Abstract
Medulloblastoma is the most common malignant pediatric brain tumor. Unfortunately, conventional therapies have led to long-term side effects in the majority of survivors. Therefore, novel therapies inhibiting specific target molecules seem promising. However, the efficacy of these novel drug treatments targeting specific tyrosine kinases is often hampered, leading to tumor recurrence within several months of treatment. The aim of this study is to examine whether various growth factors, present in the tumor micro-environment, are able to influence the viability of medulloblastoma cell lines during tyrosine kinase inhibitor (TKI) treatment, providing a possible explanation for the recurrence of the tumor.
To test this hypothesis, five different medulloblastoma cell lines (DAOY, RES256, UW402, UW426, and UW473) were treated with the tyrosine kinase inhibitor canertinib or crizotinib, targeting the Epidermal Growth Factor Receptor (EGFR) and MET-receptor, respectively. Upon treatment, cells were stimulated with the growth factor HGF, EGF, FGF, VEGF or PDGF. Subsequently, a cell viability assay was used to measure cell survival upon growth factor stimulation, compared to non-growth factor stimulated cells. Expression levels of respective receptor tyrosine kinases (RTK's) were analyzed using flow cytometry and phosphorylation status was visualized using western blot analysis. Effects on downstream signaling effectors were examined using western blot analysis and human phospho-kinase arrays.
The cell viability assays showed increased cell viability upon HGF stimulation in canertinib treated medulloblastoma cell lines. The respective RTK's, MET and EGFR, were highly expressed on the cell surface of all medulloblastoma cell lines. Furthermore, western blotting and phospho-kinase arrays showed that HGF and EGF were able to phosphorylate their respective RTK's and activate downstream signaling effectors. As expected from the cell survival results, stimulation with HGF during canertinib treatment led to re-activation of downstream signaling effectors Akt and/or ERK1/2. Surprisingly, stimulation with EGF during crizotinib treatment also led to downstream re-activation but did not however, lead to an increase in overall tumor cell survival. Enhanced tumor cell survival due to HGF during canertinib treatment could be reversed by combined treatment with a low additional dose of the MET-inhibitor crizotinib. This decrease in cell viability during combined treatment with crizotinib was also mirrored by the inhibition of downstream signaling effectors.
In this study we show that the availability of HGF can lead to enhanced tumor cell survival and re-activation of downstream signaling effectors in canertinib treated medulloblastoma cell lines. This redundancy in RTK-activation by alternative RTK-ligands might provide a possible explanation for the recurrence of the tumor. Therefore, a combined treatment with tyrosine kinase inhibitors, targeting for example MET and EGFR, would be rational in the treatment of medulloblastoma.
Citation Format: Walderik W. Zomerman, Sabine L.A. Plasschaert, Sander H. Diks, Harm Jan Lourens, Eelco W. Hoving, Wilfred F.A. den Dunnen, Eveline S.J.M. de Bont. HGF enhances tumor cell survival in canertinib-treated medulloblastoma cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B10.
