Insulin-Like Growth Factor-I Receptor/Human Epidermal Growth Factor Receptor 2 Heterodimerization Contributes to Trastuzumab Resistance of Breast Cancer Cells

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.

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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). The digests were then dried IGF-IR interaction with integrin h3 was similar between parental down to 20 AL. Five microliters of each sample were analyzed by liquid and pool 1 cells and less in pool 2 cells. PDGFR did not interact chromatography-tandem mass spectrometry (LC-MS/MS). Briefly, the with IGF-IR in any of the lysates. These data show that IGF-IR peptides were separated by high-performance liquid chromatography specifically interacts with HER-2 in resistant cells as association of (Ultimate, LC Packings, Sunnyvale, CA) using a 150 Am Â 10 cm reversed- IGF-IR with a nontyrosine kinase receptor transmembrane protein phase C18 column (Grace Vydac, Hesperia, CA). The peptides were eluted (integrin h3) or non-EGFR tyrosine kinase receptor (PDGFR) was with an increasing gradient of buffer B (60% acetonitrile/20% isopropanol/ not increased in resistant cells relative to parental cells. In 0.01% TFA) from 5% to 60% in 45 minutes. Eluted peptides from the column were ionized by electrospray and analyzed by ion-trap MS addition, total levels of proteins with phosphotyrosine (monoclo- (LCQdecaXP, Thermo Finnigan, Waltham, MA). The data were searched nal PT66) and total and phosphorylated HER-2 were similar among against the National Center for Biotechnology Information human protein parental cells and pools (Fig. 1F). These data show that IGF-IR and database with the SonarMSMS software (Genomic Solutions). HER-2 interact exclusively in trastuzumab-resistant cells. Insulin-like growth factor-I stimulation. Parental SKBR3 and Evidence of cross talk between insulin-like growth factor-I resistant cells were serum starved overnight using serum-free DMEM receptor and human epidermal growth factor receptor 2 in medium. The next day, 100 ng/mL IGF-I was added directly to culture trastuzumab-resistant cells. Next, we examined the effect of medium for the indicated time points. At each time point, medium was IGF-IR on HER-2 phosphorylation in SKBR3 parental and resistant aspirated, cells were washed with PBS, and 150 AL of lysis buffer cells. Cells were serum starved overnight and stimulated with (described in Immunoblotting) were added to cells for 30 minutes. Cells IGF-I (100 ng/mL) for the indicated time points. Within 5 minutes, were scraped and collected and stimulation of IGF-IR phosphorylation was examined by immunoblotting using BioSource polyclonal p-Tyr1162/ IGF-IR phosphorylation increased in parental cells (Fig. 2A). 1163 IR/IGF-IR at 1:500. Phosphorylation of HER-2 on Tyr1248, a major phosphorylation Cell survival assays. Parental cells or resistant pools were seeded at site involved in the oncogenic function of HER-2, was slightly a density of 5 Â 104 cells per well in 12-well dishes. The next day, cells decreased in IGF-I-stimulated parental cells, possibly due to were treated with either a-IR3 (0.25, 0.5, or 1 Ag/mL) or I-OMe-AG538 feedback mechanisms because IGF-IR and HER-2 share common (1, 5, or 10 Amol/L). After 24 hours, medium was aspirated and replaced signaling pathways. Downstream of IGF-IR, IRS-1, phosphatidyli- with medium containing trastuzumab at concentrations of 0, 1, 5, or nositol 3-kinase/Akt, and MAPK signaling were activated as shown 20 Ag/mL for an additional 72 hours. Parallel cultures were treated with by increased phosphorylation of Ser789 on IRS-1, Ser473 on Akt, a-IR3 alone for 24 hours, I-OMe-AG538 alone for 24 hours, or trastuzumab and Thr202 and Tyr204 on p42 MAPK and p44 MAPK. In addition, alone for 72 hours, each at the same doses as stated above. Additionally, IGF-I stimulation of SKBR3 cells resulted in decreased levels of parental and trastuzumab-resistant cells were treated for 5 days with p27kip1, consistent with data reported by Lu et al. (10). 2-fold serial dilutions of pertuzumab ranging from 3.125 to 50 Ag/mL. For each experiment, cells were trypsinized and stained with trypan blue IGF-I stimulation of resistant cells increased phosphorylation of dye and viable cells were counted under a microscope. All experiments IGF-IR within 1 minute (Fig. 2B). In contrast to parental cells, HER-2 were done in triplicate and repeated at least twice. Cell growth inhibition phosphorylation on Tyr1248 increased in trastuzumab-resistant cells for all experiments is expressed as the percentage of viable cells compared within 15 minutes of IGF-I exposure. Total levels of IGF-IR and HER-2 with untreated cells. were unaffected. Similar to parental cells, IRS-1, Akt, and MAPK www.aacrjournals.org 11119 Cancer Res 2005; 65: (23). December 1, 2005

