Trastuzumab Therapy for Tamoxifen-Stimulated Endometrial Cancer

Trastuzumab Therapy for Tamoxifen-Stimulated Endometrial Cancer

Clodia Osipo,1Kathleen Meeke,

1Hong Liu,

1Dong Cheng,

1Sherry Lim,

1

Alyssa Weichel,1and V. Craig Jordan

1,2

1Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinoisand 2Fox Chase Cancer Center, Philadelphia, Pennsylvania

Abstract

A novel in vivo model of tamoxifen-stimulated endometrialcancer was developed and the role of HER-2/neu investigatedby using trastuzumab. Tamoxifen-stimulated tumors (ECC-1TAM) were growth stimulated by 17B-estradiol (E2),tamoxifen, or raloxifene. Trastuzumab inhibited growth of E2-stimulated ECC-1E2 tumors by 50% and tamoxifen-stimulatedECC-1TAM tumors by 100%. ECC-1 tumors expressed func-tional estrogen receptor A (ERA) as measured by induction ofpS2 and c-myc mRNAs. E2 induced pS2 and c-myc mRNAs upto 40-fold in ECC-1E2 and ECC-1TAM. Tamoxifen induced pS2and c-myc mRNAs up to 5-fold in ECC-1E2 tumors and up to10-fold in ECC-TAM tumors. Trastuzumab blocked E2-inducedpS2 mRNA (P < 0.01) in ECC-1E2 by 50% and tamoxifen-induced c-myc mRNA (P < 0.1) in ECC-1TAM tumors by 70%.Trastuzumab decreased phosphorylated and total HER-2/neuprotein in ECC-1E2 and ECC-1TAM tumors. However, onlyphospho-ERK-1/2 and not phospho-Akt protein was decreasedby trastuzumab in tamoxifen-treated ECC-1TAM tumors. Theinsulin-like growth factor (IGF-I) signaling pathway alsoactivates extracellular signal-related kinase (ERK)-1/2 andcould block the efficacy of trastuzumab in ECC-1E2 tumors.The results showed that IGF-I, IGF-IR mRNAs, and phospho-insulin receptor substrate-1 (IRS-1) protein were decreasedin ECC-1TAM compared with ECC-1E2 tumors. The resultsshow that trastuzumab is an effective therapy for both E2-stimulated and tamoxifen-stimulated endometrial cancer. Thedata suggest estrogenic activities of E2 and tamoxifen at ERA-regulated pS2 and c-myc genes are in part mediated by HER-2/neu . However, trastuzumab is a better growth inhibitor ofECC-1TAM tumors where there is diminished IGF-I signalingallowing for complete blockade of the downstream phospho-ERK-1/2 signal. (Cancer Res 2005; 65(18): 8504-13)

Introduction

Tamoxifen has been the standard of care for estrogen receptor a(ERa)–positive breast cancer in premenopausal and postmenopaus-al women for over 15 years (1). Five years of adjuvant tamoxifentherapy reduces the incidence of contralateral breast cancer (2),improves overall survival (2), and also reduces the risk of breastcancer in women at high risk (3). However, tamoxifen is not a pureantiestrogen but a selective estrogen ERa modulator (4, 5). It hasantiestrogenic affects on the breast epithelium but estrogenic affectson bone density (6, 7), circulating cholesterol and cardiovascular

events (8), and the uterus (9). As a result of tamoxifen’s selectiveestrogen receptor modulator activity around a woman’s body, itincreases the incidence of endometrial cancer by 0.1% inpostmenopausal women (9). The exact mechanism by whichtamoxifen induces endometrial cancer during breast cancer therapyis as yet unclear.Numerous studies during the last 10 years have focused on

formation of DNA adducts by metabolites of tamoxifen in theendometrium of rats and humans. Recently, Liehr showed that 17-estradiol (E2) is a dual mutagen and carcinogen in the immaturerat lacking expression of ER (10). Tamoxifen might also be amutagen/carcinogen as it has estrogenic activity in the uterus.However, the data to date shows that tamoxifen is a potent rat livercarcinogen causing the formation of tamoxifen-DNA adducts andliver cancer (11). However, little conclusive data exists showing adirect affect of tamoxifen or its metabolites on DNA adductformation in the rat uterus to cause endometrial cancer. Moststudies lack convincing evidence for tamoxifen-DNA adductformation in rat and human endometrium (11–18), suggestingthat the estrogenic action of tamoxifen at ERa-regulated genesis likely to be the more important factor in development ofendometrial cancer in women undergoing tamoxifen therapy.Shang and Brown (19) showed, using in vitro cell culture models,

that tamoxifen is estrogenic in endometrial cancer cells bystimulating the recruitment of coactivators, in particular SRC-1, amember of the p160 family of coactivators, to a certain subset ofgenes regulated by the ERa at the transcriptional level. Theinvestigators concluded that the SERM activity of tamoxifen in theendometrium could be due to differences in the recruitment ofcoregulators to certain ER-regulated promoters. Furthermore, Shahand Rowen (20) recently showed that the nonreceptor tyrosinekinase, src, enhances the agonist activity of tamoxifen inendometrial cancer cells by phosphorylation of Ser167 andstabilization of ER promoter interaction. However, neither of thesestudies has been confirmed using preclinical in vivo models oftamoxifen-stimulated endometrial tumors.In addition, resistance to adjuvant tamoxifen therapy for ERa-

positive breast cancer is a very common occurrence. Resistance totamoxifen is defined as the lack of tumoristasis and/or regressionor might be evidenced by growth stimulation. Subsets of breasttumors that do not initially respond (i.e., are not growth arrestedor do not regress) to tamoxifen are intrinsically resistant totamoxifen therapy. In contrast, acquired resistance to tamoxifenduring z5 years of adjuvant tamoxifen therapy (21) could resultfrom growth stimulation in response to continuous treatmentwith tamoxifen. Osborne et al. (22) showed that postmenopausalwomen with ERa-positive breast cancer do not initially respondto tamoxifen therapy when their tumors overexpress both HER-2/neu , a member of the epidermal growth factor receptor family ofreceptor tyrosine kinases, and the gene amplified in breastcancer-1 (AIB1), a member of the p160 family of coactivators.

