Co-targeting ER and HER Family Receptors Induces Apoptosis in HER2-normal or Overexpressing Breast Cancer Models

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1 Co-targeting ER and HER Family Receptors Induces Apoptosis in HER2-normal or Overexpressing Breast Cancer Models ASHOK K. CHAKRABORTY, RANEE MEHRA and MICHAEL P. DIGIOVANNA Departments of Internal Medicine (Section of Medical Oncology) and Pharmacology, and the Yale Cancer Center/Smilow Cancer Hospital, Yale University School of Medicine, New Haven, CT, U.S.A. Abstract. Background: Estrogen receptor (ER) and human epidermal growth factor receptor (HER) family receptors interact in breast cancer; co-targeting these receptors is of interest. We previously reported on a synergistic growth inhibition for the combination of trastuzumab plus tamoxifen in HER2 + /ER + BT474 cells, but no induction of apoptosis. Herein we describe the effects of co-targeting in models of differing HER2 overexpression status (MCF7 HER2-normal/ER +, BT474 HER2-overexpressing/ER + ). Materials and Methods: Assays of proliferation were carried-out using WST-1, cell cycle using flow cytometry, and apoptosis by determination of sub-g 1 population and by annexin-v. Results: Combining a dual HER2/EGFR kinase inhibitor with anti-estrogens induces apoptosis of BT474 cells. Furthermore, in MCF7 cells, despite HER2-normal status and lack of response to single-agent HER2 inhibitors, addition of HER2 inhibitors or dual HER2/EGFR inhibitor to antiestrogens augments the anti proliferative effect of anti-estrogens, and converts the drug effect from cytostatic to apoptosisinducing. Conclusion: ER-HER co-targeting enhanced the antitumor effects and can bring about effects of targeting HER2 in models of HER2-normal breast cancer. A large body of literature has demonstrated interactions between the estrogen receptor (ER) and human epidermal growth factor receptor (HER) family of receptors in breast cancer. Hence, co-targeting these two receptors for therapeutic gain has been the subject of intense interest. We have shown Correspondence to: Michael P. DiGiovanna, Department of Internal Medicine (Section of Medical Oncology), and the Yale Cancer Center/Smilow Cancer Hospital, Yale University School of Medicine, 300 George St., Suite 120, New Haven, CT 06510, U.S.A. michael.digiovanna@yale.edu Current address: Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 10111, U.S.A. Key Words: Breast cancer, estrogen receptor (ER), HER2, EGFR, tamoxifen, trastuzumab (Herceptin), fulvestrant (Faslodex), GW2974, proliferation, apoptosis. that in the ER +, HER2-overexpressing human breast cancer cell line BT474, the combination of trastuzu mab plus tamoxifen results in synergistic anti-proliferative effects, enhanced effects on the cell cycle, and reduction of clonogenicity, but no induction of apoptosis (1), indicating a purely cytostatic effect in vitro. Given the known importance of the multiple HER family receptors in HER2 signaling effects, the implication of HER2 and epidermal growth factor receptor (EGFR) in acquired endocrine resistance, and the clinical availability of dual-targeting drugs, in the current study we explored the antitumor effect of HER2-specific and HER2/EGFR dualtargeting drugs in combination with anti-estrogen therapies, in HER2-overexpressing and HER2-normal cell lines. Materials and Methods Cell culture. BT474 cells, obtained from the American Type Culture Collection (Manassas, VA, USA), and MCF7 cells, a gift from Marc Lippman (University of Michigan), were cultured as described previously (2). To sensitize MCF7 cells to the effects of estrogen and anti-estrogens, before each experiment, cells were cultured for five days in estrogen-free medium; estradiol was then added, with or without drugs, at the start of each experiment. Drugs. 4-Hydroxy-tamoxifen (4HT) was purchased from Sigma (St. Louis, MO, USA). Fulvestrant (Faslodex, ICI 182,780) was obtained from the Yale Cancer Center Medical Oncology pharmacy. Both drugs were diluted in 100% ethanol to make stock concentrations of 10 and 2 mm, respectively. The stocks were further diluted in sterile phosphate-buffered saline (PBS) and/or culture media before addition to cells in culture. Trastuzumab (Herceptin) was also obtained from the Yale Cancer Center Medical Oncology pharmacy; a 20 mg/ml stock preparation was kept at 4 C and was further diluted in sterile PBS before addition to cells in culture. AG825, a HER2 specific tyrosine kinase inhibitor (TKI), and GW2974, a dual HER2/EGFR TKI, were purchased from Calbiochem (San Diego, CA, USA) and Sigma, respectively. All TKIs were dissolved in dimethyl sulfoxide to make 10 mm stock solutions. Assays of proliferation, cell cycle, and apoptosis. Assays of proliferation using the WST-1 tetrazolium salt colorimetric growth method, cell-cycle distribution by flow cytometry, and apoptosis by sub-g 1 population analysis and annexin-v assay were performed as /2015 $