Figure 1. Heterodimerization between IGF-IR and HER-2 occurs uniquely in trastuzumab-resistant cells. A, relative levels of total IGF-IR were assessed in parental cells and resistant pools by immunoblotting. B, IGF-IR was immunoprecipitated (200 Ag) from whole-cell lysates of parental and resistant cells and immunoblotted for phosphotyrosine (monoclonal PT66). A 90-kDa band corresponding to phospho-IGF-IR (p-IGF-IR) was detected in all immunoprecipitates. A 185-kDa phosphoprotein (asterisk) was also observed and was found exclusively in trastuzumab-resistant cells (pool 1 and pool 2). Immunoblotting for IGF-IR confirmed equal levels of IGF-IR in all immunoprecipitates. C, the 185-kDa band found exclusively in the resistant cells was manually cut from a silver-stained gel. After trypsin digestion, peptides were extracted and analyzed by LC-MS/MS. Tryptic peptide coverage map; boldfaced sequences, identified by MS and matched HER-2 (gi|4758298|). D, to confirm the HER-2 identity, IGF-IR was immunoprecipitated from whole-cell lysates and immunoblotted for HER-2. Conversely, immunoprecipitation of HER-2 with immunoblotting for IGF-IR reveals the same exclusivity of the IGF-IR/HER-2 interaction in resistant cells. E, to determine the specificity of IGF-IR interaction with HER-2 in resistant cells, IGF-IR was immunoprecipitated from parental and resistant cells and immunoblotted for EGFR (a related family member of HER-2), integrin h3 (a nontyrosine kinase receptor membrane protein), or PDGFR (a non-EGFR tyrosine kinase receptor). Interaction between IGF-IR and EGFR was not observed in any of the immunoprecipitates. (Faint bands observed in resistant cell immunoprecipitates likely represent cross-reactivity of the EGFR antibody with HER-2.) As a control for EGFR immunoblotting, HER-2 was immunoprecipitated and immunoblotted for EGFR. As expected, EGFR was detected in the HER-2 immunoprecipitates of all lysates. IGF-IR interacted with integrin h3 in all cells but not any more so in resistant cells versus parental cells. PDGFR was not detected in any of the IGF-IR immunoprecipitates. (The control for PDGFR was 3T3 cell lysate.) F, whole-cell lysates from parental and resistant cells were immunoblotted for total phosphotyrosine (monoclonal PT66), HER-2 phosphorylated on tyrosine (Y) 1248, and total HER-2, all of which were expressed at similar levels in parental and resistant cells.

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Figure 2. Evidence of receptor cross talk: IGF-I induces phosphorylation of HER-2 in trastuzumab-resistant cells. A, parental SKBR3 cells were serum starved overnight and then stimulated with IGF-I (100 ng/mL) for 0, 1, 5, 10, or 15 minutes. Protein lysates (50 Ag) were examined by immunoblotting for p-IGF-IR (Tyr1163), total IGF-IR, p-HER-2 (Tyr1248), total HER-2, and actin. IGF-I stimulated phosphorylation of IGF-IR within 5 minutes. HER-2 phosphorylation was not significantly altered by IGF-I stimulation in parental cells. In addition, downstream signaling was studied by immunoblotting protein lysates (50 Ag) for p-IRS-1 (Ser789), total IRS-1, p-Akt (Ser473), total Akt, p-MAPK (Thr202/Tyr204 p42/p44 MAPK), total MAPK (p42/p44), p27kip1, and actin. Within 10 minutes of IGF-I stimulation, downstream signaling was activated with increased phosphorylation of IRS-1, Akt, and MAPK and down-regulation of p27kip1. B, resistant pool 2 cells were serum starved overnight and then stimulated with IGF-I (100 ng/mL) for 0, 1, 5, 10, 15, or 20 minutes. Protein lysates (50 Ag) were examined by immunoblotting for p-IGF-IR (Tyr1163), total IGF-IR, p-HER-2 (Tyr1248), total HER-2, and actin. Rapid phosphorylation of IGF-IR was evident within 1 minute. Importantly, IGF-I stimulated phosphorylation of HER-2 in resistant cells within 15 minutes. Downstream signaling was studied by immunoblotting protein lysates (50 Ag) for p-IRS-1 (Ser789), total IRS-1, p-Akt (Ser473), total Akt, p-MAPK (Thr202/Tyr204 p42/p44 MAPK), total MAPK (p42/p44), p27kip1, and actin. Within 1 minute of IGF-I stimulation, downstream signaling was activated with increased phosphorylation of IRS-1, Akt, and MAPK and down-regulation of p27kip1. C, parental and resistant pool 2 cells were serum starved overnight and then stimulated with IGF-I (100 ng/mL) for 0, 5, 10, or 15 minutes. IGF-IR was immunoprecipitated (200 Ag) and immunoblotted for HER-2 or IGF-IR. IGF-I stimulation did not affect the HER-2/IGF-IR heterodimer in resistant cells and seemed to promote interaction in parental cells. signaling increased in resistant cells on IGF-I stimulation. However, Additional evidence supporting cross talk between IGF-IR and in contrast to parental cells, these downstream signaling pathways HER-2 was obtained by inhibiting IGF-IR tyrosine kinase activity were stimulated more rapidly, within 1 minute versus 5 minutes in and examining HER-2 phosphorylation. Parental and resistant parental cells. These data suggest that cross talk occurs between cells were treated overnight with 1 or 10 Amol/L of the IGF-IR IGF-IR and HER-2 in trastuzumab-resistant cells. tyrosine kinase inhibitor I-OMe-AG538. Inhibition of IGF-IR Reduced levels of p27kip1 were previously reported to phosphorylation was observed in both parental and resistant contribute to trastuzumab resistance in these cells (9). Stimulation cells but was more pronounced in resistant cells (Fig. 3A). Total of serum-starved resistant cells with IGF-I resulted in down- IGF-IR levels were unchanged. Phosphorylation of HER-2 was regulation of p27kip1 (Fig. 2B), similar to parental cells (Fig. 2A). unaffected by I-OMe-AG538 in parental, trastuzumab-sensitive Hence, increased IGF-IR signaling is a potential upstream cells. In contrast, I-OMe-AG538 inhibited phosphorylation of HER-2 mechanism by which p27kip1 levels are reduced in resistant cells. in resistant cells whereas total levels of HER-2 remained Interaction between HER-2 and IGF-IR in resistant cells was unchanged. Although it is feasible that reduced phosphorylation unaffected by IGF-I stimulation (Fig. 2C). Interestingly, in parental of HER-2 by I-OMe-AG538 is due to inhibition of other tyrosine cells, IGF-I stimulation seemed to promote heterodimerization to kinases including autophosphorylation by HER-2, I-OMe-AG538 a small degree as low levels of HER-2 were detected in IGF-IR did not change phospho-HER-2 levels in parental cells, showing immunoprecipitates from IGF-I-stimulated cells. These data that this inhibitor does not directly inhibit HER-2 phosphoryla- suggest that IGF-IR signaling may promote interaction between tion. Consistent with the finding that I-OMe-AG538 inhibited IGF-IR and HER-2. IGF-IR to a greater degree in resistant cells versus parental cells, www.aacrjournals.org 11121 Cancer Res 2005; 65: (23). December 1, 2005

