Synergistic Interactions between Tamoxifen and Trastuzumab (Herceptin) · tured in the presence of...

Synergistic Interactions between Tamoxifen andTrastuzumab (Herceptin)

Athanassios Argiris, Chun-Xia Wang,Steve G. Whalen, and Michael P. DiGiovannaYale University School of Medicine, Departments of InternalMedicine (Section of Medical Oncology) and Pharmacology, and theYale Cancer Center, New Haven, Connecticut

ABSTRACTPurpose: HER-2/neu and estrogen receptor (ER) are

critical in the biology of breast carcinoma, and both arevalidated therapeutic targets. Extensive interactions be-tween the signaling pathways of these receptors have beendemonstrated. This suggests that targeting both receptorssimultaneously may have a dramatic effect on the biology ofbreast cancer. This hypothesis was tested in cell cultureexperiments.

Experimental Design: ER-positive, HER-2/neu-overex-pressing BT-474 human breast carcinoma cells were cul-tured in the presence of the anti-HER-2/neu therapeuticantibody trastuzumab (Herceptin), the antiestrogen tamox-ifen, or both. The effects on cell growth, cell cycle distribu-tion, clonogenicity, survival, and the level and activity ofHER-2/neu were examined.

Results: The combination of tamoxifen and Herceptinresulted in synergistic growth inhibition and enhancementof cell accumulation in the G0-G1 phase of the cell cycle, witha decrease in cells in S phase. Clonogenicity was inhibited inthe presence of each drug and more so by the combination,although prior exposure to drugs did not affect subsequentclonogenicity in drug-free media, and neither drug nor thecombination induced apoptosis. Herceptin, but not tamox-ifen, inhibited signaling by HER-2/neu.

Conclusions: The combination of tamoxifen and Her-ceptin is formally demonstrated to result in synergisticgrowth inhibition and enhancement of G0-G1 cell cycle ac-cumulation. In vitro, the individual drugs or combination

produces a cytostatic effect. These results suggest that com-bined inhibition of ER and HER-2/neu signaling may rep-resent a powerful approach to the treatment of breastcancer.

INTRODUCTIONApproximately 25% of invasive human breast tumors have

amplification of the HER-2/neu gene and/or overexpression ofHER-2/neu protein, which has been found to be an adverseprognostic factor (reviewed in Refs. 1 and 2). Trastuzumab(Herceptin) is a humanized monoclonal antibody against theextracellular domain of HER-2/neu (3–5) that has shown sig-nificant therapeutic activity in the treatment of patients withHER-2/neu-overexpressing metastatic breast cancer, both as asingle agent (6–8) and in enhancing antitumor activity in com-bination with cytotoxic chemotherapy (9–11). Signaling by theestrogen/estrogen receptor (ER) pathway is also important inbreast cancer tumorigenesis and biology. Antiestrogen therapyhas long been a cornerstone of the treatment of breast cancer. Alarge body of literature has now demonstrated extensive inter-actions between the ER signaling pathway and growth factorreceptor (including HER-2/neu) signaling, both in terms ofdownstream effects as well as regulation of each other’s activity.

HER-2/neu and ER are capable of down-regulating eachother. Clinically, an inverse relationship between HER-2/neuand ER expression is well appreciated (1, 2). Regulatory ele-ments exist within the HER-2/neu gene which allow estrogen tonegatively regulate its transcription (12–18); inhibiting estrogenaction can increase HER-2/neu levels (12, 13, 17, 19–21).Conversely, cross-activation of HER-2/neu by the neuregulins(ligands for HER-3 and HER-4) results in decreased ER levels(22–25) via unknown mechanisms.

In clinical practice, it has been observed in retrospectivestudies that tumors that overexpress HER-2/neu, even when ERpositive, may have reduced responsiveness to antiestrogen ther-apy (Refs. 26–33 but see Ref. 34). Although there is an inverserelationship between the degree of ER positivity and HER-2/neuexpression, a not insignificant proportion of human breast can-cers are both ER positive and overexpress HER-2/neu (26–28).Growth factor signaling alters steroid hormone responsiveness/sensitivity. Stimulation of growth factor signaling can lead toestrogen independence in ER-positive cells that are otherwiseestrogen dependent (23, 24). Growth factor signaling can alsolead to antiestrogen resistance in such cells (24, 35, 36). Suchresults suggest that growth factor signaling can rescue estrogen-dependent cells from the effects of estrogen deprivation.

The ER antagonist tamoxifen has been the most widelyused antiestrogen in clinical practice. One component of theantiestrogen mechanism of tamoxifen is believed to be inhibi-tion of the binding of coactivators to ER (37) and, possibly evenmore importantly, strengthening of the interaction of ER withcorepressors such as N-CoR (38). Acquired tamoxifen resist-

Received 9/4/02; revised 10/14/03; accepted 10/31/03.Grant support: Grants from the American Cancer Society, GreenwichBreast Cancer Alliance, the Hellman Family Fellowship (to M. P. D.),and the United States Public Health Service (Grant CA-16359) from theNational Cancer Institute.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Note: Present address for Dr. Argiris is The Feinberg School of Med-icine and the Robert H. Lurie Comprehensive Cancer Center of North-western University, 676 North St. Clair, Suite 850, Chicago, IL 60611.Requests for reprints: Michael P. DiGiovanna, Yale University Schoolof Medicine, Departments of Internal Medicine (Section of MedicalOncology) and Pharmacology, and the Yale Cancer Center, 333 CedarStreet, Room NSB 288, New Haven, CT 06510. Phone (203): 737-5240;Fax: (203) 785-7531; E-mail: [email protected].

