Response to Trastuzumab, Erlotinib, and Bevacizumab, Alone and In

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Response to trastuzumab, erlotinib, and bevacizumab, alone and in combination, is correlated with the level of human epidermal growth factor receptor-2 expression in human breast cancer cell lines

David R. Emlet, Kathryn A. Brown, not increase apoptosis substantially. These studies sug- Deborah L. Kociban, Agnese A. Pollice, gest that the effects of two and three-drug combinations Charles A. Smith, Ben Brian L. Ong, of trastuzumab, erlotinib, and bevacizumab might offer and Stanley E. Shackney potential therapeutic advantages in HER2-overexpressing breast cancers, although these effects are of low Laboratory of Cancer Cell Biology and Genetics, Department of magnitude, and are likely to be transient. [Mol Cancer Human Oncology, Drexel University College of Medicine and the Ther 2007;6(10):2664–74] Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania Introduction Human epidermal growth factor receptor-2 (HER2), a Abstract member of the epidermal growth factor receptor (EGFR) Human epidermal growth factor receptor-2 (HER2) and family of tyrosine kinases, is overexpressed in 25% to 30% epidermal growth factor receptor (EGFR) heterodimerize to of human breast cancers (1). It has been implicated in activate mitogenic signaling pathways. We have shown cancer progression (2, 3), and has been identified as a previously, using MCF7 subcloned cell lines with graded prognostic and predictive marker for breast cancer out- levels of HER2 expression, that responsiveness to trastu- come (4). Clinically, chemotherapeutic regimens that zumab and AG1478 (an anti-EGFR agent), varied directly include trastuzumab (Herceptin), a humanized monoclonal with levels of HER2 expression. HER2 and EGFR up- antibody directed against the extracellular domain of regulate vascular endothelial growth factor (VEGF), a HER2, prolong the time to progression and increase overall growth factor that promotes angiogenesis and participates survival in patients with advanced disease whose tumors in autocrine growth-stimulatory pathways that might be amplify/overexpress HER2 (5, 6). In the adjuvant setting, in vitro active . Here, we show that trastuzumab, erlotinib, trastuzumab increases disease-free survival and overall and bevacizumab, individually and in combination, inhibit survival in patients whose tumors amplify/overexpress cell proliferation in a panel of unrelated human breast HER2 (7, 8). cancer cell lines, in proportion to their levels of HER2 EGFR is overexpressed in 15% to 35% of breast cancers, expression. The combination of all three drugs provided a and EGFR overexpression is associated with a poor greater suppression of growth than any single drug or two- prognosis (9, 10). Agents that target EGFR, including drug combination in the high HER2–expressing cell lines erlotinib (Tarceva/OSI-774), are currently approved for P ( < 0.001). Combination index analysis suggested that the management of non–small cell lung cancer, colon the effects of these drugs in combination were additive. cancer, pancreatic, and head and neck cancers (11, 12). The pretreatment net level of VEGF production in each Responses are seen in 8% to 20% of patients with non–small cell line was correlated with the level of HER2 expression cell lung cancer, with higher response rates in a subset of r P ( = 0.883, = 0.016). Trastuzumab and erlotinib each patients whose tumors contain mutations involving the reduced total net VEGF production in all cell lines. kinase domain of EGFR (13, 14). Although preclinical cell Multiparameter flow cytometry studies indicated that culture and xenograft studies suggest that EGFR-targeted erlotinib alone and the triple drug combination produced therapies may be active in breast cancer, clinical trials have a prolonged but reversible blockade of cells in G , but did 1 shown responses in <10% of patients (15). HER2 and EGFR heterodimerize with one another to drive mitogenic signaling pathways, and this interaction is

Received 2/5/07; revised 7/27/07; accepted 8/30/07. likely to play a key role in cancer progression in breast Grant support: Agreement no. 41331809 with Beckman Coulter. cancer cells that overexpress HER2 and express EGFR. In The costs of publication of this article were defrayed in part by the preclinical studies, combinations of EGFR and HER2 payment of page charges. This article must therefore be hereby marked targeting agents were found to be more effective than advertisement in accordance with 18 U.S.C. Section 1734 solely to single targeted therapy agents. Combinations of EGFR- indicate this fact. targeted agents erlotinib or gefitinib (Iressa/ZD1839) with Requests for reprints: Stanley E. Shackney, Allegheny General Hospital, 10ST320 East North Avenue, Pittsburgh, PA 15212. Phone: 412-359- the HER2-targeted agents trastuzumab or pertuzumab 4306; Fax: 412-359-3238. E-mail: [email protected] (Omnitarg/2C4) have shown additive or synergistic effects Copyright C 2007 American Association for Cancer Research. on growth inhibition of cancer cells in vitro and/or in vivo doi:10.1158/1535-7163.MCT-07-0079 (16–19). Clinical results with a combination of erlotinib and