Figure 3. Evidence of receptor cross talk: IGF-IR tyrosine kinase inhibition reduces HER-2 phosphorylation in trastuzumab-resistant cells. A, SKBR3 parental and resistant pool 2 cells were treated with the IGF-IR tyrosine kinase inhibitor I-OMe-AG538 overnight at 0, 1, or 10 Amol/L. Western blot analysis was done for p-IGF-IR (Tyr1163), total IGF-IR, p-HER-2 (Tyr1248), and total HER-2. I-OMe-AG538 inhibited IGF-IR phosphorylation in parental and resistant cells although resistant cells showed a more significant inhibition of IGF-IR phosphorylation. IGF-IR inhibition was associated with reduced HER-2 phosphorylation in resistant cells. Downstream signaling was studied by immunoblotting protein lysates (50 Ag) for p-IRS-1 (Ser789), total IRS-1, p-Akt (Ser473), total Akt, p-MAPK (Thr202/Tyr204 p42/p44 MAPK), total MAPK (p42/p44), p27kip1, and actin. Consistent with the finding that resistant cells showed a greater decrease in IGF-IR phosphorylation on I-OMe-AG538 treatment, resistant cells showed a greater decrease in signaling through IRS-1 and Akt. MAPK signaling was unaffected. P27kip1 was modestly increased after I-OMe-AG538 treatment. B, resistant cells were treated overnight with 0, 1, or 10 Amol/L I-OMe-AG538. IGF-IR was immunoprecipitated (200 Ag) and immunoblotted for HER-2 or IGF-IR. IGF-IR inhibition by I-OMe-AG538 did not affect the HER-2/IGF-IR heterodimer. C, pool 2 cells were serum starved overnight and then stimulated with 100 ng/mL IGF-I for 15 or 20 minutes, treated with 10 Amol/L I-OMe-AG538 for 6 hours, or pretreated with 10 Amol/L I-OMe-AG538 for 6 hours followed by 15 or 20 minutes of 100 ng/mL IGF-I stimulation. Lysates were immunoblotted for p-HER-2 (Tyr1248), total HER-2, p-IGF-IR (Tyr1163), and total IGF-IR. IGF-I stimulated phosphorylation of HER-2 and IGF-IR. I-OMe-AG538 at 6 hours partially blocked IGF-I-mediated IGF-IR phosphorylation. HER-2 phosphorylation was blocked in response to IGF-I when cells were pretreated with the IGF-IR kinase inhibitor. D, parental and resistant cells were pretreated with I-Ome-AG538 overnight at 0, 1, 5, or 10 Amol/L. Medium was then replaced with medium alone or medium containing trastuzumab at 0, 1, 5, or 20 Ag/mL for 72 hours. Cell viability was measured by trypan blue exclusion assay. Cell viability is given as a percentage of untreated cells. All experiments were repeated at least twice. Points, mean of three iterations; bars, SD. Trastuzumab-resistant cells were highly sensitive to the IGF-IR tyrosine kinase inhibitor I-OMe-AG538 and this sensitivity was increased even further when combined with trastuzumab. Statistical significance between cell viability of trastuzumab alone versus trastuzumab combined with I-OMe-AG538 was P = 0.0355 for parental and P = 0.0023 for pool 2 cells (Student’s t test).

