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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.

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.

MAP4K4 has been correlated with increased cell motility and reduced proliferation in mammalian cells. Consequences and regulation of this dichotomous functionality of MAP4K4 in tumor cells remained elusive. We find that MAP4K4 interacts with the STRN3 protein in medulloblastoma and that STRN3 and MAP4K4 exert opposing functions in Hippo tumor suppressor signaling and clonal growth. However, depletion of either STRN3 or MAP4K4 reduces migration and tissue invasion, and concomitant loss of both proteins halts tumor cell growth in the cerebellar tissue. Mechanistically, STRN3 couples MAP4K4 to the protein phosphatase 2A, which inactivates growth repressing activities of MAP4K4. In parallel, STRN3 enables growth factor-induced PKCθ activation and the direct phosphorylation of VASPS157 by MAP4K4, which are necessary for efficient migration and tissue invasion. Thus, STRN3 controls MAP4K4 functionality in growth factor-activated tumor cells, and the cooperation of the two proteins promotes invasiveness through nPKCs and VASP and increases proliferation via inactivation of Hippo signaling. Targeting of the STRN3-MAP4K4 cooperation could reactivate Hippo signaling and repress invasiveness in growth factor-driven tumors.Graphical abstract

Hypoxia inducible transcription factors (HIFs) are the main regulators of adaptive responses to hypoxia and are often activated in solid tumors, but their role in leukemia is less clear. In acute myeloid leukemia (AML), in particular, controversial new findings indicate that HIF-1α can act either as an oncogene or a tumor suppressor gene, and this may depend on the stage of leukemia development and/or the AML sub-type. In this study, we find that HIF-1α promotes leukemia progression in the acute monocytic leukemia sub-type of AML through activation of an invasive phenotype. By applying a list of validated HIF-1α-target genes to different AML sub-types, we identified a HIF-1α signature that typifies acute monocytic leukemia when compared with all other AML sub-types. We validated expression of this signature in cell lines and primary cells from AML patients. Interestingly, this signature is enriched for genes that control cell motility at different levels. As a consequence, inhibiting HIF-1α impaired leukemia cell migration, chemotaxis, invasion and transendothelial migration in vitro, and this resulted in impaired bone marrow homing and leukemia progression in vivo. Our data suggest that in acute monocytic leukemia an active HIF-1α-dependent pro-invasive pathway mediates the ability of leukemic cells to migrate and invade extramedullary sites and may be targeted to reduce leukemia dissemination.

The molecular mechanisms contributing to distant dissemination and local recurrence of medulloblastoma, the most common malignant brain tumor in childhood, are poorly understood and no targeted anti-invasion therapies exist till date. We explored regulators and effectors of MAP4K4, a pro-invasive kinase overexpressed in MB and associated with metastatic progression in different solid malignancies. MAP4K4 is upregulated both at mRNA and protein levels in primary pediatric brain tumors compared to normal cerebellum. MAP4K4 is required for growth factor- and irradiation-induced migration and invasion of medulloblastoma cells. It furthermore promotes turnover and activation of the receptor tyrosine kinase c-Met and of the ß1 integrin adhesion receptor 1. To characterize these clinically relevant consequences and to identify druggable targets of MAP4K4 function, we profiled the interactome of MAP4K4 in starved and growth factor stimulated medulloblastoma cells. To systematically address MAP4K4 impact on receptor expression and turnover, we determined the MAP4K4-dependent surface proteome in medulloblastoma cells. We found that MAP4K4 is part of the striatin-interacting phosphatase and kinase (STRIPAK) complex and that STRIPAK component striatin 4 is controlling cell motility and invasiveness in medulloblastoma cells. Invasiveness of medulloblastoma cells is abrogated by a truncation mutant of MAP4K4 lacking the striatin 4 interaction domain. We furthermore found that MAP4K4 mediates growth factor-induced surface expression of solute carriers and immunomodulatory proteins involved in chemoresistance and immune evasion. Thus, our study identified MAP4K4 as a missing link between pro-tumorigenic growth factor signaling and tumor cell functions relevant for disease progression. It may help identifying druggable vulnerabilities in medulloblastoma cells to restrict tumor growth and dissemination. 1. Tripolitsioti, D. et al., Oncotarget9, 23220–23236 (2018).

Tumor-secreted extracellular vesicles (EVs) have been identified as mediators of cancer&ndash, host intercellular communication and shown to support pre-metastatic niche formation by modulating stromal cells at future metastatic sites. While osteosarcoma, the most common primary malignant bone tumor in children and adolescents, has a high propensity for pulmonary metastases, the interaction of osteosarcoma cells with resident lung cells remains poorly understood. Here, we deliver foundational in vitro evidence that osteosarcoma cell-derived EVs drive myofibroblast/cancer-associated fibroblast differentiation. Human lung fibroblasts displayed increased invasive competence, in addition to increased &alpha, smooth muscle actin expression and fibronectin production upon EV treatment. Furthermore, we demonstrate, through the use of transforming growth factor beta receptor 1 (TGFBR1) inhibitors and CRISPR-Cas9-mediated knockouts, that TGF&beta, 1 present in osteosarcoma cell-derived EVs is responsible for lung fibroblast differentiation. Overall, our study highlights osteosarcoma-derived EVs as novel regulators of lung fibroblast activation and provides mechanistic insight into how osteosarcoma cells can modulate distant cells to potentially support metastatic progression.

Local tissue infiltration of Medulloblastoma (MB) tumor cells precedes metastatic disease but little is still known about intrinsic regulation of migration and invasion in these cells. We found that MAP4K4, a pro-migratory Ser/Thr kinase, is overexpressed in 30% of primary MB tumors and that increased expression is particularly associated with the frequently metastatic SHH β subtype. MAP4K4 is a driver of migration and invasion downstream of c-Met, which is transcriptionally up-regulated in SHH MB. Consistently, depletion of MAP4K4 in MB tumor cells restricts HGF-driven matrix invasion in vitro and brain tissue infiltration ex vivo. We show that these pro-migratory functions of MAP4K4 involve the activation of the integrin β-1 adhesion receptor and are associated with increased endocytic uptake. The consequent enhanced recycling of c-Met caused by MAP4K4 results in the accumulation of activated c-Met in cytosolic vesicles, which is required for sustained signaling and downstream pathway activation. The parallel increase of c-Met and MAP4K4 expression in SHH MB could predict an increased potential of these tumors to infiltrate brain tissue and cause metastatic disease. Molecular targeting of the underlying accelerated endocytosis and receptor recycling could represent a novel approach to block pro-migratory effector functions of MAP4K4 in metastatic cancers.

The Ducal Palace in Sabbioneta, commissioned by Vespasiano Gonzaga in the end of 16th century, keeps four precious wooden ceilings inside. Rough wooden planks nailed to a skeleton structure creates the support for fine wood decorative elements. The requirement of a conservation work has prompted a diagnosis phase about the ceilings as a whole. This was carried out by closed up observations, drilling tests and laboratory analysis.