Real-time sensing of MAPK signaling in medulloblastoma cells reveals cellular evasion mechanism counteracting dasatinib blockade of ERK activation during invasion.

Aberrantly activated kinase signaling pathways drive invasion and dissemination in medulloblastoma (MB). A majority of tumor-promoting kinase signaling pathways feed into the mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) pathway. The activation status of ERK1/2 during invasion of MB cells is not known and its implication in invasion control unclear. We established a synthetic kinase activation relocation sensor (SKARS) for the MAPK ERK1/2 pathway in MB cells for real-time measuring of drug response. We used 3D invasion assays and organotypic cerebellum slice culture to test drug effects in a physiologically relevant tissue environment. We found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) caused rapid nuclear ERK1/2 activation in MB cells, which persisted for several hours. Concomitant treatment with the BCR/ABL kinase inhibitor dasatinib completely repressed nuclear ERK1/2 activity induced by HGF and EGF but not by bFGF. Increased nuclear ERK1/2 activity correlated positively with speed of invasion. Dasatinib blocked ERK-associated invasion in the majority of cells, but we also observed fast-invading cells with low ERK1/2 activity. These ERK1/2-low, fast-moving cells displayed a rounded morphology, while ERK-high fast-moving cells displayed a mesenchymal morphology. Dasatinib effectively blocked EGF-induced proliferation while it only moderately repressed tissue invasion, indicating that a subset of cells may evade invasion repression by dasatinib through non-mesenchymal motility. Thus, growth factor-induced nuclear activation of ERK1/2 is associated with mesenchymal motility and proliferation in MB cells and can be blocked with the BCR/ABL kinase inhibitor dasatinib.

Crosstalk between SHH and FGFR Signaling Pathways Controls Tissue Invasion in Medulloblastoma.

In the Sonic Hedgehog (SHH) subgroup of medulloblastoma (MB), tumor initiation and progression are in part driven by smoothened (SMO) and fibroblast growth factor (FGF)-receptor (FGFR) signaling, respectively. We investigated the impact of the SMO-FGFR crosstalk on tumor growth and invasiveness in MB. We found that FGFR signaling represses GLI1 expression downstream of activated SMO in the SHH MB line DAOY and induces MKI67, HES1, and BMI1 in DAOY and in the group 3 MB line HD-MBO3. FGFR repression of GLI1 does not affect proliferation or viability, whereas inhibition of FGFR is necessary to release SMO-driven invasiveness. Conversely, SMO activation represses FGFR-driven sustained activation of nuclear ERK. Parallel activation of FGFR and SMO in ex vivo tumor cell-cerebellum slice co-cultures reduced invasion of tumor cells without affecting proliferation. In contrast, treatment of the cells with the SMO antagonist Sonidegib (LDE225) blocked invasion and proliferation in cerebellar slices. Thus, sustained, low-level SMO activation is necessary for proliferation and tissue invasion, whereas acute, pronounced activation of SMO can repress FGFR-driven invasiveness. This suggests that the tumor cell response is dependent on the relative local abundance of the two factors and indicates a paradigm of microenvironmental control of invasion in SHH MB through mutual control of SHH and FGFR signaling.