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. IGF-IR/HER-2 Contributes to Trastuzumab Resistance we observed decreased phosphorylation of IRS-1 and Akt not sufficient to inhibit phosphorylation of HER-2. These results downstream of IGF-IR in resistant cells (Fig. 3A). In addition, suggest that HER-2 phosphorylation can occur independent of p27kip1 was modestly increased in resistant cells treated with I- heterodimer formation with IGF-IR, most likely due to phosphor- OMe-AG538 in contrast to parental cells, supporting the concept ylation by other tyrosine kinases. However, when HER-2 is in that IGF-IR signaling may contribute to p27kip1 down-regulation complex with IGF-IR, IGF-IR seems to be a major contributor of in trastuzumab-resistant cells and that inhibition of IGF-IR may HER-2 phosphorylation as stimulation or inhibition of IGF-IR increase p27kip1 levels. Immunoprecipitation of IGF-IR from signaling was associated with increased or decreased HER-2 resistant cells showed that dimerization between HER-2 and IGF- phosphorylation, respectively. IR was unaffected by I-OMe-AG538 (Fig. 3B). Treatment of trastuzumab-resistant cells with a-IR3 resulted in We next examined the effect of IGF-IR kinase inhibition on IGF- reduced signaling downstream of IGF-IR. Within 60 minutes of I-stimulated cells. Resistant pool 2 cells were serum starved exposure to a-IR3, there was reduced phosphorylation of Ser789 on overnight and then either stimulated with IGF-I, treated with IRS-1, Ser473 on Akt, and Thr202 and Tyr204 on p42 MAPK and I-OMe-AG538, or pretreated with I-OMe-AG538 for 6 hours, p44 MAPK (Fig. 4B). However, expression of p27kip1 was followed by IGF-I stimulation. Immunoblotting showed that IGF- unaffected within 60 minutes of treatment with a-IR3. These I stimulated phosphorylation of both HER-2 and IGF-IR within 15 results indicate that IGF-IR signaling can be inhibited in resistant and 20 minutes (Fig. 3C). Cells were exposed to I-OMe-AG538 cells using an IGF-IR–targeted antibody. for 6 hours, which only partially blocked IGF-I-mediated IGF-IR We next sought to determine the biological importance of the phosphorylation, in contrast to overnight I-OMe-AG538 treatment IGF-IR/HER-2 heterodimer in the context of trastuzumab (Fig. 3A). IGF-I-mediated phosphorylation of HER-2 was blocked in resistance. The biological effect of blocking IGF-IR with a-IR3 resistant cells when pretreated with the IGF-IR kinase inhibitor. was studied by trypan blue exclusion cell viability assay. Pool 1 We next determined if inhibition of IGF-IR tyrosine kinase (Fig. 4C) and pool 2 (Fig. 4D) resistant cells were treated with activity by I-OMe-AG538 could restore trastuzumab sensitivity 0.25, 0.5, or 1 Ag/mL a-IR3 for 24 hours, at which point medium to resistant cells. Parental and resistant cells were treated with was replaced with medium containing trastuzumab at 1, 5, or 20 I-OMe-AG538 at 0, 1, 5, or 10 Amol/L for 24 hours, at which point Ag/mL. Trypan blue cell viability assays were done after 72 medium was aspirated and replaced with medium containing hours. Pretreatment of cells with a-IR3 dramatically restored trastuzumab at 0, 1, 5, or 20 Ag/mL, doses at which these cells were trastuzumab sensitivity to resistant cells whereas a-IR3 alone did previously shown to be resistant (9). Cell viability was measured not significantly decrease cell survival. Hence, blockade of after 72 hours by trypan blue exclusion assay. Parental cells IGF-IR, such that it can no longer bind HER-2, facilitated showed similar sensitivity to trastuzumab and I-OMe-AG538 and trastuzumab-mediated cytotoxicity in resistant cells. These pretreatment with I-OMe-AG538 slightly increased trastuzumab results suggest that the IGF-IR/HER-2 heterodimer contributes sensitivity at the higher doses (Fig. 3D). Resistant cells were highly to trastuzumab resistance and that disruption of this complex sensitive to I-OMe-AG538 and sensitivity to trastuzumab was may be a valid therapeutic approach in trastuzumab-resistant increased when cells were pretreated with I-OMe-AG538. These cells. data suggest that the IGF-IR tyrosine kinase may be an important The human epidermal growth factor receptor 2–targeted molecular target in trastuzumab-resistant breast cancer cells. antibody pertuzumab partially disrupts the insulin-like Thus, evidence supporting cross talk between IGF-IR and HER-2 growth factor-I receptor/human epidermal growth factor in trastuzumab-resistant cells, but not in the parental trastuzu- receptor 2 heterodimer and decreases viability of resistant mab-sensitive cells, includes the following: (a) IGF-I stimulated cells. In addition to examining the response of resistant cells to HER-2 phosphorylation in resistant cells, (b) inhibition of IGF-IR IGF-IR blockade, we also examined HER-2 blockade as a potential tyrosine kinase decreased HER-2 phosphorylation in resistant cells, strategy for disrupting heterodimerization and reverting trastu- and (c) IGF-IR inhibition suppressed IGF-I-mediated effects on zumab resistance. The HER-2-targeted monoclonal antibody HER-2 phosphorylation. pertuzumab partially disrupted the interaction between IGF-IR Blockade of insulin-like growth factor-I receptor disrupts and HER-2 (Fig. 5A) without affecting total levels of IGF-IR and insulin-like growth factor-I receptor/human epidermal HER-2 (Fig. 5B). Pertuzumab did not inhibit phosphorylation of growth factor receptor 2 heterodimerization and restores HER-2 on Tyr1248 (Fig. 5B), again suggesting that additional trastuzumab sensitivity. The anti–IGF-IR monoclonal antibody sources of HER-2 phosphorylation exist when HER-2 is not bound a-IR3 blocks the receptor-binding domain of IGF-IR and inhibits to IGF-IR and that these sources are still active at these doses of serum-dependent growth of breast cancer cells (11). Resistant cells pertuzumab. Phosphorylation of IGF-IR (Fig. 5B) and the IGF-IR were treated with 0.5 Ag/mL a-IR3 for 30 or 60 minutes, followed downstream molecule IRS-1 (Fig. 5C) was not significantly altered by immunoprecipitation of IGF-IR and immunoblotting for HER-2 by pertuzumab. Phosphorylation of Akt, which lies downstream of (Fig. 4A). Levels of HER-2 bound to IGF-IR were reduced as early as HER-2, was reduced in resistant cells whereas MAPK phosphor- 30 minutes after a-IR3 treatment whereas total IGF-IR and HER-2 ylation was unaffected, which is consistent with results observed levels were unchanged (Fig. 4B), indicating that IGF-IR blockade by in another HER-2-overxpressing breast cancer cell line (12). In a-IR3 partially disrupts the IGF-IR/HER-2 heterodimer. Phosphor- addition, p27kip1 was slightly induced by pertuzumab in resistant ylation of IGF-IR was decreased within 60 minutes in both pools. cells. The biological effect of pertuzumab was studied by trypan However, a-IR3 did not decrease phosphorylation of HER-2 on blue viability assay in trastuzumab-resistant cells (Fig. 5D). Tyr1248 (Fig. 4B) or on other phosphorylation sites of HER-2 (not Although not statistically significant, resistant cells showed a shown) when cells were treated up to 60 minutes. Thus, although trend of increased sensitivity to pertuzumab relative to parental direct inhibition of the IGF-IR tyrosine kinase by I-OMe-AG538 cells, particularly at lower doses. Thus, trastuzumab-resistant cells correlated with decreased HER-2 phosphorylation, IGF-IR anti- may retain the ability to respond to different HER-2-targeted body-mediated disruption of the IGF-IR/HER-2 heterodimer was therapies. www.aacrjournals.org 11123 Cancer Res 2005; 65: (23). December 1, 2005