Requests for reprints: V. Craig Jordan, OBE, PhD, Dsc. Vice President andResearch Director for Medical Science, Alfred G. Knudson Chair of Cancer Research.Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111-2497. E-mail:[email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-04-4107

Cancer Res 2005; 65: (18). September 15, 2005 8504 www.aacrjournals.org

Research Article

More recently, Shou et al. (23) concluded that tamoxifen’sestrogenic activity is partly due to crosstalk between HER-2/neuand ERa in MCF-7 cancer cells that express high levels of AIB1and stably overexpress HER-2/neu . These studies showed thatoverexpression of HER-2/neu and AIB1 in ERa-positive breastcancer cells can cause intrinsic resistance to tamoxifen. However,only f10% of all ERa-positive breast cancer cells overexpressHER-2/neu . Therefore, it is unclear whether HER-2/neu plays arole in the development of acquired resistance to tamoxifenduring the 5 years of adjuvant therapy.Studies suggest that overexpression of HER-2/neu leads to

tamoxifen-resistant breast cancer (22, 24). A recent report showedthat src tyrosine kinase potentiates the agonist activity of tamoxifenin endometrial cancer cells (20). In addition, overexpression of HER-2/neu correlates with high-grade endometrial cancer (25–27).However, exact mechanisms leading to tamoxifen-stimulated breastand endometrial cancers are not well understood. Currently,inhibitors to epidermal growth factor receptor (i.e., gefitinib), HER-2/neu (i.e., trastuzumab), Ras/mitogen-activated protein kinase (i.e.,farnesyltransferase inhibitor, tipifarnib), and protein kinase B (Akt;i.e., an mammalian target of rapamycin inhibitor, CCI 779) are beinginvestigated either alone or in combination with tamoxifen toprevent and/or treat resistance to tamoxifen therapy. The questionthat remains to be answered is if HER-2/neu is important fortamoxifen-resistant breast cancer, then does HER-2/neu play a rolein the development of endometrial cancer during long-term tamox-ifen therapy? We have developed a novel in vivo model oftamoxifen-stimulated endometrial cancer (ECC-1TAM) by treatingtamoxifen-naive, E2-stimulated ECC-1E2 tumors with E2 plus tamox-ifen for 24 weeks. We investigated the role of HER-2/neu in thegrowth of both ECC-1E2 and ECC-1TAM tumors using thehumanized monoclonal antibody to HER-2/neu , trastuzumab.

Materials and Methods

Cell culture. The human endometrial cancer cell line, ECC-1, was a

generous gift from Dr. Myles Brown at the Dana-Farber Cancer Institute,

Harvard Medical School. ECC-1 cells were maintained at 37jC in a 95%humidified/5% CO2 atmosphere in phenol red–containing DMEM supple-

mented with 10% fetal bovine serum, 6 ng/mL insulin solution, 1%

L-glutamine, 1% nonessential amino acids, 1% of a combination of penicillinstreptomycin, and antimycotic (Life Technologies, Long Island, NY).

Growth of ECC-1E2 tumors in vivo . The ECC-1E2 tumors used in these

experiments were derived by bilateral inoculation of 0.10 mL of 1 � 107

suspended ECC-1 cells in sterile PBS into the mammary fat pads of 4- to6-week-old ovariectomized BALB/c nu/nu athymic mice (Harlan Sprague-

Dawley, Madison, WI). ECC-1 cells-inoculated into athymic mice were

supplemented with 0.30-cm E2 (Sigma, St. Louis, MO) silastic capsules

(Baxter Health Care, Mundelein, IL) to achieve E2-stimulated tumor growth(ECC-1E2; refs. 28–33). Tumors were maintained by serial passage of solid

tumors into athymic mice as described previously (29). Thirty mice were

bitransplanted with parental ECC-1E2 tumors and groups (10 mice) were

treated as follows: vehicle, 0.30-cm E2 capsules, and E2 plus 1.5 mg/dtamoxifen for 5 days (given orally by gavage) for 24 weeks. In a separate

experiment, 50 new athymic mice were serially bitransplanted with 1-mm

ECC-1E2 tumors portions and grown to a mean cross sectional area of 0.28cm2 and randomly separated into five groups of 10 and treated with the

following: vehicle, 0.30-cm E2 capsule, 1.5 mg/d tamoxifen, 10 mg

fulvestrant (dissolved in 100% ethanol and diluted in sterile-filtered peanut

oil, 5 mg/0.10 mL injected s.c., twice weekly), and 0.30-cm E2 + 30 mg/kgtrastuzumab (15 mg/kg injected i.p., twice weekly).

Growth of ECC-1TAM tumors in vivo . Long-term tamoxifen-stimulated

tumors (ECC-1TAM) were developed by retransplanting the surviving E2 +

1.5 mg tamoxifen-treated ECC-1E2 tumors into 40 new athymic mice, and

treating groups (10 mice) with vehicle, 1.5 mg/d tamoxifen, 0.30-cm E2capsules, or 1.5 mg/d Raloxifene (obtained from the pharmacy; refs. 29, 30, 34)

for 12 weeks. In another experiment, 50 animals were bitransplanted with

ECC-1TAM tumors and grown to a mean cross-sectional area of 0.31 cm2 and

randomly separated into groups of 10 mice that were then subsequentlytreated as follows: vehicle, 1.5 mg tamoxifen, 0.30-cm E2 capsules, 10 mg

fulvestrant (5 mg given s.c., twice weekly), and 1.5 mg tamoxifen + 30 mg/kg

trastuzumab. In a final in vivo experiment, 30 mice were bilaterally

transplanted with 1-mm tamoxifen-treated ECC-1TAM tumors fromFig. 1D and 10 mice per group were treated as follows: control, 1.5 mg

tamoxifen, and 1.5 mg tamoxifen plus 5 mg fulvestrant (twice weekly).

