Annals of Oncology Advance Access published online on July 17, 2008
Annals of Oncology, doi:10.1093/annonc/mdn406
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Synergic antitumoral effect of an IGF-IR inhibitor and trastuzumab on HER2-overexpressing breast cancer cells
1 Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca
2 Servicio de Oncología Médica, Complejo Hospitalario Universitario de Albacete, Spain
* Correspondence to: Dr A. Pandiella, Instituto de Biología Molecular y Celular del Cáncer, Campus Miguel de Unamuno, 37007-Salamanca, Spain. Tel/Fax: +34-923-294815; E-mail: atanasio{at}usal.es
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Abstract |
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Background: Receptor tyrosine kinases play an important role in breast cancer. One of them, the type I insulin-like growth factor, has been linked to resistance to trastuzumab (Herceptin), an agent that targets human epidermal growth factor receptor 2. Here, we show that the insulin-like growth factor-I receptor (IGF-IR) antagonist NVP-AEW541 inhibits proliferation of breast cancer cells and synergizes with trastuzumab.
Patients and methods: Patient samples and breast cancer cell lines were evaluated for IGF-IR expression or activation by western blotting. 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) uptake assays and Annexin V staining were used for the analyses of cell proliferation/apoptosis. Biochemical and genomic studies were carried out to gain insights into the mechanism of action of NVP-AEW541.
Results: The IGF-IR was expressed above normal levels in a number of breast cancer samples. Activation of this receptor was inhibited by NVP-AEW541 that also decreased cell proliferation and increased apoptosis. NVP-AEW541 decreased the amount of pAkt and increased the level of p27. Combination studies with several drugs used in the breast cancer clinic showed that NVP-AEW541 synergistically increased the action of trastuzumab.
Conclusions: Our results show the anti-breast cancer action of NVP-AEW541 and support the clinical development of anti-IGF-IR agents, especially in combination with trastuzumab.
breast cancer, IGF-IR, trastuzumab
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Receptor tyrosine kinases (RTKs) have been associated with the development/progression of different types of cancer [1, 2]. In breast cancer, overexpression of one of these RTKs, termed ErbB2/human epidermal growth factor receptor 2 (HER2), occurs approximately in 25% of patients and is associated with shorter survival [3, 4]. These findings led to the development of agents that target HER2. One of these compounds is trastuzumab, a monoclonal antibody that acts on the extracellular domain of HER2 [5, 6]. In HER2-overexpressing breast cancer tumors, treatment with trastuzumab increases survival in metastatic and early-stage breast cancer [7, 8].
In spite of its effectiveness in breast cancer, a substantial number of HER2-positive patients do not respond to trastuzumab or relapse in a short period of time. This resistance to trastuzumab has been attributed to various mechanisms, including phosphatase and tensin homolog (PTEN) inactivation [9], the presence of HER2-truncated forms [10], or the activation of other tyrosine kinase receptors such as the insulin-like growth factor-I receptor (IGF-IR) [11]. IGF-IR is a heterotetrameric transmembrane tyrosine kinase receptor that is widely expressed in normal human tissues [12, 13]. After binding to IGF-I, the receptor phosphorylates the insulin receptor substrate 1 (IRS-1) that is responsible for mediating the activation of multiple downstream signaling networks, including the phosphatidylinositol-3-kinase or the Ras/Raf/mitogen-activated protein kinase pathways. Activation of these signaling pathways leads to stimulation of cell cycle progression and resistance to cell death. In addition, a second adaptor protein, termed IRS-2, is also activated by the IGF-IR and participates in mediating motility signals.
