Trastuzumab Therapy for Tamoxifen-Stimulated Endometrial Cancer

Research Article

Clodia Osipo,1 Kathleen Meeke,1 Hong Liu,1 Dong Cheng,1 Sherry Lim,1 Alyssa Weichel,1 and V. Craig Jordan1,2

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

Abstract events (8), and the uterus (9). As a result of tamoxifen’s selective A novel in vivo model of tamoxifen-stimulated endometrial estrogen receptor modulator activity around a woman’s body, it cancer was developed and the role of HER-2/neu investigated increases the incidence of endometrial cancer by 0.1% in by using trastuzumab. Tamoxifen-stimulated tumors (ECC- postmenopausal women (9). The exact mechanism by which 1TAM)weregrowthstimulatedby17B-estradiol (E2), tamoxifen induces endometrial cancer during breast cancer therapy tamoxifen, or raloxifene. Trastuzumab inhibited growth of E2- is as yet unclear. stimulated ECC-1E2 tumors by 50% and tamoxifen-stimulated Numerous studies during the last 10 years have focused on ECC-1TAM tumors by 100%. ECC-1 tumors expressed func- formation of DNA adducts by metabolites of tamoxifen in the tional estrogen receptor A (ERA) as measured by induction of endometrium of rats and humans. Recently, Liehr showed that 17- pS2 and c-myc mRNAs. E2 induced pS2 and c-myc mRNAs up estradiol (E2) is a dual mutagen and carcinogen in the immature to 40-fold in ECC-1E2 and ECC-1TAM. Tamoxifen induced pS2 rat lacking expression of ER (10). Tamoxifen might also be a and c-myc mRNAs up to 5-fold in ECC-1E2 tumors and up to mutagen/carcinogen as it has estrogenic activity in the uterus. 10-fold in ECC-TAM tumors. Trastuzumab blocked E2-induced However, the data to date shows that tamoxifen is a potent rat liver pS2 mRNA (P < 0.01) in ECC-1E2 by 50% and tamoxifen- carcinogen causing the formation of tamoxifen-DNA adducts and induced c-myc mRNA (P < 0.1) in ECC-1TAM tumors by 70%. liver cancer (11). However, little conclusive data exists showing a Trastuzumab decreased phosphorylated and total HER-2/neu direct affect of tamoxifen or its metabolites on DNA adduct protein in ECC-1E2 and ECC-1TAM tumors. However, only formation in the rat uterus to cause endometrial cancer. Most phospho-ERK-1/2 and not phospho-Akt protein was decreased studies lack convincing evidence for tamoxifen-DNA adduct by trastuzumab in tamoxifen-treated ECC-1TAM tumors. The formation in rat and human endometrium (11–18), suggesting insulin-like growth factor (IGF-I) signaling pathway also that the estrogenic action of tamoxifen at ERa-regulated genes activates extracellular signal-related kinase (ERK)-1/2 and is likely to be the more important factor in development of could block the efficacy of trastuzumab in ECC-1E2 tumors. endometrial cancer in women undergoing tamoxifen therapy. The results showed that IGF-I, IGF-IR mRNAs, and phospho- Shang and Brown (19) showed, using in vitro cell culture models, insulin receptor substrate-1 (IRS-1) protein were decreased that tamoxifen is estrogenic in endometrial cancer cells by in ECC-1TAM compared with ECC-1E2 tumors. The results stimulating the recruitment of coactivators, in particular SRC-1, a show that trastuzumab is an effective therapy for both E2- member of the p160 family of coactivators, to a certain subset of stimulated and tamoxifen-stimulated endometrial cancer. The genes regulated by the ERa at the transcriptional level. The data suggest estrogenic activities of E2 and tamoxifen at ERA- investigators concluded that the SERM activity of tamoxifen in the regulated pS2 and c-myc genes are in part mediated by HER-2/ endometrium could be due to differences in the recruitment of neu. However, trastuzumab is a better growth inhibitor of coregulators to certain ER-regulated promoters. Furthermore, Shah ECC-1TAM tumors where there is diminished IGF-I signaling and Rowen (20) recently showed that the nonreceptor tyrosine allowing for complete blockade of the downstream phospho- kinase, src, enhances the agonist activity of tamoxifen in 167 ERK-1/2 signal. (Cancer Res 2005; 65(18): 8504-13) endometrial cancer cells by phosphorylation of Ser and stabilization of ER promoter interaction. However, neither of these Introduction studies has been confirmed using preclinical in vivo models of tamoxifen-stimulated endometrial tumors. Tamoxifen has been the standard of care for estrogen receptor a In addition, resistance to adjuvant tamoxifen therapy for ERa- (ERa)–positive breast cancer in premenopausal and postmenopaus- positive breast cancer is a very common occurrence. Resistance to al women for over 15 years (1). Five years of adjuvant tamoxifen tamoxifen is defined as the lack of tumoristasis and/or regression therapy reduces the incidence of contralateral breast cancer (2), or might be evidenced by growth stimulation. Subsets of breast improves overall survival (2), and also reduces the risk of breast tumors that do not initially respond (i.e., are not growth arrested cancer in women at high risk (3). However, tamoxifen is not a pure or do not regress) to tamoxifen are intrinsically resistant to antiestrogen but a selective estrogen ERa modulator (4, 5). It has tamoxifen therapy. In contrast, acquired resistance to tamoxifen antiestrogenic affects on the breast epithelium but estrogenic affects during z5 years of adjuvant tamoxifen therapy (21) could result on bone density (6, 7), circulating cholesterol and cardiovascular from growth stimulation in response to continuous treatment with tamoxifen. Osborne et al. (22) showed that postmenopausal a Requests for reprints: V. Craig Jordan, OBE, PhD, Dsc. Vice President and women with ER -positive breast cancer do not initially respond Research Director for Medical Science, Alfred G. Knudson Chair of Cancer Research. to tamoxifen therapy when their tumors overexpress both HER-2/ Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111-2497. E-mail: neu, a member of the epidermal growth factor receptor family of [email protected]. I2005 American Association for Cancer Research. receptor tyrosine kinases, and the gene amplified in breast doi:10.1158/0008-5472.CAN-04-4107 cancer-1 (AIB1), a member of the p160 family of coactivators.

