Cellular origins and genetic factors governing the genesis and maintenance of glioblastomas (GBM) are not well understood. Here, we report a pathogenetic role of the developmental regulator Id4 (inhibitor of differentiation 4) in GBM. In primary murine Ink4a/Arf −/− astrocytes, and human glioma cells, we provide evidence that enforced Id4 can drive malignant transformation by stimulating increased cyclin E to produce a hyperproliferative profile and by increased Jagged1 expression with Notch1 activation to drive astrocytes into a neural stem-like cell state. Thus, Id4 plays an integral role in the transformation of astrocytes via its combined actions on two-key cell cycle and differentiation regulatory molecules.Supplemental material is available at http://www.genesdev.org. The self-renewal, differentiative, and proliferative states of tissue stem and progenitor cells are maintained by a variety of well-defined cell fate determinant factors (Keller 2005). Disregulation of such key developmental regulators and acquisition and maintenance of an immature differentiation phenotype are common themes across a broad spectrum of cancer types, including primary brain cancers (Garraway and Sellers 2006). Indeed, a growing body of evidence supports the view that developmental regulators play direct roles in driving aspiring cancer cells toward a malignant phenotype, one that is endowed with stem-like cell properties including robust renewal potential (Beachy et al. 2004). This cellular phenotype, embodied in the cancer stem cell paradigm (Pardal et al. 2003), has been well documented in the highly malignant brain cancer, primary glioblastoma multiforme (GBM) (Galli et al. 2004;Singh et al. 2004). The cellular origins and specific genetic elements involved in the genesis and maintenance of these so-called brain cancer stem cells (BCSCs) remain areas of intensive investigation. Results and Discussion Id4 is overexpressed human GBM, and can drive transformation of murine Ink4a/Arf −/− astrocytesGiven the biological significance of stem cell fate determinants in the regulation of normal self-renewal and differentiation, we hypothesized that the genetic or epigenetic alterations in classical cell fate determinants might cooperate with prototypical oncogenes and tumor suppressor genes to both effect malignant transformation and endow stem cell-like renewal activity in these tumors (Leung et al. 2004;Ligon et al. 2007). We were particularly focused on the expression and functional activity of inhibitor of differentiation 4 (Id4) in GBM given its prominent role in governing neural stem cell (NSC) fate decisions (Yun et al. 2004). Id4 mRNA levels were found to be increased in nine of 15 GBM samples as compared with human normal brain tissue (Supplemental Fig. S1A) and abundant Id4 mRNA and protein levels were detected in five of seven human glioma cell lines (A172, LN229, LN18, U87MG, and T98G) and an immortalized human NSC line (HB1.F3) (Cho et al. 2002), but not in the normal human astrocytes (NHA) (Supplemental Fig. S1B). Tumor ...

Keywords:MERS HPV CrPV IRES RNA Adjuvant Vaccine Th1/Th2 Alum a b s t r a c t An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as liveattenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

Signal transducer and activator of transcription 3 (STAT3) and telomerase are considered attractive targets for anticancer therapy. The in vitro anticancer activity of the gold(I) compound auranofin was investigated using MDA-MB 231 human breast cancer cells, in which STAT3 is constitutively active. In cell culture, auranofin inhibited growth in a dose-dependent manner, and N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), markedly blocked the effect of auranofin. Incorporation of 5-bromo-2’-deoxyuridine into DNA and anchorage-independent cell growth on soft agar were decreased by auranofin treatment. STAT3 phosphorylation and telomerase activity were also attenuated in cells exposed to auranofin, but NAC pretreatment restored STAT3 phosphorylation and telomerase activity in these cells. These findings indicate that auranofin exerts in vitro antitumor effects in MDA-MB 231 cells and its activity involves inhibition of STAT3 and telomerase. Thus, auranofin shows potential as a novel anticancer drug that targets STAT3 and telomerase. [BMB Reports 2013; 46(1): 59-64]

Abstract. Auranofin (2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosato-S-[triethylphosphine] gold) is a gold(I)-containing antirheumatic drug that possesses anti-inflammatory properties. The pharmacological activity of this drug is associated with its ability to induce heme oxygenase-1 (HO-1). However, the mechanism underlying auranofin-mediated HO-1 induction remains unclear. We investigated the action of auranofin on activation of nuclear factor erythroid 2-related factor 2 (Nrf2), an activator of HO-1. Auranofin elevated cellular levels of Nrf2 by increasing protein stability but not transcriptional activation. Coimmunoprecipitation and Western blot analysis indicated that auranofin inhibited Nrf2 degradation by inducing the dissociation of the Nrf2 / Kelchlike ECH-associated protein 1 (Keap1) complex, which resulted in nuclear accumulation of Nrf2. In addition, auranofin treatment activated cellular Rac1 and induced inducible nitric oxide synthase (iNOS) expression. An inhibitor of Rac1 (NSC23766) blocked the iNOS induction as well as Nrf2 activation and HO-1 expression. N G -nitro-L-arginine methyl ester and aminoguanidine, inhibitors of iNOS, diminished the auranofin-induced Nrf2 activation and HO-1 expression. Phosphorylation of mitogen-activated protein kinases (MAPKs) was increased by auranofin treatment, and inhibitors of MAPKs partially diminished the Nrf2 activation. A chromatin immunoprecipitation assay showed that the Nrf2 activated by auranofin was involved in transactivation of the HO-1 gene. These findings indicate that auranofin leads to HO-1 upregulation by activating Keap1/Nrf2 signaling via Rac1/iNOS induction and MAPK activation.

A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf(-/-) astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf(-/-) astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.

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