Targeting multiple signalling pathways showed high anti-tumour potential in two mouse phaeochromocytoma cell lines
S. Nölting1, E. Garcia1, G. Alusi1, M. Korbonits1 & A. Grossman1,2
Introduction: Since there is no completely effective therapy available for malignant phaeochromocytomas (PCCs) and paragangliomas, we have been investigating novel targeted therapies utilising one more benign (MPC) and one more malignant (MTT) PCC cell line. We have previously shown that the IGF1-receptor-inhibitor NVP-AEW541 led to compensatory ERK and mTORC1 up-regulation at suboptimal doses and that the dual PI3K/mTORC1-inhibitor NVP-BEZ235 also resulted in compensatory ERK activation at low doses. Therefore we speculated that the efficacy of NVP-BEZ235 may be enhanced by combination with the established agent lovastatin which inhibits ERK-signalling.
Methods/Design: Both cell lines, generated from heterozygous Neurofibromin-1 knock-out-mice, were treated with varying concentrations of lovastatin, NVP-BEZ235, or the combination of both drugs, for different time periods. Metabolic viability was assessed by cell proliferation assay (MTS). Protein levels of phospho-(p) and total AKT, ERK and p70S6K were investigated by Western blotting in MPC cells. Apoptosis was measured by a caspase assay (Caspase-Glo®3/7) in MPC and MTT cells.
Results: Lovastatin significantly dose- and time-dependently decreased MPC and MTT cell viability and diminished pAKT and pERK, but increased pp70S6K. Pre-treatment for 24 h with 10 uM lovastatin followed by addition of 50nM NVP-BEZ235 for 48 h showed a marked additive effect in both cell lines. Combination treatment decreased both pAKT and pp70S6K without ERK up-regulation. Single treatment with NVP-BEZ235 did not induce apoptosis. However, lovastatin significantly enhanced apoptosis compared to the vehicle. Combination treatment increased apoptosis compared to single NVP-BEZ235 treatment but decreased apoptosis compared to single lovastatin treatment.
Conclusions: Targeting PI3K/AKT-, mTORC1/p70S6K- and ERK-signalling simultaneously suggests a novel therapeutic approach for malignant PCCs. Targeting only one or two of these pathways results in compensatory activation of the remaining ones. ERK inhibition appears to specifically increase apoptosis.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details are unavailable.