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More recently, Shou et al. (23) concluded that tamoxifen’s treating groups (10 mice) with vehicle, 1.5 mg/d tamoxifen, 0.30-cm E2 estrogenic activity is partly due to crosstalk between HER-2/neu capsules, or 1.5 mg/d Raloxifene (obtained from the pharmacy; refs. 29, 30, 34) a for 12 weeks. In another experiment, 50 animals were bitransplanted with and ER in MCF-7 cancer cells that express high levels of AIB1 2 and stably overexpress HER-2/neu. These studies showed that ECC-1TAM tumors and grown to a mean cross-sectional area of 0.31 cm and randomly separated into groups of 10 mice that were then subsequently overexpression of HER-2/neu and AIB1 in ERa-positive breast treated as follows: vehicle, 1.5 mg tamoxifen, 0.30-cm E2 capsules, 10 mg cancer cells can cause intrinsic resistance to tamoxifen. However, fulvestrant (5 mg given s.c., twice weekly), and 1.5 mg tamoxifen + 30 mg/kg f only 10% of all ERa-positive breast cancer cells overexpress trastuzumab. In a final in vivo experiment, 30 mice were bilaterally HER-2/neu. Therefore, it is unclear whether HER-2/neu plays a transplanted with 1-mm tamoxifen-treated ECC-1TAM tumors from role in the development of acquired resistance to tamoxifen Fig. 1D and 10 mice per group were treated as follows: control, 1.5 mg during the 5 years of adjuvant therapy. tamoxifen, and 1.5 mg tamoxifen plus 5 mg fulvestrant (twice weekly). Studies suggest that overexpression of HER-2/neu leads to Real-time reverse transcription-PCR for human pS2,c-myc, HER-2/ tamoxifen-resistant breast cancer (22, 24). A recent report showed neu, insulin-like growth factor-I, and insulin-like growth factor-IR that src tyrosine kinase potentiates the agonist activity of tamoxifen mRNAs in tumors. Total RNA is extracted from the tumors using the RNeasy in endometrial cancer cells (20). In addition, overexpression of HER- Mini Kit (Qiagen, Stanford Valencia, CA) according to the manufacturer’s instructions. The total RNA is reverse transcribed using Taqman reverse 2/neu correlates with high-grade endometrial cancer (25–27). transcription reagents (PE Applied Biosystems, Hayward, CA) with the use of However, exact mechanisms leading to tamoxifen-stimulated breast random hexamers as the primers according to the manufacturer’s and endometrial cancers are not well understood. Currently, instructions. Primers and probes for human pS2, HER-2/neu, insulin growth inhibitors to epidermal growth factor receptor (i.e., gefitinib), HER- factor-1 (IGF-I), and IGF-IR are designed using Primer ExpressTM1.5 2/neu (i.e., trastuzumab), Ras/mitogen-activated protein kinase (i.e., software set at default variables to select the most optimized primer and farnesyltransferase inhibitor, tipifarnib), and protein kinase B (Akt; probe sets for this system. The sequences for the forward and reverse primers i.e., an mammalian target of rapamycin inhibitor, CCI 779) are being for human pS2 are 5V-AGGCCCAGACAGAGACGTG-3V and 5V-CCCTGCA- investigated either alone or in combination with tamoxifen to GAAGTGTCTAAAATTCA-3V, respectively. The sequence for the pS2 probe is prevent and/or treat resistance to tamoxifen therapy. The question 5V-CTGCTGTTTCGACGACACCGTTCG-3Vwhere the FAM is the reporter and that remains to be answered is if HER-2/neu is important for QSY7 is the quencher (MegaBases, Inc., Chicago, IL). The c-myc primer and probe mixture was purchased from Perkin-Elmer Applied Biosystems (PE- tamoxifen-resistant breast cancer, then does HER-2/neu play a role ABI, Stanford Valencia, CA) and used according to the manufacturing in the development of endometrial cancer during long-term tamox- instructions. The sequences for the forward and reverse primers for human ifen therapy? We have developed a novel in vivo model of HER-2/neu are 5V-ACTGCAGAGGCTGCGGATT-3Vand 5V-ACGGCCAGGGCA- tamoxifen-stimulated endometrial cancer (ECC-1TAM) by treating TAGTTGT-3V, respectively. The sequence for the human HER-2/neu probe is tamoxifen-naive, E2-stimulated ECC-1E2 tumors with E2 plus tamox- 5V-TGCGAGGCACCCAGCTCTTTGA-3V where the FAM is the reporter ifen for 24 weeks. We investigated the role of HER-2/neu in the and QSY7 is the quencher (MegaBases). The sequences for the forward and growth of both ECC-1E2 and ECC-1TAM tumors using the reverse primers for human IGF-I are 5V-TGCTTCCGGAGCTGTGATC-3Vand humanized monoclonal antibody to HER-2/neu, trastuzumab. 