Real-time reverse transcription-PCR for human pS2 , c-myc , HER-2/neu , insulin-like growth factor-I, and insulin-like growth factor-IRmRNAs in tumors. Total RNA is extracted from the tumors using the RNeasy

Mini Kit (Qiagen, Stanford Valencia, CA) according to the manufacturer’s

instructions. The total RNA is reverse transcribed using Taqman reversetranscription reagents (PE Applied Biosystems, Hayward, CA) with the use of

random hexamers as the primers according to the manufacturer’s

instructions. Primers and probes for human pS2, HER-2/neu , insulin growth

factor-1 (IGF-I), and IGF-IR are designed using Primer ExpressTM1.5software set at default variables to select the most optimized primer and

probe sets for this system. The sequences for the forward and reverse primers

for human pS2 are 5V-AGGCCCAGACAGAGACGTG-3V and 5V-CCCTGCA-GAAGTGTCTAAAATTCA-3V, respectively. The sequence for the pS2 probe is5V-CTGCTGTTTCGACGACACCGTTCG-3Vwhere the FAM is the reporter and

QSY7 is the quencher (MegaBases, Inc., Chicago, IL). The c-myc primer and

probe mixture was purchased from Perkin-Elmer Applied Biosystems (PE-ABI, Stanford Valencia, CA) and used according to the manufacturing

instructions. The sequences for the forward and reverse primers for human

HER-2/neu are 5V-ACTGCAGAGGCTGCGGATT-3Vand 5V-ACGGCCAGGGCA-TAGTTGT-3V, respectively. The sequence for the human HER-2/neu probe is5V-TGCGAGGCACCCAGCTCTTTGA-3V where the FAM is the reporter

and QSY7 is the quencher (MegaBases). The sequences for the forward and

reverse primers for human IGF-I are 5V-TGCTTCCGGAGCTGTGATC-3Vand5V-AGCTGACTTGGCAGGCTTGA-3V, respectively. The sequence for thehuman IGF-I probe is 5V-AGGAGGCTGGAGATGTATTGCGCACC-3V where

the FAM is the reporter and QSY7 is the quencher (MegaBases). The probe

and primers for human IGF-IR were purchased from Perkin-Elmer AppliedBiosystems (PE-ABI, Stanford Valencia, CA). The quantity of human 18s RNA

was also measured in each total cDNA sample for normalization purposes.

The probe and primers for 18s RNA were purchased from Perkin-Elmer

Applied Biosystems (Stanford Valencia, CA). The PCR portion of the reactionwas done with the use of the Taqman PCR Core Reagent Kit (Perkin-Elmer

Applied Biosystems). In a total volume of 25 AL, 50 ng of total cDNA,

100 nmol/L probe, and 200 nmol/L primers were used in the PCR reaction.

Real-time PCR was done using the ABI-Prism 7700 Sequence DetectionSystem. The PCR conditions were 50jC for 2 minutes, 95jC for 10 minutes

followed by 40 cycles of 95jC for 15 seconds and 60jC for 1 minute.

Western blot analyses. Tumors were homogenized by grinding in liquid

nitrogen and resuspending in lysis buffer [1% Triton X-100, 1 mmol/L EDTA,150 mmol/L NaCl, 50 mmol/L Tris base (pH 7.4), 25 mg/mL phenyl-

methanesulfonyl fluoride, 10 (g/mL leupeptin, 10 mg/mL aprotinin,

10 mg/mL pepstatin, 10 mg/mL TLCK, 10 mg/mL N-tosyl-L-phenylalaninechloromethyl ketone, 100 mmol/L NaF, 10 mmol/L ortho-vanadate; Sigma].

The extractwas subsequently sonicated and then centrifuged for 5minutes at

5,000 � g at 4jC. The supernatant was collected and protein concentration

was measured using the Bradford assay (Bio-Rad Laboratories, Inc., SantaCruz, CA). Equal amounts of protein (25-50 Ag) were loaded onto a 7%

polyacrylamide/bisacrylamide gel for SDS-PAGE followed by Western

blotting. The following proteins were detected by Western blot: Tyr1248-

phosphorylatedHER-2/neu (1:2,000Rabbit anti-human, UpstateBiotechnology,New York, NY), HER-2/neu (1:200 mouse anti-human, Ab-11, Neomarkers,

Fremont, CA), Ser473-phosphorylated Akt (1:1,000 rabbit anti-human, Cell

Signaling Technology, Beverly, MA), Akt (1:1,000 rabbit anti-human, CellSignaling Technology), phosphorylated-ERK-1/2 (1:1,000 mouse anti-

human, Cell Signaling Technology), extracellular signal-related kinase

(ERK)-1/2 (1:1,000 mouse anti-human, Cell Signaling Technology), h-actin

HER-2/neu Signaling and Endometrial Cancer

www.aacrjournals.org 8505 Cancer Res 2005; 65: (18). September 15, 2005

(1:20,000 mouse anti-human, Sigma-Aldrich, St. Louis, MO), tyrosine-

phosphorylated insulin receptor substrate (IRS-1; 1 Ag/mL rabbit anti-human, Upstate Biotechnology), and IRS-1 (2 Ag/mL mouse anti-human,

Upstate Biotechnology). The appropriate secondary antibody conjugated

to horseradish peroxidase was used to detect the primary antibody (eithergoat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase, Santa

Cruz Biotechnology, Santa Cruz, CA). The blot was developed using an

enhanced chemiluminescence kit (Amersham Corp., Arlington Heights, IL).

The membrane was exposed to Kodak X-OMAT film for 10 to 30 seconds.Densitometry was done using the Scion program to quantify the intensity

of bands from three independent Western blots.

Statistical analysis. Tumor growth curves were analyzed longitudinally

using a two-factor ANOVA comparing all tumor cross sectional areas withintreatments in a time-dependent manner. Tumor growth curves represent

meansF SE of tumor cross-sectional areas. A two-sided Student’s t test was

used to analyze differences in mRNA levels as detected by real-time PCR ofthe treatments to the control group. The error bars for the measurement of

mRNA copy number represent SE calculated using Excel program.