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trastuzumab suggests that this combination is well tolera- cell lines (American Type Culture Collection) were cul- ted, and the addition of erlotinib may be able to overcome tured in the same media without geneticin. The BT474 cell resistance to trastuzumab, but the in vitro activity of the line (American Type Culture Collection) was maintained in combination has not yet been borne out in the clinic (20). DMEM containing 100 units/mL of penicillin and strepto- Breast cancer cell lines and primary breast cancers can mycin, 0.25 Ag/mL of amphotericin B, and 10% fetal bovine express vascular endothelial growth factor (VEGF), or its serum. The MDA-361 cell line (American Type Culture receptors (21–25), and increased VEGF expression in breast Collection) was maintained in Leibovitz’s L-15 Media cancers is associated with tumor progression and increased containing 100 units/mL of penicillin and streptomycin, risk of recurrence (26). Although the antiangiogenic effects 0.25 Ag/mL of amphotericin B, and 10% fetal bovine serum of agents targeted against VEGF have been emphasized in in a CO2-free incubator. cancer (27–29), tumors that express both VEGF and its Immunoblot Analysis receptors can participate in autocrine/paracrine feedback Cell lysate (50 Ag) was subjected to SDS-PAGE on 4% to loops (30–32), and the specific effects of anti-VEGF agents 20% tris/glycine gels, transferred to nitrocellulose filters, on these autocrine loops can be studied in vitro, where and blocked for 30 min in TTBS [100 mmol/L Tris (pH 7.5), angiogenesis is not at issue. 0.9% NaCl, 0.1% Tween 20] with 5% nonfat dry milk. A HER2 can regulate VEGF production in human breast monoclonal antibody against HER2 (CB11; Novacastra cancer cell lines (33, 34), and VEGF levels are correlated Labs.) was used at a 1:1,000 dilution. The secondary with HER2 expression in primary breast cancers (35, 36). antibody was goat anti-mouse horseradish peroxidase– EGFR can also up-regulate the expression of VEGF in cancer conjugated antibody (Santa Cruz Biotechnology), and was cell lines (37–39), and inhibition of EGFR activity reduces used at a 1:1,000 dilution. For the supplemental data, a VEGF production and angiogenesis in vivo (38, 40, 41). polyclonal antibody against the insulin-like growth factor- Given that HER2 and EGFR can heterodimerize to initiate IR (Santa Cruz Biotechnology) was used at a 1:200 dilution, signal transduction, and that each can regulate VEGF and the secondary antibody was goat anti-rabbit horserad- production, it would seem reasonable to compare the ish peroxidase–conjugated antibody (Santa Cruz Biotech- effects of inhibition of EGFR, HER2, and VEGF, individu- nology), used at a 1:1,000 dilution. Antibody signal was ally and in combination, in breast cancer cell lines. We have visualized using the SuperSignal West Pico Chemilumi- shown previously that responses to AG1478 (a small nescent Substrate (Pierce) exposed to Kodak BioMax XAR molecule EGFR inhibitor) alone, or in combination with film. Densitometry was done using a Personal Densitom- trastuzumab, were correlated with levels of expression of eter SI (Molecular Dynamics), and the data were analyzed HER2 in a series of MCF7 breast cancer cell line subclones using Scion Image for Windows (Scion Corporation). that expressed graded levels of HER2 (42). Growth Inhibition Assays In this study, we examine this relationship in genetically Trastuzumab and bevacizumab were provided by Gen- unrelated human breast cancer cell lines using clinically entech, erlotinib was provided by OSI Pharmaceuticals, relevant concentrations of the EGFR inhibitor erlotinib, and and the anti–insulin-like growth factor-IR antibody IMC- we have extended our studies to include the VEGF- A12 was provided by ImClone Systems. Trastuzumab, inhibiting antibody bevacizumab (Avastin). We show that bevacizumab, and IMC-A12 were stored at 4jC. Erlotinib, the triple combination of erlotinib, trastuzumab, and VEGF receptor (VEGFR) inhibitor (Calbiochem), and bevacizumab provided a greater suppression of growth etoposide (Calbiochem) were stored as 10 mmol/L stocks than any of these drugs alone or any possible two-drug in DMSO at 20jC. Cell stocks were grown to 80% combination. The pretreatment net level of VEGF produc- confluence, trypsinized, and plated in duplicate into 24- tion in each cell line was correlated with the level of HER2 well tissue culture plates at 1 to 2 104 cells/well. The expression, and trastuzumab and erlotinib each reduced following day, duplicate wells (for day 0 counts) were net VEGF production in all cell lines. Multiparameter flow trypsinized and each well was counted with a hemocy- cytometry studies indicated that all three drugs produced a tometer. The remaining wells were fed on days 0 and 4 prolonged, reversible blockade of cells in G1 but did not with regular cell culture media supplemented with the increase apoptosis substantially at clinically relevant con- inhibitors, DMSO, or human IgG as vehicle controls. On centrations. day 6, the cells were trypsinized and counted with a hemocytometer, and the data were graphed in Excel. The data presented are from at least three separate experiments. Materials and Methods VEGF Analysis by ELISA Cell Culture Endogenous VEGF production and secretion from the The HER2 stable transfectant cell lines NH29, NH47, and cell lines was assayed using the Quantikine human VEGF NH27 (derivatives of the MCF7 breast cancer cell line), immunoassay (R&D Systems). Cells were plated and were generated as described previously (42), and were grown as for the growth inhibition assays above (no drug, maintained in RPMI 1640 containing 100 units/mL of or 1 Ag/mL trastuzumab, or 2 Amol/L erlotinib), and prior penicillin and streptomycin, 0.25 Ag/mL of amphotericin B, to cell counting, the medium from each well was removed, and 10% fetal bovine serum, supplemented with 500 Ag/mL spun down to remove particulate matter, and used for the of geneticin. MCF7, MDA-231, and SKBR3 breast cancer immunoassay according to the manufacturer’s instructions,