5V-AGCTGACTTGGCAGGCTTGA-3V, respectively. The sequence for the human IGF-I probe is 5V-AGGAGGCTGGAGATGTATTGCGCACC-3V where the FAM is the reporter and QSY7 is the quencher (MegaBases). The probe Materials and Methods and primers for human IGF-IR were purchased from Perkin-Elmer Applied Cell culture. The human endometrial cancer cell line, ECC-1, was a Biosystems (PE-ABI, Stanford Valencia, CA). The quantity of human 18s RNA generous gift from Dr. Myles Brown at the Dana-Farber Cancer Institute, was also measured in each total cDNA sample for normalization purposes. Harvard Medical School. ECC-1 cells were maintained at 37jC in a 95% The probe and primers for 18s RNA were purchased from Perkin-Elmer humidified/5% CO2 atmosphere in phenol red–containing DMEM supple- Applied Biosystems (Stanford Valencia, CA). The PCR portion of the reaction mented with 10% fetal bovine serum, 6 ng/mL insulin solution, 1% was done with the use of the Taqman PCR Core Reagent Kit (Perkin-Elmer L-glutamine, 1% nonessential amino acids, 1% of a combination of penicillin Applied Biosystems). In a total volume of 25 AL, 50 ng of total cDNA, streptomycin, and antimycotic (Life Technologies, Long Island, NY). 100 nmol/L probe, and 200 nmol/L primers were used in the PCR reaction. Growth of ECC-1E2 tumors in vivo. The ECC-1E2 tumors used in these Real-time PCR was done using the ABI-Prism 7700 Sequence Detection experiments were derived by bilateral inoculation of 0.10 mL of 1 Â 107 System. The PCR conditions were 50jC for 2 minutes, 95jC for 10 minutes suspended ECC-1 cells in sterile PBS into the mammary fat pads of 4- to followed by 40 cycles of 95jC for 15 seconds and 60jC for 1 minute. 6-week-old ovariectomized BALB/c nu/nu athymic mice (Harlan Sprague- Western blot analyses. Tumors were homogenized by grinding in liquid Dawley, Madison, WI). ECC-1 cells-inoculated into athymic mice were nitrogen and resuspending in lysis buffer [1% Triton X-100, 1 mmol/L EDTA, supplemented with 0.30-cm E2 (Sigma, St. Louis, MO) silastic capsules 150 mmol/L NaCl, 50 mmol/L Tris base (pH 7.4), 25 mg/mL phenyl- (Baxter Health Care, Mundelein, IL) to achieve E2-stimulated tumor growth methanesulfonyl fluoride, 10 (g/mL leupeptin, 10 mg/mL aprotinin, (ECC-1E2; refs. 28–33). Tumors were maintained by serial passage of solid 10 mg/mL pepstatin, 10 mg/mL TLCK, 10 mg/mL N-tosyl-L-phenylalanine tumors into athymic mice as described previously (29). Thirty mice were chloromethyl ketone, 100 mmol/L NaF, 10 mmol/L ortho-vanadate; Sigma]. bitransplanted with parental ECC-1E2 tumors and groups (10 mice) were The extract was subsequently sonicated and then centrifuged for 5 minutes at treated as follows: vehicle, 0.30-cm E2 capsules, and E2 plus 1.5 mg/d 5,000 Â g at 4jC. The supernatant was collected and protein concentration tamoxifen for 5 days (given orally by gavage) for 24 weeks. In a separate was measured using the Bradford assay (Bio-Rad Laboratories, Inc., Santa experiment, 50 new athymic mice were serially bitransplanted with 1-mm Cruz, CA). Equal amounts of protein (25-50 Ag) were loaded onto a 7% ECC-1E2 tumors portions and grown to a mean cross sectional area of 0.28 polyacrylamide/bisacrylamide gel for SDS-PAGE followed by Western cm2 and randomly separated into five groups of 10 and treated with the blotting. The following proteins were detected by Western blot: Tyr1248- following: vehicle, 0.30-cm E2 capsule, 1.5 mg/d tamoxifen, 10 mg phosphorylated HER-2/neu (1:2,000 Rabbit anti-human, Upstate Biotechnology, fulvestrant (dissolved in 100% ethanol and diluted in sterile-filtered peanut New York, NY), HER-2/neu (1:200 mouse anti-human, Ab-11, Neomarkers, oil, 5 mg/0.10 mL injected s.c., twice weekly), and 0.30-cm E2 + 30 mg/kg Fremont, CA), Ser473-phosphorylated Akt (1:1,000 rabbit anti-human, Cell trastuzumab (15 mg/kg injected i.p., twice weekly). Signaling Technology, Beverly, MA), Akt (1:1,000 rabbit anti-human, Cell Growth of ECC-1TAM tumors in vivo. Long-term tamoxifen-stimulated Signaling Technology), phosphorylated-ERK-1/2 (1:1,000 mouse anti- tumors (ECC-1TAM) were developed by retransplanting the surviving E2 + human, Cell Signaling Technology), extracellular signal-related kinase 1.5 mg tamoxifen-treated ECC-1E2 tumors into 40 new athymic mice, and (ERK)-1/2 (1:1,000 mouse anti-human, Cell Signaling Technology), h-actin www.aacrjournals.org 8505 Cancer Res 2005; 65: (18). September 15, 2005