Results

Growth of ECC-1E2 and ECC-1TAM tumors in vivo . Toelucidate the mechanism of action by which tamoxifen stimulatesgrowth of endometrial cancer cells under physiologic conditions tomimic the clinical situation, we developed a novel tamoxifen-stimulated endometrial cancer model in vivo by treating theparental ECC-1E2 tumors originated from cell lines (ECC-1)derived from the human EnCa101 tumor with postmenopausallevels of E2 plus tamoxifen for 24 weeks. The results showed thatthe parental ECC-1E2 tumors grew faster in response to E2 (P <0.0001) compared with control (Fig. 1A). At 24 weeks, ECC-1E2tumors treated with E2 plus tamoxifen were larger (P < 0.0001)than control (Fig. 1A). Upon serial bitransplantation of ECC-1E2tumors treated with E2 plus tamoxifen into new generations ofathymic mice, these tumors were growth stimulated more by E2alone (P < 0.01), tamoxifen alone (P = 0.48), or raloxifene alone

Figure 1. Development of tamoxifen (TAM )–stimulated endometrial cancer in vivo and the effect of trastuzumab on growth. A, thirty athymic mice were bitransplantedwith 1-mm ECC-1E2 tumors. Mice were randomly treated as follows: 10 mice received no treatment as Controls, 20 mice received 0.30-cm E2 capsules that wereimplanted intrascapularly under the skin for silastic release of E2. The capsules were replaced every 8 weeks to maintain a blood concentration of 83 to 100 pg/mL.Of the 20 mice receiving E2, 10 were also fed 1.5 mg/d tamoxifen orally by gavage 5 days a week. The length and width of tumors were measured weekly up to24 weeks using Vernier calipers. B, at week 24, the E2 plus tamoxifen tumors were excised and retransplanted into 40 new athymic mice and 10 mice per group weretreated as follows: Control, 1.5 mg/d tamoxifen, 0.30-cm E2 capsules, or 1.5 mg/d raloxifene (RAL , fed orally by gave, 5 days a week). The tumors were grown up to12 weeks and measured weekly as described above. C, fifty athymic mice were bitransplanted with 1-mm ECC-1E2 tumors and treated with 0.30-cm E2 capsules untilthey reached a mean cross-sectional area of 0.28 cm2 at week 4. Then, 10 mice per group were randomly treated as follows: Control, E2, tamoxifen, 5 mg/0.1 mLfulvestrant injected twice per week s.c., or E2 plus 15 mg/kg trastuzumab injected i.p. twice per week. The tumor sizes were measured weekly as described in (A) upto 9 weeks. D, fifty athymic mice were bilaterally transplanted with 1-mm tamoxifen-treated ECC-1TAM tumors from (B) and continued to be treated with tamoxifen untilthe mean cross-sectional area of the tumors reached 0.31 cm2. Then, 10 mice per group were randomly treated as follows: Control, tamoxifen, E2, fulvestrant, ortamoxifen plus trastuzumab. The tumor sizes were measured as described in (A) up to 11 weeks. The cross-sectional area of each tumor was calculated usingthe following formula: cross sectional area = (length � width � p)/4. Points, mean cross-sectional areas of tumors; bars, FSE. *, statistical significance of meancross-sectional areas of tumors at the end of each growth study compared with Control. **, statistical significance of mean cross-sectional areas of tumors at the endof each growth study compared with the E2-treated group in (C ) or to the tamoxifen-treated group in (D).

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(P = 0.35) compared with control (Fig. 1B). These data show thedevelopment of a novel in vivo model of tamoxifen-stimulatedendometrial tumor that continues to grow in response to E2 and iscross-resistant with raloxifene.Growth of ECC-1E2 and ECC-1TAM tumors in response to

trastuzumab. We investigated the consequences of using a HER-2/neu inhibitor, trastuzumab, on growth of ECC-1E2 and ECC-1TAMtumors in vivo . The results showed that ECC-1E2 tumors that havereached an initial mean cross sectional area of 0.28 cm2 at week 4 didnot grow in response to vehicle control, tamoxifen, or fulvestrantcompared with the E2-treated group (P = 0.02; Fig. 1C). Interestingly,trastuzumab partially blocked E2-induced growth of ECC-1E2tumors by almost 50% (P = 0.06) compared with tumors treatedwith E2 alone (Fig. 1C).ECC-1TAM tumors that were initially a mean cross sectional area

of 0.31 cm2 at week 6 grew with E2 (P < 0.01) or tamoxifen (P = 0.1)compared with control (Fig. 1D). Interestingly, fulvestrant modestlystimulated growth of ECC-1TAM tumors compared with control.More importantly, tamoxifen-stimulated ECC-1TAM tumors werecompletely growth inhibited by trastuzumab (P = 0.002) comparedwith tamoxifen alone (Fig. 1D). These results suggest for the firsttime that tamoxifen stimulates growth of endometrial cancer at leastin part through HER-2/neu .To determine whether tamoxifen stimulated growth of ECC-

1TAM tumors through ERa, a separate experiment was done usingthe combination of tamoxifen plus fulvestrant. The results showedthat fulvestrant inhibited growth of tamoxifen-treated ECC-1TAMtumors (Fig. 2) indicating that the growth stimulatory effect oftamoxifen is mediated by ERa.Expression of estrogen-responsive genes, pS2 and c-myc , in

ECC-1E2 and ECC-1TAM tumors. The results from the growthstudies showed that fulvestrant blocked tamoxifen-stimulated ECC-1TAM tumors (Fig. 2) thus suggesting that tamoxifen is estrogenicthrough ERa. In addition, Shang and Brown previously showed thattamoxifen is estrogenic at ERa-mediated promoters for c-myc andIGF-I genes (19). Therefore, we investigated ERa transcriptionalactivity at pS2 (classic promoter-driven gene) and c-myc (tethered-promoter driven gene) in both ECC-1E2 and ECC-TAM tumors. Theresults in Fig. 3A showed that E2 increased pS2 mRNA expression in

ECC-1E2 (25.06-fold over control, P < 0.0001) and ECC-1TAM (34.59-fold over control, P < 0.01) tumors. Trastuzumab inhibited E2-induced pS2 mRNA expression (P < 0.1) by almost 50% in parentalECC-1E2 tumors (Fig. 3A). Tamoxifen induced pS2mRNA expressionby 3.94-fold over control (P = 0.045) in ECC-1E2 tumors and 7-fold inECC-1TAM tumors over control (P = 0.045; Fig. 3A) and trastuzumabhad no effect on tamoxifen-mediated increase of pS2 mRNAexpression in ECC-1TAM tumors (Fig. 3A).mRNA for the c-myc gene was increasedwith E2 treatment in both