2 Figure 1. Effect of antagonists of human epidermal growth factor receptor 2 and estrogen receptor on MCF7 cell proliferation. MCF7 cells in exponential growth phase were seeded on day -1 in 96-well plates at cells/well. They were allowed to adhere overnight and then treated with drugs the following day (day 0) with the indicated doses. After six days of incubation, the WST-1 proliferation assay was performed. Results are expressed as a percentage of that of the control (vehicle-treated cells). Data are the mean±sd of four wells from at least three experiments. described previously (3). See individual figure legends for controls, number of replicates and statistical analyses. Results Cell line models. Two ER + human breast cancer cell lines that differ in their HER2 expression level were chosen for these studies. BT474 is estrogen-dependent for growth, and highly overexpresses HER2 and EGFR in association with HER2 gene amplification. MCF7 is ER + and estrogendependent for growth but expresses a low level (nonoverexpressed) of HER2 and EGFR. Effect of HER2 inhibitors alone and in combination with antiestrogens on proliferation of MCF7 cells. Dose-response curves for the effect on cell proliferation were generated for antagonists of HER2 and ER. We found that in MCF7 cells, which do not have HER2 overexpression, as expected, growth was not inhibited by the HER2 antagonists trastuzumab or AG825 (Figure 1C and D). However, as expected, these cells were sensitive to anti-estrogens, with IC 50 s for 4-HT and fulvestrant of ~5.0 μm and 0.7 μm, respectively (Figure 1A and B). When drug combinations were examined, although single-agent HER2 inhibitors had no growth-inhibitory activity, they enhanced the growth inhibition elicited by the ER antagonists (Figure 2); this was the case for both the monoclonal therapeutic antibody trastuzumab (Figure 2A) and the HER2 TKI AG825 (Figure 2B). The dual HER2/EGFR TKI GW2974 did show some single-agent growth-inhibitory activity against MCF7 cells 1244

3 Chakraborty et al: Co-targeting ER and HER Family Receptors Figure 2. Effect of human epidermal growth factor receptor 2 (HER2) antagonists in combination with estrogen receptor antagonists on MCF7 cell proliferation. HER2 antagonists trastuzumab (Herceptin; H in panel A) or AG825 (A in panel B) were combined with either 4-hydroxy-tamoxifen (4- HT; 1 μm) or fulvestrant (Fulv; 0.5 μm), as indicated. After six days of drug treatment WST-1 proliferation assay was performed. Results are expressed to a percentage of that of the control (vehicle-treated cells; V). Data are the mean±sd of four wells from at least three experiments. Significantly different at * p<0.05 and ** p<0.01. Numbers in designations indicate concentrations in corresponding units. (Figure 3A), albeit with less potency than for inhibition of HER2-overexpressing BT474 cells (Figure 3D). The addition of GW2974 to the anti-estrogens dramatically enhanced growth inhibition in both the MCF7 and BT474 cell lines (Figure 3B, C, E and F); in BT474 cells, the effect saturated at higher concentrations of GW2974. Effect of drugs on the cell cycle. Anti-estrogens are known to cause cell-cycle arrest in hormone-dependent breast cancer cells. In MCF7 cells, we found that both of the antiestrogens caused a small increase in the percentage of cells in the G 0 -G 1 phase of cell cycle, accompanied by a small decrease in the percentage of cells in the S phase. The addition of trastuzumab to the anti-estrogens caused a small further reduction in the percentage of cells in the S phase; the addition of AG825 caused a small further reduction in percentage of cells in the S phase when combined with 4- HT but not when combined with fulvestrant (Figure 4A). This result was also seen, to a somewhat greater degree, when the dual HER2/EGFR inhibitor GW2974 was added to the anti-estrogens for the MCF7 cell line (Figure 5A), although the result seemed to be saturated for the BT474 cell line (Figure 5D). Effect of drugs on apoptosis. Apoptosis was assayed after four days of drug treatment by analyzing the percent of cells in the sub-g 1 peak and by the annexin-v assay. We previously showed that neither trastuzumab, tamoxifen, nor their combination induced apoptosis of BT474 cells (1). Here we found that likewise, in MCF7 cells, antagonists of HER2 (trastuzumab and AG825) or ER (4-HT or fulvestrant) as single agents did not induce apoptosis; however, the combination of HER2 plus ER targeting induced a significant level of apoptosis (Figure 4 B and C). More striking levels of apoptosis were observed with the combination of dual EGFR/HER2 inhibitor plus anti-estrogen in both MCF7 and BT474 cells (Figure 5 B-F). These results are particularly remarkable because the MCF7 cell line does not overexpress either HER2 or EGFR. Discussion Signaling interactions between ER and HER family receptors has long been known and has implicated the HER family receptors in endocrine resistance. Conversely, one report showed that ER signaling can be a mechanism of resistance to lapatinib (4). This has resulted in significant interest in co-targeting these receptors. We previously showed that in the ER +, HER2-overexpressing BT474 cell line, the combination of trastuzumab plus tamoxifen resulted in synergistic antiproli ferative effects and enhanced effects on cell cycle and reduction of clonogenicity, but caused no induction of apoptosis (1), indicating a purely cytostatic effect in vitro. We also explored their 1245