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ance may be related to a change in the balance of association ofER with coactivators or corepressors. One study focused on whythe forced overexpression of HER-2/neu in estrogen-dependent,tamoxifen-sensitive cells results in tamoxifen resistance (39).These investigators found that a HER-2/neu tyrosine kinaseinhibitor restored tamoxifen sensitivity to this cell line. Theimportant mechanistic insight was that this was accompanied byrestoration of tamoxifen-induced association of ER with thecorepressor N-CoR in the transfected cells, which had beenimpaired compared with the parental line. Treatment with acombination of the tyrosine kinase inhibitor and tamoxifenmarkedly inhibited the growth of the transfected cell line as axenograft, whereas the transfected line was known to be other-wise tamoxifen resistant in vivo.

Mechanistically, it has been found that growth factor sig-naling may result in ligand-independent activation of ER. Ac-tivation of the ras/mitogen-activated protein kinase pathway,known to lie downstream of growth factor receptor signaling,can cause ligand-independent activation of ER and even activa-tion of ER in the presence of tamoxifen (40–43); this appears toinvolve the phosphorylation of ER Ser118 by mitogen-activatedprotein kinase (40, 43), a site normally phosphorylated in re-sponse to estrogen itself by a mitogen-activated protein kinase-independent mechanism (44). Another study found that theactivation of ER by epidermal growth factor was mediated byphosphatidylinositol 3�-kinase/Akt signaling (45), a survivalpathway also activated by HER-2/neu. These studies suggestadditional mechanistic explanations for how signaling bygrowth factor receptors can result in hormone independence/tamoxifen resistance.

Conversely, estrogen treatment can lead to activation ofgrowth factor signaling pathways. Estrogen is known to induceepidermal growth factor-related peptides, including transform-ing growth factor-� (46, 47), and medroxyprogesterone acetateinduces neuregulin production (48). It therefore appears that sexhormones may exert their mitogenic response by inducing re-lease of peptide growth factors that then activate receptors suchas HER-2/neu and epidermal growth factor receptor. Directassociation of ER with the cytoplasmic tyrosine kinase src hasalso been observed, resulting in activation of src and src-dependent activation of the ras/mitogen-activated protein kinasepathway (49, 50). The ER has also been found to bind to the p85regulatory subunit of phosphatidylinositol 3�-kinase in a ligand-dependent manner, resulting in activation protein kinase B/Akt(51).

Hence, activation of ER leads to a rapid activation ofpathways typically activated by peptide growth factors, andactivation of growth factor signaling pathways by peptidegrowth factors can activate ER directly and independently ofestrogen. This biology and apparent redundancy/positive feed-back argues that targeting both ER and growth factor receptorssimultaneously could be a fruitful approach to the treatment ofbreast cancer. Preliminary work on this concept (52, 53) hasshown encouraging results. Our goal was to further explore thecombined effect of antiestrogen and anti-HER-2/neu treatmentin vitro, using two clinically useful drugs in the treatment ofbreast cancer, tamoxifen and Herceptin. Because both tamoxifenand Herceptin are active as single agents in breast cancer andhave a favorable toxicity profile compared with cytotoxic chem-

otherapy, our results may translate into efficacious and safetherapy for this malignancy.

MATERIALS AND METHODSDrugs. Herceptin was a gift of Genentech (South San

Francisco, CA). A 5 mg/ml stock preparation was kept at 4°Cand was further diluted in sterile PBS before addition to cells inculture. Tamoxifen was purchased from Sigma (St. Louis, MO)and dissolved in 100% ethanol at a stock concentration of 10mg/ml; the stock was further diluted in PBS and/or culturemedia before addition to cells in culture. Tamoxifen stocks werekept light protected.

Cell Culture. BT-474 (54) is a human breast cancer cellline that is positive for ER (low; Ref. 23), is estrogen dependentfor growth (23), and highly overexpresses HER-2/neu in asso-ciation with gene amplification (55, 56). Cells were cultured inRPMI 1640 supplemented with 10% fetal bovine serum, gluta-mine, 10 �g/ml insulin, and penicillin at 37°C in 5% CO2

humidified air.Growth Curves. BT-474 cells in exponential growth

were seeded on day �1 in 12-well plates at 2 � 105 cells/wellin 3 ml of culture volume and allowed to adhere overnight.Drugs were added on day 0. For time point 0 (24 h afterseeding), and subsequently for all other time points, cells werewashed twice in PBS, trypsinized, collected, and counted usinga Coulter ZBI particle counter with a multichannel analyzer,with counts confirmed by counting with a hemocytometer.Growth curves were analyzed for cells that were treated witheither 1 �M tamoxifen, 10 �g/ml Herceptin, or both. As con-trols, cells were also examined when grown in drug-free media(“No Addition”) or 10 �g/ml irrelevant antibody (rabbit anti-mouse IgG; Vector Laboratories, Inc., Burlingame, CA) plusethanol (the vehicle for tamoxifen, at identical concentration;“Vehicle”). The data represent two individual experiments per-formed in duplicate and averaged together.

WST-1 Colorimetric Growth Assay. BT-474 cells wereplated in 96-well plates at a density of 104 cells/100-�l culture/well. They were allowed to adhere overnight and then either leftuntreated or treated with Herceptin at doses of �10 �g/ml,tamoxifen at doses of �8 �M, or various combinations of thetwo. After a 5- or 6-day incubation period, the WST-1 tetrazo-lium salt colorimetric growth assay (Boehringer Mannheim Bio-chemicals, Mannheim, Germany) was performed by adding 1/10volume of WST-1 solution and incubating for 2.5 h at 37°C.Absorbance at 450 nm was determined using a microplatereader (Molecular Devices Corp., Sunnyvale, CA). Results wereexpressed as a percentage of control (cells without drug). Themean and SE values of four or more wells in at least twoexperiments for each point are reported.