Figure 1. Development of tamoxifen (TAM)–stimulated endometrial cancer in vivo and the effect of trastuzumab on growth. A, thirty athymic mice were bitransplanted with 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 were implanted 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 to 24 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 were treated 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 to 12 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 until they 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 mL fulvestrant 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)up to 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 until the 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, or tamoxifen plus trastuzumab. The tumor sizes were measured as described in (A) up to 11 weeks. The cross-sectional area of each tumor was calculated using the following formula: cross sectional area = (length Â width Â p)/4. Points, mean cross-sectional areas of tumors; bars, FSE. *, statistical significance of mean cross-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 end of each growth study compared with the E2-treated group in (C) or to the tamoxifen-treated group in (D).

(1:20,000 mouse anti-human, Sigma-Aldrich, St. Louis, MO), tyrosine- Results phosphorylated insulin receptor substrate (IRS-1; 1 Ag/mL rabbit anti- Growth of ECC-1E2 and ECC-1TAM tumors in vivo. To human, Upstate Biotechnology), and IRS-1 (2 Ag/mL mouse anti-human, Upstate Biotechnology). The appropriate secondary antibody conjugated elucidate the mechanism of action by which tamoxifen stimulates to horseradish peroxidase was used to detect the primary antibody (either growth of endometrial cancer cells under physiologic conditions to goat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase, Santa mimic the clinical situation, we developed a novel tamoxifen- Cruz Biotechnology, Santa Cruz, CA). The blot was developed using an stimulated endometrial cancer model in vivo by treating the enhanced chemiluminescence kit (Amersham Corp., Arlington Heights, IL). parental ECC-1E2 tumors originated from cell lines (ECC-1) The membrane was exposed to Kodak X-OMAT film for 10 to 30 seconds. derived from the human EnCa101 tumor with postmenopausal Densitometry was done using the Scion program to quantify the intensity levels of E2 plus tamoxifen for 24 weeks. The results showed that of bands from three independent Western blots. the parental ECC-1E2 tumors grew faster in response to E2 (P < Statistical analysis. Tumor growth curves were analyzed longitudinally 0.0001) compared with control (Fig. 1A). At 24 weeks, ECC-1E2 using a two-factor ANOVA comparing all tumor cross sectional areas within treatments in a time-dependent manner. Tumor growth curves represent tumors treated with E2 plus tamoxifen were larger (P < 0.0001) means F SE of tumor cross-sectional areas. A two-sided Student’s t test was than control (Fig. 1A). Upon serial bitransplantation of ECC-1E2 used to analyze differences in mRNA levels as detected by real-time PCR of tumors treated with E2 plus tamoxifen into new generations of the treatments to the control group. The error bars for the measurement of athymic mice, these tumors were growth stimulated more by E2 mRNA copy number represent SE calculated using Excel program. alone (P < 0.01), tamoxifen alone (P = 0.48), or raloxifene alone

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Figure 2. Growth of ECC-1TAM tumors in response to tamoxifen (TAM) plus fulvestrant. Thirty mice were bilaterally transplanted with 1-mm tamoxifen-treated ECC-1TAM tumors from Fig. 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). The cross-sectional area of each tumor was calculated using the following formula: cross sectional area = (length Â width Â p)/4. Points, mean cross-sectional area of tumors; bars, FSE. *, statistical significance of the tamoxifen + fulvestrant treated group at the end of the study compared with tamoxifen alone.

