvant treatment. The adjuvant treat-nt of group III consisted of aromataseGPa G

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www.AJOG.org Basic Science: Gynecology Researchnt (n 8) with or without chemo-rapy. In group I, the median duration

tween the start of tamoxifen treatmentd the vaginal bleeding and/or suspi-us endometrial sonography was 27nths (range, 559 months).he most common histopathologic

IGURE 3PR30 is important for tamoxifen-indroliferation in endometrial cells

HEC-1A cells were treated with 100 nM tamoisense oligonucleotides (GPR30/AS) or with contrre counted on day 5. Cells treated only with mresents the mean SD of 6 independent meassfected with GPR30 antisense oligonucleotidesotides (Con/AS) was detected using immunoblot30, G-protein-coupled receptor.

tov. GPR30 and tamoxifen-induced endometrial pathology.gnosis in the whole study population whs normal or atrophic endometrium incases, followed by endometrial hyper-sia in 36 cases, endometrial polyps incases and endometrial carcinoma in 1e (Table 1). The most cases of endo-trial hyperplasia and polyps were ob-ved in group I and II, namely, patients

ed

after transfection of the cells with GPR30crambled oligonucleotides (Con/AS). The cellsium were used as control. Each data pointments. B, GPR30 expression in HEC-1A cellsR30/AS) or with control scrambled oligonu-. -actin used as loading control.

J Obstet Gynecol 2010.o received tamoxifen therapy. The exp

DECEMBER 2010 American Jst common histologic diagnosis ob-ved in group III was normal/atrophicdometrium.

R30 protein expressiond tamoxifen-induceddometrial abnormalitiesirty-eight (40.0%) of the investigatedcimens were negative, 30 (31.6%)wed low-grade protein expression,ereas the rest of 27 (28.4%) cases

monstrated high-grade GPR30 im-norectivity (Table 2). There was a sig-cant relationship between GPR30 ex-ssion and the tamoxifen-induced

dometrial pathology (P .006). In 2227 (81.5%) cases with high grade and18 of 30 (60.0%) cases with low-gradeR30 expression, an abnormal endo-trial histology was observed (Table 2).contrast, endometrial pathology was

tected only in 13 of 38 (34.2%)R30-negative slides. The staining wasnificantly stronger in the tamoxifenup (group I), where 38 of 48 (79.2%)es were GPR30 positive, high or lowde. In groups II and III, the GPR30sitivity was observed in 9 of 22.9%) and in 10 of 25 (40.0%) sec-

ns, respectively (Table 3). These re-ts suggested involvement of GPR30 inoxifen-induced endometrial pathol-

y in breast cancer patients.oreover, GPR30 expression inten-

correlated significantly with the timetween the start of tamoxifen treatmentd the development of an endometrialnormality (Figure 4). For the patientsth high-grade GPR30 expression thedian duration of the tamoxifen treat-nt to the presenting of the symptomss 27 months (95% confidence intervalI], 2331 months) and for low-gradeR30 expression, it was 33 months% CI, 1353 months). This was a sig-cantly shorter (P .019) period untilptoms developed, compared withcases with negative GPR30 expres-

n. In this cases, the mean duration ofoxifen treatment until the develop-

nt of symptomology was 59 months% CI, 46 66 months) (Figure 4). Anrease in GPR30 expression grade wasersely associated with the symptom-e time length of adjuvant tamoxifenwa42pla16casmeser

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59MMENTthe current study, we provided addi-nal evidence of the role of GPR30 in

oxifen-agonistic activity in endome-al cell lines and, for the first time, con-

ed them in endometrial tissue fromast cancer patients treated withoxifen.arger epidemiological studies havewn that tamoxifen not only signifi-tly reduces breast cancer recurrence

d mortality rate,22 but also preventsast cancer in high-risk populations.9

erefore, tamoxifen is 1 of the mosten prescribed antineoplastic drugs.ce the initial report of Fornander at

3 many works have shown that in therus, tamoxifen is characterized withnistic properties and increased inci-

nce of endometrial abnormalities suchpolyps, endometrial hyperplasia, anddometrial carcinoma.10,11 This risk in-ased with the duration of tamoxifen

ABLE 1istribution of endometrial histology

istologyTamoxifGroup I

ormal/atrophic endometrium 3.........................................................................................................................

ndometrial hyperplasia 29.........................................................................................................................

ndometrial polyp 15.........................................................................................................................

ndometrial carcinoma 1.........................................................................................................................

otal 48 (50.5.........................................................................................................................

gnatov. GPR30 and tamoxifen-induced endometrial patholo

ABLE 2ssociation of GPR30 expressionnd endometrial abnormalities

PR30xpression

Endometrial histology

Normaln (%)

Abnormaln (%)

PR30 negn 38)

25 (65.8) 13 (34.2)

..........................................................................................................

PR30 lown 30)

12 (40.0) 18 (60.0)

..........................................................................................................

PR30 highn 27)

5 (15.5) 12 (81.5)

..........................................................................................................