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Figure 1. HER2 expression levels in breast cancer cell lines, and dose response effects of targeted agents on cell lines with low and high HER2 expression. A, representative immunoblot of HER2 expression in MCF7 cells, HER2 expression vector – stably transfected MCF7 subclones (NH29, NH47, NH27), and unrelated breast cancer cell lines with varying levels of endogenous HER2 expression (MDA-231, MDA-361, SKBR3, BT474). B, HER2 densitometry values from three separate immunoblots of each cell line were normalized to those of the accompanying BT474 cell line. Cell lines were grouped according to their HER2 expression levels; low (LOWHER2 ), intermediate (INT HER2),andhigh(HIGH HER2). Columns, means; bars, SE. P < 0.001, HER2 signals of each group are significantly different from one another. C, dose response effects on various cell lines for (1) trastuzumab (0, 0.01, 0.1, 1.0, 10, and 50 Ag/mL); (2) erlotinib (0, 0.1, 0.5, 2.0, 10, and 50 Amol/L); (3) bevacizumab (0, 0.01, 0.1, 1.0, 10, and 50 Ag/mL); and (4) the anti – insulin-like growth factor-IR antibody IMC-A12 (0, 0.01, 0.1, 1.0, and 10 Ag/mL). Points, means; bars, SE.

using fresh medium as a negative control, and a concen- methanol for DNA analysis and the other aliquot was fixed tration standard provided with the immunoassay. The in 3.0 mL of 0.5% paraformaldehyde at room temperature assay was analyzed using an ELx800 microplate reader for 15 min, washed once in PBS, and resuspended in 1.0 mL (BioTek). The amount of VEGF (pg/mL) in each cell line of cold PBS and 3.0 mL of cold 90% methanol. Samples was derived using the provided standard, then divided by were stored at 20jC until the time of staining and the number of cells to obtain the value (pg/mL/cell), and analysis. the data were graphed in Excel. The data presented are For DNA histogram analysis, cells fixed with 90% from at least three separate experiments. methanol were stained with 50 Ag/mL of propidium iodide Flow Cytometry Studies of Cell Cycle and Apoptosis and 1 mg/mL of RNase (Sigma) for 1 h at 4jC, and analyzed SKBR3 cells were plated into T75 flasks at 1.65 106 by flow cytometry. For multiparameter flow cytometry cells/flask. The following day, the cells were treated with apoptosis studies, paraformaldehyde/methanol-fixed sam- 1 Ag/mL of trastuzumab, 2 Amor10Amol/L of erlotinib, ples were first subjected to the terminal transferase dUTP 1 Ag/mL of bevacizumab, or a combination of all three, nick end labeling method to detect apoptosis using the with or without 500 nmol/L of etoposide for 0, 12, 24, 36, 48 APO-DIRECT KIT (BD Biosciences), according to the manu- or 72 h. After drug treatment, the media were collected, facturer’s instructions. The samples were washed in PBS adherent cells were trypsinized and pooled with their and stained with 4¶,6¶-diamidino-2-phenylindole (Sigma) for respective media, spun down, and washed once with PBS. 1hat4jC to measure cell DNA content. Samples were divided into two aliquots, and one aliquot Measurements were made on each of several thousand was fixed in 1.0 mL of cold PBS and 3.0 mL of cold 90% cells using an EPICS ELITE flow cytometer (Beckman-

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Coulter) using a 15-mW air-cooled argon laser emitting ment data were analyzed by one-way ANOVA followed at 488 nm with a 550 nm dichroic long-pass filter and a by a two-sided Dunnett’s test post hoc for the determina- 525 nm band pass filter for FITC, and a water-cooled 5 W tion of differences between groups. Pearson correlation laser with UV capabilities emitting at 325 nm with a 381 nm coefficients of linear regression were calculated for the band pass filter for 4¶,6¶-diamidino-2-phenylindole. All mea- log of surviving cell fraction versus relative HER2 expres- surements were collected and stored in listmode. DNA sion and net VEGF production versus relative HER2 measurements were collected in 1,024 channels and later expression. converted to a 256-channel format, and the FITC measure- The combination index (CI) for the triple drug effect in ments were collected in 256 channels. The data presented each cell line was calculated from the experimentally are from at least three separate experiments. determined surviving cell fractions of cells treated with Data Analysis each drug alone, SFA,SFB, and SFC, and the surviving cell Statistical analyses were done using SPSS version 12.0 fraction for the triple drug combination, SFABC. The CI was for Windows. HER2 expression, cell growth, and drug treat- calculated as the observed surviving fraction/expected