Figure 4. Blockade of IGF-IR by anti–IGF-IR monoclonal antibody a-IR3 disrupts IGF-IR/HER-2 interaction and reduces downstream signaling. SKBR3-derived pools 1 and 2 were treated for 0, 30, or 60 minutes with 0.5 Ag/mL a-IR3. A, IGF-IR was immunoprecipitated from protein lysates (200 Ag) and immunoblotted for HER-2 and IGF-IR. HER-2/IGF-IR heterodimerization was disrupted in cells treated with a-IR3. B, total lysates (50 Ag) were immunoblotted for p-Tyr1248-HER-2, total HER-2, p-Tyr1163-IGF-IR, and total IGF-IR. Phosphorylation of IGF-IR was reduced by 60 minutes whereas total levels of IGF-IR and HER-2 were unaffected. Phosphorylation of HER-2 was unaffected by a-IR3-mediated heterodimer disruption. Total lysates (50 Ag) were also immunoblotted for p-IRS-1 (Ser789), total IRS-1, p-Akt (Ser473), total Akt, p-MAPK (Thr202/Tyr204 p42/p44 MAPK), total MAPK (p42/p44), p27kip1, and actin. Reduced IGF-IR signaling was observed within 60 minutes with decreased p-IRS-1, p-Akt, and p-MAPK. P27kip1 levels were unaffected by 60 minutes of a-IR3 treatment. Pool 1 (C) and pool 2 (D) cells were pretreated with 0.25, 0.5, or 1 Ag/mL of a-IR3 for 24 hours. Medium was then replaced with medium alone or medium containing 0, 1, 5, or 20 Ag/mL of trastuzumab for 72 hours. Cell viability was measured by trypan blue exclusion assay and is given as a percentage of untreated cells. All experiments were repeated at least twice. Points, mean of three iterations; bars, SD. Trastuzumab sensitivity was dramatically increased when resistant cells were pretreated with a-IR3. Statistical significance between cell viability of trastuzumab alone versus trastuzumab combined with a-IR3 was P = 0.0213 for pool 1 and P = 0.0219 for pool 2 cells (Student’s t test).

