Sensitivity of breast cancer cell lines to the novel insulin-like growth factor-1 receptor (IGF-1R) inhibitor NVP-AEW541 is dependent on the level of IRS-1 expression

Abstract

To investigate the potential value of targeting insulin-like growth factor-1 receptor (IGF-1R) in breast cancer, we examined the effects of NVP-AEW541, a selective small-molecule inhibitor of the IGF-1R tyrosine kinase, in a panel of 16 breast cancer cell lines. All cell lines expressed IGF-1R, but MCF-7 expressed much higher levels of insulin receptor substrate-1 (IRS-1) than the others. NVP-AEW541 was more potent at inhibiting growth of MCF-7 cells as compared to the others (IC₅₀, 1 μM vs. ≈7 μM). Comparing MCF-7 to T47D cells, which express IGF-1R at a level identical to MCF-7 but have less than 1/30 the amount of IRS-1, NVP-AEW541 caused cell-cycle arrest at the G1–S boundary, reduced in vitro cell migration, and enhanced the cytotoxic effects of vinorelbine and paclitaxel in MCF-7, but not in T47D. While NVP-AEW541 decreased the phosphorylation of IGF-1R in both cell lines, it inhibited phosphorylation of Akt and disrupted the IRS-1/PI3 K complex only in MCF-7. These findings suggest that inhibiting IGF-1R may be an effective therapeutic strategy for breast cancers that co-express IGF-1R and IRS-1 at high levels.

Introduction

With approximately a million new cases annually, breast cancer is the leading cause of cancer death among women worldwide [1], [2]. Once the disease metastasizes, it is no longer curable despite use of various systemic treatments including cytotoxic chemotherapies, endocrine manipulations, and anti-HER2 monoclonal antibodies. Therefore, novel therapeutic approaches are needed.

Since 1990s, molecularly-targeted drugs have been vigorously developed for cancer treatment. Today, receptor tyrosine kinases (RTKs) are the most promising therapeutic targets. In fact trastuzumab, an anti-HER2 monoclonal antibody used to treat HER2-overexpressing breast cancers, was one of the first therapeutic agents targeting RTKs in solid tumors. For treatment of HER2-overexpressing metastatic breast cancers, trastuzumab combined with conventional chemotherapy has significantly higher efficacy than chemotherapy alone [3]. The use of trastuzumab has recently been extended to adjuvant or neo-adjuvant treatment for operable breast cancer [4], [5], [6]. Among other solid tumors, small-molecule tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR) and Kit have provided new treatment options in patients with non-small cell lung cancer and gastrointestinal stromal tumor, respectively [7].

Insulin-like growth factor-1 receptor (IGF-1R) is another RTK characterized by its contribution to a variety of oncogenic properties, including cell proliferation, cell survival, cell motility, angiogenesis, and metastasis [8], [9], [10]. The ligands of IGF-1R are insulin-like growth factor-1 (IGF-1) and IGF-2. Once the ligands bind to IGF-1R’s extracellular domain, the receptor is autophosphorylated and becomes active as a tyrosine kinase. Subsequently, adaptor molecules, such as insulin receptor substrate-1 (IRS-1) or IRS-2, are tyrosine-phosphorylated and mediate downstream signaling. This includes activation of the phosphatidylinositol 3-kinase (PI3 K)/Akt and Ras-Raf-MEK-ERK1/2 pathways [8], [10]. In breast cancer cell lines, it was reported that IRS-1, but not IRS-2, is the predominant signaling molecule activated by IGF-1 [11]. Several mechanisms able to enhance the PI3 K pathway in malignancies have been identified. In breast cancer, these include mutation of p110, which is the catalytic subunit of PI3 K, and the loss of PTEN [12]. Recent studies suggested that these PI3 K-enhancing alterations may cause resistance to anti-RTK agents, such as trastuzumab and erlotinib [13], [14], [15].

Protein expression of IGF-1R is elevated in a majority of breast cancer cell lines and in 39–93% of breast cancer tumors [8], [16]. Elevated levels of circulating IGF-1 have been found to be associated with an increased risk of developing breast cancer [17]. IRS-1 is reported to be frequently phosphorylated in breast cancer tumors [18] and the level of IRS-1 is correlated with shorter disease-free survival in a subgroup of breast cancer patients [19]. Further, Lu, et al. have proposed a potential mechanism of resistance to trastuzumab in vitro, which consists of alternative cell signaling triggered by IGF-1R instead of HER2 in the presence of trastuzumab [20]. Based on these findings, IGF-1R is considered to be a rational therapeutic target in breast cancer.

Several different methods for targeting IGF-1R are under investigation, including oligodeoxynucleotides, monoclonal antibodies, and small-molecule tyrosine kinase inhibitors [8]. Among these, small-molecule inhibitors have the advantage of possible oral administration. NVP-AEW541 is a selective inhibitor for IGF-1R tyrosine kinase [21], [22]. A previous study showed that it inhibited anchorage-independent growth of MCF-7 breast cancer cells [22]. Here, as part of our investigations into protocol designs for clinical trials of NVP-AEW541 in patients with breast cancer, we evaluated the efficacy of this agent in a large panel of breast cancer cell lines, its activities against oncogenic processes other than cell growth, details of its mechanism of action, and methods for identifying tumors likely to respond to it.

In this study, we show that NVP-AEW541 inhibits cell growth and motility, and enhances the induction of apoptosis by chemotherapeutic agents in vitro. These effects were observed only in MCF-7 breast cancer cells, the only line among those tested that expresses both IGF-1R and IRS-1 at high level. We also showed that NVP-AEW541 inhibits Akt phosphorylation by causing dissociation of IRS-1/PI3 K complex, which appears to be an important mechanism of action. These results suggest that cells that co-express IGF-1R and IRS-1 are likely to be dependent on the signaling pathway from IGF-1R to PI3 K, and therefore sensitive to NVP-AEW541, which disrupts this pathway.