The level of insulin growth factor-1 receptor expression is directly correlated with the tumor uptake of 111In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin)

doi:10.1016/j.nucmedbio.2008.05.010

Nuclear Medicine and Biology

Volume 35, Issue 6, August 2008, Pages 645-653

https://doi.org/10.1016/j.nucmedbio.2008.05.010 Get rights and content

Abstract

Introduction

Our objective was to define the relationships between tumor uptake of [¹¹¹In]-IGF-1 and [¹¹¹In]-IGF-1(E3R), an analogue which does not bind insulin growth factor-1 (IGF-1) binding proteins (i.e., IGFBP-3), and the level of IGF-1 receptor (IGF-1R) expression on human breast cancer (BC) xenografts in athymic mice, as well as the feasibility for tumor imaging. A second objective was to correlate IGF-1R (and HER2 density) with the cytotoxicity of trastuzumab in the absence/presence of IGFBP-3 or the IGF-1R tyrosine kinase inhibitor, AG1024.

Methods

The tumor and normal tissue uptake of [¹¹¹In]-IGF-1 and [¹¹¹In]-IGF-1(E3R) were determined at 4 h postinjection in mice implanted subcutaneously with MDA-MB-231, H2N, HR2 or MCF-7/HER2-18 human BC xenografts (8.5×10⁴, 1.4×10⁴, 4.0×10⁴ and 1.0×10⁵ IGF-1R/cell, respectively). The effect of co-injection of IGF-1 (50 μg) or IGFBP-3 (2 or 25 μg) was studied. The relationship between tumor uptake of [¹¹¹In]-IGF-1(E3R) and IGF-1R density was examined. MicroSPECT/CT imaging was performed on mice with MCF-7/HER2-18 tumors injected with [¹¹¹In]-IGF-1(E3R). The surviving fraction of BC cells exposed to trastuzumab (67.5 μg/ml) in the absence/presence of IGFBP-3 (1 μg/ml) or the IGF-1R kinase inhibitor, AG1024 (1 or 5 μg/ml), was determined.

Results

[¹¹¹In]-IGF-1 was specifically taken up by MCF-7/HER2-18 xenografts; tumor uptake was decreased twofold when co-injected with IGF-1 (1.9±0.1 vs. 1.0±0.1 %ID/g). Co-injection of IGBP-3 decreased kidney uptake of [¹¹¹In]-IGF-1 up to twofold and increased circulating radioactivity threefold. There was a strong linear correlation (r²=0.99) between the tumor uptake of ¹¹¹In-IGF-1(E3R) and IGF-1R density. Tumor uptake ranged from 0.4±0.05 %ID/g for H2N to 2.5±0.5 %ID/g for MCF-7/HER2-18 xenografts. MCF-7/HER2-18 tumors were visualized by microSPECT/CT. Resistance of BC cells to trastuzumab was directly associated with IGF-1R expression, despite co-expression of HER2. The resistance of HR2 cells could be partially reversed by IGFBP-3 or AG1024.

Conclusion

Imaging of IGF-1R expression using [¹¹¹In]-IGF-1(E3R) may be useful for identifying HER2-positive tumors in BC patients that are resistant to trastuzumab through this mechanism.

Introduction

Trastuzumab (Herceptin) is a humanized IgG₁ monoclonal antibody (mAb) that is used for immunotherapy of HER2-amplified breast cancer (BC). The response rate to trastuzumab is 11–23% administered as a single agent, but up to 41% when given in combination with anthracyclines or paclitaxel [1]. Nevertheless, almost one in two patients with HER2-positive tumors does not respond to the drug. Moreover, almost all responding patients develop resistance to trastuzumab within a year [2]. One proposed mechanism of resistance is overexpression of insulin growth factor-1 receptors (IGF-1R). The role of IGF-1R in mediating resistance to trastuzumab was first described by Lu et al. [3] in 2001, who showed that SKBR-3 human BC cells that are HER2 amplified were resistant to trastuzumab in vitro when transfected with the IGF-1R gene (SKBR3/IGF-1R), in contrast to nontransfected cells which were sensitive. Moreover, resistance in SKBR-3/IGF-1R cells was reversed in the presence of IGF binding protein-3 (IGFBP-3) which sequesters IGF-1. Similar results were obtained by Benz et al. [4] for MCF-7 cells which naturally express IGF-1R transfected with the HER2 gene (MCF-7/HER2-18). These cells were growth inhibited by trastuzumab only if IGF-1R was blocked by anti-IGF-1R (α-IGF-1R) mAbs or IGF-1 was sequestered by IGFBP-3 [4]. The mechanism of IGF-1R-mediated resistance of BC cells to trastuzumab is thought to involve the PI3K-Akt pathway and down-regulation of the cyclin-dependent kinase inhibitor, p27^Kip1 [5].