ECC-1E2 and ECC-1TAMtumors by 41.72-fold (P = 0.0001) and 29.98-fold (P < 0.01), respectively, compared with controls (Fig. 3B).Tamoxifen induced expression of c-myc mRNA in ECC-1E2 by only3.32-fold (P = 0.25) while increasing c-myc mRNA by 10.06-fold (P <0.001) in ECC-1TAM tumors compared with control. Interestingly,trastuzumab inhibited the tamoxifen-induced increase of c-mycmRNA in ECC-1TAM tumors by 70% (P < 0.1; Fig. 3B). These resultsconfirm that tamoxifen is estrogenic at ERa-regulated genes pS2 andc-myc in ECC-1 endometrial tumors. More importantly, the datasuggest that estrogenic affects that correlate directly to growthstimulation by either E2 in ECC-1E2 tumors or tamoxifen in ECC-1TAMtumors at specific genes such as pS2 or c-myc , respectively, areat least in part regulated by HER-2/neu .Effectiveness of trastuzumab in inhibiting HER-2/neu ,

mitogen-activated protein kinase, and Akt activities. HER-2/neu was not overexpressed at the mRNA level in ECC-1TAMtumors compared with ECC-1E2 tumors (data not shown). Thus,an increase in HER-2/neu expression could not explain theeffectiveness of trastuzumab in blocking growth of ECC-1TAMtumors compared with ECC-1E2 tumors. To determine the affectof trastuzumab on HER-2/neu activity, we measured tyrosine-phosphorylated HER-2/neu , total HER-2/neu , and downstreameffectors, phosphorylated ERK-1/2, total ERK-1/2, phosphorylatedAkt, and total Akt protein levels by Western blot analyses. Theresults showed that total HER-2/neu protein levels wereunchanged in ECC-1TAM tumors compared with ECC-1E2tumors (Fig. 3C). However, basal levels of phospho-HER-2/neuprotein were increased in ECC-1TAM by 2.7-fold versus ECC-1E2tumors (Fig. 3C) as determined by densitometry of threeindependent Western blots. However, trastuzumab decreased

Figure 2. Growth of ECC-1TAM tumors inresponse to tamoxifen (TAM ) plus fulvestrant.Thirty mice were bilaterally transplanted with 1-mmtamoxifen-treated ECC-1TAM tumors from Fig. 1Dand 10 mice per group were treated as follows:Control, 1.5 mg tamoxifen, and 1.5 mg tamoxifenplus 5 mg fulvestrant (twice weekly). Thecross-sectional area of each tumor was calculatedusing the following formula: cross sectionalarea = (length � width � p )/4. Points, meancross-sectional area of tumors; bars, FSE.*, statistical significance of the tamoxifen +fulvestrant treated group at the end of the studycompared with tamoxifen alone.



tyrosine-phosphorylated HER-2/neu and total HER-2/neu proteinto almost undetectable levels in both ECC-1E2 and ECC-1TAM(Fig. 3C). Furthermore, trastuzumab had little effect on phospho-ERK-1/2, total ERK-1/2, phospho-Akt, or total Akt protein levelsin ECC-1E2 tumors (Fig. 3C). In contrast, phospho-Akt protein

was increased in ECC-1TAM tumors treated with tamoxifencompared with control but this increase was not affected bytrastuzumab. Interestingly and in contrast to ECC-1E2 tumors,E2 or fulvestrant increased phospho-ERK-1/2 levels in ECC-1TAMtumors compared with control (Fig. 3C). More importantly,

Figure 3. Expression of estrogen-responsive genes, HER-2/neu , Akt, and ERK-1/2 proteins in ECC-1E2 and ECC-1TAM tumors. A, ECC-1E2 tumors treatedwith Control, E2, tamoxifen (TAM), fulvestrant (F), or E2 + trastuzumab were excised at week 9 (see Fig. 1C ). ECC-1TAM tumors-treated with Control, tamoxifen, E2,fulvestrant, or tamoxifen + trastuzumab were excised at week 11 (see Fig. 1D). Total RNA was extracted from both sets of tumors as described in Materials andMethods. Total RNA was reverse transcribed to total cDNA and real-time PCR was done using the Applied Biosystems ABI 7700 Taqman PCR instrument to detectexpression of the human pS2 mRNA as described in Materials and Methods. 18S RNA was used as a loading control in all samples. pS2 mRNA cycle threshold(C t) values were normalized to C t values for 18S RNA by subtracting the C t18s from the C tpS2. Columns, mean pS2 mRNA copy numbers relative to Control in threeindependent tumors with three replicates per tumor. *, statistical significance of pS2 mRNA copy number compared with Control. **, statistical significance of pS2 mRNAcopy number of E2 + trastuzumab compared with E2 alone in ECC-1E2 tumors. B, the same sets of cDNA and protocol used to detect the human pS2 mRNA was usedto detect the human c-myc mRNA in ECC-1E2 and ECC-1TAM tumors. *, statistical significance of c-myc mRNA copy number compared with Control. **, statisticalsignificance of c-myc mRNA copy number of tamoxifen + trastuzumab compared with tamoxifen alone in ECC-1TAM tumors. C, total protein was extracted from ECC-1E2 (at week 9, Fig. 1C ) and ECC-1TAM (at week 11, Fig. 1D ) tumors as previously described in Materials and Methods. Extracts were vigorously vortexed followed by5-second ultrasonic pulses to disrupt cellular membranes. The total mixture was centrifuged for 5 minutes at 5,000 � g at 4 jC. Fifty micrograms of supernatant fromeach sample were boiled in 2� Leammli buffer and loaded onto a 7% polyacrylamide/bisacrylamide gel followed by SDS-PAGE. Western blot analyses to detecttyrosine (Y1248) phosphorylated HER-2/neu , total HER-2/neu , phosphorylated Ser473-Akt, total Akt, phosphorylated ERK-1 and ERK-2, total ERK-1 and ERK-2, andh-actin proteins were done as described in Materials and Methods. The experiment was repeated at least thrice using three independent tumors. *, 2.7 F 0.3-foldincrease of basal levels of PY-HER2 protein as determined by densitometry in ECC-1TAM tumors compared with ECC-1E2 tumors.

