Abstract 4025: Exogenous HGF bypasses ErbB inhibition on tumor cell viability in medulloblastoma cell lines
Authors: Zomerman Walderik W. , Plasschaert Sabine LA , Diks Sander H. , Lourens Harm-Jan , Meeuwsen-de Boer Tiny , Hoving Eelco W. , den Dunnen Wilfred FA , de Bont Eveline SJM 
Published: August 2015

Abstract
Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed limited clinical responses in medulloblastoma. An emerging concept is the role of the tumor microenvironment in bypassing targeted therapies by producing ligands that can compensate for the drug-inhibited RTK via alternative routes of pathway activation. The aim of this study is to investigate the role of well-known CNS expressed growth factors in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (EGFR, ErbB2-4) inhibition on tumor cell viability and downstream signaling in medulloblastoma cell lines.
Five different medulloblastoma cell lines (DAOY, RES256, UW402, UW426, and UW473) were treated with crizotinib or canertinib, targeting MET or EGFR, ErbB2-4, respectively. Upon treatment, cells were stimulated with the CNS expressed growth factor VEGF-A PDGF-AB, HGF, FGF-2 or EGF basic. Subsequently, a cell viability assay was used to measure cell viability upon growth factor stimulation, compared to non-growth factor stimulated cells. Expression levels of respective receptor tyrosine kinases (RTK’s) and growth factors were analyzed using flow cytometry and human growth factor antibody arrays. Phosphorylation status of RTK's and downstream signaling effectors was visualized using western blot analysis and human phospho-kinase antibody arrays.
We observed high MET and EGFR cell surface expression levels. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and resulted in downstream phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Whereas crizotinib and canertinib inhibited cell viability and phosphorylation of Akt and ERK1/2, addition of HGF to canertinib significantly enhanced cell viability and phosphorylation of Akt and ERK1/2. The HGF induced bypass of canertinib was reversed by the addition of crizotinib. HGF protein was hardly released by medulloblastoma cell lines. This suggests that MET activation is mainly dependent on paracrine HGF contribution. In conclusion, these data point to the bypassing capacity of well-known CNS expressed paracrine growth factors (e.g. HGF) in medulloblastoma cell lines and provides a potential explanation for the limited clinical response of single RTK inhibitors in medulloblastoma clinical trials. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of tyrosine kinase inhibitors, targeting for example MET and EGFR in medulloblastoma.
Citation Format: Walderik W. Zomerman, Sabine LA Plasschaert, Sander H. Diks, Harm-Jan Lourens, Tiny Meeuwsen-de Boer, Eelco W. Hoving, Wilfred FA den Dunnen, Eveline SJM de Bont. Exogenous HGF bypasses ErbB inhibition on tumor cell viability in medulloblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2015-4025