Increased levels of IGF-I in the blood are associated with an increased risk of breast cancer [14], and overexpression of IGF-IR and its ligands has been associated to the development and maintenance of breast cancer [13,15–17]. In addition, ligand-induced stimulation of the IGF-IR inhibits the action of trastuzumab [11]. These facts led to efforts aimed at developing treatments to decrease IGF-IR function to be used directly to treat breast cancer and to help in the reversion of trastuzumab resistance. During the last years, several types of agents that target the IGF-IR have been developed, including small tyrosine kinase inhibitors, monoclonal antibodies, or IGF-I-binding proteins. Although monoclonal antibodies are in clinical development [18], some studies indicate that some of these antibodies can also down-regulate the insulin receptor and may provoke metabolic disorders such as ‘hyperglycemia’ [19, 20]. Furthermore, monoclonal antibodies or other protein antagonists have the inconvenience of parenteral administration that requires treatment of the patient at the clinic. To circumvent these problems, specific IGF-IR small molecule tyrosine kinase inhibitors that do not affect glucose metabolism are in clinical development. One of these compounds, the pyrrolopyrimidine derivative NVP-ADW742, has demonstrated potent antitumor activity against several tumoral cells [21]. A more recently developed analog, termed NVP-AEW541, has shown a 27-fold inhibitory selectivity for IGF-IR with respect to the insulin receptor and is more specific than NVP-ADW742 [20, 22]. Moreover, NVP-AEW541 prevented the growth of different tumoral cell lines in vitro and in vivo [20, 22, 23]. However, its action on breast cancer cells has not been explored in detail.
Given the importance of the IGF-IR in breast cancer and the potential clinical benefit of agents that target this signaling pathway, we explored the action of NVP-AEW541 on breast cancer cells. Here, we show that IGF-IR overexpression is frequent in patients with breast cancer. Studies using cell lines showed that NVP-AEW541 reduces proliferation and acts synergistically with trastuzumab in restricting proliferation of breast cancer cells. This latter finding is of particular importance as it opens the possibility of combining anti-IGF-IR therapies such as NVP-AEW541 with trastuzumab for the more efficient treatment of trastuzumab-sensitive breast cancers.
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reagents and immunochemicals
NVP-AEW541 was from Novartis Pharma (Basel, Switzerland). Other generic chemicals, as well as docetaxel and vincristine were from Sigma Chemical Co. (St. Louis, MO), Roche Biochemicals (Indianapolis, IN), or Merck (Darmstadt, Germany). IGF-I was from Strathmann (Hamburg, Germany). Trastuzumab and Neuregulin (NRG) were a generous gift of Dr Mark X. Sliwkowski (Genentech, San Francisco, CA). The anti-phosphotyrosine, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-cyclin D1, the rabbit polyclonal anti-p21, anti-p27, anti-IGF-IR, anti-Cdk2, and anti-cyclin D3 were from Santa Cruz Biotechnology (Sta. Cruz, CA). The anti-phospho-IGF-IR antibody used for western blotting was from Cell Signalling (Beverly, MA). We observed that the latter antibody also reacts with anti-pHER2 in western blots (data not shown). Horseradish peroxidase conjugates of anti-rabbit and anti-mouse immunoglobulin G were from Bio-Rad Laboratories (Cambridge, MA). The anti-pAkt Ser473 antibody was generated against the sequence RPHFPQFpS473YSAS.
cell culture, immunoprecipitation, and western blotting
All cell lines were cultured at 37°C in a humidified 5% CO2/95% air atmosphere. Cells were grown in Dulbecco's modified Eagle's medium containing high glucose (4500 mg/l) and antibiotics (penicillin 100 units/ml, streptomycin 100 µg/ml) and supplemented with 10% foetal bovine serum. Immunoprecipitation and western blotting were carried out as described [24]. Conditions for the analysis of the cell cycle and proliferation in breast cancer cells have been reported [25].
apoptosis analyses
For apoptosis analyses, trypsinized cells were washed twice with cold phosphate-buffered saline (PBS) and then resuspended in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 x 106 cells/ml. In total, 100 000 cells were incubated for 15 min in the dark with 5 µl of Annexin V–fluorescein isothiocyanate (FITC) (BD Biosciences) and 10 µl of propidium iodide (5 µg/ml, final concentration). Finally, 400 µl of binding buffer was added. Apoptosis analyses were carried out on a FACSCalibur machine using the CellQuest software (BD Biosciences, Franklin Lakes, NJ).