(P = 0.35) compared with control (Fig. 1B). These data show the ECC-1E2 (25.06-fold over control, P < 0.0001) and ECC-1TAM (34.59- development of a novel in vivo model of tamoxifen-stimulated fold over control, P < 0.01) tumors. Trastuzumab inhibited E2- endometrial tumor that continues to grow in response to E2 and is induced pS2 mRNA expression (P < 0.1) by almost 50% in parental cross-resistant with raloxifene. ECC-1E2 tumors (Fig. 3A). Tamoxifen induced pS2 mRNA expression Growth of ECC-1E2 and ECC-1TAM tumors in response to by 3.94-fold over control (P = 0.045) in ECC-1E2 tumors and 7-fold in trastuzumab. We investigated the consequences of using a HER-2/ ECC-1TAM tumors over control (P = 0.045; Fig. 3A) and trastuzumab neu inhibitor, trastuzumab, on growth of ECC-1E2 and ECC-1TAM had no effect on tamoxifen-mediated increase of pS2 mRNA tumors in vivo. The results showed that ECC-1E2 tumors that have expression in ECC-1TAM tumors (Fig. 3A). reached an initial mean cross sectional area of 0.28 cm2 at week 4 did mRNA for the c-myc gene was increased with E2 treatment in both not grow in response to vehicle control, tamoxifen, or fulvestrant ECC-1E2 and ECC-1TAM tumors by 41.72-fold (P = 0.0001) and 29.98- compared with the E2-treated group (P = 0.02; Fig. 1C). Interestingly, fold (P < 0.01), respectively, compared with controls (Fig. 3B). trastuzumab partially blocked E2-induced growth of ECC-1E2 Tamoxifen induced expression of c-myc mRNA in ECC-1E2 by only tumors by almost 50% (P = 0.06) compared with tumors treated 3.32-fold (P = 0.25) while increasing c-myc mRNA by 10.06-fold (P < with E2 alone (Fig. 1C). 0.001) in ECC-1TAM tumors compared with control. Interestingly, ECC-1TAM tumors that were initially a mean cross sectional area trastuzumab inhibited the tamoxifen-induced increase of c-myc of 0.31 cm2 at week 6 grew with E2 (P < 0.01) or tamoxifen (P = 0.1) mRNA in ECC-1TAM tumors by 70% (P < 0.1; Fig. 3B). These results compared with control (Fig. 1D). Interestingly, fulvestrant modestly confirm that tamoxifen is estrogenic at ERa-regulated genes pS2 and stimulated growth of ECC-1TAM tumors compared with control. c-myc in ECC-1 endometrial tumors. More importantly, the data More importantly, tamoxifen-stimulated ECC-1TAM tumors were suggest that estrogenic affects that correlate directly to growth completely growth inhibited by trastuzumab (P = 0.002) compared stimulation by either E2 in ECC-1E2 tumors or tamoxifen in ECC- with tamoxifen alone (Fig. 1D). These results suggest for the first 1TAM tumors at specific genes such as pS2 or c-myc, respectively, are time that tamoxifen stimulates growth of endometrial cancer at least at least in part regulated by HER-2/neu. in part through HER-2/neu. Effectiveness of trastuzumab in inhibiting HER-2/neu, To determine whether tamoxifen stimulated growth of ECC- mitogen-activated protein kinase, and Akt activities. HER-2/ 1TAM tumors through ERa, a separate experiment was done using neu was not overexpressed at the mRNA level in ECC-1TAM the combination of tamoxifen plus fulvestrant. The results showed tumors compared with ECC-1E2 tumors (data not shown). Thus, that fulvestrant inhibited growth of tamoxifen-treated ECC-1TAM an increase in HER-2/neu expression could not explain the tumors (Fig. 2) indicating that the growth stimulatory effect of effectiveness of trastuzumab in blocking growth of ECC-1TAM tamoxifen is mediated by ERa. tumors compared with ECC-1E2 tumors. To determine the affect Expression of estrogen-responsive genes, pS2 and c-myc,in of trastuzumab on HER-2/neu activity, we measured tyrosine- ECC-1E2 and ECC-1TAM tumors. The results from the growth phosphorylated HER-2/neu, total HER-2/neu, and downstream studies showed that fulvestrant blocked tamoxifen-stimulated ECC- effectors, phosphorylated ERK-1/2, total ERK-1/2, phosphorylated 1TAM tumors (Fig. 2) thus suggesting that tamoxifen is estrogenic Akt, and total Akt protein levels by Western blot analyses. The through ERa. In addition, Shang and Brown previously showed that results showed that total HER-2/neu protein levels were tamoxifen is estrogenic at ERa-mediated promoters for c-myc and unchanged in ECC-1TAM tumors compared with ECC-1E2 IGF-I genes (19). Therefore, we investigated ERa transcriptional tumors (Fig. 3C). However, basal levels of phospho-HER-2/neu activity at pS2 (classic promoter-driven gene) and c-myc (tethered- protein were increased in ECC-1TAM by 2.7-fold versus ECC-1E2 promoter driven gene) in both ECC-1E2 and ECC-TAM tumors. The tumors (Fig. 3C) as determined by densitometry of three results in Fig. 3A showed that E2 increased pS2 mRNA expression in independent Western blots. However, trastuzumab decreased www.aacrjournals.org 8507 Cancer Res 2005; 65: (18). September 15, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research tyrosine-phosphorylated HER-2/neu and total HER-2/neu protein was increased in ECC-1TAM tumors treated with tamoxifen to almost undetectable levels in both ECC-1E2 and ECC-1TAM compared with control but this increase was not affected by (Fig. 3C). Furthermore, trastuzumab had little effect on phospho- trastuzumab. Interestingly and in contrast to ECC-1E2 tumors, ERK-1/2, total ERK-1/2, phospho-Akt, or total Akt protein levels E2 or fulvestrant increased phospho-ERK-1/2 levels in ECC-1TAM in ECC-1E2 tumors (Fig. 3C). In contrast, phospho-Akt protein tumors 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 treated with 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 and Methods. Total RNA was reverse transcribed to total cDNA and real-time PCR was done using the Applied Biosystems ABI 7700 Taqman PCR instrument to detect expression 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 (Ct) 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 three independent tumors with three replicates per tumor. *, statistical significance of pS2 mRNA copy number compared with Control. **, statistical significance of pS2 mRNA copy 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 used to detect the human c-myc mRNA in ECC-1E2 and ECC-1TAM tumors. *, statistical significance of c-myc mRNA copy number compared with Control. **, statistical significance 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 by 5-second ultrasonic pulses to disrupt cellular membranes. The total mixture was centrifuged for 5 minutes at 5,000 Â g at 4 jC. Fifty micrograms of supernatant from each sample were boiled in 2Â Leammli buffer and loaded onto a 7% polyacrylamide/bisacrylamide gel followed by SDS-PAGE. Western blot analyses to detect tyrosine (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, and h-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-fold increase of basal levels of PY-HER2 protein as determined by densitometry in ECC-1TAM tumors compared with ECC-1E2 tumors.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. HER-2/neu Signaling and Endometrial Cancer trastuzumab decreased phospho-ERK-1/2 protein levels in ECC- trastuzumab would be less effective in blocking the activity of 1TAM compared with tamoxifen alone (Fig. 3C). These results ERK-1/2 or Akt from HER-2/neu if the IGF-IR signaling pathway suggest that trastuzumab is effective at down-regulating HER-2/ was also active. A recent study has shown that the IGF-IR neu protein and tyrosine phosphorylation of HER-2/neu in ECC- signaling pathway blocks the effectiveness of trastuzumab to 1E2 and ECC-1TAM tumors in the absence of HER-2/neu inhibit growth of breast cancer cells and possibly lead to overexpression. However, the difference in trastuzumab’s efficacy resistance (35). In addition, the IGF-IR pathway is increased in on growth of ECC-1TAM versus ECC-1E2 tumors might be due response to E2 and leads to activation of ERK-1/2 and Akt to differences in the regulation of activity of HER-2/neu and signaling in breast cancer cells (36). Therefore, we measured ERK-1/2, as the phosphorylation of HER-2/neu was elevated in mRNA expression of both human IGF-I and IGF-IR in ECC-1E2 ECC-1TAM tumors and ERK-1/2 was inhibited by trastuzumab and ECC-1TAM tumors. In addition, to measure the cellular only in ECC-1TAM tumors. activity of the IGF-I/IGF-IR signaling pathway in the tumors, we Measurement of insulin-like growth factor-I and insulin-like measured tyrosine-phosphorylated IRS-1, and total IRS-1, a growth factor-IR mRNA and insulin receptor substrate-1 downstream adaptor protein that is recruited to the activated protein expression. Both HER-2/neu and insulin-like growth IGF-IR upon IGF-I ligand binding (37). The results showed that factor-1 receptor (IGF-IR) activate ERK-1/2 and Akt. Thus, E2 increased IGF-I mRNA in ECC-1E2 tumors (4.7-fold, P < 0.01)

