Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness.

The unfolded protein response (UPR) aids cellular recovery by increasing the capacity and decreasing the protein load of the endoplasmic reticulum (ER). Although the main pathways of the UPR are known, the mechanisms of UPR-associated transcriptional repression have not been explored in mammalian cells. Previous studies indicate that endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency decrease when the UPR is activated. In the present study, we demonstrate that inhibition of CFTR expression under ER stress leads to reduced cAMP-activated chloride secretion in epithelial monolayers, an indication of diminished CFTR function. Moreover, ER stress and the UPR obliterate endogenous ⌬F508 CFTR mRNA expression in CFPAC-1 cells without affecting recombinant ⌬F508 CFTR mRNA levels or mRNA half-life. These results emphasize that transcriptional repression of CFTR under ER stress, in concert with decreased CFTR maturation efficiency, leads to diminished function. Using human CFTR promoter reporter constructs, we confined the ER stress-associated CFTR transcriptional repression to the minimal promoter. Chromatin immunoprecipitation assays established the binding of the UPR-activated ATF6 transcription factor to this region during ER stress, which links the repression to the UPR. Methylation-specific PCR (MSP) revealed hypermethylation of CpG sites inside and in the vicinity of the MAZ transcription factor binding region of CFTR, demonstrating methylation-dependent repression. Using pharmacological inhibitors, we show that both DNA methylation and histone deacetylation contribute to CFTR transcriptional inhibition. These studies provide novel insight into the mechanism of gene repression during the mammalian UPR.In eukaryotic cells, the endoplasmic reticulum (ER) 3 is the site of protein folding and assembly. The unfolded protein response (UPR) can result from ER stress brought on by any number of insults (1-3), including depletion of ER Ca 2ϩ stores (1), proteasome blockade (4), increase in the concentration of reactive oxygen species (5, 6), inflammation (7), overexpression of secretory proteins (2, 8), or altered glycosylation (9). In addition to increasing the capacity of the ER by enhancing the synthesis of membrane components and chaperones (10), the UPR also decreases the ER protein load by enhancing ERAD (11) and to some extent by inhibiting transcription and translation (10,12). Although the principal mechanisms of the UPR have been studied extensively, only limited information is available regarding the extent and specificity of transcriptional repression during the UPR. In yeast, the limited number of genes that are transcriptionally repressed by the UPR encode secreted or cell surface proteins (6). Importantly, neither the extent nor the mechanisms of UPRassociated transcriptional repression have been investigated in mammalian cells.The cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane glycoprotein expressed in the ap...

Malignant gliomas are diffusively infiltrative and remain among the deadliest of all cancers. NF-κB is a transcription factor that mediates cell growth, migration and invasion, angiogenesis and resistance to apoptosis. Normally, the activity of NF-κB is tightly regulated by numerous mechanisms. However, in many cancers, NF-κB is constitutively activated and may function as a tumor promoter. Herein, we show that in gliomas, NF-κB is constitutively activated and the levels of cIAP2, Bcl-2, Bcl-xL and Survivin are elevated. These genes are regulated by NF-κB and can inhibit apoptosis. To understand the potential role of NF-κB p65 in suppressing apoptosis, we generated human glioma cell lines that inducibly express shRNA molecules specific for p65. We demonstrate that in the absence of p65, TNF-α induced cIAP2 expression is significantly reduced while the levels of Bcl-2, Bcl-xL and Survivin are not affected. These data suggest that of these genes, only cIAP2 is a direct target of p65. Using RT-PCR and chromatin immunoprecipitation assays, we have confirmed that cIAP2 is a transcriptional target of NF-κB p65. As a consequence of reduced p65 and cIAP2 levels, we demonstrate that the levels of RIP poly-ubiquitination are reduced, and that p65-deficient glioma cells are more sensitive to the cytotoxic effects of TNF-α than glioma cells expressing p65. Specifically, in the presence of TNF-α, glioma cells lacking p65 showed cellular proliferation defects and underwent apoptosis. Moreover, glioma cells were similarly sensitized to the effects of TNF-α if the levels of cIAP2 were reduced through lentivirus shRNA expression. These data suggest that NF-κB and/or cIAP2 may be therapeutically relevant targets for the treatment of malignant gliomas.

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Japan Glioblastoma (GBM), one of the most frequently occurring malignancies in the central nervous system, still has a very poor prognosis. To improve the prognosis of GBM patients, various attempts have been made. Immunotherapy targeting Wilms' tumor 1 (WT1) has proved to be effective in GBM (Izumoto et al. 2008). However, the functional roles of WT1 in GBM have not been intensively studied. In this study, we aim to examine the functional roles of WT1 in GBM. We established WT1 shRNA knocked down GBM cell lines (U87 and U251) and examined the functional roles in vitro and in vivo. In cell-proliferation assays, we plated 3 × 10 4 cells in 12-well plates. On day 4 the numbers of the proliferated cells were (39 + 7.8) ×10 4 cells in the U87 control shRNA, U87 WT1 shRNA, U251 control shRNA, and U251 WT1 shRNA, respectively (P , 0.05). Furthermore, an Annexin V apoptosis assay showed the numbers of the apoptotic cells per 30 times magnified microscopic field were 2.5 + 0.6, 15.3 + 5.7, 3.0 + 1.7, and 9.0 + 1.0 cells in the U87 control shRNA, U87 WT1 shRNA, U251 control shRNA, and U251 WT1 shRNA, respectively (P , 0.05). In the in vivo experiment, U87 control shRNA and U87 WT1 shRNA cells were intracranially injected into newborn pups of immunodeficient mice. At day 30, all of the mice transplanted with U87MG control shRNA developed GBM, whereas none of the mice transplanted with U87MG WT1 shRNA developed GBM. These results suggest that WT1 is involved in GBM cell proliferation, apoptosis, and tumor formation. Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Glioma invasion is strongly associated with the resistance of these tumors to therapy, but the mechanisms that underlie this association are poorly understood. Targeting soluble factors triggering invasion and resistance could substantially affect the difficult-to-reach, infiltrative glioma cells that are a major source of recurrence. Fibulin-3, a matrix protein absent in normal brain tissue but upregulated in gliomas, promotes tumor invasion by unknown mechanisms. We show here that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor of Notch, and promotes tumor-cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we demonstrate that controlled downregulation of fibulin-3 reduces Notch signaling and leads to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. Finally, we show that fibulin-3 expression correlates with expression levels of Notch-dependent genes (Hes1, Hes5) and is a marker of Notch activation in clinical gliomas. These results underscore a major role of the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this is the first description of a noncanonical, soluble activator of Notch in a cancer model and a demonstration of how Not...