89Zr-trastuzumab PET visualises HER2 downregulation by the HSP90 inhibitor NVP-AUY922 in a human tumour xenograft
Introduction
Heat shock protein 90 (HSP90) is a 90 kDa molecular chaperone protein which is involved in the conformation, activation, functionality and stability of over 100 client proteins. Client proteins of HSP90 are involved in all hallmarks of oncogenesis: tumour cell growth, invasion, metastasis, angiogenesis, evading apoptosis and insensitivity to anti-growth signals.1 Tumour cells overexpress HSP90 2- to 10-fold compared to normal cells of the related tissue and are more dependent on HSP90 than normal counterparts.2 In tumour cells HSP90 is predominantly present in an active complexed state with a higher ATPase activity and ATP affinity.3 These characteristics make HSP90 a target with high potential for cancer therapy, as the inhibition of HSP90 results in targeting multiple signalling pathways crucial for tumour maintenance.
Several HSP90 inhibitors are currently in clinical development. Most pre-clinical and clinical experience has been obtained with the geldanamycin class of HSP90 inhibitors of which 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; tanespimycin) is the best-studied family member. Therapeutic effects (disease stabilization, tumour responses) of 17-AAG were seen in several phase I and II trials, including a phase II trial in patients refractory to trastuzumab therapy.4, 5 However, clinical application of 17-AAG is hampered due to hepatotoxicity and formulation difficulties.
A new interesting class of HSP90 inhibitors consists of the pyrazole resorcinols, of which NVP-AUY922 is in vitro the most potent family member.6 Pre-clinical activity of NVP-AUY922 has been reported recently7, 8 and NVP-AUY922 is currently being investigated in two phase I–II clinical trials.
Monitoring the pharmacodynamic effects of HSP90 inhibitors would facilitate the clinical development of these drugs. Currently there is no proven early biomarker for evaluating HSP90 inhibition. In a clinical phase I study with 17-AAG, tumour biopsies were taken in which Western blot analysis revealed c-RAF1 inhibition, CDK4 depletion and HSP70 induction.4 The limited feasibility of repeated tumour biopsies, however, has driven the search for non-invasive pharmacodynamic monitoring of HSP90 inhibitors.9
One of the most potent oncogenic client proteins of HSP90 is the human epidermal growth factor receptor-2 (HER2). HER2 is a key player in oncogenic transformation in a variety of cancer types and is overexpressed in 20–25% of breast cancers.10 The rapid but transient HER2 degradation induced by HSP90 inhibition has been shown in vitro and in vivo in several pre-clinical reports.7, 8, 11, 12
Molecular imaging techniques are well suited for non-invasive monitoring of the rapid molecular changes induced by tumour-targeting therapies. Serial HER2 positron emission tomography (PET) imaging is potentially attractive as biomarker as it allows the visualisation and quantification of the molecular tumour response to the drug early during treatment.
HER2 PET imaging with a 68Ga-labelled trastuzumab F(ab′)2 fragment (68Ga-DCHF) has preclinically shown to be able to monitor the HER2 downregulation after 17-AAG treatment.13 In the search for a clinical usable PET tracer, we labelled the HER2 full length antibody trastuzumab with the long-lived PET isotope zirconium-89 (89Zr) for HER2 PET imaging. The pre-clinical kinetics and biodistribution of 89Zr-trastuzumab have recently been described.14 Our clinical experience with this tracer for HER2 imaging in metastatic breast cancer patients showed excellent feasibility.15 In the present study, we aim to use 89Zr-trastuzumab PET imaging for non-invasive quantification of the HER2 downregulation by the HSP90 inhibitor NVP-AUY922 in a HER2 positive xenograft model.
Section snippets
Cell line and reagents
The HER2 overexpressing human ovarian cancer cell-line SKOV-3 was obtained from American Type Culture Collection. Cells were cultured in a humidified incubator at 5% CO2 and 37 °C in D-MEM high glucose, supplemented with 10% FCS. NVP-AUY922 was provided by Novartis. 17-AAG was purchased from LC Laboratories and was used as a reference for the in vitro effects of NVP-AUY922. For the in vitro experiments NVP-AUY922 and 17-AAG were dissolved in DMSO and stored at –80 °C or –20 °C, respectively. For
NVP-AUY922 downregulates HER2 expression in SKOV-3 cells in vitro
NVP-AUY922 induced HER2 downregulation is shown in Fig. 1A. Treatment with 30 and 100 nM NVP-AUY922 for 24 h resulted in HER2 downregulation of 72.5 ± 2.4% and 80.1 ± 1.4%, respectively, compared with untreated control cells. Treatment with 30, 100 and 500 nM 17-AAG resulted in HER2 downregulation of 11.4 ± 3.6%, 41.6 ± 3.7% and 82.0 ± 1.2%, respectively, compared with untreated control cells. NVP-AUY922 and 17-AAG isomolar concentrations of 30 and 100 nM resulted in a more pronounced HER2 downregulation
Discussion
In the present study, we successfully demonstrate the feasibility of 89Zr-trastuzumab HER2 PET as a biomarker for HSP90 inhibition in a HER2 positive xenograft model. Currently there is no clinically proven early biomarker for evaluating the pharmacodynamic effects of HSP90 inhibition. Serial molecular imaging can provide information about the molecular alterations as an early response to HSP90 inhibition. Because many oncoproteins are clients of HSP90,18, 19 there are several molecular imaging
Conflict of interest statement
M.R.J. and C.Q. are employees of Novartis.
Role of the funding source
Personnel (TOM) and materials were paid from the Dutch Cancer Society grant. Novartis provided NVP-AUY922.
Acknowledgements
The authors would like to thank Kirsten van Huisstede and Esther van Straten for their technical assistance. This work was supported by grant 2007-3739 of the Dutch Cancer Society.
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Contributed equally.