Analysis of Synergy. Data from the WST-1 colorimetricgrowth assay were analyzed using the method of Chou andTalalay (57), using commercially available software (CalcuSyn;Ref. 58). This method is based on the median-effect equation fordose-effect relationship: fa/fu � (D/Dm)m, which can be linearlytransformed as log (fa/fu) � m log (D) � m log (Dm), where fa

� fraction affected, fu � fraction unaffected, D � dose, Dm �dose that produces median-effect (IC50), and m � coefficientsignifying the sigmoidicity of the curve (or slope in linear

1410 Synergy between Tamoxifen and Herceptin



transformation). For the model to be applicable, the linearcorrelation coefficient (r) should be �0.9. For examining theeffect of multiple drugs, a Combination Index (CI) is calculatedbased on the doses that have equivalent effects, according to theformula: CI � (D1/D1c) � (D2/D2c), where D1 and D2 are thedoses for each drug alone that inhibit x%, and D1c and D2c arethe doses for each drug in a combination that inhibits the samepercentage x%. The CI is a quantitative measure of the degreeof interaction between two or more drugs. When it equals to 1,it denotes additivity, �1 antagonism, 1 synergism, and 0.3strong synergism. The CI was calculated under the assumptionof a mutually nonexclusive drug interaction.

Analysis of Cell Cycle by Flow Cytometry. BT-474cells in exponential growth were seeded at a confluency of50% in 100-mm dishes. After overnight incubation, cells wereeither left untreated or treated with 1 �M tamoxifen, 10 �g/mlHerceptin, or the combination. At indicated time points, cellswere trypsinized, washed with cold PBS, resuspended in 2 ml ofice cold PBS, fixed by three stepwise additions of 2 ml each of95% ethanol, and stored at 4°C for cell cycle analysis (59).Subsequently, cells were resuspended in 1 mg/ml RNase(Sigma) in PBS and stained with 0.05 mg/ml propidium iodide(Sigma P5264) for 1 h on ice. Flow cytometric analysis wasperformed with a FACS Vantage flow cytometer (Becton-Dick-inson, San Jose, CA). A minimum of 15,000 cells was analyzedfor each sample. Data analysis was performed using Modfit 5.2analysis software (Verity Software House, Topsham, ME). Eachpoint represents at least duplicate samples from between oneand three experiments or triplicate samples from a single exper-iment.

Apoptosis Assays. To measure apoptosis, BT-474 cellswere plated at 2.5 � 106 cells/100-mm dish and cultured in thepresence of 1 �M tamoxifen, 10 �g/ml Herceptin, both drugs, nodrug, or vehicle for 3 days. Both adherent and floating cellswere harvested and used for the following apoptosis assays. Forthe DNA ladder assay, DNA was prepared using the ApoptoticDNA Ladder Kit (Roche Diagnostics, Mannheim, Germany)and electrophoresed through 1% agarose. For the poly(ADP-ribose) polymerase (PARP) cleavage assay, whole cell lysateswere prepared by boiling in lysis buffer [100 mM Tris (pH 6.8),4% SDS, 20% glycerol, 6 M urea, 0.2% Bromphenol Blue, 10%�-mercaptoethanol, and 7 mM EDTA] followed by 3 min ofwater bath sonication. Protein concentrations of the lysates weredetermined by using a dendritic cell protein assay kit (Bio-Rad,Hercules, CA); 100 �g of protein from each lysate were re-solved in 7.5% SDS-polyacrylamide gels and then transferred tonitrocellulose membranes. Western blotting was carried outusing an anti-PARP antibody from Cell Signaling Technology(Beverly, MA). Apoptosis was detected by monitoring proteol-ysis of the Mr 116,000 native PARP enzyme to the apoptosis-specific Mr 89,000 fragment. The flow cytometric terminaldeoxynucleotidyl transferase-mediated nick end labeling assaywas performed using the APO-bromodeoxyuridine kit (PhoenixFlow Systems, San Diego, CA). The percentage of FITC-posi-tive apoptotic cells in the gated area was quantitated by flowcytometry.

Soft Agar Colony Growth Assay. To prepare baseplates, 1 ml of RPMI 1640 supplemented with 10% FCS, 2 mM

L-glutamine, 10 �g/ml insulin, 10 mM HEPES, and 0.8% agar-

ose was added into each 35-mm dish. The dishes were placed at4°C to solidify the agarose and then transferred to a 37°Cincubator. BT-474 cells were mixed with RPMI 1640 supple-mented with the same components in the presence of 1 �M

tamoxifen, 10 �g/ml Herceptin, both drugs, no drug (“Con-trol”), or vehicle, then plated on top of the base at a density of2 � 104 cells/35-mm dish in triplicate. Dishes were incubated at37°C for 15 days. Clusters of greater than or equal to six cellswere scored as colonies, and single cells as well as clusters offewer than six cells were scored as single cells. Five micro-scopic fields (�60) of each dish were scored, and the colonyformation percentage of each dish was calculated by dividingthe number of colonies by the total of colonies plus single cells.Final results were expressed as the percentage of untreated(“Control”) cells.

