Research Article Insulin-like Growth Factor-I Receptor/Human Epidermal Growth Factor Receptor 2 Heterodimerization Contributes to Trastuzumab Resistance of Breast Cancer Cells Rita Nahta,1 Linda X.H. Yuan,1 Bing Zhang,1 Ryuji Kobayashi,2 and Francisco J. Esteva1,3,4 Departments of 1Breast Medical Oncology, 2Molecular Pathology, and 3Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center and 4The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas Abstract against the extracellular domain of the human epidermal growth The majority of breast cancer patients who achieve an factor (EGF) receptor 2 (HER-2) tyrosine kinase receptor (2). initial therapeutic response to the human epidermal growth Clinical studies established that trastuzumab is active against factor receptor 2 (HER-2)–targeted antibody trastuzumab HER-2-overexpressing metastatic breast cancers (3–5), leading to will show disease progression within 1 year. We previously its approval in 1998 by the U.S. Food and Drug Administration. reported the characterization of SKBR3-derived trastuzumab- However, the objective response rates to trastuzumab mono- resistant pools. In the current study, we show that HER-2 therapy are quite low, ranging from 12% to 34% for a median interacts with insulin-like growth factor-I receptor (IGF-IR) duration of 9 months (4, 5). The majority of patients who achieve an initial response to trastuzumab-based therapy acquire uniquely in these resistant cells and not in the parental trastuzumab-sensitive cells. The occurrence of cross talk resistance within 1 year of treatment initiation (6, 7). Elucidating between IGF-IR and HER-2 exclusively in resistant cells is the mechanisms by which tumors escape the cytotoxic properties evidenced by the IGF-I stimulation resulting in increased of trastuzumab is critical to improving the survival of metastatic phosphorylation of HER-2 in resistant cells, but not in breast cancer patients whose tumors overexpress HER-2. parental cells, and by the inhibition of IGF-IR tyrosine Trastuzumab resistance has been associated with overexpres- kinase activity leading to decreased HER-2 phosphorylation sion of the insulin-like growth factor-I receptor (IGF-IR; ref. 8). only in resistant cells. In addition, inhibition of IGF-IR We previously reported the characterization of a pair of tyrosine kinase activity by I-OMe-AG538 increased sensitivity trastuzumab-resistant pools derived from the SKBR3 HER-2- of resistant cells to trastuzumab. HER-2/IGF-IR interaction overexpressing breast cancer cell line (9). In the current study, we extend this characterization by showing that (a) IGF-IR was disrupted on exposure of resistant cells to the anti–IGF- IR antibody A-IR3 and, to a lesser extent, when exposed to interacts with HER-2 exclusively in trastuzumab-resistant cells; the anti-HER-2 antibody pertuzumab. Heterodimer disrup- (b) IGF-I induces phosphorylation of HER-2 in resistant cells, tion by A-IR3 dramatically restored sensitivity to trastuzu- but not parental cells; and (c) The IGF-IR tyrosine kinase mab and resistant cells showed a slightly increased inhibitor I-OMe-AG538 reduces HER-2 phosphorylation only in sensitivity to pertuzumab versus parental cells. Neither the resistant cells. These data suggest that cross talk occurs A-IR3 nor pertuzumab decreased HER-2 phosphorylation, between IGF-IR and HER-2 in trastuzumab-resistant breast suggesting that additional sources of phosphorylation other cancers that show HER-2/IGF-IR heterodimerization. Disruption than IGF-IR exist when HER-2 and IGF-IR are not physically of the IGF-IR/HER-2 heterodimer by the anti–IGF-IR antibody a bound. Our data support a unique interaction between HER-2 -IR3 or the anti-HER-2 antibody pertuzumab did not affect and IGF-IR in trastuzumab-resistant cells such that cross talk HER-2 phosphorylation, suggesting that additional sources of occurs between IGF-IR and HER-2. These data suggest that HER-2 phosphorylation exist in the absence of dimerization the IGF-IR/HER-2 heterodimer contributes to trastuzumab with IGF-IR. Importantly, IGF-IR–targeted agents restored trastuzumab sensitivity to resistant cells, supporting further resistance and justify the need for further studies examining this complex as a potential therapeutic target in breast cancers study of such agents for use in the context of trastuzumab- that have progressed while on trastuzumab. (Cancer Res 2005; resistant breast cancers. 