Dolores Hambardzumyan

PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells

In normal brain, the side population (SP) phenotype is generated by ABC transporter activity and identifies stem cell and endothelial cell subpopulations by dye exclusion. By drug efflux, the ABCG2 transporter provides chemoresistance in stem cells and contributes to the blood brain barrier (BBB) when active in endothelial cells. We investigated the SP phenotype of mouse and human gliomas. In glioma endothelial cells, ABC transporter function is impaired, corresponding to disruption of the BBB in these tumors. By contrast, the SP phenotype is increased in nonendothelial cells that form neurospheres and are highly tumorigenic. In this cell population, Akt, but not its downstream target mTOR, regulates ABCG2 activity, and loss of PTEN increases the SP. This Akt-induced ABCG2 activation results from its transport to the plasma membrane. Temozolomide, the standard treatment of gliomas, although not an ABCG2 substrate, increases the SP in glioma cells, especially in cells missing PTEN.

Glioblastoma Subclasses Can Be Defined by Activity among Signal Transduction Pathways and Associated Genomic Alterations

Background Glioblastoma multiforme (GBM) is an umbrella designation that includes a heterogeneous group of primary brain tumors. Several classification strategies of GBM have been reported, some by clinical course and others by resemblance to cell types either in the adult or during development. From a practical and therapeutic standpoint, classifying GBMs by signal transduction pathway activation and by mutation in pathway member genes may be particularly valuable for the development of targeted therapies. Methodology/Principal Findings We performed targeted proteomic analysis of 27 surgical glioma samples to identify patterns of coordinate activation among glioma-relevant signal transduction pathways, then compared these results with integrated analysis of genomic and expression data of 243 GBM samples from The Cancer Genome Atlas (TCGA). In the pattern of signaling, three subclasses of GBM emerge which appear to be associated with predominance of EGFR activation, PDGFR activation, or loss of the RAS regulator NF1. The EGFR signaling class has prominent Notch pathway activation measured by elevated expression of Notch ligands, cleaved Notch receptor, and downstream target Hes1. The PDGF class showed high levels of PDGFB ligand and phosphorylation of PDGFRβ and NFKB. NF1-loss was associated with lower overall MAPK and PI3K activation and relative overexpression of the mesenchymal marker YKL40. These three signaling classes appear to correspond with distinct transcriptomal subclasses of primary GBM samples from TCGA for which copy number aberration and mutation of EGFR, PDGFRA, and NF1 are signature events. Conclusions/Significance Proteomic analysis of GBM samples revealed three patterns of expression and activation of proteins in glioma-relevant signaling pathways. These three classes are comprised of roughly equal numbers showing either EGFR activation associated with amplification and mutation of the receptor, PDGF-pathway activation that is primarily ligand-driven, or loss of NF1 expression. The associated signaling activities correlating with these sentinel alterations provide insight into glioma biology and therapeutic strategies.

The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo

Activated oncogenic signaling is central to the development of nearly all forms of cancer, including the most common class of primary brain tumor, glioma. Research over the last two decades has revealed the particular importance of the Akt pathway, and its molecular antagonist PTEN (phosphatase and tensin homolog), in the process of gliomagenesis. Recent studies have also demonstrated that microRNAs (miRNAs) may be responsible for the modulation of cancer-implicated genes in tumors. Here we report the identification miR-26a as a direct regulator of PTEN expression. We also show that miR-26a is frequently amplified at the DNA level in human glioma, most often in association with monoallelic PTEN loss. Finally, we demonstrate that miR-26a-mediated PTEN repression in a murine glioma model both enhances de novo tumor formation and precludes loss of heterozygosity and the PTEN locus. Our results document a new epigenetic mechanism for PTEN regulation in glioma and further highlight dysregulation of Akt signaling as crucial to the development of these tumors.

PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo

Medulloblastomas are brain tumors that arise in the cerebellum of children and contain stem cells in a perivascular niche thought to give rise to recurrence following radiation. We used several mouse models of medulloblastomas in parallel to better understand how the critical cell types in these tumors respond to therapy. In our models, the proliferating cells in the tumor bulk undergo radiation-induced, p53-dependent apoptotic cell death. Activation of Akt signaling via PTEN loss transforms these cells to a nonproliferating extensive nodularity morphology. By contrast, the nestin-expressing perivascular stem cells survive radiation, activate PI3K/Akt pathway, undergo p53-dependent cell cycle arrest, and re-enter the cell cycle at 72 h. Furthermore, the ability of these cells to induce p53 is dependent on the presence of PTEN. These cellular characteristics are similar to human medulloblastomas. Finally, inhibition of Akt signaling sensitizes cells in the perivascular region to radiation-induced apoptosis.