Figure 2. Effects of drugs and drug combinations on cell proliferation, and correlation between growth inhibition and HER2 expression level. A, cells were plated and treated in duplicate with DMSO or human IgG (controls), 2 Amol/L of erlotinib alone (red columns), 1 Ag/mL of trastuzumab alone (yellow columns), 1 Ag/mL bevacizumab alone (light blue columns), erlotinib + trastuzumab (burgundy columns), erlotinib + bevacizumab (orange columns), trastuzumab + bevacizumab (dark blue columns), or a combination of all three drugs (black columns) for 6 days. Final cell numbers from the drug-treated samples were normalized to those of control cells treated with vehicle only. Columns, means; bars, SE. B, drug-induced growth inhibition, expressed as log surviving cell fraction, from trastuzumab (1 Ag/mL), erlotinib (2 Amol/L), bevacizumab (1 Ag/mL), IMC-A12 (1 Ag/mL), and the triple drug combination (trastuzumab at 1 Ag/mL, erlotinib at 2 Amol/L, bevacizumab at 1 Ag/mL) was plotted against relative HER2 expression levels of each cell line to determine the correlation between drug effects and HER2 expression level. Drug effect, as log of surviving cell fraction, is plotted on the ordinate, and mean HER2 expression is plotted on the abscissa. Top, the correlation coefficient (r) and P value (P) for each correlation. Bars, confidence intervals.

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Table 1. Comparisons of effects of drug combinations on surviving cell fraction by HER2 expression level

HER2 expression level One drug to two-drug regimen

E–ET E–EB E–TB T–ET T–EB T–TB B–ET B–EB B–TB

Low EEETTTBBB 0.86 F 0.02 0.86 F 0.02 0.86 F 0.02 0.95 F 0.02 0.95 F 0.02 0.95 F 0.02 0.85 F 0.03 0.85 F 0.03 0.85 F 0.03 ET EB TB ET EB TB ET EB TB 0.89 F 0.01 0.81 F 0.04 0.87 F 0.03 0.89 F 0.01 0.81 F 0.04 0.87 F 0.03 0.89 F 0.01 0.81 F 0.04 0.87 F 0.03 P = 0.487 P = 0.319 P = 0.749 P = 0.074 P = 0.026 P = 0.016 P = 0.439 P = 0.572 P = 0.603 Intermediate E E E T T T B B B 0.72 F 0.03 0.72 F 0.03 0.72 F 0.03 0.74 F 0.03 0.74 F 0.03 0.74 F 0.03 0.76 F 0.02 0.76 F 0.02 0.76 F 0.02 ET EB TB ET EB TB ET EB TB 0.57 F 0.02 0.68 F 0.02 0.66 F 0.02 0.57 F 0.02 0.68 F 0.02 0.66 F 0.02 0.57 F 0.02 0.68 F 0.02 0.66 F 0.02 P = 0.001 P = 0.212 P = 0.073 P < 0.001 P = 0.104 P = 0.014 P < 0.001 P = 0.036 P = 0.007 High EEETTTBBB 0.45 F 0.03 0.45 F 0.03 0.45 F 0.03 0.57 F 0.04 0.57 F 0.04 0.57 F 0.04 0.61 F 0.05 0.61 F 0.05 0.61 F 0.05 ET EB TB ET EB TB ET EB TB 0.39 F 0.01 0.46 F 0.03 0.44 F 0.03 0.39 F 0.01 0.46 F 0.03 0.44 F 0.03 0.39 F 0.01 0.46 F 0.03 0.44 F 0.03 P = 0.011 P = 0.166 P = 0217 P < 0.001 P = 0.112 P = 0.002 P = 0.005 P = 0.007 P = 0.017

NOTE: Paired t tests were done on the data from Fig. 2A to compare the results of treatment regimens by HER2 expression level group. The means (F SE), and P value (P) of single-drug treatments are compared with those of the two-drug regimens (One-drug to two-drug regimen), and to the three-drug regimen (One drug to three-drug regimen); also, the results of the two-drug regimens are compared with the three-drug regimen (Two-drug to three-drug regimen), and the data are grouped according to HER2 expression level (Low, Intermediate, High). Comparisons that are statistically significant (P < 0.05) are in boldface. Abbreviations: E, erlotinib; T, trastuzumab; B, bevacizumab; ET, erlotinib + trastuzumab; EB, erlotinib + bevacizumab; TB, trastuzumab + bevacizumab; ETB, erlotinib + trastuzumab + bevacizumab.