Figure 4. Expression of human IGF-I and IGF-IR mRNAs and PY-IRS-1 and total IRS-1 proteins. A, ECC-1E2 tumors treated 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 (see Fig. 1D). Total RNA was extracted from both sets of tumors as described in Materials and Methods. Real-time PCR was done as described in Materials and Methods. 18S RNA was used as a loading control in all samples. IGF-I mRNA cycle threshold (C t) values were normalized to Ct values for 18S RNA by subtracting the Ct18s from the C tIGF-I. Columns, mean IGF-I mRNA copy numbers relative to Control in three independent tumors with three replicates per tumor. B, the same total cDNA and protocol were used to detect human IGF-IR mRNA in ECC-1E2 and ECC-1TAM tumors. *, statistical significance of IGF-I or IGF-IR mRNA copy number relative with Control. #, statistical significance of IGF-I or IGF-IR mRNA copy number in ECC-1TAM tumors compared with the same treatments in ECC-1E2 tumors. C, Western blot analyses of tyrosine-phosphorylated IRS-1, total IRS-1, and h-actin proteins were done as described previously in Materials and Methods. Representative of three independent experiments. D, densitometry was done on three independent Western blots. Columns, mean of the percentage of PY-IRS-1 calculated as follows: %PY-IRS-1 = (mean density of PY-IRS-1 / mean density of total IRS-1) Â 100; bars, FSE.

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

Experiment treatment Growth pS2* c-myc* Phospho- Phospho- Phospho- IGF, IGF-IR, mRNA mRNA HER-2/neu Akt ERK-1/2 phospho-IRS-1

ECC-1E2 tumor Control À 1.00 1.00 +++ +++ +++ +++ E2 ++++ 25.06 41.72 +++ +++ +++ +++++++ E2 + trastuzumab ++ 12.00 37.50 + +++ +++ +++++++ Tamoxifen À 3.94 3.32 +++ +++ +++ +++ Fulvestrant À 0.75 1.05 +++ ++ +++ +++ ECC-1TAM tumor Control + 1.50 1.14 +++ + ++ ++ E2 ++++ 34.59 29.98 +++ + ++++ ++++ Tamoxifen +++ 7.00 10.06 +++ ++ ++ ++ Tamoxifen + trastuzumab À 4.89 3.25 + ++ À ++ Fulvestrant ++ 4.58 1.08 +++ ++ ++++ ++