Discussion be a suitable molecular target in breast cancers that have The following novel findings are described in this study: (a) progressed while on trastuzumab. IGF-IR and HER-2 heterodimerize uniquely in trastuzumab- Baseline phosphorylation of HER-2 is present in the parental trastuzumab-sensitive cells and is likely to be due to autophos- resistant cells. (b) IGF-I stimulates phosphorylation of HER-2 phorylation or kinases other than IGF-IR as IGF-I did not enhance exclusively in the resistant cells. (c) IGF-IR tyrosine kinase HER-2 phosphorylation in these cells and I-OMe-AG538 did not inhibition blocks HER-2 phosphorylation only in the resistant reduce HER-2 phosphorylation. However, in resistant cells, IGF-IR cells. (d) Antibody-mediated blockade of IGF-IR disrupts IGF-IR and HER-2 physically interact and IGF-I stimulates phosphoryla- interaction with HER-2 and restores trastuzumab sensitivity. To a tion of IGF-IR and HER-2. In addition, reduced IGF-IR phosphor- lesser degree, HER-2 blockade also disrupts heterodimerization ylation mediated by I-OMe-AG538 was associated with significantly and decreases viability of resistant cells. In addition, resistant cells reduced levels of phospho-HER-2 in resistant cells even when cells are highly sensitive to the IGF-IR tyrosine kinase inhibitor. These were stimulated with IGF-I. Thus, we propose a model in which results suggest that cross talk exists between IGF-IR and HER-2 trastuzumab-sensitive cells do not show interaction between HER- when these receptors are physically bound and that IGF-IR may 2 and IGF-IR and HER-2 phosphorylation is due to sources other

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than IGF-IR whereas in trastuzumab-resistant cells, HER-2 and IGF-IR interact, facilitating cross talk between IGF-IR and HER-2. Hence, IGF-I stimulation does not affect HER-2 phosphorylation in sensitive parental cells but increases HER-2 phosphorylation in resistant cells (Fig. 6A) whereas IGF-IR inhibition blocks HER-2 phosphorylation in resistant cells with no effect in sensitive cells (Fig. 6B). Importantly, IGF-I signaling may promote the interaction between HER-2 and IGF-IR as parental cells stimulated with IGF-I showed low levels of heterodimerization. Physical interaction between IGF-IR and HER-2 was previously reported (13). However, the association of this receptor heterodimer with trastuzumab resistance is a novel and potentially clinically important finding. In contrast to Lu et al. (8), we did not find an association between total IGF-IR levels and resistance. Nonetheless, our studies support the concept that the IGF-IR may be an important molecular target in trastuzumab-resistant disease as the anti–IGF-IR antibody a-IR3 disrupted HER-2/IGF-IR interaction, blocked IGF-IR signaling, and restored trastuzumab sensitivity in resistant cells. Treatment of cells with a-IR3 for 24 hours was previously shown to down-regulate IGF-IR (14). However, when used over a shorter time course, we show that total IGF-IR levels are unaffected by a-IR3, indicating that the decreased levels of HER-2 in IGF-IR immunoprecipitates are truly due to disrupted heterodimerization and not caused by IGF-IR down- regulation. Although a-IR3 blocked IGF-IR signaling and disrupted dimerization with HER-2 in resistant cells, HER-2 phosphorylation was unaffected by 60 minutes of a-IR3 treatment. Hence, we propose that on heterodimer disruption, HER-2 phosphorylation can occur independent of IGF-IR, most likely due to phosphorylation by other tyrosine kinases, such as autophosphorylation by HER-2 and phosphorylation by EGFR (Fig. 6C and data not shown). However, when HER-2 is in complex with IGF-IR, IGF-IR seems to be a major contributor of HER-2 phosphorylation as IGF- I-mediated stimulation of IGF-IR signaling was associated with increased HER-2 phosphorylation and IGF-IR kinase inhibition was associated with decreased HER-2 phosphorylation. Blockade of IGF-IR increased trastuzumab sensitivity to a greater degree than did HER-2 blockade. Although not statistically significant, resistant cells treated with pertuzumab did show a trend toward decreased cell viability versus parental cells, which was associated with slightly decreased phosphorylation of Akt downstream of HER-2. We previously reported synergistic cytotoxicity between trastuzumab and pertuzumab in another breast cancer cell line (12). Our current results extend these findings by suggesting that pertuzumab may remain an effective mediator of Figure 5. Blockade of HER-2 by anti-HER-2 monoclonal antibody pertuzumab cell death once trastuzumab resistance develops. Pertuzumab has partially disrupts IGF-IR/HER-2 dimerization and slightly decreases viability of resistant cells. SKBR3-derived resistant cells were treated for 72 hours with been shown to block dimerization between HER-2 and EGFR or 0, 1, or 10 Ag/mL of pertuzumab. A, IGF-IR was immunoprecipitated from protein HER-3 (15). The current study provides the first description of lysates (200 Ag) and immunoblotted for HER-2 or IGF-IR. HER-2/IGF-IR heterodimerization was partially disrupted in cells treated with pertuzumab. pertuzumab-mediated inhibition of HER-2 dimerization with a B, total lysates (50 Ag) were immunoblotted for p-Tyr1248 HER-2, total HER-2, non-erbB family member (IGF-IR). Thus, alternate forms of anti- p-Tyr1163-IGF-IR, and total IGF-IR. Pertuzumab treatment did not affect HER-2 therapy may still be beneficial in patients resistant to phosphorylation or total levels of IGF-IR or HER-2. C, total lysates (50 Ag) were immunoblotted for p-IRS-1 (Ser789), total IRS-1, p-Akt (Ser473), total Akt, trastuzumab. However, as molecular profiles will vary among p-MAPK (Thr202/Tyr204 p42/p44 MAPK), total MAPK (p42/p44), p27kip1, and different tumors, it is unlikely that all trastuzumab-resistant actin. Signaling to Akt was partially inhibited and p27kip1 was induced. D, tumors will benefit from another HER-2-targeted therapy. Some parental cells and pools were treated in triplicate with 2-fold serial dilutions of pertuzumab ranging from 3.125 to 50 Ag/mL for 5 days in the presence of tumors may show cross-resistance, as suggested by Tanner et al. 4 Ag/mL trastuzumab. Cells were trypsinized, stained with trypan blue dye, and (16), who did not observe any benefit from pertuzumab in their viable cells were counted by microscopic examination. Cell viability is given as a percentage of untreated cells. All experiments were repeated at least twice. trastuzumab-resistant JIMT-1 cell model. Interestingly, however, Points, mean of three iterations; bars, SD. Resistant cells maintained on their cell model showed decreased binding of trastuzumab to HER-2 trastuzumab showed a slightly increased sensitivity to pertuzumab relative to whereas pertuzumab was still able to bind to HER-2 (17). Thus, parental cells (P > 0.05). Statistical significance between cell viability of parental cells versus pool 1 cells was P = 0.1835 and for parental versus pool 2 cells different HER-2-targeted agents seem to have different effects on was P = 0.0572 (Student’s t test). trastuzumab-resistant cells. www.aacrjournals.org 11125 Cancer Res 2005; 65: (23). December 1, 2005