surviving fraction, in which the expected surviving fraction values were used to correlate drug effect with HER2 was the product of the measured surviving fractions for expression. each of the component drugs. Dose Response Effects of Targeted Agents Dose response curves were obtained in different cell ¼ =ð Þ lines for trastuzumab, erlotinib, bevacizumab, and the CI SFABC SFA SFB SFC anti–insulin-like growth factor-IR antibody IMC-A12 (Fig. 1C). For trastuzumab, the lowest drug concentration Triplicate analyses were subjected to two-sided statistical that produced the maximum or near-maximum drug tests (one group, two-tailed test with 2 df) to determine if effect was 1 Ag/mL (Fig. 1C1). For erlotinib, in the most the mean CI value for each cell line was significantly sensitive cell lines studied (SKBR3 and NH27), the IC different from a CI of 1.0 at P < 0.05. Synergy was defined 50 was close to 1 Amol/L (Fig. 1C2). For bevacizumab, SKBR3 by a CI value that was significantly less than 1.0, and was the most sensitive cell line, with an IC of close to antagonism was defined by a CI value significantly greater 50 1 Ag/mL (Fig. 1C3), with no further increase in drug effect than 1.0. Effects were considered additive if the CI was not at higher concentrations. In contrast to all other drugs, significantly different from 1.0 (43). IMC-A12 was most sensitive in the low HER2–expressing cell lines MCF7 and MDA-231, where the IC50 concentra- Results tion was 1 Ag/mL (Fig. 1C4). In subsequent studies, HER2 Expression in Human Breast Cancer Cell Lines trastuzumab, bevacizumab, and IMC-A12 were each used A panel of human breast cancer cell lines expressing at 1 Ag/mL. Erlotinib was used at 2 Amol/L, in keeping graded levels of HER2 was constituted from a group of with previously reported clinical steady state plasma unrelated cell lines obtained from the American Type concentrations in the range of 2 to 4 Amol/L (44, 45). Culture Collection (MDA-231, MCF7, MDA-361, SKBR3, These studies suggest that high HER2–expressing breast and BT 474), and HER2-stably transfected cell lines (NH29, cancer cell lines exhibit greater growth inhibition in res- NH47, and NH27) that were derived from parental MCF7 ponse to trastuzumab, erlotinib, or bevacizumab, than the cells as previously described (42). A representative blot of low HER2–expressing cell lines over a range of concen- HER2 expression level in each cell line is shown in Fig. 1A. trations. On the other hand, the low HER2–expressing The mean HER2 expression level in each cell line was breast cancer cell lines exhibit the greatest growth derived from densitometry measurements of multiple suppression with IMC-A12. The effects of IMC-A12 are HER2 immunoblots, normalized to the signal of BT474, shown here solely for contrast purposes, to demonstrate the highest HER2-expressing cell line (Fig. 1B). The cell that not all ligand or receptor-targeted drugs suppress lines were categorized into low, intermediate, and high growth in proportion to HER2 expression level. However, HER2–expressing groups, and the mean HER2 expression although we suggest here that the combinations of drugs,

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Table 1. Comparisons of effects of drug combinations on surviving cell fraction by HER2 expression level (Cont’d)

One drug to three-drug regimen Two-drug to three-drug regimen

E–ETB T–ETB B–ETB ET–ETB EB–ETB TB–ETB

E T B ETEBTB 0.86 F 0.02 0.95 F 0.02 0.85 F 0.03 0.89 F 0.01 0.81 F 0.04 0.87 F 0.03 ETB ETB ETB ETB ETB ETB 0.79 F 0.03 0.79 F 0.03 0.79 F 0.03 0.79 F 0.03 0.79 F 0.03 0.79 F 0.03 P = 0.159 P = 0.004 P = 0.229 P = 0.039 P = 0.538 P = 0.044 ETBETEBTB 0.72 F 0.03 0.74 F 0.03 0.76 F 0.02 0.57 F 0.02 0.68 F 0.02 0.66 F 0.02 ETB ETB ETB ETB ETB ETB 0.57 F 0.03 0.57 F 0.03 0.57 F 0.03 0.57 F 0.03 0.57 F 0.03 0.57 F 0.03 P = 0.003 P = 0.001 P < 0.001 P = 0.784 P = 0.005 P = 0.001 ETBETEBTB 0.45 F 0.03 0.57 F 0.04 0.61 F 0.05 0.39 F 0.01 0.46 F 0.03 0.44 F 0.03 ETB ETB ETB ETB ETB ETB 0.23 F 0.02 0.23 F 0.02 0.23 F 0.02 0.23 F 0.02 0.23 F 0.02 0.23 F 0.02 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001