*Fold induction of mRNA compared with control. and ECC-1TAM tumors (3.0-fold, P = 0.45) compared with between HER-2/neu through ERK-1/2 and ERa contributes to control (Fig. 4A). More importantly, IGF-I mRNA was decreased the natural development of tamoxifen-stimulated endometrial in ECC-1TAM control (60% less, P = 0.02), E2-treated (70% less, cancer. P = 0.14), tamoxifen-treated (67% less, P = 0.11), or fulvestrant- Five years of adjuvant tamoxifen therapy has been the standard of treated tumors (70% less, P = 0.04) compared with ECC-1E2 care for ERa-positive breast cancer for over 15 years (1). The major tumors (Fig. 4A). IGF-IR mRNA was increased by E2 in ECC-1E2 serious side effect of tamoxifen treatment is the increased risk of tumors (5.51-fold, P = 0.03) and ECC-1TAM tumors (3.5-fold, P = endometrial cancer by 0.1% (9). To date, little is known about the 0.04) compared with controls (Fig. 4B). Similarly to the IGF-I mechanism of tamoxifen-stimulated endometrial cancer cell mRNA results, IGF-IR mRNA was decreased in ECC-1TAM growth. However, Shang and Brown (19) showed that tamoxifen control tumors (56% less, P = 0.02), E2-treated (150% less, P = was more estrogenic in endometrial cancer cells compared with 0.21), tamoxifen-treated (50% less, P = 0.03), or fulvestrant- MCF-7 breast cancer cells in vitro by inducing transcription of ERa- treated (70% less, P < 0.01; Fig. 4B). The activity of the IGF-I/ regulated genes, c-myc and IGF-I. Our study examined the function IGF-IR signaling pathway was assessed by measuring tyrosine of the ERa in a novel model of tamoxifen-stimulated endometrial phosphorylation of IRS-1. E2, tamoxifen, or fulvestrant increased cancer developed in vivo under physiologic conditions by measuring phosphorylated IRS-1 protein in ECC-1E2 tumors (Fig. 4C). In expression of two estrogen-responsive genes, pS2 and c-myc. The addition, E2 induced phosphorylated IRS-1 protein in ECC-1TAM results clearly showed that tamoxifen treatment increased expres- tumors whereas tamoxifen had no effect on phospho-IRS-1 sion of pS2 and c-myc up to 4-fold in parental ECC-1E2 tumors and status compared with control (Fig. 4C). However, the overall up to 10-fold in ECC-1TAM tumors (Fig. 3A and B). These data extent of phosphorylated IRS-1 was less in ECC-1TAM tumors showed that tamoxifen is a weak agonist initially in tamoxifen-naive compared with ECC-1E2 tumors as determined by densitometry ECC-1E2 tumors and then a stronger agonist in tamoxifen- of three independent Western blots (Fig. 4D). These results taken stimulated ECC-1TAM tumors. The results suggested that the together suggest that the effectiveness of trastuzumab to inhibit increased tamoxifen-induced transcriptional activity might play an growth of ECC-1TAM tumors by blocking the activities of HER- important role in the development of tamoxifen-stimulated 2/neu, downstream ERK-1/2, and ERa-mediated c-myc mRNA endometrial cancer. More importantly, our data showed that expression could be due to the diminished IGF-I/IGF-IR trastuzumab, the HER-2/neu inhibitor, blocked tamoxifen-induced signaling pathway (results summarized on Table 1 and con- expression of c-myc by 70% in ECC-1TAM tumors, indicating that the clusions summarized in Fig. 5). increased agonist activity of tamoxifen on c-myc mRNA expression in these tumors might be due to the activity of the HER-2/neu signaling pathway. Discussion In addition to the estrogenic activity of tamoxifen at ERa- The results show, for the first time, that trastuzumab can regulated genes, tamoxifen could potentially exert nongenomic completely block the growth of tamoxifen-stimulated endome- activity by binding to membrane-associated ERa-activating ERK- trial cancer by inhibiting HER-2/neu signaling through ERK-1/2 1/2 and/or Akt directly. It has been shown previously that E2 and by blocking tamoxifen-induced transcription of c-myc activates ERK-1/2 or Akt within minutes of treatment suggesting (summarized in Table 1 and Fig. 5). We also showed that a nongenomic activity for ERa (38). Moreover, Song et al. showed trastuzumab is more efficacious in tamoxifen-stimulated endo- that ERa is recruited to the membrane by interacting with Shc metrial cancer than the parental E2-stimulated endometrial and IGF-IR where it can bind E2 or tamoxifen (39). Thus, cancer possibly due to increased activity of HER-2/neu and the increase in phospho-ERK-1/2 by E2 or fulvestrant or phopsho- decreased expression and signaling of the IGF-I/IGF-IR signaling Akt by tamoxifen or fulvestrant in ECC-1TAM tumors (Fig. 3C) pathway. These results strongly indicate that enhanced crosstalk could be a result of interaction with membrane ERa. Interestingly,