4 Figure 3. Effect of dual epidermal growth factor receptor/ human epidermal growth factor receptor 2 tyrosine kinase inhibitor GW2974 (G) and estrogen receptor antagonists on MCF7 and BT474 cell proliferation. Cells in exponential growth phase were seeded on day -1 in 96-well plates at cells/well (for MCF7; A-C) and cells/well (for BT474; D-F). They were allowed to adhere overnight and then treated with drugs the following day (day 0) at the indicated doses. After six days of incubation, the WST-1 proliferation assay was performed. Results are expressed as the percentage of that of the control (vehicle-treated cells; V). Data are mean±sd of four wells from at least three experiments. Significantly different at * p<0.05, ** p<0.01 and *** p< HT: 4-Hydroxy-tamoxifen; Fulv: fulvestrant. Numbers in designations indicate concentrations (μm). 1246

5 Chakraborty et al: Co-targeting ER and HER Family Receptors Figure 4. Effect of human epidermal growth factor receptor 2 antagonists in combination with estrogen receptor antagonists on cell cycle (A) and apoptosis (B, C) of MCF7 cells. A: The cell-cycle distribution after two days of drug treatment. Results are expressed as the mean±sd from three different data points and expressed as percentage of cells in G 0 -G 1, S and in G 2 -M phases. B: SubG 1 population size after six days of drug treatment. Results are expressed as the mean±sd from three independent experiments. C: Annexin-V staining results. Results are expressed as mean±sd from three different experiments. Significantly different at * p<0.05 and *** p<0.005 when compared with vehicle control (V). Trast: Trastuzumab (Herceptin), 5 μg/ml; 4-HT: 4-hydroxy-tamoxifen, 1 μm; Fulv: fulvestrant, 0.5 μm; AG825, 10 μm. combination in vivo using BT474 xenografts, and found an enhanced inhibition of tumor growth for the combination compared to both as single agents (5). Given the above considerations and the clinical availability of HER2/EGFR dual-targeting drugs, in the current work we explored the effect of HER2-specific and HER2/EGFR dual inhibition in combination with anti-estrogen therapies in HER2-overexpressing and HER2-non-overexpressing cell lines. Our results showed that the dual HER2/EGFR inhibitor GW2974, an analog of the clinically used drug lapatinib, when combined with anti-estrogens, does induce high levels of apoptosis of BT474 cells. More dramatic results were observed in MCF7 cells: despite their HER2-normal status, we found that addition of HER2 inhibitors or the dual HER2/EGFR inhibitor to anti-estrogens augmented the antiproliferative effect of anti-estrogens, and induced apoptosis of these HER2-normal cells. Two large randomized clinical trials of co-targeting HER2 and ER have been conducted. In the TAnDEM study, the aromatase inhibitor anastrazole was given with or without trastuzumab for patients with metastatic ER + and HER2 + breast cancer (6). The response rate to the single-agent anastrazole was poor, at 6.8%, likely reflecting the known sub-optimal response of HER2 + breast cancer to endocrine therapy. The study met its primary endpoint in that the addition of trastuzumab did increase the progression-free survival significantly, from 2.4 months to 4.8 months, however, the result was not as robust as one might have hoped for based on aforementioned pre-clinical synergy; indeed, the response rate for the combination (20.3%) was not very different from that reported for single-agent trastuzumab (35%) in a similar patient population (7). Perhaps the lack of more robust results is mirrored in the lack of apoptosis-inducing activity for the trastuzumab/ tamoxifen combination in our BT474 model. 1247

6 Figure 5. Influence of dual epidermal growth factor receptor/human epidermal growth factor receptor 2 tyrosine kinase inhibitor GW2974 (G) alone and in combination with estrogen receptor antagonists on cell-cycle distribution and apoptosis of MCF7 (A-C) and BT474 (D-F) cells. Cell-cycle distribution after two days of drug treatment for MCF7 (A) and BT474 (D) cells. Results are expressed as the percentage of cells in G 0 -G 1, S and in G 2 -M phases. SubG 1 cell population sizes for MCF7 (B) and BT474 (E) cells are shown after four days of drug treatment. C and F: Annexin-V staining results. 4-HT: 4-Hydroxy-tamoxifen (A-C: 1 μm, D-F: 5 μm); Fulv: fulvestrant (A-C: 0.5 μm, D-F: 1 μm); GW2974: A-C: 0.5 μm, D-F: 0.1 μm. Data are expressed as the mean±sd from three independent experiments. Significantly different at * p<0.05 and ** p<0.01 when compared to the vehicle control (V). 1248