the effects of which positively correlate with HER2 statistically significantly greater inhibition of cell prolifera- expression, should be considered for combination therapy, tion over any single drug or two-drug combination (Table 1), the effects of IMC-A12 suggest that one should not rule and whereas the overall magnitude of growth suppression out investigations of other combinations. IMC-A12 shows was relatively minor, produced the greatest growth a negative correlation with HER2 expression (Fig. 2), but inhibition overall in these experiments (Fig. 2A). can provide additional benefits in high HER2–expressing Correlation of growth-inhibitory effect of individual breast cancer cell lines in combination with trastuzumab drugs to HER2 expression level showed statistically (Supplemental Data),1 in keeping with the suggestion that significant (P = 0.05) and strong correlations in the different the insulin-like growth factor-IR can contribute to resis- cell lines in response to trastuzumab, erlotinib, and tance to trastuzumab (46). bevacizumab, and an inverse correlation between the Growth-Inhibitory Effects of Targeted Agent Combi- degree of growth inhibition and level of HER2 expression nations and Correlations of Drug Effects with Levels of in response to IMC-A12 (Fig. 2B). However, the strongest HER2 Expression correlation was achieved with the three-drug combination We compared the growth-inhibitory effects of individual (r = 0.974, P = 0.0001; Fig. 2B). CI were done for the three- erlotinib, trastuzumab, and bevacizumab with the effects of drug combination (ref. 43; Table 2). Overall, the CIs suggest all possible two-drug combinations and the three-drug that the combined drug effects were additive, but in the combination in a larger panel of human breast cancer cell low and intermediate HER2–expressing groups, in which lines (Fig. 2A). In the low HER2–expressing cell lines, the there were no statistically significant differences in drug single agents and the two-drug combinations produced effect between the single agents and the combinations, the growth inhibition that was in the range of 30% or less, and CI results are difficult to interpret. the three-drug combination provided no advantage over Evidence for VEGF Autocrine Loops in Breast Cancer the best single drug or two-drug combination. In cell lines Cell Lines with intermediate and high levels of HER2 overexpression, Although the role of bevacizumab as an antiangiogenic trastuzumab, erlotinib, and bevacizumab were progres- agent has been emphasized in studies in vivo (27–29), other sively more effective in inhibiting tumor cell growth, mechanisms must be invoked to account for its effects especially when used in combination. In the intermediate in vitro. Autocrine loops involving VEGF have been HER2–expressing cell line group, although the three-drug identified in human tumor cell lines and in various cancers combination and many two-drug combinations provided (30–32). To investigate the role of such autocrine loops in greater inhibition of proliferation over the single agents, not our studies, we did dose response studies on these cell lines all of the possible one-drug to two-drug comparisons were using a VEGFR inhibitor that has specificity for the VEGFRs statistically significant (Table 1). In the high HER2– Flt1 and KDR (Fig. 3A). This VEGFR inhibitor, like expressing group, the three-drug combination produced a bevacizumab, suppressed cell growth to a greater degree in high HER2–expressing cell lines (SKBR3, NH27, BT474), than in low HER2–expressing cell lines (MDA-231, MCF7). 1 Supplementary material for this article is available at Molecular Cancer Figure 3B shows the net production of VEGF (pg/mL/cell), Therapeutics Online (http://mct.aacrjournals.org/). secreted into the culture medium over 6 days of growth by

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the cell lines, which suggests that the cells expressing high reveals a greater effect of the triple drug combination over levels of HER2 produced the highest levels of VEGF per erlotinib alone (arrows, Fig. 4A5 and A6). cell. As shown in Fig. 3C, there is a statistically significant Quantitative comparisons of the cell cycle effects are correlation between VEGF production and the level of shown in Fig. 4B. The triple drug combination with erlotinib HER2 expression per cell (r = 0.883, P = 0.016). Trastuzu- at 2 Amol/L produced a modest increase of up to 1.2-fold in mab and erlotinib each reduced net VEGF production to the G1 cell fraction over the 72 h treatment period (Fig. 4B1), varying degrees in comparison with untreated controls and an f0.7-fold decrease in the fraction of cells in S phase (Fig. 3D), but there was no significant correlation between (Fig. 4B2), but there were no clear changes in the fraction of the degree of reduction of VEGF and HER2 expression. The cells in G2-M with either erlotinib alone or in the triple drug VEGF-reducing effect of bevacizumab could not be combination (Fig. 4B3). Because erlotinib has been used at determined by ELISA because the drug itself interferes conservative doses in the clinic (44, 45), we increased the with the assay. concentration of erlotinib to 10 Amol/L and examined its Inhibition of Cell Cycle Progression and Apoptosis effects alone and in the three-drug combination. G1 The inhibition of tumor cell growth may be due to the blockade (Fig. 4B4), and the decrease in the fraction of cells inhibition of cell cycle progression, due to apoptosis, or in S phase (Fig. 4B5) are more pronounced during the 72- both. We compared the effects of the three-drug combina- h observation period, and a late decrease in the fraction of tion to the effects of erlotinib alone on these processes in the cells in G2-M can be observed (Fig. 4B6). high HER2–expressing cell line SKBR3 (Fig. 4). Serial DNA Figure 4C shows the normalized cell concentration and histograms obtained over 72 h from the initiation of the corresponding apoptotic fractions at the end of 72 h in continuous drug exposure are shown for untreated control control cells and triple drug–treated cells with erlotinib at cells (Fig. 4A1), erlotinib alone at a concentration of 2 and 10 Amol/L. There was no substantial net decrease 2 Amol/L (Fig. 4A2), and the three-drug combination with in total cell concentration, even with the high erlotinib erlotinib at 2 Amol/L (Fig. 4A3). The effects of erlotinib concentration (Fig. 4C1), suggesting that much of the alone on the G1 cell fraction over time seem relatively small growth-inhibitory effects observed in our studies might (compare Fig. 4A2 with Fig. 4A1), but they persist in be due to transient cell cycle arrest rather than to apoptosis. replicate studies (see Fig. 4B1). To better appreciate the The time course of the apoptotic cell fraction measured contribution of erlotinib to G1 cell cycle blockade, we used by the terminal transferase dUTP nick end labeling assay etoposide, a topoisomerase II inhibitor, which blocks cells (see Materials and Methods), showed that considerable in the G2 phase of the cell cycle. This agent prevents cells apoptosis was observed after only 24 h with exposure to the from proceeding through mitosis and reentering G1, where drug combination with the 10 Amol/L erlotinib concentra- they might obscure the G1 blocking effects of the targeted tion (Fig. 4C2). agents. When the effects of erlotinib plus etoposide are compared with those of etoposide alone, the G1 blocking effect of erlotinib is shown more clearly by 72 h of Discussion treatment (see arrows at 72 h in Fig. 4A4 and A5). When In a previous study using a panel of clonally related breast comparing the G1 blocking effects of the three-drug cancer cell lines (42) we showed that a combination of combination to erlotinib alone, adding etoposide also trastuzumab and AG1478 (an EGFR inhibitor), produced

