The molecular pathology of malignant paragangliomas and phaeochromocytomas:Identification of signalling pathways being involved in the tumourigenesis of malignant phaeochromocytomas and paragangliomas in order to explore new molecularly targeted therapy options
Final Report Abstract
Malignant phaeochromocytomas (PCCs) and paragangliomas (PGLs) are progressive cancers with few current therapeutic options. The aim of this project was to find new molecular-targeted therapy options for PCCs and PGLs, based on the investigation of relevant signalling pathways and potential mechanisms of resistance of the cancer cell to these agents. Since there is no human PCC cell line available at present, two novel complementary mouse PCC cell lines from heterozygous NF1 knock-out mice, MPC cells (more benign) and MTT cells (more malignant), were utilised for our in vitro studies. PCC and PGL promoting gene mutations can be separated in two major groups (clusters) – cluster 1 and cluster 2 – depending on their transcriptional pattern and underlying gene mutations (reviewed in http://www.ncbi.nlm.nih.gov/pubmed/22391976). Cluster 1 mutations are associated with pseudo-hypoxia, while cluster 2 mutations are associated with aberrant activation of signalling pathways (PI3K/AKT, mTORC1/2 and RAS/RAF/ERK). The NF1 mutation of our cell line model belongs to cluster 2. These in vitro data have recently been published in the Journal of Molecular Endocrinology under http://www.ncbi.nlm.nih.gov/pubmed/22715163. We first investigated the IGF1 receptor inhibitor NVP-AEW541 in both cell lines, and showed a significant reduction of MPC and MTT cell viability at relatively high doses; there was a significant inhibition of PI3K/AKT-signalling at low doses. However, there was an up-regulation of ERK- and p70S6K/mTORC1-signalling at suboptimal doses. Therefore, we next tested the dual PI3K/mTORC1/2-inhibitor NVP-BEZ235 and reported a significant decrease of MPC and MTT cell viability at therapeutically-relevant low doses. However, dual PI3K/mTORC1/2-inhibition was accompanied by compensatory ERK activation. Thus we next investigated the established, well-tolerated and approved drug lovastatin, since this has been described to inhibit ERK-signalling. Lovastatin alone significantly decreased MPC and MTT cell viability at therapeutically-relevant doses and significantly inhibited AKT- and ERK-signalling, but interestingly led to mTORC1/p70S6K activation. Consequently, we subsequently tested the combination of NVP-BEZ235 and lovastatin. Finally, combination treatment led to a highly significantly and much stronger decrease of cell viability compared to each drug separately: the effect was not just additive, but synergistic. Combination treatment significantly inhibited PI3K/AKT- and mTORC1/p70S6K-signalling and prevented the ERK activation which was observed after single treatment with NVP-BEZ235. Lovastatin significantly induced apoptosis in MPC and MTT cells, while NVP-BEZ235 slightly decreased apoptosis after combination treatment compared to treatment with lovastatin alone. ERK up-regulation seems to prevent apoptosis. Each drug singly led to a G1-cell-cycle arrest while combination treatment showed a much stronger inhibitory effect on the cell-cycle with induction of G1-cell-cycle arrest. We concluded that there is a need to target PI3K/AKT-, mTORC1/2- and ERK-signalling simultaneously to overcome resistance.
Publications
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Combined blockade of signalling pathways shows marked anti-tumour potential in phaeochromocytoma cell lines. J Mol Endocrinol. 49(2): 79-96, 2012
Nölting S, Garcia E, Alusi G, Giubellino A, Pacak K, Korbonits M, Grossman AB
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Signaling pathways in pheochromocytomas and paragangliomas: Prospects for future therapies. Endocr Pathol. 23(1): 21-33, 2012
Nölting S, Grossman AB
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Combined inhibition of mTORC1 and mTORC2 signaling pathways is a promising therapeutic option in inhibiting pheochromocytoma tumor growth: In Vitro and in vivo studies in female athymic nude mice. Endocrinology. 154(2): 646-655, 2013
Giubellino A, Bullova P, Nölting S, Turkova H, Powers JF, Liu Q, Guichard S, Tischler AS, Grossman AB, Pacak K