Perivascular Nitric Oxide Activates Notch Signaling and Promotes Stem-like Character in PDGF-Induced Glioma Cells

eNOS expression is elevated in human glioblastomas and correlated with increased tumor growth and aggressive character. We investigated the potential role of nitric oxide (NO) activity in the perivascular niche (PVN) using a genetic engineered mouse model of PDGF-induced gliomas. eNOS expression is highly elevated in tumor vascular endothelium adjacent to perivascular glioma cells expressing Nestin, Notch, and the NO receptor, sGC. In addition, the NO/cGMP/PKG pathway drives Notch signaling in PDGF-induced gliomas in vitro, and induces the side population phenotype in primary glioma cell cultures. NO also increases neurosphere forming capacity of PDGF-driven glioma primary cultures, and enhances their tumorigenic capacity in vivo. Loss of NO activity in these tumors suppresses Notch signaling in vivo and prolongs survival of mice. This mechanism is conserved in human PDGFR amplified gliomas. The NO/cGMP/PKG pathways promotion of stem cell-like character in the tumor PVN may identify therapeutic targets for this subset of gliomas.

Seeding and propagation of untransformed mouse mammary cells in the lung.

The acquisition of metastatic ability by tumor cells is considered a late event in the evolution of malignant tumors. We report that untransformed mouse mammary cells that have been engineered to express the inducible oncogenic transgenes MYC and KrasD12, or polyoma middle T, and introduced into the systemic circulation of a mouse can bypass transformation at the primary site and develop into metastatic pulmonary lesions upon immediate or delayed oncogene induction. Therefore, previously untransformed mammary cells may establish residence in the lung once they have entered the bloodstream and may assume malignant growth upon oncogene activation. Mammary cells lacking oncogenic transgenes displayed a similar capacity for long-term residence in the lungs but did not form ectopic tumors.

The role of microglia and macrophages in glioma maintenance and progression

There is a growing recognition that gliomas are complex tumors composed of neoplastic and non-neoplastic cells, which each individually contribute to cancer formation, progression and response to treatment. The majority of the non-neoplastic cells are tumor-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, that create a supportive stroma for neoplastic cell expansion and invasion. TAMs are recruited to the glioma environment, have immune functions, and can release a wide array of growth factors and cytokines in response to those factors produced by cancer cells. In this manner, TAMs facilitate tumor proliferation, survival and migration. Through such iterative interactions, a unique tumor ecosystem is established, which offers new opportunities for therapeutic targeting.

Sonic Hedgehog Pathway Activation Is Induced by Acute Brain Injury and Regulated by Injury-Related Inflammation

The adult mammalian brain responds to injury by activating a program of cell proliferation during which many oligodendrocyte precursors, microglia, and some astrocytes proliferate. Another common response to brain injury is the induction of reactive gliosis, a process whereby dormant astrocytes undergo morphological changes and alter their transcriptional profiles. Although brain injury-induced reactive gliosis is concurrent with the proliferation of surrounding cells, a functional relationship between reactive gliosis and this cell proliferation has not been clearly demonstrated. Here, we show that the mitogen sonic hedgehog (SHH) is produced in reactive astrocytes after injury to the cerebral cortex and participates in regulating the proliferation of Olig2-expressing (Olig2+) cells after brain injury. Using a cortical freeze injury to induce reactive gliosis in a Gli–luciferase reporter mouse, we show that the SHH pathway is maximally active 3 d after brain injury and returns to baseline levels by 14 d. SHH expression parallels Gli activation and localizes to glial fibrillary acidic protein-expressing reactive astrocytes. Inhibition of the SHH pathway with cyclopamine blocks the Gli response and significantly reduces both the proliferating and overall number of Olig2+ cells in the injured cortex. To provide mechanistic insight into SHH pathway activation in astrocytes, we show that proinflammatory stimuli activate SHH-expressing reactive astrocytes, whereas inhibition of inflammation-induced reactive gliosis by macrophage depletion abolishes SHH activation after brain injury and dampens cell proliferation after injury. Our data describes a unique reactive astrocyte-based, SHH-expressing niche formed in response to injury and inflammation that regulates the proliferation of Olig2+ cells.

Mathematical modeling of pdgf-driven glioblastoma reveals optimized radiation dosing schedules

Glioblastomas (GBMs) are the most common and malignant primary brain tumors and are aggressively treated with surgery, chemotherapy, and radiotherapy. Despite this treatment, recurrence is inevitable and survival has improved minimally over the last 50 years. Recent studies have suggested that GBMs exhibit both heterogeneity and instability of differentiation states and varying sensitivities of these states to radiation. Here, we employed an iterative combined theoretical and experimental strategy that takes into account tumor cellular heterogeneity and dynamically acquired radioresistance to predict the effectiveness of different radiation schedules. Using this model, we identified two delivery schedules predicted to significantly improve efficacy by taking advantage of the dynamic instability of radioresistance. These schedules led to superior survival in mice. Our interdisciplinary approach may also be applicable to other human cancer types treated with radiotherapy and, hence, may lay the foundation for significantly increasing the effectiveness of a mainstay of oncologic therapy. PAPERCLIP:

Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor-induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show beta-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity.