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Highlights • Growth factor signaling causes sustained nuclear ERK1/2 activation. • The SCR and BCR/ABL inhibitor dasatinib blocks ERK1/2 and represses cell invasion. • EGF-stimulated cells may escape dasatinib inhibition of invasion through mesenchymal to amoeboid transition. • Combined inhibition of SRC and Rho-kinase signaling is necessary to completely block EGF-induced 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.

Rational targeting of proteins involved in controlling cancer cell behavior with small bioactive compounds can accelerate anti-cancer drug discovery. We report the identification of a new small molecule compound inhibitor of the FGFR adaptor protein FRS2. Pharmacophore-based computational screening combined with functional, biophysical, and structural binding analyses, led to the identification of low-molecular weight ligands that interact with the PTB domain of FRS2. By assessing the compounds’ anti-invasion activity in human FGFR-driven cancer cell models, three chemically distinct bioactive molecules were shortlisted for further analysis. A lead compound was selected that specifically repressed FGFR-driven MAPK activation and matrix invasion and displayed on-target activity in cells. Proteome-wide off-target activity of the primary lead was determined and functional in vivo efficacy in an FGFR driven ovarian cancer model confirmed. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven human cancers.

The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the fast-endophilin-mediated endocytosis effector endophilin-A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells.

The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the FEME effector Endophilin A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells.Graphical abstractc-MET activation upon HGF stimulation induces c-MET internalization and induces downstream MAP4K4 activity. (1) MAP4K4 is required downstream of activated c-MET for the maintenance of surface presentation of CD155 in activated cells. CD155 expression is required for MB cell migration, invasion and proliferation in the tissue context. (2) MAP4K4 is required downstream of activated c-MET to maintain membrane depolarization, possibly by regulating the surface localization of several ion channels and transporters. (3) MAP4K4 is required downstream of activated c-MET cause PM-proximal localization of FEME effector CIP4, FBP17 and CIN85. The FEME effector endophilin A is necessary for MB cell migration, invasion and dissemination.

Medulloblastoma (MB) is the most common malignant brain tumour in children and long-term sequelae in survivors caused by the harsh, non-targeted treatments is common. Current screening protocols for the detection of novel druggable vulnerabilities rely on 2D and 3D cell culture systems using established or primary tumour cells. However, these assays do neither provide the cellular, chemical and biophysical context of the tissue, nor reveal drug effects on the invasive behavior of the tumour cells. To overcome these limitations, we have established ex vivo Organotypic Cerebellum Slice Co-culture (OCSC) models for SHH and group 3 MB tumours, which provide spatio-temporal insights in the growth and dissemination behaviour of the tumour cells. OCSCs represent an excellent translational model for patient sample profiling, drug testing, and microenvironmental or explorative target identification studies in a physiologically relevant tissue context. We found that the AURKB inhibitor Barasertib (AZD-1152), which was ineffective to reduce ONS-76 cell invasion in vitro, effectively reduced tumour volume and proliferation of these SHH tumour cells in the tissue context. The AURKB inhibitor thus displays higher efficacy ex vivo, indicating an impact of the microenvironment on its functional activity. We further validated tumour suppressive activity of AURKB inhibition with Barasertib at low nanomolar concentration or with siRNA-mediated depletion of AURKB in Grp3 MB tumour cells and in primary tumour cells ex vivo, thereby confirming the therapeutic potential of AURKB inhibition against MB progression. We furthermore found that the combination of AURKB inhibition with irradiation or with the SRC inhibitor Dasatinib, lead to a near complete eradication of the tumour cells. Thus, OCSCs are a clinically relevant model where tumour material can be efficiently tested for drug sensitivities, which allowed us to identify AURKB inhibition as an effective treatment against tissue invasive MB.

Bis-cyclometalated iron(II) complex [Fe(κC,N-phpy)2(CO)2] (1) (phpyH = 2-phenylpyridine) has been prepared in good yield from [Fe(CO)5] and [Hg(phpy)2] in the presence of dibromine, which is unexpe...

International audience; [Fe(NCN)2]PF6 (1·PF6) [NCHN = 1,3-di(pyridin-2-yl)benzene] was readily obtained by a transmetalation reaction between [Fe3(CO)12] and Hg(NCN)Cl followed by a metathesis reaction with KPF6. X-ray diffraction, electron paramagnetic resonance spectroscopy, and cyclic voltammetry studies confirmed the proposed structure. Cytotoxic assays in human colon cancer (HCT-15), lung cancer (SKLU), and gastric cancer (AGS, KATOIII) cells were performed, and the IC50 data obtained for all cell lines showed that 1·PF6 has a much higher activity than cisplatin.