Visualising dual downregulation of insulin-like growth factor receptor-1 and vascular endothelial growth factor-A by heat shock protein 90 inhibition effect in triple negative breast cancer

Abstract

Purpose

Triple negative breast cancer (TNBC) is biologically characterised by heterogeneous presence of molecular pathways underlying it. Insulin-like growth factor receptor-1 (IGF-1R) expression and vascular endothelial growth factor-A (VEGF-A) have been identified as key factors in these pathways in TNBC. In this study, we aimed at in vivo PET imaging the effect of heat shock protein (Hsp) 90 inhibition by means of NVP-AUY922 on these pathways, with zirconium-89 (⁸⁹Zr) labelled antibodies targeting IGF-1R and VEGF-A.

Materials and methods

In vitro NVP-AUY922 effects on cellular IGF-1R expression and VEGF-A secretion were determined in MCF-7 and MDA-MB-231 cell lines. Moreover human TNBC bearing MDA-MB-231 mice received 50 mg/kg NVP-AUY922 or vehicle q3d intraperitoneally for 21 days. PET scans with ⁸⁹Zr-MAB391 and ⁸⁹Zr-bevacizumab for visualisation of IGF-1R and VEGF-A were performed before and during treatment. Ex vivo biodistribution and correlative tissue analyses were performed.

Results

NVP-AUY922 treatment reduced IGF-1R expression and VEGF-A excretion in both cell lines. Hsp90 inhibition lowered tumour uptake on ⁸⁹Zr-MAB391-PET by 37.3% (P < 0.01) and on ⁸⁹Zr-bevacizumab-PET by 44.4% (P < 0.01). This was confirmed by ex vivo biodistribution with a reduction of 41.3% injected dose (ID)/g for ⁸⁹Zr-MAB391 and 37.8% ID/g for ⁸⁹Zr-bevacizumab, while no differences were observed for other tissues. This coincided with reduced IGF-1R expression and mean vessel density in the NVP-AUY922 treated tumours.

Conclusion

⁸⁹Zr-MAB391 and ⁸⁹Zr-bevacizumab PET reflect effect of Hsp90 inhibitors and can therefore potentially be used to monitor therapeutic effects of Hsp90 inhibitor therapy in TNBC.

Introduction

Triple negative breast cancer (TNBC) accounts for ∼15% of the invasive breast cancers [1]. These tumours lack oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER) 2 expression. TNBC has an aggressive phenotype with an affinity for onset at premenopausal age, early dissemination and high frequency of relapse. Regardless of disease stage, women with TNBC have a worse survival than other breast cancer patients [2]. Breast cancers found in BRCA1 mutation carriers are frequently triple negative and basal like. Effective systemic treatment is limited to chemotherapy and this type of breast cancer lacks effective targeted treatment for standard use [1], [2]. The search for effective targeted agents has been hampered by the heterogeneous presence of molecular mechanisms in TNBC, which is much more complex compared to other breast cancer types [1], [2]. It is unlikely that one driver process can be identified and targeted in this setting, to produce a straightforward effective anti-tumour treatment. Moreover, much effort is ongoing with sophisticated techniques to identify relevant pathways in TNBC. Insulin-like growth factor receptor-1 (IGF-1R) and vascular endothelial growth factor-A (VEGF-A) which regulates angiogenesis are key factors in these pathways [3], [4]. IGF-1R is important for tumour growth and survival and plays a major role in the multistep metastatic process where it regulates migration, invasion and angiogenesis [5].

Therefore an agent with a broad spectrum anti-tumour activity, is of interest in TNBC. Targeting heat shock protein 90 (Hsp90) allows such a broad focus, by inhibiting multiple pathways in cancer. Hsp90 is a widely expressed molecular chaperone, required for proper folding and activation of a multitude of key oncogenic proteins [6]. Hsp90 supports in tumour cells the activated or metastasising forms of oncoproteins and buffers cellular stresses induced by the malignant environment [7]. Hsp90 client proteins such as IGF-1R, hypoxia-inducible factor 1α (HIF-1α), epidermal growth factor receptor (EGFR) and HER2, are involved in all hallmarks of cancer, including tumour cell growth, invasion, metastasis and angiogenesis [8], [9]. VEGF is a downstream product of various Hsp90 client proteins. All of these processes are potentially affected by Hsp90 inhibition, and Hsp90 inhibitors are currently among the most actively pursued cancer drug targets by the pharmaceutical industry.

This rationale for Hsp90 inhibition in TNBC, is supported by two preclinical studies which showed efficacy in TNBC models [10], [11]. Clinical trials have so far focused on non-TNBC subtypes with well known Hsp90 client proteins, such as HER2 overexpressing breast cancer. Absence of clinical trials in TNBC might be related to the lack of biomarkers for Hsp90 inhibition effect in TNBC. Molecular imaging in vivo, a technique that is also applicable in the human setting, can be used to examine Hsp90 targets as potential biomarkers.

In the present study, we have therefore visualised dual therapeutic effects in TNBC that can be influenced by Hsp90 inhibition with NVP-AUY922 in vitro and in vivo, namely IGF-1R expression and angiogenesis, driven by VEGF-A.