Table 2. Mean survival fractions for the individual drugs and the three-drug combination, and CI for the three-drug combination treatment

Mean survival Mean survival Mean survival Mean expected Mean observed Mean CI P value fraction with fraction with fraction with survival fraction survival fraction with the (CI) erlotinib trastuzumab bevacizumab (for additivity) erlotinib + trastuzumab + bevacizumab combination

MCF7 0.863 F 0.076 0.948 F 0.055 0.841 F 0.098 0.698 F 0.168 0.839 F 0.035 1.260 F 0.333 0.215 MDA-231 0.852 F 0.052 0.971 F 0.053 0.870 F 0.024 0.718 F 0.024 0.703 F 0.108 0.978 F 0.140 0.778 NH29 0.749 F 0.043 0.805 F 0.021 0.833 F 0.036 0.502 F 0.022 0.572 F 0.092 1.134 F 0.141 0.153 NH47 0.773 F 0.014 0.768 F 0.045 0.736 F 0.031 0.438 F 0.047 0.592 F 0.028 1.370 F 0.206 0.091 MDA-361 0.656 F 0.083 0.654 F 0.063 0.712 F 0.051 0.309 F 0.083 0.566 F 0.135 1.860 F 0.422 0.072 NH27 0.428 F 0.066 0.639 F 0.035 0.611 F 0.078 0.170 F 0.051 0.195 F 0.081 1.126 F 0.306 0.550 SKBR3 0.485 F 0.059 0.643 F 0.055 0.490 F 0.095 0.155 F 0.046 0.252 F 0.091 1.710 F 0.730 0.095 BT474 0.583 F 0.094 0.410 F 0.029 0.794 F 0.065 0.188 F 0.026 0.157 F 0.069 0.807 F 0.256 0.230

NOTE: Mean survival fraction values for the individual drug treatments and the three-drug treatment, and CI values for the three-drug treatment were obtained for each cell line from triplicate studies shown Fig. 2A. The P values for each cell line indicate the level of statistical significance of the CI compared with a CI of 1.0. Mean CI values significantly greater than 1.0 (P < 0.05), indicate antagonism, values not significantly different from 1.0 (P < 0.05) indicate additivity, and values less than 1.0 (P < 0.05) indicate synergy (FSE).

Mol Cancer Ther 2007;6(10). October 2007

Figure 3. VEGF and VEGFR inhibition. A, dose response effects of a VEGFR inhibitor with activity against flt-1 and KDR (Calbiochem) used at 0, 0.1, 0.5, 2.0, 10, and 50 Amol/L. Points, means; bars, SE. B, medium from cell lines cultured for 6 days was tested for VEGF by ELISA. Net VEGF production (pg/mL/cell) was derived using a standard provided in the kit and cell counts. Columns, means; bars, SE. C, correlation between VEGF production and HER2 expression level. Net VEGF production (pg/mL/cell) was plotted against the normalized HER2 expression levels from the immunoblot data (see Fig. 1). Top, the correlation coefficient (r) and P value (P). Bars, confidence intervals. D, cell lines were cultured as in (B) with the addition of treatment with 2 Amol/L of erlotinib (black columns)or 1 Ag/mL of trastuzumab (gray columns). Net VEGF production in the drug-treated samples was normalized to the net VEGF production in untreated samples. Columns, means; bars, SE.

