Sunitinib Targets in Pheochromocytomas and Paragangliomas
Clarissa Cassol, Daniel Winer, Wei Liu, Miao Guo, Sylvia Asa. University of Toronto, Toronto, ON, Canada; University Health Network, Toronto, ON, Canada; Ontario Cancer Institute, Toronto, ON, Canada
Background: Sunitinib, a multitargeted tyrosine kinase inhibitor (TKi), has been reported as a successful treatment alternative for metastatic pheochromocytoma (PCT)/paraganglioma (PGL). To determine whether this is due to its well-known anti-angiogenic properties or to direct anti-proliferative/pro-apoptotic effects on tumour cells, we created an in vitro model of Sunitinib treatment using MPC 4/30 cells. Potential Sunitinib targets were also investigated in human tumors using a tissue microarray (TMA) and correlated with outcomes.
Design: MPC 4/30 cells were treated with 0uM, 2.5uM and 5.0uM Sunitinib for 72h. Treatment effect were analysed by flow cytometry (FACSCalibur) and gene expression profiling (Gene 1.0 ST Array). Results were examined using DAVID Bioinformatic Resources 6.7 and String protein association network software. IHC expression of Sunitinib targets (VEGFR1, VEGF2, PDGFRα, PDGFRβ, C-KIT) and new potential targets identified through gene expression profiling - Aurora Kinases A (ArkA) and B (ArkB) - were evaluated on a TMA from 39 PCTs and 76 PGLs. Outcome correlations were investigated by logistic regression.
Results: Cell-cycle analysis showed Sunitinib decreases MPC 4/30 proliferation (13.5% of untreated cells on M2 phase versus 5.5% in 5uM treated; p=0.001) and increases apoptosis (14.5% apoptotic debris in untreated cultures versus 24% in 2.5uM and 19.5% in 5uM treated; p non-significant). Gene expression profiling revealed Sunitinib strongly down-regulates cell cycle associated genes (37% of all genes with >2fold downregulation), with more than 50% of these affecting M phase. IHC showed VEGFR2 and PDGFRα to be overexpressed in both tumors, PDGFRβ and VEGFR1 overexpressed only in PGLs and C-KIT overexpressed only in PCTs*. ArkA was underexpressed in PGLs* and ArkB expression was negligible. Increased risk of metastasis was associated with membranous expression of PDGFRα (OR=13.712,*) and VEGFR1 (OR=8.009;*).*p<0.05
Conclusions: Sunitinib decreases cell proliferation in PCT cells mainly by targeting cell cycle, as well DNA metabolism, and cell organization genes. Membranous expression of PDGFRα and VEGFR1 correlated with increased risk of metastastic disease. In contrast with most malignancies, ArkA was underexpressed PGLs. This was unexpected given Aurora's downregulation by Sunitinib in PCT cells in vitro. The findings provide novel insights into mechanistic actions of Sunitinib and identify potential new therapeutic targets for PCTs/PGLs.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 81, Monday Morning