Mitochondrial membrane potential was analyzed by the incorporation of tetramethylrhodamine ethyl ester (TMRE). In total, 220 000 BT474 cells were plated in 60-mm dishes, allowed to attach during 24 h and treated with different doses of NVP-AEW541 for 72 h. TMRE (1 µM) was added to the plate for 30 min, and then cells were trypsinized and resuspended in 500 µl of PBS before fluorescence quantification on a FACSCalibur cytometer (BD Biosciences, Franklin, NJ).
analyses of synergism by the CalcuSyn program
In order to determine whether the combination of NVP-AEW541 and trastuzumab was additive, antagonist, or synergistic, we used the CalcuSyn software program (Biosoft, Ferguson, MO). This program allows the calculation of the combination index (CI) following the algorithm of Chou and Talalay [26]. CI values <1.0 indicate synergism, CI = 1 indicates an additive effect, whereas values >1 correspond to antagonism. CIs from two different experiments were generated with the CalcuSyn software for each combination and graphically represented as the mean ± standard deviation.
microarray RNA analyses
Total RNA was extracted and purified with RNeasy Mini Kit (Qiagen, CA). Double-stranded complementary DNA and afterwards biotinylated complementary RNA were synthesized using a T7-polyT primer and the BioArray RNA labeling kit (Enzo, NY), respectively. The labeled RNA was then fragmented and hybridized according to Affymetrix protocols to HU-133 Plus 2.0 oligonucleotide arrays (Affymetrix, CA). The expression values for each probe set were calculated using the dChip 2006 software. All samples had a scaling factor lower than two-fold and a 3'–5' of GAPDH probe set <1.5. Gene expression levels were transformed to base two logarithms. A median normalization approach was applied.
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expression of IGF-IR in patient samples and human breast cancer cell lines
We evaluated the presence of the IGF-IR in 22 samples from breast cancer patients, using as a control normal glandular breast tissue (Figure 1A). The anti-IGF-IR antibody used identified a 97-kDa form, together with two additional bands of 180 and 200 kDa (asterisks in Figure 1A). The 97-kDa band migrated with the expected molecular mass of the β chain of the IGF-IR, while the upper molecular mass forms may correspond to the unprocessed immature receptor. Levels of IGF-IR substantially higher than those present in control breast tissue were present in nine (40%) of the samples. Amounts of IGF-IR analogous to those of control normal breast epithelium were observed in eight (37%) samples.
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action of NVP-AEW541 on breast cancer cell lines
To explore the action of NVP-AEW541 in breast cancer, we used several cell lines representative of distinct molecular pathological situations. Two of them, BT474 and SKBR3, are frequently used as model cell lines of HER2 overexpression, while the MCF7 cell line contains a low to normal HER2 level [27]. MDA-MB231 cells are representative of the triple-negative genotype [28], characterized by the absence of hormonal receptors and HER2 amplification. The different breast cancer cell lines were treated with a fixed dose of the drug, and tyrosine phosphorylation IGF-IR was analyzed by western blotting. On sufficiently long exposure times, and upon enrichment of the IGF-IR by immunoprecipitation, resting tyrosine phosphorylation of the IGF-IR was observed in BT474, MCF7, and SKBR3 cells (Figure 1B). Addition of NVP-AEW541 inhibited the constitutive tyrosine phosphorylation of the IGF-IR in MCF7, BT474, and SKBR3 cells. No resting level of tyrosine-phosphorylated IGF-IR was detected in MDA-MB231 cells.
We evaluated the anti-proliferative activity of NVP-AEW541 in the four breast cancer cell lines. In MCF7 cells, treatment with NVP-AEW541 inhibited the MTT absorbance of these cells in a dose-dependent manner (Figure 1C). In BT474 cells, the growth inhibitory effect of NVP-AEW541 was less marked than in MCF7 cells for doses up to 2.5 µM. SKBR3 cells that express much lower IGF-IR levels than the other cell lines analyzed were less sensitive to the action of NVP-AEW541, and higher doses of the compound were required to inhibit their growth. Interestingly, NVP-AEW541 also decreased MTT metabolization in the triple-negative cell line MDA-MB231.
The decrease in the MTT uptake could be caused by reduced proliferation or cell death. To investigate the potential contribution of these biological responses to the action of NVP-AEW541, we carried out propidium iodide staining followed by fluorescence-activated cell sorter analysis of BT474 cells. Addition of NVP-AEW541 provoked a dose-dependent increase in the G0/G1 phases and a decrease in the G2/M and S phases (Figure 2A). Biochemical evaluation of the action of NVP-AEW541 on signaling routes and proteins involved in the control of cell cycle progression was also carried out. NVP-AEW541 provoked an increase in p27, together with a decrease in p21 (Figure 2B). No significant changes were observed in the amounts of cyclin-dependent kinase 2 (CDK2) or cyclin D1. NVP-AEW541 decreased pAkt levels.