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trastuzumab decreased phospho-ERK-1/2 protein levels in ECC-1TAM compared with tamoxifen alone (Fig. 3C). These resultssuggest that trastuzumab is effective at down-regulating HER-2/neu protein and tyrosine phosphorylation of HER-2/neu in ECC-1E2 and ECC-1TAM tumors in the absence of HER-2/neuoverexpression. However, the difference in trastuzumab’s efficacyon growth of ECC-1TAM versus ECC-1E2 tumors might be dueto differences in the regulation of activity of HER-2/neu andERK-1/2, as the phosphorylation of HER-2/neu was elevated inECC-1TAM tumors and ERK-1/2 was inhibited by trastuzumabonly in ECC-1TAM tumors.Measurement of insulin-like growth factor-I and insulin-like

growth factor-IR mRNA and insulin receptor substrate-1protein expression. Both HER-2/neu and insulin-like growthfactor-1 receptor (IGF-IR) activate ERK-1/2 and Akt. Thus,

trastuzumab would be less effective in blocking the activity ofERK-1/2 or Akt from HER-2/neu if the IGF-IR signaling pathwaywas also active. A recent study has shown that the IGF-IRsignaling pathway blocks the effectiveness of trastuzumab toinhibit growth of breast cancer cells and possibly lead toresistance (35). In addition, the IGF-IR pathway is increased inresponse to E2 and leads to activation of ERK-1/2 and Aktsignaling in breast cancer cells (36). Therefore, we measuredmRNA expression of both human IGF-I and IGF-IR in ECC-1E2and ECC-1TAM tumors. In addition, to measure the cellularactivity of the IGF-I/IGF-IR signaling pathway in the tumors, wemeasured tyrosine-phosphorylated IRS-1, and total IRS-1, adownstream adaptor protein that is recruited to the activatedIGF-IR upon IGF-I ligand binding (37). The results showed thatE2 increased IGF-I mRNA in ECC-1E2 tumors (4.7-fold, P < 0.01)

Figure 4. Expression of human IGF-I andIGF-IR mRNAs and PY-IRS-1 and totalIRS-1 proteins. A, ECC-1E2 tumorstreated with Control, E2, tamoxifen (TAM ),fulvestrant (F ), or E2 + trastuzumab (trast )were excised at week 9 (see Fig. 1C ).ECC-1TAM tumors-treated with Control,tamoxifen, E2, fulvestrant, or tamoxifen +trastuzumab were excised at week 11 (seeFig. 1D ). Total RNA was extracted fromboth sets of tumors as described inMaterials and Methods. Real-time PCRwas done as described in Materials andMethods. 18S RNA was used as a loadingcontrol in all samples. IGF-I mRNA cyclethreshold (C t) values were normalized toC t values for 18S RNA by subtracting theC t18s from the C tIGF-I. Columns, meanIGF-I mRNA copy numbers relative toControl in three independent tumors withthree replicates per tumor. B, the sametotal cDNA and protocol were used todetect human IGF-IR mRNA in ECC-1E2and ECC-1TAM tumors. *, statisticalsignificance of IGF-I or IGF-IR mRNA copynumber relative with Control. #, statisticalsignificance of IGF-I or IGF-IR mRNA copynumber in ECC-1TAM tumors comparedwith the same treatments in ECC-1E2tumors. C, Western blot analyses oftyrosine-phosphorylated IRS-1, totalIRS-1, and h-actin proteins were done asdescribed previously in Materials andMethods. Representative of threeindependent experiments. D, densitometrywas done on three independent Westernblots. Columns, mean of the percentageof PY-IRS-1 calculated as follows:%PY-IRS-1 = (mean density of PY-IRS-1 /mean density of total IRS-1) � 100;bars, FSE.



and ECC-1TAM tumors (3.0-fold, P = 0.45) compared withcontrol (Fig. 4A). More importantly, IGF-I mRNA was decreasedin ECC-1TAM control (60% less, P = 0.02), E2-treated (70% less,P = 0.14), tamoxifen-treated (67% less, P = 0.11), or fulvestrant-treated tumors (70% less, P = 0.04) compared with ECC-1E2tumors (Fig. 4A). IGF-IR mRNA was increased by E2 in ECC-1E2tumors (5.51-fold, P = 0.03) and ECC-1TAM tumors (3.5-fold, P =0.04) compared with controls (Fig. 4B). Similarly to the IGF-ImRNA results, IGF-IR mRNA was decreased in ECC-1TAMcontrol tumors (56% less, P = 0.02), E2-treated (150% less, P =0.21), tamoxifen-treated (50% less, P = 0.03), or fulvestrant-treated (70% less, P < 0.01; Fig. 4B). The activity of the IGF-I/IGF-IR signaling pathway was assessed by measuring tyrosinephosphorylation of IRS-1. E2, tamoxifen, or fulvestrant increasedphosphorylated IRS-1 protein in ECC-1E2 tumors (Fig. 4C). Inaddition, E2 induced phosphorylated IRS-1 protein in ECC-1TAMtumors whereas tamoxifen had no effect on phospho-IRS-1status compared with control (Fig. 4C). However, the overallextent of phosphorylated IRS-1 was less in ECC-1TAM tumorscompared with ECC-1E2 tumors as determined by densitometryof three independent Western blots (Fig. 4D). These results takentogether suggest that the effectiveness of trastuzumab to inhibitgrowth of ECC-1TAM tumors by blocking the activities of HER-2/neu , downstream ERK-1/2, and ERa-mediated c-myc mRNAexpression could be due to the diminished IGF-I/IGF-IRsignaling pathway (results summarized on Table 1 and con-clusions summarized in Fig. 5).

