1. Angiotensin signalling in GBM: AT2R as a novel therapeutic target
Renziehausen A.1, Eschbacher J.2, Tzakos A.3, O'Neill K.1, Crook T.4 and Syed N.1
1John Fulcher’ Molecular Neuro-oncology Laboratory, Imperial College, London, UK
2Barrow Neurological Institute, Phoenix, AZ, USA
3University of Ioannina, Ioannina, Greece
4Southend University Hospital, London, UK
Acknowledgments
This work is funded by the Brain Tumour Research Campaign and the Barrow Neurological Foundation
Introduction
Locally expressed angiotensin II type 1 and type 2 receptors (AT1R and AT2R respectively) of the renin-angiotensin system have been implicated in cancer progression. While the involvement of AT1R has been well studied, the role of AT2R in cancer is largely unknown. The aim of this study is to investigate the influence of angiotensin signalling on the growth, migration and induction of angiogenesis in GBM, as well as determine whether commonly used drugs targeting angiotensin signalling to manage blood pressure have the potential to be repurposed as adjuvant therapies for GBM.
8MG UC
8MG
PD+Los
Methods
The AT1R antagonist Losartan (Los) and AT2R antagonist PD (PD) were used alone or in combination with the ligand angiotensin II (AngII) in:
Colorimetric proliferation assays
Wound healing assays
hCMEC/D3 endothelial cell tube formation assays
Downstream gene expression analysis using qPCR
Fig.3. AT2R inhibition disrupts endothelial cell network formation. Particularly mesh formation, a determinant of network integrity, is significantly reduced upon PD treatment.
Results A B
Fig.4. Downstream gene analysis following 6h treatment with AT2R inhibitor PD. CYR61, which has been implicated in promoting GBM growth and angiogenesis, is down-regulated and so is Lamb1, which is known to enhance cell migration. Interestingly, pro-angiogenic VEGF is down-regulated while the anti-angiogenic factor THBS1 is up-regulated, which indicates that AT2R signalling can influence major angiogenic pathways in GBM.
Summary & Conclusion
Contrary to reports in other cancer types, inhibition of AT1R signalling via Losartan had no negative effect on the growth of GBM
On the other hand, inhibition of AT2R significantly reduced GBM cell proliferation, migration and the induction of angiogenesis.
This effect was accompanied by differential expression of downstream genes implicated in these processes.
Therefore, inhibition of AT2R signalling should be considered as a new potential adjuvant treatment of GBM
Fig.1. Colorimetric proliferation assays to determine the role of AT1R and AT2R on GBM cell growth: A) Ang-II treatment increases cell number significantly in cell lines expressing both AT1R and AT2R. Inhibition of AT2R with PD can significantly reduce cell number both with and without exogenous Ang-II.
B) Patient derived primary cells that express AT2R are susceptible to significant growth reduction upon AT2R inhibition with PD. T0
T24 UC
T24 PD
T24 PD+AngII
T24 PD+Los
8MG:
Clinical relevance
A recent retrospective study on 81 GBM patients highlighted improvements in clinical outcomes of GBM patients treated with Ang-II inhibitors in combination with RT and TMZ. Patients on Ang-II inhibitors remained functionally independent significantly longer and overall survival was increased by 3.8 months on average2.
A novel highly selective AT2R antagonist that is predicted to cross the BBB is currently under clinical development by Novartis. Previous studies have shown that it is around 250-fold more potent than PD in relieving neuropathic pain. With its high potency and good safety profile this would be a great candidate drug to test for GBM.
Fig.2. AT2R inhibition reduces GBM cell migration, particularly when AT1R is inhibited simultaneously using Los.
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2Januel E, Ursu R, Alkhafaji A, Marantidou A, Doridam J, Belin C, Levy-Piedbois C, Carpentier AF (2015) Impact of renin-angiotensin blockade on clinical outcome in glioblastoma. Eur J Neurol.;22(9):1304-9