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Analyses of apoptotic cell death by Annexin V–FITC staining indicated that NVP-AEW541 provoked an increase in BT474 apoptotic cells (Figure 2C). In parallel, we also measured the mitochondrial membrane potential, using TMRE staining (Figure 2D). NVP-AEW541 significantly affected the mitochondrial membrane potential.
synergistic effect of NVP-AEW541 in combination with trastuzumab
We next evaluated the potential synergistic effect of NVP-AEW541 and trastuzumab on breast cancer cells. In BT474 and SKBR3 cells, treatment with this drug combination resulted in a significant inhibitory effect when compared with the drug being added alone (Figure 3A). In MCF7 cells, that are insensitive to trastuzumab [29], addition of both compounds did not significantly increase the effect of the individual drug treatments. The increased efficiency of the combined treatment in BT474 cells was already evidenced within 2 days and persisted for as long as 8 days (Figure 3B). To assess whether this potentiation effect was synergistic or additive, we used the CI algorithm of Chou and Talalay. In BT474 cells, and as shown in Figure 3C, a clear synergistic effect of NVP-AEW541 and trastuzumab was obtained at 1, 1.5, and 2 µM doses of NVP-AEW541 when combined with a fixed and maximal dose (10 nM) of trastuzumab.
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We also explored whether NVP-AEW541 could potentiate the action of other compounds commonly used in the treatment of breast cancer. To this end, we first evaluated the antitumoral effect of a taxane (docetaxel) and a vinca alkaloid (vincristine) on BT474 cells, to select for an adequate dose to be combined with NVP-AEW541. Both compounds had a potent anti-proliferative/proapoptotic effect on BT474 cells both at 3 or at 6 days of treatment, with IC50 values of 5 nM for docetaxel and 0.5 nM for vincristine (Figure 4A). The quadruple combination of these agents (trastuzumab + docetaxel + vincristine + NVP-AEW541) was more efficient than the addition of these compounds in double or triple combinations (Figure 4B, and data not shown).
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To gain insights into the mechanism of potentiation of the action of trastuzumab by NVP-AEW541, we carried out biochemical and genomic profiling studies. For these experiments, BT474 cells were treated with a saturating dose of trastuzumab, and a suboptimal dose of NVP-AEW541 for 3 or 6 days, and then MTT assays carried out to check efficient synergic action of both compounds. In parallel, cell lysates were prepared from sister cultures, and protein and messenger RNA (mRNA) extracted. Biochemical studies indicated that the combined treatment decreased the levels of pAkt and increased p27 more profoundly than either treatment alone. The levels of cyclin D1 and cyclin D3 remained unchanged, while the amount of p21 slightly decreased by the single or the combined treatments (Figure 4C and data not shown).
Microarray studies identified 119 mRNAs selectively up- or down-regulated by the combined treatment. Neither the amount of these mRNAs was also modified by trastuzumab or NVP-AEW541 nor their levels were substantially higher or lower when compared with the individual treatments (Table 1). The mostly up-regulated mRNA corresponded to the prolactin-induced protein (PIP), and the mostly down-regulated mRNA corresponded to fascin homolog 1. In addition, p8, a protein recently described to be involved in the control of apoptosis, was up-regulated [30]. The increases in the mRNA for PIP or p8 proteins were also verified by quantitative RT-PCR (Figure 4D). Some mRNAs related to cell cycle progression, such as E2F4, E2F5, TFDP1, or cyclin G, were also specifically modified by the combined treatment. A significant number of mRNAs related to the control of proteolytic processes, including proteases and protease inhibitors, as well as mRNAs coding for proteins involved in metabolic regulation were also controlled by the combined treatment.
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The present study was initiated with the purpose of evaluating the action of the IGF-IR antagonist NVP-AEW541 on breast cancer cells. The rationale for this study was on the basis of several data that linked the IGF-IR to tumor generation/progression [13, 15], together with the favorable antitumoral action of protein antagonists of the IGF-IR system [20, 31]. Furthermore, as IGF-IR has been associated to trastuzumab resistance, the combination of an anti-IGF-IR antagonist with trastuzumab could help to overcome resistance to the latter [11, 32].