.006.PR30, G-protein-coupled receptor.

gnatov. GPR30 and tamoxifen-induced endometrial

athology. Am J Obstet Gynecol 2010. I

5.e14 American Journal of Obstetrics& Gynecolograpy. The mechanism of tamoxifennistic activity in endometrium is not

ly understood.sing human endometrial carcinoma

C-1A cells, we demonstrated that ta-xifen can induce cell growth in aR30-dependent manner. After activa-n by tamoxifen, GPR30 leads to

ulation of cell proliferation, moressibly by crosstalks with EGFR and ac-ation of MAPK phosphorylation.13,15

cently, 2 studies on the role of GPR30tamoxifen-induced proliferation in

dometrial carcinoma cells have beenblished.14,16 These works demon-ated results similar to ours. Recently,ith et al17 found a significant correla-n between GPR30 and EGFR expres-n in 47 patients with endometrial car-oma, providing further evidence of

potential role of the nonclassicalR30/EGFR-MAPK signaling in endo-trial pathology. In our cohort of 95

tients, we observed a significant corre-ion between GPR30 expression and

tamoxifen-induced endometrial pa-logy (P .002), which is in agree-nt with our results observed in vitro.

the study groups

Control groups

Group II Group III

16 23...........................................................................................................

5 2...........................................................................................................

1 0...........................................................................................................

0 0...........................................................................................................

22 (23.2%) 25 (26.3%)...........................................................................................................

m JObstet Gynecol 2010.

ABLE 3istribution of GPR30 expression in d

PR30 intensityTamoxifenGroup I n (%)

PR30 Neg n 41 10 (20.8).........................................................................................................................

PR30 Low n 25 17 (35.4).........................................................................................................................

PR30 High n 29 21 (43.8).........................................................................................................................

.002.PR30, G-protein-coupled receptor.gnatov. GPR30 and tamoxifen-induced endometrial pathology. A

y DECEMBER 2010the best of our knowledge, this is thet study evaluating the association ofR30 and tamoxifen-induced endo-trial abnormalities in vivo and assess-the GPR30-mediated tamoxifen ago-

tic effects. GPR30 immunoreactivitys detected in the cytoplasm and in thecleus of the cells and has already beenscribed by different research groups inast cancer.12,21,24,25 These resultsght help us to definitely resolve the ex-ssion pattern of GPR30 in differentues.igh-grade GPR30 staining was asso-

ted with an increased rate of endome-al polyps or hyperplasia and was sig-cantly lower in normal and atrophic

dometrial tissue. It is in agreementth the work of Smith et al,17 whond low levels of GPR30 expression in

nign endometrial tissue. Another factderlying the role of GPR30 in tamox-n-induced endometrial proliferationhe observation that patients with highels of GPR30 expression had an endo-trial abnormality develop signifi-tly faster. GPR30 expression showed

everse correlation to the length of timetween the start of tamoxifen therapyd the development of symptoms.

he effect of tamoxifen agonist activ-on serum estradiol has been contro-sially discussed in the literature. Inmenopausal women, it has beennd that tamoxifen increases thesma estrogen level, whereas it slightlyuced the free estradiol in postmeno-

usal women.26,27 In this context, ta-xifen can act as an agonist only in

stmenopausal women. However,e other reports have not found any

nificant difference in endometrial ab-

erent groups

Control

Group II n (%) Group III n (%)

13 (59.1) 15 (60.0)...........................................................................................................

6 (27.3) 7 (28.0)...........................................................................................................

3 (13.6) 3 (12.0)...........................................................................................................me

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www.AJOG.org Basic Science: Gynecology Researchrmalities in pre- and postmenopausalmen receiving tamoxifen.28,29 Al-ugh not significant, several in our co-

rt who showed an endometrial pa-logy under tamoxifen therapy, weremenopausal. This is in agreement

th our recent results observed in vitro,ere E2 induced GPR30 up-regulationMCF-7 breast cancer cell line.15 Le-nc et al30 have recently confirmed thatR30 mRNA expression in endome-

al cell lines increased significantly inestrogen-rich milieu. In this respect,controversial data in the literatureld be explained by the influence of

ferent environmental factors on ta-xifen agonist effect. However, E2-in-

ced up-regulation of GPR30 in vitrold explain our findings in vivo that

dometrial abnormalities were moreen observed in premenopausal womenh GPR30 immunoreactivity.aken together, these results suggestt GPR30 might be an important

IGURE 4aplan-Meier plot for symptom-freeccording to the GPR30 expression s

R30 neg, negative receptor expression; GPR30 lh-grade receptor expression. The log rank test w30, G-protein-coupled receptor.

tov. GPR30 and tamoxifen-induced endometrial pathology.mponent in the molecular mecha-7. LChm of tamoxifen-induced endome-al abnormalities. f

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595.e16 American Journal of Obstetrics& Gynecology DECEMBER 2010

Role of GPR30 in endometrial pathology after tamoxifen for breast cancerMATERIALS AND METHODSMaterialsCell cultureExperiments with GPR30 antisense oligonucleotidesWestern blottingPatients and study designImmunohistochemistryEvaluation of GPR30 immunoreactivityStatistical analysis

RESULTSE2, G1, and tamoxifen stimulate cell growth and MAPK phosphorylation in endometrial cellsTamoxifen-induced cell growth is reversed by knocking down GPR30Endometrial pathology in the study groupGPR30 protein expression and tamoxifen-induced endometrial abnormalities

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Role of GPR30 in Endometrial Pathology After Tamoxifen for Breast Cancer