Anchorage-Dependent (Monolayer) Clonogenicity As-says. Monolayer colony assays A and AA were performed inthe continuous presence of drugs, whereas assay B was per-formed in drug-free media after prior exposure to drugs. Formonolayer colony assay A, cells were seeded at a density of 5 �103 cells/60-mm dish in triplicate in the presence of 1 �M

tamoxifen, 10 �g/ml Herceptin, both drugs, no drug, or vehicle.Dishes were incubated at 37°C for 30 days. Media were ex-changed for fresh media containing drugs every 6 days. Disheswere stained with 1 ml of 0.005% crystal violet for 30 min andwashed three times with double-distilled water. Solid cell clus-ters of �0.4 mm in diameter were scored as colonies. Clustersof cells that were not solid, but more dispersed, were sometimesalso observed. In assay AA, these “loose” clusters were alsoscored as colonies. The trends for the effect of drug treatment,compared with control untreated cells, were the same whetherthese dispersed clusters were scored as colonies or not. Resultsare expressed as a percentage of control (untreated) colonynumber.

For monolayer colony assay B, cells were seeded on day�1 in 100-mm dishes at a density of 2.5 � 106 cells/dish andallowed to adhere overnight. On day 0, cells were exposed to 1�M tamoxifen, 10 �g/ml Herceptin, both drugs, no drug, orvehicle. After 4-day incubation in the presence of drugs, cellswere trypsinized and seeded at a density of 5 � 103 cells/60-mmdish in triplicate in drug-free RPMI 1640 and incubated at 37°Cfor 30 days. Media were exchanged for fresh (drug free) mediaevery 6 days. Dishes were stained with 1 ml of 0.005% crystalviolet for 30 min and washed three times with double-distilledwater. Solid cell clusters of �0.4 mm in diameter were scoredas colonies. Results are expressed as a percentage of control(untreated) colony number.

Immunoblot Analysis. Cell lysates were prepared fromBT-474 cells cultured in the presence of 1 �M tamoxifen, 10�g/ml Herceptin, both drugs, no drug, or vehicle in 100-mmplates. Media were aspirated, and cells were washed twice withice-cold PBS. Subsequently, 0.8 ml of ice-cold lysis buffer [50mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP40, 2 mM EDTA,1 mM �-glycerol phosphate, 25 mM NaF, 1 mM Na3VO4, 1 mM

phenylmethylsulfonyl fluoride, 5 �g/ml leupeptin, 10 �g/mlaprotinin, and 5 �g/ml pepstatin] was added into each plate andspread evenly. After incubating the plate on ice for 10 min, cellswere scraped off the plate using a rubber policeman, transferredinto a 1.5-ml centrifuge tube, and lysed for another 15 min on

1411Clinical Cancer Research



ice. The lysate was centrifuged at 12,000 � g for 30 min at 4°Cto remove insoluble material. Protein concentrations of thelysates were determined by using a Bio-Rad protein assay kit(Bio-Rad). Protein (50 �g) was separated on a 7.5% SDS-polyacrylamide gel followed by transfer to a single nitrocellu-lose membrane as described previously (60). Membranes wereblocked for 1 h in Tris-buffered saline Superblock (Pierce)containing 0.1% Tween 20. Primary antibodies used were eitheranti-HER2/neu polyclonal antibody sc-284 (Santa Cruz Bio-technology, Inc., Santa Cruz, CA) or anti-phospho-HER-2/neumonoclonal antibody clone PN2A (c-erbB-2/HER-2/neu phos-pho-specific Ab-18; NeoMarkers, Fremont, CA). Anti-HER2/neu and anti-phospho-HER-2/neu antibodies were diluted inblocking buffer at a concentration of 1:2000 and 1:200, respec-tively. Membranes were incubated at 4°C overnight and washedfor 1 h at room temperature. Secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc.) was usedat a dilution of 1:2000. Membranes were washed and processedusing enhanced chemiluminescence (Amersham, Little ChalfontBuckinghamshire, United Kingdom).

RESULTSEffect of Herceptin, Tamoxifen, and the Combination

on Cell Growth. Growth curves for BT-474 cells that wereeither untreated or cultured in the presence of 1 �M tamoxifen,10 �g/ml Herceptin, or the combination, are shown in Fig. 1.Additionally shown are data for cells treated with irrelevantantibody at 10 �g/ml plus ethanol vehicle (“Vehicle”). Eachindividual drug at these physiologically relevant concentrationscaused partial growth inhibition, whereas the combination re-

sulted in nearly complete growth inhibition. The data representtwo individual experiments performed in duplicate and averagedtogether and is reflective of multiple similar experiments.

The effect of the addition of varying concentrations oftamoxifen or Herceptin to a given concentration of the otherdrug on cell growth using the WST-1 colorimetric growth assayafter a 5–6-day exposure period is shown in Fig. 2. These areclinically relevant concentrations of Herceptin that can beachieved in patients (6, 7). For Herceptin alone, near maximalgrowth inhibition is seen at 1 �g/ml; higher concentrations didnot significantly further inhibit cell growth (Fig. 2, top panel).The IC50 for Herceptin was 0.4 �g/ml. The addition of tamox-ifen at all doses in the range of 1–8 �M significantly enhancedthe growth inhibition of any given concentration of Herceptin.For single agent tamoxifen, increasing growth inhibition is seenin a dose-response fashion at concentrations ranging from 0 to 8�M (Fig. 2, bottom panel). The IC50 for tamoxifen was 1 �M.The addition of Herceptin at concentrations as low as 0.375 or1 �g/ml significantly enhanced the growth inhibition of anygiven concentration of tamoxifen.

An analysis of synergism was conducted, using the methodof Chou and Talalay (Ref. 57; see “Materials and Methods”) and

Fig. 1 Growth curves. BT-474 cells in exponential growth were seededon day �1 in 12-well plates at 2 � 105 cells/well in 3 ml of culturevolume and allowed to adhere overnight. Drugs were added on day 0. Attime 0 (24 h after seeding), and subsequently for all other time points,cells were washed, trypsinized, and counted. Growth curves were ana-lyzed for cells that were treated either with 1 �M tamoxifen, 10 �g/mlHerceptin, or both. No Addition, drug-free conditions; Vehicle, 10 �g/mlirrelevant antibody plus ethanol at equivalent concentration as used forthe tamoxifen vehicle. The data represent two individual experimentsperformed in duplicate and averaged.