Discussion

The results show, for the first time, that trastuzumab cancompletely block the growth of tamoxifen-stimulated endome-trial cancer by inhibiting HER-2/neu signaling through ERK-1/2and by blocking tamoxifen-induced transcription of c-myc(summarized in Table 1 and Fig. 5). We also showed thattrastuzumab is more efficacious in tamoxifen-stimulated endo-metrial cancer than the parental E2-stimulated endometrialcancer possibly due to increased activity of HER-2/neu anddecreased expression and signaling of the IGF-I/IGF-IR signalingpathway. These results strongly indicate that enhanced crosstalk

between HER-2/neu through ERK-1/2 and ERa contributes tothe natural development of tamoxifen-stimulated endometrialcancer.Five years of adjuvant tamoxifen therapy has been the standard of

care for ERa-positive breast cancer for over 15 years (1). The majorserious side effect of tamoxifen treatment is the increased risk ofendometrial cancer by 0.1% (9). To date, little is known about themechanism of tamoxifen-stimulated endometrial cancer cellgrowth. However, Shang and Brown (19) showed that tamoxifenwas more estrogenic in endometrial cancer cells compared withMCF-7 breast cancer cells in vitro by inducing transcription of ERa-regulated genes, c-myc and IGF-I . Our study examined the functionof the ERa in a novel model of tamoxifen-stimulated endometrialcancer developed in vivo under physiologic conditions by measuringexpression of two estrogen-responsive genes, pS2 and c-myc . Theresults clearly showed that tamoxifen treatment increased expres-sion of pS2 and c-myc up to 4-fold in parental ECC-1E2 tumors andup to 10-fold in ECC-1TAM tumors (Fig. 3A and B). These datashowed that tamoxifen is a weak agonist initially in tamoxifen-naiveECC-1E2 tumors and then a stronger agonist in tamoxifen-stimulated ECC-1TAM tumors. The results suggested that theincreased tamoxifen-induced transcriptional activity might play animportant role in the development of tamoxifen-stimulatedendometrial cancer. More importantly, our data showed thattrastuzumab, the HER-2/neu inhibitor, blocked tamoxifen-inducedexpression of c-myc by 70% in ECC-1TAMtumors, indicating that theincreased agonist activity of tamoxifen on c-myc mRNA expressionin these tumors might be due to the activity of the HER-2/neusignaling pathway.In addition to the estrogenic activity of tamoxifen at ERa-

regulated genes, tamoxifen could potentially exert nongenomicactivity by binding to membrane-associated ERa-activating ERK-1/2 and/or Akt directly. It has been shown previously that E2activates ERK-1/2 or Akt within minutes of treatment suggestinga nongenomic activity for ERa (38). Moreover, Song et al. showedthat ERa is recruited to the membrane by interacting with Shcand IGF-IR where it can bind E2 or tamoxifen (39). Thus,the increase in phospho-ERK-1/2 by E2 or fulvestrant or phopsho-Akt by tamoxifen or fulvestrant in ECC-1TAM tumors (Fig. 3C)could be a result of interaction with membrane ERa. Interestingly,

Table 1. Summary of growth and signaling for ECC-1 tumors

Experiment treatment Growth pS2*

mRNA

c-myc*

mRNA

Phospho-

HER-2/neu

Phospho-

Akt

Phospho-

ERK-1/2

IGF, IGF-IR,

phospho-IRS-1

ECC-1E2 tumor

Control � 1.00 1.00 +++ +++ +++ +++E2 ++++ 25.06 41.72 +++ +++ +++ +++++++

E2 + trastuzumab ++ 12.00 37.50 + +++ +++ +++++++

Tamoxifen � 3.94 3.32 +++ +++ +++ +++Fulvestrant � 0.75 1.05 +++ ++ +++ +++

ECC-1TAM tumor

Control + 1.50 1.14 +++ + ++ ++

E2 ++++ 34.59 29.98 +++ + ++++ ++++Tamoxifen +++ 7.00 10.06 +++ ++ ++ ++

Tamoxifen + trastuzumab � 4.89 3.25 + ++ � ++

Fulvestrant ++ 4.58 1.08 +++ ++ ++++ ++

*Fold induction of mRNA compared with control.

Cancer Research


trastuzumab inhibited growth of tamoxifen-stimulated ECC-1TAMtumors (Fig. 1D), induction of c-myc mRNA (Fig. 3B), andphospho-ERK-1/2 (Fig. 3C) but did not block tamoxifen-inducedphospho-Akt (Fig. 3C). Therefore, the function of the membraneERa in activating Akt in terms of growth is unclear for tamoxifen-stimulated ECC-1TAM tumors. However, E2, tamoxifen, orfulvestrant stimulated growth and induced phospho-ERK-1/2 inECC-1TAM tumors, suggesting that a direct interaction of ligandwith membrane ERa is possible. In addition, fulvestrant had littleeffect on pS2 and c-myc mRNA expression (Fig. 3A and B),increased phospho-HER-2/neu , phospho-Akt, and -ERK-1/2 (Fig.3C), and modestly increased growth of ECC-1TAM tumors (Fig.1D). These results suggest that fulvestrant might act throughmembrane ERa. This result is not surprising as previously wereported that fulvestrant in combination with E2 stimulatedgrowth and HER-2/neu signaling of tamoxifen-stimulated MCF-7breast tumors independent of ERa transcriptional activity (40).Surprisingly, the results from the present study also showed that

trastuzumab therapy is effective in blocking tamoxifen-stimulatedendometrial tumor growth in the absence of HER-2/neu over-expression. However, the basal levels of phospho-HER-2/neu wereincreased in ECC-1TAM tumors by 2.7-fold (Fig. 3C) suggesting thathyperactivity of HER-2/neu could be important for the completegrowth inhibitory effect of trastuzumab. Clinical studies have