We analyzed the presence of IGF-IR in breast cancer tumors from patients by western blotting. Overexpression of IGF-IR was observed in 40% of the samples studied. It is important to mention that normal breast epithelial tissue expresses low levels of IGF-IR (Figure 1A and [13]). This increased expression, together with the demonstrated role of IGF-IR in stimulating breast cancer cell proliferation, positions IGF-IR as a candidate target for breast cancer treatment. In support of the latter are our in vitro data with NVP-AEW541, as well as data from other groups using alternative anti-IGF-IR strategies [11, 31, 32]. In our experimental setting, NVP-AEW541 substantially decreased MTT uptake of all four breast cancer cell lines studied. It should be mentioned that these cell lines represent prototypic models of three phenotypically different molecular situations: the estrogen receptor positive, but HER2-negative status (MCF7), the HER2-overexpressing situation (BT474 and SKBR3), and the triple-negative, basal-like type of breast cancer, characterized by the absence of hormone and HER2 receptors (MDA-MB231). Of those here studied, the MCF7 cell line was the one that expressed the highest levels of IGF-IR and was also the most sensitive to NVP-AEW541. However, in musculoskeletal tumors other authors did not observe a correlation between the level of expression of the IGF-IR and the sensitivity to NVP-AEW541, even though correlation was found when analyzing the degree of tyrosine phosphorylation [22]. It is unlikely that the effect of NVP-AEW541 on breast cancer cells is due to nonspecific inhibition of other kinases. In fact, activation of HER2 by NRG was largely unaffected by doses of NVP-AEW541 as high as 10 µM, while activation of the IGF-IR was fully prevented at 1 µM (Esparís-Ogando A, unpublished observations).
Interestingly, expression of activated pIGF-IR was observed under resting conditions. Although the reason for such an activation has not been explored, resting IGF-IR activation may be due to IGF-I availability, either produced autocrinally or supplied by serum. The mechanism of action of NVP-AEW541 on breast cancer cells is complex and involves cell cycle arrest and apoptosis. NVP-AEW541 induced an increase in the G0/G1 and a decrease in S phase, and biochemical studies demonstrated that this effect may be due to an increase in p27 levels. These findings are in line with other reports that have shown that IGF-IR-transfected cells express lower levels of p27 [11]. The increase in p27 levels could be secondary to Akt inactivation, as this kinase plays a predominant role in regulating p27 stability and subcellular distribution [33]. Analogous decreases in pAkt levels have been described in neuroblastoma or sarcoma cells treated with NVP-AEW541 [22, 23]. By contrast, NVP-AEW541 produced a decrease in p21. This finding may in principle result paradoxical due to the proposed role of p21 as a negative regulator of cell cycle progression [34]. However, previous observations indicate that up-regulation of p21 is required for IGF-IR-induced proliferation in MCF7 cells [35].
We also observed an increase in Annexin V staining and a decrease in the mitochondrial membrane potential, both signs of apoptotic cell death. The changes in the mitochondrial membrane potential were detected at high doses of NVP-AEW541. Although the degree of induction of Annexin V staining was low and only observed at high concentrations of the drug, our results indicate that apoptotic cell death may play a role in the action of NVP-AEW541 on breast cancer cells. Indeed, a recent report in myeloid leukemia cells has shown that NVP-AEW541 induces apoptosis as the principal mechanism of action [36]. In a similar manner, other anti-IGF-IR strategies such as monoclonal antibodies have shown to produce a proapoptotic effect which was partially attributed to alterations in the ratio of proapoptotic and anti-apoptotic (Bax/Bcl-2) proteins [37].
High expression of IGF-IR in breast cancer cells has been linked to trastuzumab resistance [11]. This opens the interesting possibility of using therapies that target IGF-IR to favor responses to trastuzumab. Following this reasoning, we studied the anti-proliferative effect of both drugs administered in combination. Addition of NVP-AEW541 and trastuzumab was synergistic, and this occurred in the trastuzumab-sensitive cell lines BT474 and SKBR3, but not in trastuzumab-resistant MCF7 cells. In contrast, combination of NVP-AEW541 with other agents commonly used in the breast cancer clinic, such as docetaxel or vincristine, was not effective in terms of potentiation. Interestingly, the combination of NVP-AEW541 and trastuzumab produced the same anti-proliferative effect than the combination of trastuzumab and docetaxel. It is important to mention that the combination of trastuzumab and taxanes is one of the gold standards in the treatment of breast cancer patients with HER2-overexpressing tumors [38]. In addition to our findings, precedents of increased action of trastuzumab when combined with anti-IGF-IR antibodies or IGF-I-binding proteins indicate that the combination of trastuzumab with anti-IGF-IR agents is active and could be further explored in phase Ib-II clinical trials. Furthermore, the potential combination of anti-IGF-IR antagonists with lapatinib (a dual epidermal growth factor receptor and HER2 inhibitor that is reaching the breast cancer clinic) may prevent the discomfort and expenses of drugs that require parenteral administration.
