Diana Munoz

Loss of p53 cooperates with K‐ras activation to induce glioma formation in a region‐independent manner

Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morphological features. These differences, between and within varying grades of gliomas, have not been explained solely on the grounds of an oncogenic stimulus. Interactions with the tumor microenvironment as well as inherent characteristics of the cell of origin are likely a source of this heterogeneity. There is an ongoing debate over the cell of origin of gliomas, where some suggest a progenitor, while others argue for a stem cell origin. Thus, it is presumed that neurogenic regions of the brain such as the subventricular zone (SVZ) containing large numbers of neural stem and progenitor populations are more susceptible to transformation. Our studies demonstrate that K‐rasG12D cooperates with the loss of p53 to induce gliomas from both the SVZ and cortical region, suggesting that cells in the SVZ are not uniquely gliomagenic. Using combinations of doxycycline‐inducible K‐rasG12D and p53 loss, we show that tumors induced by the cooperative actions of these genes remain dependent on active K‐ras expression, as deinduction of K‐rasG12D leads to complete tumor regression despite absence of p53. These results suggest that the interplay between specific combinations of genetic alterations and susceptible cell types, rather than the site of origin, are important determinates of gliomagenesis. Additionally, this model supports the view that, although several genetic events may be necessary to confer traits associated with oncogenic transformation, inactivation of a single oncogenic partner can undermine tumor maintenance, leading to regression and disease remission. GLIA 2013;61:1862–1872

Developmental profile and regulation of the glycolytic enzyme hexokinase 2 in normal brain and glioblastoma multiforme

Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is metabolized to lactate rather than by oxidative metabolism, even in the presence of oxygen. This metabolic adaptation provides a survival advantage and facilitates synthesis of biosynthetic precursors required for continued cellular proliferation. An important mediator of aerobic glycolysis is our demonstration that in malignant gliomas there is over-expression of the glycolytic enzyme hexokinase 2 (HK2), phosphorylating glucose as the first step of the glycolytic pathway. In contrast, normal brain preferentially expresses HK1 and undergoes oxidative glucose metabolism. In this study, we examine whether this switch in HK isoform also occurs in the developing embryo and central nervous system (CNS). Bioinformatic analysis of available microarray data, including that of The Cancer Genome Atlas, demonstrated a ~17% overlap in metabolic-related genes in blastocyst stage embryo and human GBM tissue, including upregulation of HK2 and downregulation of HK1. Quantitative RT-PCR on mouse brains isolated at different embryonic and postnatal development time-points demonstrated HK2 expression was highest in the early embryo, while HK1 expression increased with CNS maturation. The downstream glycolytic enzymes PKM2 and LDHA had similar temporal profiles as HK2. Expression of the HK2 isoform was due in part to epigenetic regulation of HK2. In support, adult normal human brain and the few human GBM cell lines with low HK2 expression had methylation of CpG islands within intron 1 of HK2. In contrast, developing human fetal brain and GBM tissue expressing HK2 demonstrated significantly lower percent methylation. Furthermore, treatment of GBM cells lacking HK2 with 5-aza-2-deoxycytidine restored HK2 transcript expression. Overall, our results demonstrate that proliferative states including the developing embryo and malignant gliomas, which rely on aerobic glycolysis, preferentially express the HK2 isoform, found to be regulated in part epigenetically.

In Situ Analysis of Mutant EGFRs Prevalent in Glioblastoma Multiforme Reveals Aberrant Dimerization, Activation, and Differential Response to Anti-EGFR Targeted Therapy

Aberrations in epidermal growth factor receptor (EGFR/ErbB1) are the most common oncogenic alterations in glioblastoma multiforme (GBM), the most common primary brain tumor. Interactions between wild-type (wt) and mutant EGFRs and their subsequent activation are of biologic and potential therapeutic importance in GBMs. We recently showed that in situ proximity ligation assay (PLA) allows for quantitative evaluation of EGFR dimerization and activation in intact cells. Using this in situ platform, we show the aberrant homo-/heterodimeric properties of EGFRvIII and EGFRc958 mutants, the two most common EGFR mutants in GBMs. In addition, dimer phosphoactivation status could be detected by PLA with superior signal–noise ratio (>17-fold) and sensitivity (>16-fold) than immunofluorescence-based phospho-EGFR measurements. Dimer activation analysis indicated quantitative activation differences of mutant dimers. These aberrant features were not overexpression dependent but appeared independent of cellular expression levels, suggesting inherent properties of the mutant receptors. Moreover, we observed in situ detection of EGFRwt-EGFRvIII heterodimerization in GBM specimens, supporting our cell line observations. Notably, currently used anti-EGFR therapeutics, such as cetuximab, matuzumab, and panitumumab, could effectively block EGFRwt dimerization and activation but did not equally impair EGFRvIII homodimers, EGFRwt-EGFRvIII, or EGFRvIII-EGFRc958 heterodimers. EGFRvIII appears to have intrinsic phosphoactivation independent of dimerization as matuzumab blockade of homodimerization had no effect on receptor phosphorylation levels. These data suggest differences in the dimerization-blocking efficacy of EGFR monoclonal antibodies as mutant EGFR dimer configurations prevalent in GBMs can evade blockade by anti-EGFR treatments. Further studies are warranted to evaluate whether this evasion contributes to poor therapeutic response or resistance. Mol Cancer Res; 10(3); 428–40. ©2012 AACR.