greater growth inhibition than the effects of either drug response rates as a first line agent (20), and has increased the individually, and that the magnitudes of the effect of these time to progression in combination with capecitabine in agents correlated with the levels of HER2 expression. In this patients with breast cancer refractory to trastuzumab. article, we have examined the effects of trastuzumab, The rationale for pursuing bevacizumab in combination erlotinib (a clinically relevant EGFR inhibitor), and the with other agents has been based largely on its known VEGF-inhibiting antibody bevacizumab on cell prolifera- antiangiogenic effects (27–29). In addition, there are active tion, cell cycle arrest, and apoptosis in relation to levels of autocrine growth loops involving VEGF in various tumor HER2 expression in a panel of unrelated human breast cell types including breast cancer cells (30–32). Because the cancer cell lines. We have shown that erlotinib (like AG1478), antiangiogenic effects of bevacizumab are not applicable produces growth inhibition that varies directly with the level in vitro, the growth-inhibitory effects of this agent in our of HER2 expression (Fig. 2A and B). This finding addition- studies (Figs. 1 and 2) might well be due to interference ally supports the possibility that HER2 expression level with autocrine VEGF signaling loops. Our findings that the might be useful as a clinical marker for the combination of effects of an agent targeted against VEGFR were similar to anti-HER2 and anti-EGFR therapies in breast cancer. those of bevacizumab support this premise (Fig. 3A). That Clinical trials investigating the effects of various two-drug is, they suggest that VEGFRs are present and active in our combinations of trastuzumab, erlotinib, and bevacizumab cell lines, and that interference with their function are currently under way in several different cancer types, produced effects that that were comparable to those with promising results (20). An early trial of the combination produced by interfering with VEGF itself. The finding that of erlotinib and trastuzumab in patients with trastuzumab- both agents were more effective in cell lines with high resistant breast cancer suggested that the two-drug combi- HER2 overexpression suggests the possibility that VEGF nation might overcome trastuzumab resistance (20). Lapa- autocrine loops might be more active in tumors with high tinib, a drug that inhibits both EGFR and HER2, has shown HER2 expression.

Mol Cancer Ther 2007;6(10). October 2007

The activation of HER2 and/or EGFR signaling pathways drug combinations or single agents in the cell lines with is known to lead to increased VEGF production (33, 34, the highest levels of HER2 expression (Fig. 2A; Table 1). CI 37, 39). This is supported by our findings that trastuzumab analysis indicates that the effect of these agents in the three- and erlotinib each reduced net VEGF production in the drug combination is neither antagonistic nor synergistic tumor cell lines (Fig. 3D). This raises the possibility that the (Table 2), suggesting that the effects are additive. Although growth-inhibitory effects of trastuzumab and erlotinib in the overall magnitude of effects of all individual drugs and breast cancer might be due in part to the disruption of drug combinations are minor, the three-drug combination VEGF autocrine loops. However, neither trastuzumab nor provided the best suppression of growth. erlotinib reduced net VEGF production in a manner that These findings support the pursuit of breast cancer directly correlated with HER2 expression level, suggesting clinical trials using the three-drug combination, or the use that the effects of these drugs on VEGF production are not of small molecule inhibitors that have shown activity the sole mechanism for their growth suppression. against HER2, EGFR and VEGFR, such as ZD6474 We have shown that the three-drug combination of (Zactima) or AEE788 (20, 47). Based on our studies, one trastuzumab, erlotinib, and bevacizumab produced a sta- might anticipate that these agents would be even more tistically significant decrease in growth over any of the two- effective against tumors that express very high levels of

Figure 4. Effects of drug treatment on the cell cycle and apoptosis. A, DNA histograms of drug effects on cell DNA content and cell cycle perturbation at 0, 12, 24, 36, 48, and 72 h of drug treatment. A1, no drug; A2, 2 Amol/L of erlotinib; A3, three-drug combination with 2 Amol/L of erlotinib; A4, etoposide alone; alone; A5, etoposide + 2 Amol/L of erlotinib; A6, etoposide + the three-drug combination with 2 Amol/L of erlotinib. Arrows, G1 peak. B, normalized drug- induced changes in the fractions of cells in the G1, S, and G2-M phases of the cell cycle. B1 – 3, changes in the fractions with cells treated with no drug, erlotinib alone at 2 Amol/L, or the triple drug combination with erlotinib at 2 Amol/L. B4 – 6, changes in the fractions with cells treated with no drug, erlotinib alone at 10 Amol/L, or the triple drug combination with erlotinib at 10 Amol/L. C, effects of the concentration of erlotinib in the triple drug combination on net cell proliferation (1) and on total apoptosis as assayed by terminal transferase dUTP nickend labeling ( 2).

Mol Cancer Ther 2007;6(10). October 2007

HER2 than trastuzumab alone. However, we would increase with erlotinib dose escalation, suggesting that dose emphasize that our in vitro studies do not take into account response may be an issue for targeted agents that might the additional antiangiogenic effects of these agents, and deserve attention in the clinical setting. the potential effects of antibody-dependent cell-mediated cytotoxicity associated with trastuzumab and other anti- Acknowledgments in vivo bodies (48), which apply only , and which may act We thankGenentech for providing the trastuzumab and bevacizumab, OSI independently of HER2 expression levels. Pharmaceuticals for providing the erlotinib, and ImClone Systems for The observed effects of therapeutic agents in the growth providing IMC-A12. inhibition assay may be due to general cytostasis, due to the transient blockade of cells in one or more phases of the cell References cycle, or due to cell death, commonly through apoptosis. In 1. 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