Figure 6. Proposed model for IGF-IR signaling and cross talk in trastuzumab resistance. A, in trastuzumab-sensitive cells, IGF-I stimulates IGF-IR signaling, resulting in phosphorylation of IGF-IR, IRS, and Akt. HER-2 is phosphorylated by other membrane receptor tyrosine kinases such as EGFR (arrows) and by autophosphorylation. In trastuzumab-resistant cells, IGF-I stimulates IGF-IR signaling with increased phosphorylation of IGF-IR, IRS, and Akt. Interaction between IGF-IR and HER-2 facilitates IGF-IR signaling to HER-2, resulting in increased HER-2 phosphorylation. B, in trastuzumab-sensitive cells, IGF-IR tyrosine kinase inhibition partially blocks phosphorylation and activation of IGF-IR signaling. HER-2 phosphorylation is still regulated by other membrane receptor tyrosine kinases and by autophosphorylation. Trastuzumab-resistant cells are more sensitive to IGF-IR tyrosine kinase inhibition, which results in a dramatic decrease in IGF-IR downstream signaling as well as significantly decreased cross-signaling to HER-2 even in the presence of IGF-I. This inhibition of IGF-IR activity results in increased trastuzumab sensitivity. C, antibody-mediated blockade of IGF-IR disrupts receptor interaction and diminishes IGF-IR signaling in trastuzumab-resistant cells. HER-2 is maintained in a phosphorylated and active state perhaps due to activity of other membrane receptor tyrosine kinases and autophosphorylation (Nahta and Esteva, unpublished results). IGF-IR blockade results in a dramatic restoration of trastuzumab sensitivity, supporting the concept that IGF-IR is a promising molecular target in this subset of breast cancers.

Several molecular mechanisms have been proposed to contrib- the authors proposed that elevated MUC-4 expression masks the ute to trastuzumab resistance. The JIMT-1 model mentioned trastuzumab-binding epitopes of HER-2, which decreases the above showed increased expression of the cell surface mucin interaction between this antibody and its therapeutic target, MUC-4 (17). The MUC-4 level was inversely correlated with the resulting in drug resistance. Interestingly, the authors also state trastuzumab-binding capacity of single cells, and knockdown of that HER-2 is unable to interact with other proteins, such as MUC-4 increased trastuzumab sensitivity of JIMT-1 cells. Thus, EGFR or HER-3, due to masking by MUC-4. This is in contrast to