Fig. 2 Growth assay. After a 5–6-day continuous exposure to drug, aWST-1 tetrazolium salt colorimetric growth assay was performed. Re-sults were expressed as a percentage of control (cells without drug) ineach experiment. The mean value and SE of the mean value of four ormore wells in at least two experiments is reported. Top panel, effectshown as a function of Herceptin concentration; bottom panel, effectshown as a function of tamoxifen concentration (same data for bothpanels).




the data from the WST-1 colorimetric growth assays. Thismodel is valid for the data collected, as indicated by the linearcoefficients (r) of �0.9 for each drug, as shown in Fig. 3. Allcombinations tested with tamoxifen in the range of 1–8 �M andHerceptin in the range of 0.375–10 �g/ml were synergistic,most strongly so with CIs of 0.35 (Table 1).

Effect on Cell Cycle. Changes in cell cycle were exam-ined by flow cytometry for BT-474 cells that were either un-treated or cultured with 1 �M tamoxifen, 10 �g/ml Herceptin, orthe combination. Both tamoxifen (61, 62) and Herceptin (63–65) individually are known to cause accumulation of cells in theG0-G1 phase. Our results showed that at 24 h (Fig. 4; Table 2)and 48 h (Fig. 5; Table 3) of drug exposure, both drugs indi-vidually resulted in a statistically significant increase in thepercentage of cells in G0-G1 compared with untreated cells (P 0.05). The combination of tamoxifen and Herceptin produced astatistically significant further increase in the percentage of cellsin G0-G1 phase compared with the effect of either drug alone

after 24 h of exposure (Fig. 4). At 24 h, there was also a decreasein the percentage of cells in S phase compared with untreatedcells or Herceptin-treated cells, although it was comparable withthe effect of single agent tamoxifen. Single agent Herceptincaused a slight increase in the percentage of cells in S phase,which might be attributable to its partial agonist activity. At48 h, the combination resulted in a high percentage of G0-G1

cells compared with control or tamoxifen-treated cells but whichwas similar to that elicited by Herceptin alone (Fig. 5). How-ever, the reduction in S phase for the combination was statisti-cally significant compared with untreated cells or cells treatedwith either agent singly.

Assays for Apoptosis. We hypothesized that the combi-nation of tamoxifen and Herceptin might cause apoptosis inBT-474 cells. Cells were cultured in the presence of 1 �M

Fig. 4 Effect on cell cycle at 24 h. BT-474 cells in exponential growthwere plated at a confluency of 50% in 100-mm dishes. After overnightincubation, 1 �M tamoxifen, 10 �g/ml Herceptin, neither (Control), orthe combination was added to the media. After a 24-h incubation, cellswere trypsinized, washed with cold PBS, fixed in ethanol, and stored at4°C. Subsequently, cells were resuspended in RNase and subjected topropidium iodide. Samples were analyzed by flow cytometry as de-scribed in “Materials and Methods.” �, P 0.05 compared with control(t test); ��, P 0.05 for all comparisons (t test).

Table 1 Fraction affected (Fa) and combination index (CI) forvarious combinations of tamoxifen and herceptin

CI 0.3 indicates strong synergy, 0.3 CI 1 indicates synergy,CI � 1 indicates additivity, and CI � 1 indicates antagonism.

Herceptin(�g/ml)

Tamoxifen(�M) Fa CI

0.375 1 0.747 0.2500.375 2 0.885 0.0720.375 4 0.911 0.0780.375 8 0.946 0.0581 0.5 0.872 0.0621 1 0.888 0.0611 2 0.930 0.0341 4 0.893 0.1301 8 0.929 0.1043 1 0.829 0.3373 2 0.856 0.2693 4 0.894 0.1873 8 0.920 0.159

10 1 0.900 0.269

Fig. 3 Median-effect plot for tamoxifen and Herceptin, used for anal-ysis of synergy between the two. Growth inhibition data were analyzedwith the method of Chou and Talalay, using commercially availablesoftware (CalcuSyn). The tamoxifen dose is in �M, and the Herceptindose is in �g/ml. For the tamoxifen plot, the linear correlation coeffi-cient was r � 0.98, and for the Herceptin plot, the linear correlationcoefficient was r � 0.92 (�0.9), both of which suggest goodness of fitfor the data to the median-effect equation. Top panel, the untransformeddata; bottom panel, the logarithmically transformed equation. A curvefor the combination of the two drugs is not shown, because combina-tions were tested at a variety of different concentrations of the two drugs(see Table 1) that were not at a constant ratio.




tamoxifen, 10 �g/ml Herceptin, both drugs, vehicle controls, orno drug for 3 days. Adherent and floating cells were harvested,and DNA was isolated and subjected to electrophoresis to assayfor DNA fragmentation resulting in the laddering pattern char-acteristic of cells undergoing apoptosis. Camptothecin-treatedU937 cells were used as a positive control. No DNA fragmen-tation was observed for any of the conditions of treatment ofBT-474 cells (Fig. 6). To confirm this result, two additionalassays of apoptosis were used. Cells were cultured as above, andprotein extracts were analyzed for PARP cleavage by immuno-blotting with anti-PARP antibody; etoposide-treated HL-60cells were used as a positive control. No PARP cleavage wasobserved in BT-474 cells for any of the culture conditions (Fig.7). Finally, cells were cultured similarly, and apoptosis wasanalyzed using a flow cytometric terminal deoxynucleotidyltransferase-mediated nick end labeling assay (Fig. 8). For ta-moxifen, Herceptin, and the combination, the percentage ofapoptotic cells was always 1%, whereas for the positive con-trol, camptothecin-treated HL-60 cells, the corresponding valuewas 28.5%. Hence, neither tamoxifen, Herceptin, nor the com-

bination, under these culture conditions, results in apoptosis ofBT-474 cells.