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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-1 recruits and activates phosphatidylinositol 3-kinase leading to Akt activation, whereas Shc activates the Ras-Raf-Mek-ERK-1/2 pathway. ERK-1/2 phosphorylates Sp1 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 transcription of 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 to cell 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 efficient inhibitor of the HER-2/neu signaling pathway in tamoxifen (TAM)–stimulated ECC-1TAM tumors leading to decreased ERK-1/2 activity, tamoxifen/ERa-mediated c-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. trastuzumab inhibited growth of tamoxifen-stimulated ECC-1TAM shown that trastuzumab therapy is only beneficial in women with tumors (Fig. 1D), induction of c-myc mRNA (Fig. 3B), and HER-2/neu overexpressing or amplified breast tumors (41, 42). phospho-ERK-1/2 (Fig. 3C) but did not block tamoxifen-induced However, the tests for determining HER-2/neu status may need phospho-Akt (Fig. 3C). Therefore, the function of the membrane further validation as trastuzumab therapy is effective in only 40% of ERa in activating Akt in terms of growth is unclear for tamoxifen- patients with high HER-2/neu expression (42). In addition, it might stimulated ECC-1TAM tumors. However, E2, tamoxifen, or be more reasonable to measure the active HER-2/neu protein and fulvestrant stimulated growth and induced phospho-ERK-1/2 in the extent of phosphorylation of downstream effector molecules ECC-1TAM tumors, suggesting that a direct interaction of ligand such as Akt and ERK-1/2 (43–45). Keeping this in mind, there are with membrane ERa is possible. In addition, fulvestrant had little other signaling pathways in addition to HER-2/neu that converge effect on pS2 and c-myc mRNA expression (Fig. 3A and B), on Akt and ERK-1/2. The hyperactivity of the IGF-I/IGF-IR signaling increased phospho-HER-2/neu, phospho-Akt, and -ERK-1/2 (Fig. pathway has previously been shown to result in resistance to 3C), and modestly increased growth of ECC-1TAM tumors (Fig. trastuzumab therapy in breast cancer cells (35). This pathway also 1D). These results suggest that fulvestrant might act through converges on the Akt (46) and ERK-1/2 (47) proteins leading to membrane ERa. This result is not surprising as previously we activation of these intracellular molecules. Our study found that the reported that fulvestrant in combination with E2 stimulated parental E2-stimulated ECC-1E2 tumors expressed constitutively growth and HER-2/neu signaling of tamoxifen-stimulated MCF-7 active Akt and ERK-1/2 proteins and whereas trastuzumab breast tumors independent of ERa transcriptional activity (40). decreased HER-2/neu protein, it had little effect on the phosphor- Surprisingly, the results from the present study also showed that ylation status of ERK-1/2 or Akt proteins. However, trastuzumab did trastuzumab therapy is effective in blocking tamoxifen-stimulated partially inhibit E2-induced growth (Fig. 1C) and expression of pS2 endometrial tumor growth in the absence of HER-2/neu over- mRNA in E2-stimulated ECC-1E2 tumors (Fig. 3A) suggesting that expression. However, the basal levels of phospho-HER-2/neu were growth and ERa activity are in part regulated by HER-2/neu increased in ECC-1TAM tumors by 2.7-fold (Fig. 3C) suggesting that independent of ERK-1/2 and Akt activities. More importantly, the hyperactivity of HER-2/neu could be important for the complete data clearly showed that IGF-I and IGF-IR mRNAs were decreased growth inhibitory effect of trastuzumab. Clinical studies have in ECC-1TAM tumors compared with ECC-1E2 tumors (Fig. 4A www.aacrjournals.org 8511 Cancer Res 2005; 65: (18). September 15, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research and B) and that this translated to decreased signaling as measured ECC-1TAM tumors seems achieved by inhibiting activities of by the phosphorylation status of IRS-1 (Fig. 4C and D), the primary HER-2/neu, ERK-1/2, and tamoxifen-induced expression of c-myc substrate for the active IGF-IR. Therefore, it is reasonable to mRNA in an environment where the IGF-I/IGF-IR signaling conclude that the efficacy of trastuzumab to completely inhibit pathway is diminished. These data suggest that the decreased growth of ECC-1TAM tumors could be due to an overall increase of expression and activity of the IGF-I/IGF-IR signaling pathway in HER-2/neu signaling and the decrease of signaling from the IGF-I/ ECC-1TAM tumors improves the overall efficacy of trastuzumab IGF-IR pathway thereby allowing trastuzumab to block the down- to inhibit growth of tumors expressing low levels of the IGF-I stream activity of ERK-1/2-originated from HER-2/neu. ligand and receptor. To address this hypothesis, studies are ERK-1 and ERK-2 are serine/threonine kinases that are activated currently under way to investigate whether overexpression of the by phosphorylation cascades originated from receptor tyrosine IGF-IR is necessary and/or sufficient to block the efficacy of kinases such as HER-2/neu and/or IGF-IR. Once activated, they trastuzumab to inhibit growth of both breast and endometrial translocate into the nucleus, where they phosphorylate transcrip- cancer cells. Based on the current results, we suggest that tion factors, including ERa (45) and Sp1 (48–50). Activation of ERa measuring the complex network of signals such as phospho- and Sp1 leads to transcription of c-myc (51), which is associated HER-2/neu, phospho-ERK-1/2, and phospho-IRS-1 status within with increased cellular proliferation and tumor growth (52). tumors might improve response rates of patients having Transcription of the c-myc gene is regulated by ERa through tamoxifen-stimulated breast and endometrial cancer to trastu- tethering to the Sp1 transcription factor (53) which binds directly zumab treatment and possibly lead to prevention of tamoxifen- to Sp1 sites within the c-myc promoter (51). Recent studies have resistant breast cancer and tamoxifen-stimulated endometrial shown that phosphorylation of Sp1 by ERK-1/2 and/or jun kinase cancer. There is clinical significance for trastuzumab therapy for leads to transcription of target genes (50). Thus, it is reasonable to tamoxafen-stimulated endometrial cancer as a single case report conclude that trastuzumab’s inhibitory effect on tamoxifen-induced shows (54). increase of c-myc mRNA could be due to a decrease of Sp1 activity through inactivation of ERK-1/2. Further studies are currently Acknowledgments under way to elucidate the exact mechanism by which trastuzu- Received 11/18/2004; revised 5/5/2005; accepted 5/16/2005. mab inhibits c-myc transcription. Grant support: Avon Foundation, Judy Dlugie Memorial Fund Fellowship In conclusion, the results from the current study suggest that (C. Osipo), and Department of Defense Training grant DAMD17-001-0386 (C. Osipo). trastuzumab is effective at blocking growth of tamoxifen- 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 accordance stimulated endometrial cancer when the basal activity of HER- with 18 U.S.C. Section 1734 solely to indicate this fact. 2/neu is enhanced. Furthermore, complete blockade of growth of We thank Dr. Myles Brown for the generous gift of ECC-1 cells.

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