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. IGF-IR/HER-2 Contributes to Trastuzumab Resistance the mechanism we describe here in which HER-2 shows resistance in these cells (9). Thus, our current results suggest increased interaction with a non-EGFR tyrosine kinase receptor that a potential molecular mechanism by which IGF-IR/HER-2 (IGF-IR). Increased coexpression and interaction of HER-2 with contributes to trastuzumab resistance may be increased signaling EGFR family members has also been shown to affect trastu- such that p27kip1, a critical mediator of responsiveness to zumab response in SKBR3 cells (18). In addition, increased levels trastuzumab (22, 23), is down-regulated. of erbB ligands heregulin and EGF blocked trastuzumab- Future studies will examine the IGF-IR/HER-2 heterodimer as mediated growth inhibition of SKBR3 (18) and BT474 cells (19). a potential target in trastuzumab-resistant breast cancers. We The erbB kinase inhibitor RALT/MIG-6 was shown to counteract showed here that blockade of IGF-IR/HER-2 heterodimerization the ability of erbB ligands to promote trastuzumab resistance using an anti–IGF-IR antibody resulted in increased trastuzumab (20). Thus, molecular profiling of tumor cells to determine the sensitivity and that resistant cells are highly sensitive to the relative levels of other EGFR family members and their ligands IGF-IR tyrosine kinase inhibitor. These results are important as may be useful to predict sensitivity to trastuzumab. In addition, they suggest potential experimental therapeutics that can be blocking multiple growth factor receptors simultaneously may further developed preclinically for testing against trastuzumab- increase sensitivity. resistant disease. Combination regimens targeting both HER-2 Another very important and interesting potential mechanism of and IGF-IR should also be tested preclinically given our research trastuzumab resistance, described by Nagata et al. (21), is findings. Therapies that target multiple growth factor receptors decreased expression of the phosphatase and tensin homologue including IGF-IR and HER-2 have been described and seem to (PTEN). The authors show that when trastuzumab is effective, it produce benefit in trastuzumab-resistant cells (24). Our current causes dissociation of Src from HER-2, freeing PTEN to inhibit studies seek to validate the heterodimer as a marker of Akt and induce growth arrest. However, when PTEN levels are resistance to trastuzumab-based therapy in primary breast low, Akt remains active and trastuzumab response is reduced. cancer tissue. Additionally, preclinical studies using anti–IGF-IR Importantly, they validated their cell culture model in tumor agents being developed for clinical use are being done to tissues and showed that PTEN deficiency is associated with poor establish the IGF-IR/HER-2 heterodimer as a valid therapeutic response to trastuzumab. The trastuzumab-resistant model used target in trastuzumab-resistantbreastcancers.Elucidating in our current study did not show any changes in Akt activity molecular markers of trastuzumab resistance and validating relative to parental cells nor any difference in sensitivity to Akt novel therapeutic targets such as the IGF-IR/HER-2 heterodimer inhibiting agents (not shown). Thus, the mechanism contributing are critical for increasing survival rates in this subset of breast to resistance in our SKBR3-derived model is clearly different. cancer patients. Our proposed mechanism supports the findings of Lu et al. (10) in which IGF-I is shown to increase p27kip1 degradation and decrease sensitivity to trastuzumab. The authors also showed in a Acknowledgments prior publication that overexpression of IGF-IR was associated Received 10/25/2004; revised 8/19/2005; accepted 9/20/2005. with trastuzumab resistance (8). Although total IGF-IR levels were Grant support: AACR-Amgen, Inc. Fellowship in Clinical/Translational Cancer not elevated in our resistant model versus the parental cells, we Research (R. Nahta), the M.D. Anderson Cancer Center Odyssey Special Fellowship and Theodore N. Law Award for Scientific Achievement (R. Nahta), K23 CA82119 (F.J. show that IGF-IR signals to HER-2 as part of a heterodimer Esteva), the University Cancer Foundation (F.J. Esteva, R. Nahta) at the University of in resistant cells and that blockade of IGF-IR signaling is a Texas M.D. Anderson Cancer Center, the Nellie B. Connally Breast Cancer Research Fund, and NIH Cancer Center Support grant CA 16672-27 (Media Preparation Facility, potentially important therapeutic strategy in these cells. Similar Flow Cytometry and Cellular Imaging Facility, Proteomics Facility). to Lu et al. (10), we observed decreased p27kip1 levels in response The costs of publication of this article were defrayed in part by the payment of page to IGF-I in parental SKBR3 cells. Resistant cells also showed charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. p27kip1 down-regulation on IGF-I stimulation. We previously We thank Genentech for providing pertuzumab and Drs. Xiaojiang Cui and Adrian showed that p27kip1 down-regulation contributes to trastuzumab Lee for technical advice on IGF-I stimulation experiments.

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Correction: IGF-IR/HER-2 Contributes to Trastuzumab Resistance

In the article on how IGF-IR/HER-2 contributes to trastuzumab resistance in the December 1, 2005 issue of Cancer Research (1), Fig. 2A is incorrect. The corrected panel appears below.

1. Nahta R, Yuan LXH, Zhang B, Kobayashi R, Esteva FJ. Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells. Cancer Res 2005;65:11118–28.

I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-68-22-COR1

Cancer Res 2008; 68: (22). November 15, 2008 9566 www.aacrjournals.org Insulin-like Growth Factor-I Receptor/Human Epidermal Growth Factor Receptor 2 Heterodimerization Contributes to Trastuzumab Resistance of Breast Cancer Cells

Rita Nahta, Linda X.H. Yuan, Bing Zhang, et al.

Cancer Res 2005;65:11118-11128.

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