Effect on Anchorage-Dependent and -Independent Clo-nogenicity. The effect of the presence of tamoxifen, Hercep-tin, and the combination on the soft agar colony growth ofBT-474 cells was analyzed (Table 4). For these experiments,drug was present continuously during colony growth as de-scribed in “Materials and Methods.” The presence of tamoxifen

Fig. 5 Effect on cell cycle at 48 h (method as for Fig. 4.). �, P 0.05compared with control (t test); ��, P 0.05 for all comparisons (t test).

Fig. 6 DNA electrophoresis apoptosis assay. BT-474 cells were platedat 2.5 � 106 cells/100-mm dish and cultured in the presence of 1 �M

tamoxifen (T), 10 �g/ml Herceptin (H), both drugs (T/H), no drug (C,control), or vehicle (V) for 3 days. Adherent and floating cells wereharvested, and the DNA was prepared using the Apoptotic DNA LadderKit. DNA from U937 cells treated with camptothecin, supplied by themanufacturer, was used as a positive control (P).

Fig. 7 Poly(ADP-ribose) polymerase (PARP) cleavage apoptosis as-say. BT-474 cells were plated at 2.5 � 106 cells/100-mm dish andcultured in the presence of 1 �M tamoxifen (T), 10 �g/ml Herceptin (H),both drugs (T/H), no drug (C, control), or vehicle (V) for 3 days.Adherent and floating cells were harvested, and whole cell lysates wereprepared as described in “Materials and Methods.” Western blotting wascarried out using an anti-PARP antibody. Whole cell extract of humanHL-60 leukemia cells treated with the chemotherapeutic agent etopo-side, supplied by the manufacturer, was used as a positive control (P).

Table 2 Cell cycle effect at 24 h (% cells, mean � SE)

Phaseof cycle Control Tamoxifen Herceptin Combination

G0-G1 50.50 � 0.3 54.94 � 0.4a 54.31 � 0.4a 61.36 � 1.5b

S 25.62 � 2.2 22.37 � 2.7 27.74 � 3.6 18.99 � 2.6G2-M 23.88 � 2.2 22.69 � 2.3 17.94 � 3.3 19.65 � 1.7

a P 0.05 compared with control (t test).b P 0.05 for all comparisons (t test).

Table 3 Cell cycle effect at 48 h (% cells, mean � SE)

Phaseof cycle Control Tamoxifen Herceptin Combination

G0-G1 72.08 � 1.2 77.50 � 1.1a 82.36 � 0.5a 83.50 � 1.0a

S 15.10 � 1.2 11.18 � 0.8a 9.64 � 0.5a 7.94 � 0.4b

G2-M 12.81 � 0.4 11.31 � 0.6 8.00 � 0.9a 8.56 � 0.8a

a P 0.05 compared with control (t test).b P 0.05 for all comparisons (t test).




produced a modest inhibition of soft agar colony formation(85.3% of control), whereas Herceptin produced a more pro-nounced inhibition (25.8% of control) and the combination thegreatest degree of inhibition (13.5% of control).

Monolayer clonogenicity on plastic culture dishes was an-alyzed by two approaches (Table 4). In the first approach, theassay was conducted in the presence of drug. Tamoxifen re-sulted in a modest inhibition of colony formation, Herceptin amore pronounced inhibition, and the combination the greatestdegree of inhibition. The trends were the same whether onlysolid clusters were scored as colonies (Assay A) or whether thedispersed clusters (described in “Materials and Methods”) werealso scored as colonies (Assay AA). In the second approach,cells were precultured for 4 days in the presence of drug, andcells were then harvested by trysinization and replated at equiv-alent densities for assay in the absence of drug (Table 4, Mono-layer colony assay B). Pre-exposure to tamoxifen, Herceptin, orthe combination did not impair the subsequent clonogenicityafter drug was removed.

Immunoblot Analysis of HER-2/neu Expression Leveland Phosphorylation State. Changes in HER-2/neu levelsover time were analyzed by immunoblotting of cell lysates withanti-HER-2/neu antibody (Fig. 9, left panel). For cells that weretreated with 1 �M tamoxifen, 10 �g/ml Herceptin, or the com-bination, HER-2/neu levels in lysates were relatively stable overthe 5-day time course of the experiment. Because the activity ofreceptor tyrosine kinases is regulated and autophosphorylationis indicative of the signaling activity, autophosphorylated HER-2/neu levels were examined by immunoblotting with anti-phospho-HER-2/neu antibody PN2A (Fig. 9, right panel).For untreated (“C” � “Control”) and vehicle-treated (“V” �“Vehicle”) cells, an easily detectable basal level of phosphoryl-ated receptor was found, likely caused by the high level ofoverexpression. Compared with control or vehicle-treated cells,tamoxifen produced no effect on the level of phosphorylatedHER-2/neu in lysates. In contrast, Herceptin caused a markeddecrease in level of phosphorylated HER-2/neu that was evidentat the earliest time point examined (8 h) and persisted to the

Table 4 Soft agar colony growth and monolayer clonogenicity assaysResults are expressed as a percentage of control (no drugs or vehicles added). Concentrations used were 1 �M tamoxifen or 10 �g/ml Herceptin.

