SYK inhibition blocks proliferation and migration of glioma cells and modifies the tumor microenvironment.

Background: Glioblastoma (GBM) is one of the most aggressive human brain tumors, with a median survival of 15-18 months. There is a desperate need to find novel therapeutic targets. Various receptor protein kinases have been identified as potential targets; however, response rates in clinical studies have been somewhat disappointing. Targeting the spleen tyrosine kinase (SYK), which acts downstream of a range of oncogenic receptors, may therefore show more promising results. Methods: Kinase expression of brain tumor samples including GBM and low-grade tumors were compared with normal brain and normal human astrocytes by microarray analysis. Furthermore, SYK, LYN, SLP76, and PLCG2 protein expressions were analyzed by immunohistochemistry, western blot, and immunofluorescence of additional GBM patient samples, murine glioma samples, and cell lines. SYK was then blocked chemically and genetically in vitro and in vivo in 2 different mouse models. Multiphoton intravital imaging and multicolor flow cytometry were performed in a syngeneic immunocompetent C57BL/6J mouse GL261 glioma model to study the effect of these inhibitors on the tumor microenvironment. Results: SYK, LYN, SLP76, and PLCG2 were found expressed in human and murine glioma samples and cell lines. SYK inhibition blocked proliferation, migration, and colony formation. Flow cytometric and multiphoton imaging imply that targeting SYK in vivo attenuated GBM tumor growth and invasiveness and reduced B and CD11b+ cell mobility and infiltration. Conclusions: Our data suggest that gliomas express a SYK signaling network important in glioma progression, inhibition of which results in reduced invasion with slower tumor progression.

DNA damage induces GDNF secretion in the tumor microenvironment with paracrine effects promoting prostate cancer treatment resistance.

Though metastatic cancers often initially respond to genotoxic therapeutics, acquired resistance is common. In addition to cytotoxic effects on tumor cells, DNA damaging agents such as ionizing radiation and chemotherapy induce injury in benign cells of the tumor microenvironment resulting in the production of paracrine-acting factors capable of promoting tumor resistance phenotypes. In studies designed to characterize the responses of prostate and bone stromal cells to genotoxic stress, we found that transcripts encoding glial cell line-derived neurotrophic factor (GDNF) increased several fold following exposures to cytotoxic agents including radiation, the topoisomerase inhibitor mitoxantrone and the microtubule poison docetaxel. Fibroblast GDNF exerted paracrine effects toward prostate cancer cells resulting in enhanced tumor cell proliferation and invasion, and these effects were concordant with the expression of known GDNF receptors GFRA1 and RET. Exposure to GDNF also induced tumor cell resistance to mitoxantrone and docetaxel chemotherapy. Together, these findings support an important role for tumor microenvironment damage responses in modulating treatment resistance and identify the GDNF signaling pathway as a potential target for improving responses to conventional genotoxic therapeutics.

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas.

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.

Deltex-1 activates mitotic signaling and proliferation and increases the clonogenic and invasive potential of U373 and LN18 glioblastoma cells and correlates with patient survival.

Glioblastoma (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including microRNA-21- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low DTX1 levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets.

GSK3beta regulates differentiation and growth arrest in glioblastoma.

Cancers are driven by a population of cells with the stem cell properties of self-renewal and unlimited growth. As a subpopulation within the tumor mass, these cells are believed to constitute a tumor cell reservoir. Pathways controlling the renewal of normal stem cells are deregulated in cancer. The polycomb group gene Bmi1, which is required for neural stem cell self-renewal and also controls anti-oxidant defense in neurons, is upregulated in several cancers, including medulloblastoma. We have found that Bmi1 is consistently and highly expressed in GBM. Downregulation of Bmi1 by shRNAs induced a differentiation phenotype and reduced expression of the stem cell markers Sox2 and Nestin. Interestingly, expression of glycogen synthase kinase 3 beta (GSK3beta), which was found to be consistently expressed in primary GBM, also declined. This suggests a functional link between Bmi1 and GSK3beta. Interference with GSK3beta activity by siRNA, the specific inhibitor SB216763, or lithium chloride (LiCl) induced tumor cell differentiation. In addition, tumor cell apoptosis was enhanced, the formation of neurospheres was impaired, and clonogenicity reduced in a dose-dependent manner. GBM cell lines consist mainly of CD133-negative (CD133-) cells. Interestingly, ex vivo cells from primary tumor biopsies allowed the identification of a CD133- subpopulation of cells that express stem cell markers and are depleted by inactivation of GSK3beta. Drugs that inhibit GSK3, including the psychiatric drug LiCl, may deplete the GBM stem cell reservoir independently of CD133 status.

ETS transcription factor Erm controls subsynaptic gene expression in skeletal muscles.

Accumulation of specific proteins at synaptic structures is essential for synapse assembly and function, but mechanisms regulating local protein enrichment remain poorly understood. At the neuromuscular junction (NMJ), subsynaptic nuclei underlie motor axon terminals within extrafusal muscle fibers and are transcriptionally distinct from neighboring nuclei. In this study, we show that expression of the ETS transcription factor Erm is highly concentrated at subsynaptic nuclei, and its mutation in mice leads to severe downregulation of many genes with normally enriched subsynaptic expression. Erm mutant mice display an expansion of the muscle central domain in which acetylcholine receptor (AChR) clusters accumulate, show gradual fragmentation of AChR clusters, and exhibit symptoms of muscle weakness mimicking congenital myasthenic syndrome (CMS). Together, our findings define Erm as an upstream regulator of a transcriptional program selective to subsynaptic nuclei at the NMJ and underscore the importance of transcriptional control of local synaptic protein accumulation.