With respect to the mechanism of potentiation of the action of trastuzumab by NVP-AEW541, biochemical studies indicated that the combination of both drugs exerted a higher effect on the levels of pAkt and p27 than either drug administered alone. Given the importance of p27 as a mediator of the action of trastuzumab on breast cancer cells overexpressing HER2 [39, 40], it is possible that changes in the levels of p27 may play a role in the action of the combined treatment on these cells. In addition to these data, genomic studies identified several genes whose mRNAs were modified by the combined treatment. Among them, the PIP appeared to be the most highly up-regulated. Although its function in the mammary gland is unknown, PIP has been reported to be a small glycoprotein secreted by breast carcinomas [41, 42]. Another interesting finding was the profound effect of the combination on the actin-bundling protein fascin homolog 1 protein mRNA levels. This protein organizes filamentous actin into bundles present in microspikes, membrane ruffles, and stress fibers. Fascin homolog 1 has been reported to be overexpressed in breast cancer, and increased levels of the protein correlate with a poor prognosis [43]. In addition, p8, a small protein involved in stress-induced apoptotic cell death, was also up-regulated by the combined treatment [30]. Other genes that participate in cell cycle progression through binding to p130 and the control of RB function, such as E2F-4, E2F-5, or TFDP1 (which also may control apoptosis), were also specifically modified by the combined treatment. Interestingly, several proteins involved in the control of proteolytic processes were also modified by the combined treatment. Of these, several cystatins were up-regulated. Cystatins S, SN, and SA are secreted proteins that inhibit the function of cysteine proteases. The Cystatins have been reported to control multiple processes, involving cell proliferation or apoptosis, and may be useful antitumoral agents [44]. Taken together, the biochemical and genomic data indicate that the combination of trastuzumab and NVP-AEW541 may exert its action on breast cancer cells through a complex array of transcriptional and posttranscriptional modifications of proteins that control cell number.
The findings of our study have important clinical implications. We confirm that the IGF-IR is frequently overexpressed in breast tumors and that targeting of this receptor affects proliferation and survival of breast cancer cells. Although others have used anti-IGF-IR antibodies to achieve an analogous effect, these antibodies may also inhibit the insulin receptor provoking metabolic disorders [19]. It is therefore important to search for alternative ways to decrease IGF-IR activity, and in this respect, the use of specific tyrosine kinase inhibitors could overcome this problem. We further observed that the inhibition of two tyrosine kinase receptors, the IGF-IR and HER2, increases the anti-proliferative activity when compared with the effect of targeting either receptor alone, indicating that this approach should be explored in the design of clinical studies. Furthermore, the combination of these both drugs was much more active than the combination of therapeutic strategies that are currently the standard of care, as is the case of trastuzumab plus docetaxel. In summary, our results support the clinical development of anti-IGF-IR agents in breast cancer, especially in combination with agents, such as trastuzumab, that target HER2.
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Ministry of Science and Technology of Spain (BFU2006-01813/BMC to A.P.); Scientific Foundation of the Spanish Association for Cancer Research (AECC); Instituto de Salud Carlos III (PI061552 and CP04/00045 to A.E.O.); Spanish Cancer Centers Network Program (RD06/0020/0041); European Community through the regional development funding program (FEDER) to Cancer Research Institute.
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We wish to thank Dr Johan Zimmerman (Novartis Pharma, Basel, Switzerland) for comments on the manuscript and Dr Mark Andrew Pearson (Novartis Pharma) for initially providing NVP-AEW541.
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These authors contributed equally to the work. 
Received for publication March 4, 2008. Revision received May 30, 2008. Accepted for publication June 2, 2008.
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