For the soft agar assay and monolayer assays A and AA, drug was continuously present. For monolayer assay B, cells were precultured in the presenceof drug for 4 days, then replated in drug-free media for the assay. In assay A, only solid colonies were scored as such, whereas in assay AA, looselydispersed clusters were also scored as colonies as described in “Materials and Methods.”

Relative colony formation

Soft agarcolony assay

Monolayercolony assay A

Monolayercolony assay AA

Monolayercolony assay B

Control 100 � 2.61 100 � 2.57 100 � 2.39 100 � 7.90Vehicle 98.5 � 4.28 99.3 � 2.34 99.2 � 4.29 97.4 � 9.71TAM 85.3 � 3.16 26.4 � 1.12 60.5 � 5.24 109.5 � 9.17Herceptin 25.8 � 3.21 6.2 � 0.56 38.2 � 3.63 100 � 5.83TAM � Herceptin 13.5 � 2.77 1.1 � 0.56 25.5 � 2.44 108.2 � 7.58

Fig. 8 Terminal deoxynucleotidyltransferase-mediated nick end labelingassay. Apoptosis was determined by flowcytometric terminal deoxynucleotidyltransferase-mediated nick end labelingassay as described in “Materials andMethods.” BT-474 cells were plated at2.5 � 106 cells/100-mm dish and cul-tured in the presence of 1 �M tamoxifen,10 �g/ml Herceptin, both drugs, no drug(Control), or vehicle for 3 days. HL-60leukemia cells treated with camptothecin,supplied by the manufacturer, were usedas positive control. The percentage ofFITC-positive apoptotic cells in the gatedarea was quantitated by flow cytometry.




same degree throughout the 5-day time course of this experi-ment. The addition of tamoxifen to Herceptin did not have anyimpact on the Herceptin effect.

DISCUSSIONOur results demonstrate using the ER-positive, HER-2/

neu-overexpressing BT-474 cell line that the combination oftamoxifen plus Herceptin results in synergistic inhibition of cellgrowth in culture, accompanied by enhanced accumulation ofcells in the G1-G0 phase of the cell cycle and reduction in Sphase. The formal mathematical demonstration of synergy be-tween such agents (antiestrogen and anti-HER-2/neu) has notbeen demonstrated previously. In the presence of drug, bothagents inhibited anchorage-dependent and -independent clono-genicity, with the greatest degree of inhibition elicited by thecombination. However, previous culture in drug-containing me-dia did not adversely impact the subsequent anchorage-depen-dent clonogenicity in drug-free media, and neither drug nor thecombination resulted in apoptosis, indicating a purely cytostaticeffect in vitro.

Individually, tamoxifen (61, 62) and Herceptin/4D5 (63–65) have been reported to promote accumulation of cells in theG0-G1 phase of the cell cycle. We confirmed this and found aneven greater effect of the combination (at 24 h). Differenceswere also apparent at 48 h, although the combination resulted ina similar percentage of cells in G0-G1, as did single agentHerceptin; of note is that even untreated cells had �70% G0-G1

cells by 48 h. The combination, however, did produce the lowestpercentage of S phase at 48 h. In work with BT-474 cells,antibody 4D5 (the murine monoclonal antibody from whichhumanized Herceptin was derived) was found to result in in-creased levels of the cyclin-dependent kinase inhibitor p27,association of p27 with Cdk2, inactivation of cyclin-Cdk2 com-plexes, and hypophosphorylation of the retinoblastoma protein;the effect was reversible, and apoptosis was not observed (63).Similar results have been noted in the treatment of SKBR3 cells

with another anti-HER-2/neu antibody (66). How the combina-tion of anti-HER-2/neu therapy with antiestrogen impacts thecell cycle regulatory proteins is the subject of ongoing work inour laboratory.

We observed that Herceptin treatment did not affect thelevel of expression of HER-2/neu but did inhibit the signalingactivity of HER-2/neu, as indicated by a decrease in the amountof Tyr-1248 phosphorylated HER-2/neu present. This inhibitionof phosphorylation was evident at the earliest time point (8 h)examined. The inhibition of HER-2/neu activity is consistentwith previous reports, although in other studies, the effect ofHerceptin on total HER-2/neu levels has been varied. In exper-iments using 4D5, treatment has been reported to result indecreased levels of HER-2/neu using several cell lines, includ-ing SKBR3 and HER-2/neu-transfected NIH-3T3 cells (3, 67,68). However, consistent with our results, a report in whichBT-474 cells were treated with 4D5 found inhibition of phos-phorylation of HER-2/neu but no decrease in level of expression(63). The differences in different studies could be caused byinherent cell line differences or the particular experimentalconditions, such as time points examined or the density of thecells, which itself is known to impact HER-2/neu levels (69, 70).In our study, we followed cell changes for longer time pointsthan in most other studies. The long time points examined in ourstudy may better reflect steady-state changes obtained withchronic therapy in the clinical situation.