65(23): 11118-28) Introduction Materials and Methods The her-2 (erbB2/neu) gene is amplified and/or overexpressed Materials. Trastuzumab (Genentech) was dissolved in sterile water at a stock concentration of 20 mg/mL. IGF-I (Sigma-Aldrich, St. Louis, MO) in f20% to 30% of invasive breast carcinomas and is associated was dissolved at 100 Ag/mL in PBS and used at 100 ng/mL. The IGF-IR with increased metastatic potential and decreased overall survival tyrosine kinase inhibitor tyrphostin I-OMe-AG538 (Calbiochem, San Diego, (1). Trastuzumab (Herceptin; Genentech, South San Francisco, CA) was dissolved in DMSO at 10 mmol/L and used at a working CA) is a recombinant humanized monoclonal antibody directed concentration of 1 or 10 Amol/L. The anti–IGF-IR antibody clone a-IR3 was purchased from Calbiochem. Genentech provided the anti-HER-2 antibody pertuzumab (2C4), which was dissolved in sterile water at a final Requests for reprints: Rita Nahta or Francisco J. Esteva, Department of Breast concentration of 25 mg/mL. Medical Oncology, Unit 1354, The University of Texas M.D. Anderson Cancer Center, Cell culture. SKBR3 parental and trastuzumab-resistant breast cancer 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713-792-2817; Fax: 713- cells were maintained in DMEM supplemented with 10% FCS. Trastuzu- 563-0739; E-mail: [email protected] or [email protected]. I2005 American Association for Cancer Research. mab-resistant SKBR3 pools were developed as previously described (9). doi:10.1158/0008-5472.CAN-04-3841 Pools were maintained in 4 Ag/mL trastuzumab. Cancer Res 2005; 65: (23). December 1, 2005 11118 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. IGF-IR/HER-2 Contributes to Trastuzumab Resistance Immunoprecipitation. Parental and trastuzumab-resistant cells were Results lysed in buffer containing 10 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, and protease and phosphatase inhibitor Heterodimerization between insulin-like growth factor-I cocktails (Sigma Chemical Co., St. Louis, MO). IGF-IR was immunopre- receptor and human epidermalgrowthfactorreceptor cipitated (polyclonal; Cell Signaling, Beverly, MA) from total protein 2 occurs uniquely in trastuzumab-resistant cells. Reduced extracts (200 Ag) overnight, washed in PBS-0.1% Tween 20 buffer, and sensitivity to trastuzumab was previously associated with immunoblotted to detect phosphotyrosine (monoclonal PT66; Sigma increased expression of IGF-IR in the SKBR3 breast cancer cell Chemical), IGF-IR (polyclonal; Cell Signaling), or HER-2 (monoclonal line (8). Thus, we examined our SKBR3-derived trastuzumab- Ab-3; Oncogene Research Products, EMD Biosciences, Inc., San Diego, CA). resistant pools for expression and phosphorylation of IGF-IR. Immunoblotting. Cells were lysed in buffer containing 10 mmol/L SKBR3 parental cells express low levels of IGF-IR (8) and these Tris (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, and protease levels were unchanged in resistant cells (Fig. 1A). Phospho-IGF-IR and phosphatase inhibitor cocktails (Sigma Chemical). Total protein levels were also similar among parental and resistant cells as extracts (50 Ag) were immunoblotted using the following antibodies at shown by immunoprecipitation of IGF-IR followed by immuno- the indicated dilutions: polyclonal antibodies for EGF receptor (EGFR), blotting with an anti-phosphotyrosine antibody (monoclonal integrin h3, and platelet-derived growth factor receptor (PDGFR) at 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal p-Tyr1162/ PT66), which detected phospho-IGF-IR at the expected molecular 1163 IR/IGF-IR at 1:500 (BioSource); HER-2 monoclonal Ab-3 at 1:1,000 weight of 90 kDa (Fig. 1B). In addition to the 90-kDa band, a f (Oncogene Research Products); p-Ser473-Akt (monoclonal 587F11) and phosphoprotein migrating at 185 kDa (Fig. 1B, asterisk) was polyclonal antibodies against IGF-IR, insulin receptor substrate 1 (IRS-1), detected in the IGF-IR immunoprecipitates. Interestingly, this Akt, p-Thr202/Tyr204 p42/p44 mitogen-activated protein kinase (MAPK), protein was detected exclusively in the resistant cells. Peptide and total p42/p44 MAPK at 1:1,000 (Cell Signaling); polyclonal against analysis identified this 185-kDa protein as HER-2 (Fig. 1C). The p-Ser789-IRS-1 and polyclonal against p-Tyr1248-HER-2 at 1:500 (Upstate identity was confirmed by immunoprecipitating IGF-IR and h Biotechnology, Lake Placid, NY); p27 polyclonal at 1:250 and -actin immunoblotting for HER-2 (Fig. 1D). Again, HER-2 interacted monoclonal at 1:5,000 (Santa Cruz Biotechnology). Secondary antibodies with IGF-IR only in resistant cells, not in parental cells. Conversely, were chosen according to the species of origin of the primary antibody IGF-IR was detected in HER-2 immunoprecipitates from resistant and detected by enhanced chemiluminescence (Amersham-Pharmacia cells (Fig. 1D). To determine the specificity of IGF-IR interaction Biotech, Piscataway, NJ) or by using the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE). with HER-2, IGF-IR immunoprecipitates were immunoblotted h Mass spectrometry. The silver-stained gel bands were manually cut, for EGFR, integrin 3, and PDGFR (Fig. 1E). Faint bands were destained, and digested with sequencing-grade trypsin (Promega, Madison, observed in resistant cell lysates blotted for EGFR, which likely WI). Peptides were extracted with 100 AL of 50% acetonitrile/0.01% represents cross-reactivity of the EGFR antibody with HER-2. trifluoroacetyl or trifluoroacetic acid (TFA).