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
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.
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Cell lines and reagents
Cell lines MCF-7 and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC). They were cultured in RPMI 1640 and DMEM (Gibco) respectively, supplemented with 10% foetal calf serum (FCS) (Sanbio) in a humidified atmosphere with 5% CO2 at 37 °C. MDA-MB-231 medium was in addition supplemented with 1 mM glutamine. MCF-7 was used as a positive control cell line for high IGF-1R expression, MDA-MB-231 cells were chosen as TNBC cell line model with intermediate IGF-1R expression.
Hsp90 inhibition effects in vitro on IGF-1R and VEGF-A
NVP-AUY922 treatment for 24 h with 300 nM resulted in an 87.9% downregulation of IGF-1R in MCF-7 cells and an 86.1% downregulation in MDA-MB-231 cells in FACS experiments (Fig. 1A). Similar results were found with 89Zr-MAB391 (Fig. 1B). NVP-AUY922 treatment at 300 nM for 24 h down-regulated IGF-1R 84.8% in MCF-7 and 84.5% in MDA-MB-231 cells. Excess of unlabelled MAB391 reduced 89Zr-MAB391 uptake to 1.1% non-specific uptake in MCF-7 and 0.9% in MDA-MB-231 cells. NVP-AUY922 reduced IGF-1R expression
Discussion
This study visualised that Hsp90 inhibitor NVP-AUY922 has dual effects on IGF-1R and VEGF-A in TNBC. Molecular imaging of IGF-1R and VEGF-A, as read out biomarker for treatment effect of HSP90 inhibitors, may therefore further support clinical development of these agents in TNBC patients.
An agent with a broad spectrum of anti-tumour activity is of interest in TNBC. Hsp90 inhibitors interact with the N-domain ATP-binding pocket, prevent ATP binding, and stop the chaperone cycle leading to client
Conflict of interest statement
None declared.
Acknowledgements
The authors would like to thank K.J.D. Kol for technical assistance. This study was supported by grants RUG 2009-4273 and RUG 2010-4603 of the Dutch Cancer Society and supported by the Vanderes Foundation.
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