Some studies have demonstrated induction of phosphoryl-ation of HER-2/neu by Herceptin. Herceptin, 4D5, and otheranti-HER-2/neu growth-inhibiting antibodies are also known tohave the properties of being partial HER-2/neu agonists, induc-ing phosphorylation of HER-2/neu and at times transientlyactivating downstream signaling events, although these agonis-tic properties predominate at early time points, and any signal-ing occurs transiently, if at all (66, 67, 71). Using SKBR3 cells,it was shown that the F(ab) fragment of 4D5 resulted in in-creased tyrosine phosphorylation of HER-2/neu at 15 min,

Fig. 9 Immunoblot analysis ofHER-2/neu expression leveland phosphorylation state. BT-474 cells were cultured in thepresence of 1 �M tamoxifen(T), 10 �g/ml Herceptin (H), acombination of both drugs (T/H), no drug (C), or vehicle (V)as described in “Materials andMethods.” At the indicated timepoints, cell lysates were pre-pared, and 50 �g of proteinwere electrophoresed and im-munoblotted with antibody toHER2 (left panel) or phospho-rylated HER2 (right panel).




whereas treatment with 4D5 for 11–15 h resulted in reducedphosphorylation; furthermore, it was demonstrated that the de-crease in phosphorylated HER-2/neu could not be attributedsolely to down-regulation of the receptor but must have in-volved inhibition of the autophosphorylation activity itself (72),consistent with our results showing inhibition of phosphoryla-tion without changes in total expression level. Such results mayagain be dependent on the experimental design, because inanother study, 4D5 treatment of BT-474 cells was reported toresult in HER-2/neu de-phosphorylation as early as 10 min aftertreatment, with decreases in phosphorylation of downstreamErk1/2 and protein kinase B occurring concomitantly or soonthereafter (63).

Estrogen is known to cause transcriptional down-regulationof HER-2/neu (12–18), and inhibiting estrogen signaling canincrease HER-2/neu levels (12, 13, 17, 19–21). Tamoxifen hasbeen reported previously to antagonize the HER-2/neu-down-regulating effect of estrogen and up-regulate levels of HER-2/neu (19), even in nonestrogen-supplemented media (13, 17).The antiestrogen toremifene also inhibited the estradiol-inducedrepression of HER-2/neu expression at 12 and 72 h in vitro inZR-75-1 cells, and toremifene or tamoxifen inhibited estrogen-induced repression of HER-2/neu in vivo using ZR-75-1 xe-nografts when analyzed on day 10 of treatment (20). In contrast,one publication reported HER-2/neu ligand-like stimulatory ef-fects of estradiol (73), with rapid induction of phosphorylation,down-regulation of HER-2/neu, and induction of morphologicaltransformation (but not anchorage independence). In that study,tamoxifen was able to reverse the estradiol-induced phospho-rylation and morphological transformation. An additional mech-anism by which tamoxifen could inhibit activity of HER-2/neuis to antagonize the ability of estrogen to induce the autocrinesecretion of epidermal growth factor family growth factors (46,47). However, we found that tamoxifen, although enhancing theantiproliferative effect of Herceptin, did not do so via any effecton HER-2/neu expression levels or signaling activity. We hy-pothesize that the synergistic effects of Herceptin and tamoxifenmay result from signaling pathway interactions downstreamfrom the two targeted receptors, and experiments to address thisare currently underway in our laboratory.

As suggested above, in vitro effects of these drugs may becell line context dependent. Although it is of interest to extendthese results to other cell lines, BT-474 is the only available cellline that endogenously overexpresses HER-2/neu and is also ERpositive/estrogen dependent. Although it is tempting to use cellsmolecularly engineered to express these receptors, this mayresult in misleading observations, e.g., it has been found thattransfection of ER into otherwise ER-negative cells produces aphenotype in which estrogen is actually growth inhibitory ratherthan stimulatory (74–76). A number of investigations has alsostudied MCF7 cells transfected with HER-2/neu to produce highlevels of HER-2/neu, and unlike cells naturally overexpressingHER-2/neu, these transfected cells are not growth inhibited byanti-HER-2/neu antibody (35). Nonetheless, we are currentlyextending these experiments to other cell lines with varyingendogenous levels of expression of these receptors.

A previous report using BT-474 cells investigated thecombined effect of tamoxifen and 4D5 and found enhancedinhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide assay and enhanced inhibitionof [3H]thymidine incorporation into DNA, compared with eitherdrug alone (52). Another study showed that the combination ofHerceptin with ICI 182,780, a pure antiestrogen that also down-regulates ER, resulted in enhanced growth inhibition over eitherdrug alone using ML-20 cells reported to express a high level ofER and moderate level of HER-2/neu (53). An analysis ofsynergy was not performed. Either drug increased the percent-age of cells in G0-G1, although the combination was not differ-ent from the effect of the antiestrogen alone. A measurable butsmall amount of apoptosis was observed with the combinationtreatment that was greater than that of either drug alone. Neitherof these studies examined the effect on levels or activity ofHER-2/neu. Although our results and those of others support apurely cytostatic effect of these drugs in vitro, under the addedcellular stresses of in vivo conditions, cell destruction certainlyoccurs because both drugs result in tumor regressions in breastcancer patients. We are currently performing in vivo experi-ments to examine this combination using a murine BT-474xenograft model.

Targeted therapy based on the well-studied biology ofmalignancies is resulting in advances in the treatment of cancerpatients. Individual therapeutics with a defined target help tomake these strides, but major impact will likely continue tocome from the combinations of therapeutic agents. In clinicaloncology, combinations of drugs are often chosen empirically,often simply on the basis of feasibility of safe delivery whennonoverlapping toxicities are associated. The interactions be-tween the growth factor and ER signaling pathways known inbreast carcinoma provide strong biological rationale for com-bining agents that target these two pathways in particular. Theseresults may translate into improved therapy for patients withbreast carcinomas in which both of these pathways are known tobe of biological significance.

ACKNOWLEDGMENTSWe thank the services of the Yale Cancer Center Flow Cytometry

shared resource.

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2004;10:1409-1420. Clin Cancer Res Athanassios Argiris, Chun-Xia Wang, Steve G. Whalen, et al. Trastuzumab (Herceptin)Synergistic Interactions between Tamoxifen and

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