Identification of therapeutic targets for glioblastoma by network analysis.

Glioblastoma can originate from terminally differentiated astrocytes and neurons, which can dedifferentiate to a stem cell-like state upon transformation. In this study, we confirmed that transformed dedifferentiated astrocytes and neurons acquired a stem/progenitor cell state, although they still retained gene expression memory from their parental cell. Transcriptional network analysis on these cells identified upregulated genes in three main pathways: Wnt signaling, cell cycle and focal adhesion with the gene Spp1, also known as osteopontin (OPN) serving as a key common node connecting these three pathways. Inhibition of OPN blocked the formation of neurospheres, affected the proliferative capacity of transformed neurons and reduced the expression levels of neural stem cell markers. Specific inhibition of OPN in both murine and human glioma tumors prolonged mice survival. We conclude that OPN is an important player in dedifferentiation of cells during tumor formation, hence its inhibition can be a therapeutic target for glioblastoma.

Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases.

MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128's tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-α. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.

NF-kappaB and the regulation of hematopoiesis.

This review will focus on the role of nuclear factor kappaB (NF-kappaB) signaling in hematopoietic differentiation. We will also discuss several hematopoietic pathologies associated with deregulation of NF-kappaB and their potential therapies.

Gene therapy: promises and problems.

Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Based on the nature of the viral genome, these gene therapy vectors can be divided into RNA and DNA viral vectors. The majority of RNA virus-based vectors have been derived from simple retroviruses like murine leukemia virus. A major shortcoming of these vectors is that they are not able to transduce nondividing cells. This problem may be overcome by the use of novel retroviral vectors derived from lentiviruses, such as human immunodeficiency virus (HIV). The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.

Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.

Regulation of beta-catenin function by the IkappaB kinases.

Both the beta-catenin and the nuclear factor kappaB (NF-kappaB) proteins are important regulators of gene expression and cellular proliferation. Two kinases, IKKalpha and IKKbeta, are critical activators of the NF-kappaB pathway. Here we present evidence that these kinases are also important in the regulation of beta-catenin function. IKKalpha- and IKKbeta-deficient mouse embryo fibroblasts exhibited different patterns of beta-catenin cellular localization. IKKbeta decreases beta-catenin-dependent transcriptional activation, while IKKalpha increases beta-catenin-dependent transcriptional activity. IKKalpha and IKKbeta interact with and phosphorylate beta-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of beta-catenin with IKKalpha and IKKbeta may in part be responsible for regulating beta-catenin protein levels and cellular localization and integrating signaling events between the NF-kappaB and Wingless pathways.

Bimp1, a MAGUK family member linking protein kinase C activation to Bcl10-mediated NF-kappaB induction.

Bcl10 and MALT1, products of distinct chromosomal translocations in mucosa-associated lymphoid tissue lymphoma, cooperate in activating NF-kappaB. Mice lacking Bcl10 demonstrate severe immunodeficiency associated with failure of lymphocytes to activate nuclear factor kappaB (NF-kappaB) in response to antigen receptor stimulation and protein kinase C activation. We characterize Bimp1, a new signaling protein that binds Bcl10 and activates NF-kappaB. Bimp1-mediated NF-kappaB activation requires Bcl10 and IkappaB kinases, indicating that Bimp1 acts upstream of these mediators. Bimp1, Bcl10, and MALT1 form a ternary complex, with Bcl10 bridging the Bimp1/MALT1 interaction. A dominant negative Bimp1 mutant inhibits NF-kappaB activation by anti-CD3 ligation, phorbol ester, and protein kinase C expression. These results suggest that Bimp1 links surface receptor stimulation and protein kinase C activation to Bcl10/MALT1, thus leading to NF-kappaB induction.

Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that seems to be involved in transcription and DNA repair. Here we report that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3) activity, and that cancer-predisposing mutations within the BRCA1 RING domain abolish its Ub ligase activity. Furthermore, these mutants are unable to reverse gamma-radiation hypersensitivity of BRCA1-null human breast cancer cells, HCC1937. Additionally, these mutations within the BRCA1 RING domain are not capable of restoring a G(2) + M checkpoint in HCC1937 cells. These results establish a link between Ub protein ligase activity and gamma-radiation protection function of BRCA1, and provide an explanation for why mutations within the BRCA1 RING domain predispose to cancer. Furthermore, we propose that the analysis of the Ub ligase activity of RING-domain mutations identified in patients may constitute an assay to predict predisposition to cancer.

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging.

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for efficient lentiviral gene transfer, we used a fluorescence imaging system, which allows the detection of cells and tissues that express fluorescent reporter genes (e.g., green fluorescence protein) in the living animal. We show that the latest generation of lentiviral vectors efficiently transduces the murine liver. Further analysis demonstrated that neither cell-cycle activation nor division of liver cells is a prerequisite for lentiviral gene transfer in vivo.

Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors.

To facilitate the generation of SIN lentivirus vector-producer cell lines, we have developed a novel conditional SIN (cSIN) lentivirus vector, which retains its SIN properties in normal target cells yet can be produced at high titers from tetracycline-regulated packaging cell lines. The design of the cSIN vector is based on replacing the vector U3 transcription regulatory elements with the Tet-responsive element, which allows vector production exclusively in cells expressing the synthetic Tet-regulated transactivator (tTA). In contrast minimal vector production ( approximately 200 IU/ml) is obtained in target cells that do not express the tTA, even in the presence of all HIV-1 proteins. Following transduction of the Tet-regulated SODk1 lentivirus vector-packaging cell line with the cSIN vector, high titers of cSIN recombinant vector (>10(6) IU/ml) could be generated, which efficiently transduced terminally differentiated neurons in normal rat brain.