Radiopharmaceuticals that probe IGF-1R expression noninvasively in BC could be useful for identifying tumors that are intrinsically resistant to trastuzumab or that acquire this resistance over time through this mechanism. Moreover, IGF-1R and its binding proteins (IGFBPs) have been implicated in the pathogenesis of BC and, thus, imaging this receptor could be diagnostically and prognostically useful in the disease [6], [7], [8], [9]. Nevertheless, the relationship between the level of expression of IGF-1R and the progression of BC or its response/resistance to trastuzumab is currently unknown. Our objective in this study was to define the relationship between the tumor localization of [¹¹¹In]-IGF-1 and [¹¹¹In]-IGF-1(E3R), and the IGF-1R density on BC cells, as well as to explore the feasibility of imaging IGF-1R in tumors using [¹¹¹In]-IGF-1(E3R). IGF-1(E3R) is a synthetic analogue of IGF-1 that is not recognized by IGFBP-3 [10], [11]. A second objective was to correlate the level of IGF-1R (and HER2) density on these BC cells with the in vitro cytotoxicity of trastuzumab in the absence/presence of IGFBP-3 or the selective IGF-1R tyrosine kinase inhibitor, AG1024 [12].

Section snippets

Breast cancer cells

MDA-MB-231, MDA-MB-361, BT-20 and BT-474 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MDA-MB-231 and MDA-MB-361 cells were cultured in Leibovitz L15 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS) under air atmosphere at 37°C. The HER2-transfected MDA-MB-231 variant H2N and its HR2 trastuzumab-resistant clones were generously provided by Dr. Robert Kerbel (Sunnybrook and Women's College Health Science Centre,

Radioligand cell-binding assays

The K_d and B_max values for binding of [¹¹¹In]-IGF-1 or [¹¹¹In]-trastuzumab to MDA-MB-231, H2N, HR2 and MCF-7/HER2-18 cells are shown in Table 1. A representative curve for binding of [¹¹¹In]-IGF-1 to MCF-7/HER2-18 cells is shown in Fig. 1. MCF-7/HER2-18 cells are a variant of MCF-7 cells which have been transfected with the HER2 gene [4]. These cells displayed high HER2 density measured with [¹¹¹In]-trastuzumab as well as high levels of IGF-1R measured with [¹¹¹In]-IGF-1. In contrast,

Discussion

IGF-1(E3R) is an analogue of IGF-1 in which Glu³ is substituted by Arg³, disrupting its binding to IGFBPs (e.g., IGFBP-3) [10], [11]. Competition assays confirmed that the binding of [¹¹¹In]-IGF-1 to IGF-1R on MCF-7/HER2-18 cells was inhibited by IGF-1 or IGFBP-3, but binding of [¹¹¹In]-IGF-1(E3R) was blocked only by IGF-1 (Fig. 2). The K_d value for [¹¹¹In]-IGF-1 was 2 nmol/L (Table 1). [¹¹¹In]-IGF-1 or [¹¹¹In]-IGF-1(E3R) has not been previously described, but Sun et al. [17] reported a similar

Conclusion

We conclude that there was a strong linear correlation between the tumor uptake of [¹¹¹In]-IGF-1(E3R), a radiolabeled analogue of IGF-1 which does not interact with IGFBPs, and the IGF-1R expression level on human BC cells forming the tumor xenografts in athymic mice. MicroSPECT/CT imaging allowed visualization of tumor xenografts with high IGF-1R expression. We further conclude that in the panel of BC cells examined, there was a direct relationship between IGF-1R density and the resistance of

Acknowledgments

The authors would like to thank Dr. Robert Kerbel for providing the H2N and HR2 cells, Dr. Mien-Chie Hung for the MCF-7/HER2-18 cells and Ms. Deborah Scollard for her excellent technical assistance. Parts of this work were presented at the Society of Nuclear Medicine meeting, Washington, DC, June 2–6, 2007.

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^☆: This study was supported by grants from the Ontario Cancer Research Network (No. 03-NOV-0428; One Millimetre Cancer Challenge Imaging Platform) with funds from the Province of Ontario. Kristin McLarty was supported by a Canadian Institutes of Health Research (CIHR) Excellence in Radiation Research (EIRR) fellowship. Veerle Kersemans was supported by a Wyeth-CIHR postdoctoral fellowship.

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The level of insulin growth factor-1 receptor expression is directly correlated with the tumor uptake of 111In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin)☆

Abstract

Introduction

Methods

Results

Conclusion

Introduction

Section snippets

Breast cancer cells

Radioligand cell-binding assays

Discussion

Conclusion

Acknowledgments

Eur J Cancer

Lancet

Int J Rad Appl Instrum [A]

Nucl Med Biol

Ann Oncol

Clinical trials of Herceptin (trastuzumab)

Eur J Cancer

Trastuzumab and breast cancer: developments and current status

Int J Clin Oncol

Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin)

J Natl Cancer Inst

Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu

Breast Cancer Res Treat

Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells

Int J Cancer

Elevated insulin-like growth factor I receptor autophosphorylation and kinase activity in human breast cancer

Cancer Res

Expression patterns of insulin-like growth factor 1 (IGF-I) and its receptor in mammary tissues and their associations with breast cancer survival

Breast Cancer Res Treat

The level of insulin growth factor-1 receptor expression is directly correlated with the tumor uptake of ¹¹¹In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin)☆