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 ...

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.

Highlights Glioblastoma organoid cultures preserve diversity of proliferative cell phenotypes. Heterogeneous 3D cultures recapitulate resistance to clinical GBM therapeutics. Patient specimens show different behavior depending on 2D vs 3D growth.

We have established several glioma-relevant oncogeneengineered cancer cells to reevaluate the oncogeneselective cytotoxicity of previously well-characterized anticancer drugs, such as etoposide, doxorubicin, staurosporine, and carmustine. Among several glioma-relevant oncogenes (activated epidermal growth factor receptor, Ras, and Akt, as well as Bcl-2 and p53DD used in the present study), the activated epidermal growth factor receptor, Ras, and Akt exerted oncogenic transformation of Ink4a/Arf À/À murine astrocyte cells. We identified that etoposide, a topoisomerase II inhibitor, caused selective killing of myristylated Akt (Akt-myr) -transduced Ink4a/Arf À/À astrocytes and U87MG cells in a dose-and time-dependent manner. Etoposide-selective cytotoxicity in the Akt-myr -transduced cells was shown to be caused by nonapoptotic cell death and occurred in a p53-independent manner. Etoposide caused severe reactive oxygen species (ROS) accumulation preferentially in the Akt-myr -transduced cells, and elevated ROS rendered these cells highly sensitive to cell death. The etoposideselective cell death of Akt-myr -transduced cells was attenuated by pepstatin A, a lysosomal protease inhibitor.In the present study, we show that etoposide might possess a novel therapeutic activity for oncogenic Akttransduced cancer cells to kill preferentially through ROSmediated damage.

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