Differential transformation capacity of neuro-glial progenitors during development

Gliomas represent the most common type of brain tumor, but show considerable variability in histologic appearance and clinical outcome. The phenotypic differences between types and grades of gliomas have not been explained solely on the grounds of differing oncogenic stimuli. Several studies have demonstrated that some phenotypic differences may be attributed to regional differences in the neural stem cells from which tumors arise. We hypothesized that temporal differences may also play a role, with tumor phenotypic variability reflecting intrinsic differences in neural stem cells at distinct developmental stages. To determine how the tumorigenic potential of lineally related stem cells changes over time, we used a conditional transgenic system that integrates Cre-Lox–mediated and Tet-regulated expression to drive K-rasG12D expression in neuro-glial progenitor populations at different developmental time points. Using this model, we demonstrate that K-rasG12D–induced transformation is dependent on the developmental stage at which it is introduced. Diffuse malignant brain tumors develop during early embryogenesis but not when K-rasG12D expression is induced during late embryogenesis or early postnatal life. We show that differential expression of cell-cycle regulators during development may be responsible for this differing susceptibility to malignant transformation and that loss of p53 can overcome the transformation resistance seen at later developmental stages. These results highlight the interplay between genetic alterations and the molecular changes that accompany specific developmental stages; early progenitors may lack the regulatory mechanisms present at later, more lineage-restrictive, developmental time points, making them more susceptible to transformation.

Transgenic Mouse Models of CNS Tumors: Using Genetically Engineered Murine Models to Study the Role of p21-Ras in Glioblastoma Multiforme

Robust animal models have come to the forefront of understanding GBM biology and cancer biology in general. Specifically, genetically engineered murine models or GEMs have provided a great deal of understanding in investigating the role of p21-Ras in GBM. Elevation of Ras activity is a molecular hallmark of GBM and is under intense investigation. Several animal models have been engineered to express mutant forms of Ras or aberrantly express receptors, which modulate Ras activity. Embryonic stem cell transgenesis is a key methodology in engineering these mice models and so is tissue-specific targeting. We highlight several advantages of using ES-cell mediated transgenesis to generate mouse models expressing activated Ras. These animal models have been crucial in studying GBM formation, identifying novel GBM tumor suppressor genes using retroviral gene-trapping and how Ras synergizes with other signaling pathways to give rise to GBM. Lastly these models can be useful in identifying the potential cell of origin in GBM.

Inhibition of Ras Signaling for Brain Tumor Therapy

Overactive p21-Ras signaling is a major contributing factor in the initiation and progression of glioblastoma multiforme (GBM). Thirty percent of all cancers exhibit mutations in the p21-Ras gene. Elevated levels of p21-Ras activity can also arise through abnormal receptor expression as is the case in GBM. Hence, development of p21-Ras inhibitors such as farnesyltransferase inhibitors (FTIs) or other targets of p21-Ras signaling is a promising but unproven antitumor strategy. p21-Ras is a member of the small guanine nucleotide-binding protein family and plays a pivotal role in signal transduction. Activated p21-Ras and its downstream signaling cascades are involved in a variety of biological processes including cell proliferation, cell cycle progression, survival, development, and differentiation. Although mutations in p21-Ras are rare in GBM, elevated levels of activated p21-Ras are consistently seen in GBM. Current data suggest that activation of p21-Ras is a consequence of aberrant tyrosine kinase receptor activation (EGFR, PDGFR, Met, etc.), which are frequent events in GBM. Targeting p21-Ras in gliomas has not been fully exploited and a better understanding of p21-Ras and its function may lead to novel therapies that can be synergistic with other conventional and biological modalities.

Brain tumor-initiating cells and cells of origin in glioblastoma

Glioblastoma Multiforme (GBM) is the most malignant and devastating primary brain tumour with a median survival of ∼12–16 months. Although recent large scale sequencing projects have shed considerable light into the complexity of the disease, there remains much to be elucidated in the hopes of generating effective therapeutic strategies. Although these studies investigate the mutations and expression of bulk tumour they have limits with respect to cell of origin and the concept of brain tumour initiating cells (BTIC). Current research has challenged the old paradigm of the stochastic model as recent evidence suggests that a subset of cancer cells within a tumor is responsible for tumor initiation, maintenance, and resistance to therapy. To gain a better understanding of the different compartment of cells that GBM comprise of require careful and elegant experiments. In addition to studying GBM, exploring the role of normal neural stem cells and progenitors cells is essential to partially explain whether these GBM BTIC behave similarly or differently then their non transformed counterparts. Here we discuss the recent literature between the two models, candidate regions of glioma genesis, candidate cells of origin for GBM, and possible therapeutic avenues to explore.