7 Chakraborty et al: Co-targeting ER and HER Family Receptors A second clinical trial examined the addition of the dual HER2/EGFR TKI lapatinib to the aromatase inhibitor letrozole for ER + breast cancer that did or did not over - express HER2 (8). For the HER2-overexpressing population, the progression-free survival was significantly increased for the lapatinib/letrozole combination compared to letrozole mono therapy (8.2 versus 3.0 months), as was the overall response rate (28% versus 15%) (8). This somewhat better result in the ER + /HER2-overexpressing population compared to that in the TAnDEM trial may too be reflected in our current results showing a dramatic apoptosis-inducing effect for the addition of the dual inhibitor GW2974 to antiestrogens in our BT474 model, which we did not observe with trastuzumab. However, for the HER2-normal patients in the clinical trial, the addition of lapatinib to letrozole did not improve outcome; thus, our MCF7 model failed to predict a similar result in the clinic. However, in an exploratory analysis of a predefined prior anti-estrogen therapy stratification, in the patients who had less than 6 months elapse since treatment with prior adjuvant tamoxifen, there was a trend for benefit of addition of lapatinib, with clinical benefit rate increasing from 32% to 44%, and median progression-free survival increasing from 3.1 to 8.3 months; similarly, in those with progesterone receptor-negative tumors, the clinical benefit rate increased from 15% to 36%. It is disappointing that in the clinical setting, lapatinib did not improve the response or delay the development of resistance to letrozole in the ER + /HER2-normal population. This suggests that in patients with breast cancer, additional escape mechanisms from antitumor effects may be operative. One such mechanism may utilize insulin-like growth factor I receptor (IGF1R) signaling; the IGF1R interacts with both HER2 and ER signaling, and IGF1R has been implicated in resistance to both HER2-directed therapy (9-11) and in doubly anti-estrogen/ anti-her family drug resistance (12). We have reported that IGF1R inhibitors enhance the antitumor effects of anti-estrogens in both the BT474 and MCF7 models, and that in both cell lines, the addition of IGF1R inhibitors to anti-estrogens resulted in dramatic levels of apoptosis induction (2). Similarly, we reported that the combination of IGF1R inhibitors and HER2 inhibitors resulted in synergistic antiproliferative effects in both of these cell lines, including the HER2-normal MCF7, and that the combinations in both cell lines resulted in dramatic levels of apoptosis despite none of the drugs used inducing apoptosis as single agents (3). This suggests that for the HER2-normal patient population, inhibiting both the HER family and IGF family axes may be a more fruitful approach to treating patients with ER +, HER2-normal breast cancer. Our ongoing work is exploring such drug combinations in preclini cal studies. In summary, ER/HER co-targeting may be an effective strategy in yet-to-be-defined sub-populations of patients with breast cancer, or additional mechanisms might be targeted therapeutically to make this strategy more successful in the clinic. Acknowledgements This study was supported by grants from the Breast Cancer Research Foundation to MPD. We thank Dr. Mark Lippman for MCF7 cells and advice on their use, Dr. Rocco Carbone of the Yale Cancer Center Flow Cytometry shared resource (supported by US Public Health Service Grant CA from the National Cancer institute) for assistance with flow cytometry, and Cynthia Zerillo for helpful suggestions. 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8 10 Nahta R, Yuan LXH, Zhang B, Kobayashi R and Esteva FJ: Insulinlike growth factor-i receptor/human epidermal growth factor recep - tor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells. Cancer Research 65(23): , Harris LN, You F, Schnitt SJ, Witkiewicz A, Lu X, Sgroi D, Ryan PD, Come SE, Burstein HJ, Lesnikoski BA, Kamma M, Friedman PN, Gelman R, Iglehart JD and Winder EP: Predictors of resistance to preoperative trastuzumab and vinorelbine for HER2-positive early breast cancer. Clinical Cancer Research 13: , Jones HE, Goddard L, Gee JMW, Hiscox S, Rubini M, Marrow D, Knowlden JM, Williams S, Wakeling AE and Nicholson RI: Insulin-like growth factor-1 receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and pro - state cancer cells. Endocrine-Related Cancer 11(4): , Received November 20, 2014 Revised December 12, 2014 Accepted December 15,

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J Clin Oncol 27: by American Society of Clinical Oncology INTRODUCTION VOLUME 27 NUMBER 33 NOVEMBER 9 JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T From the Royal Marsden Hospital, London; GlaxoSmithKline, Middlesex, United Kingdom; Sammons Cancer Center, Dallas,