shown that trastuzumab therapy is only beneficial in women withHER-2/neu overexpressing or amplified breast tumors (41, 42).However, the tests for determining HER-2/neu status may needfurther validation as trastuzumab therapy is effective in only 40% ofpatients with high HER-2/neu expression (42). In addition, it mightbe more reasonable to measure the active HER-2/neu protein andthe extent of phosphorylation of downstream effector moleculessuch as Akt and ERK-1/2 (43–45). Keeping this in mind, there areother signaling pathways in addition to HER-2/neu that convergeon Akt and ERK-1/2. The hyperactivity of the IGF-I/IGF-IR signalingpathway has previously been shown to result in resistance totrastuzumab therapy in breast cancer cells (35). This pathway alsoconverges on the Akt (46) and ERK-1/2 (47) proteins leading toactivation of these intracellular molecules. Our study found that theparental E2-stimulated ECC-1E2 tumors expressed constitutivelyactive Akt and ERK-1/2 proteins and whereas trastuzumabdecreased HER-2/neu protein, it had little effect on the phosphor-ylation status of ERK-1/2 or Akt proteins. However, trastuzumab didpartially inhibit E2-induced growth (Fig. 1C) and expression of pS2mRNA in E2-stimulated ECC-1E2 tumors (Fig. 3A) suggesting thatgrowth and ERa activity are in part regulated by HER-2/neuindependent of ERK-1/2 and Akt activities. More importantly, thedata clearly showed that IGF-I and IGF-IR mRNAs were decreasedin ECC-1TAM tumors compared with ECC-1E2 tumors (Fig. 4A

Figure 5. Model of converging signaling pathways from HER-2/neu and IGF-IR. HER-2/neu either alone or as a partner with HER3 activates the Ras-Raf-Mek-ERK-1/2 pathway and phosphatidylinositol 3-kinase (PI3K ), which activates Akt. IGF-I ligand binds and activates the IGF-IR which recruits IRS-1 and Shc proteins. IRS-1recruits and activates phosphatidylinositol 3-kinase leading to Akt activation, whereas Shc activates the Ras-Raf-Mek-ERK-1/2 pathway. ERK-1/2 phosphorylatesSp1 transcription factor which binds to the promoter of the c-myc gene, recruiting E2:ERa complex leading to transcription of c-myc . Myc protein activates transcriptionof Cyclin D1 which activates components of the cell cycle machinery leading to proliferation and growth of the tumor cell. In addition, Akt inhibits apoptosis leading tocell survival. In parental ECC-1E2 tumors, although trastuzumab blocks phosphorylation of HER-2/neu , it is a weak inhibitor of growth and E2-induced ERa

transcriptional activity due to hyperactivity of the IGF-IR signaling pathway simultaneously activating ERK-1/2 and Akt. In contrast, trastuzumab is a more efficientinhibitor of the HER-2/neu signaling pathway in tamoxifen (TAM )–stimulated ECC-1TAM tumors leading to decreased ERK-1/2 activity, tamoxifen/ERa-mediatedc-myc transcription, and growth due to a diminished IGF-IR signaling pathway limiting activation of ERK-1/2 to the HER-2/neu signaling pathway.



and B) and that this translated to decreased signaling as measuredby the phosphorylation status of IRS-1 (Fig. 4C and D), the primarysubstrate for the active IGF-IR. Therefore, it is reasonable toconclude that the efficacy of trastuzumab to completely inhibitgrowth of ECC-1TAM tumors could be due to an overall increase ofHER-2/neu signaling and the decrease of signaling from the IGF-I/IGF-IR pathway thereby allowing trastuzumab to block the down-stream activity of ERK-1/2-originated from HER-2/neu .ERK-1 and ERK-2 are serine/threonine kinases that are activated

by phosphorylation cascades originated from receptor tyrosinekinases such as HER-2/neu and/or IGF-IR. Once activated, theytranslocate into the nucleus, where they phosphorylate transcrip-tion factors, including ERa (45) and Sp1 (48–50). Activation of ERaand Sp1 leads to transcription of c-myc (51), which is associatedwith increased cellular proliferation and tumor growth (52).Transcription of the c-myc gene is regulated by ERa throughtethering to the Sp1 transcription factor (53) which binds directlyto Sp1 sites within the c-myc promoter (51). Recent studies haveshown that phosphorylation of Sp1 by ERK-1/2 and/or jun kinaseleads to transcription of target genes (50). Thus, it is reasonable toconclude that trastuzumab’s inhibitory effect on tamoxifen-inducedincrease of c-myc mRNA could be due to a decrease of Sp1 activitythrough inactivation of ERK-1/2. Further studies are currentlyunder way to elucidate the exact mechanism by which trastuzu-mab inhibits c-myc transcription.In conclusion, the results from the current study suggest that

trastuzumab is effective at blocking growth of tamoxifen-stimulated endometrial cancer when the basal activity of HER-2/neu is enhanced. Furthermore, complete blockade of growth of

ECC-1TAM tumors seems achieved by inhibiting activities ofHER-2/neu , ERK-1/2, and tamoxifen-induced expression of c-mycmRNA in an environment where the IGF-I/IGF-IR signalingpathway is diminished. These data suggest that the decreasedexpression and activity of the IGF-I/IGF-IR signaling pathway inECC-1TAM tumors improves the overall efficacy of trastuzumabto inhibit growth of tumors expressing low levels of the IGF-Iligand and receptor. To address this hypothesis, studies arecurrently under way to investigate whether overexpression of theIGF-IR is necessary and/or sufficient to block the efficacy oftrastuzumab to inhibit growth of both breast and endometrialcancer cells. Based on the current results, we suggest thatmeasuring the complex network of signals such as phospho-HER-2/neu , phospho-ERK-1/2, and phospho-IRS-1 status withintumors might improve response rates of patients havingtamoxifen-stimulated breast and endometrial cancer to trastu-zumab treatment and possibly lead to prevention of tamoxifen-resistant breast cancer and tamoxifen-stimulated endometrialcancer. There is clinical significance for trastuzumab therapy fortamoxafen-stimulated endometrial cancer as a single case reportshows (54).

Acknowledgments

Received 11/18/2004; revised 5/5/2005; accepted 5/16/2005.Grant support: Avon Foundation, Judy Dlugie Memorial Fund Fellowship

(C. Osipo), and Department of Defense Training grant DAMD17-001-0386 (C. Osipo).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Myles Brown for the generous gift of ECC-1 cells.

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Trastuzumab Therapy for Tamoxifen-Stimulated Endometrial Cancer