Platelet-derived growth factor receptors (PDGFR) α and β have been suggested as potential targets for treatment of rhabdomyosarcoma, the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes, with PDGF-D as the solely overexpressed PDGFRβ ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRα were found in tumor cells, whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems, where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation, vessel density, and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3α and GSK-3β. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness, differentiation, senescence, and apoptosis. In summary, the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression. View Publication

Breast cancer is a heterogenous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in tumoursphere" culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence� View Publication

In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2(High) and EphA2(Low) populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications. View Publication

UNLABELLED A noninvasive technology that indiscriminately detects tumor tissue in the brain could substantially enhance the management of primary or metastatic brain tumors. Although the documented molecular heterogeneity of diseases that initiate or eventually deposit in the brain may preclude identifying a single smoking-gun molecular biomarker, many classes of brain tumors are generally avid for transferrin. Therefore, we reasoned that applying a radiolabeled derivative of transferrin ((89)Zr-labeled transferrin) may be an effective strategy to more thoroughly identify tumor tissue in the brain, regardless of the tumor's genetic background. METHODS Transferrin was radiolabeled with (89)Zr, and its properties with respect to human models of glioblastoma multiforme were studied in vivo. RESULTS In this report, we show proof of concept that (89)Zr-labeled transferrin ((89)Zr-transferrin) localizes to genetically diverse models of glioblastoma multiforme in vivo. Moreover, we demonstrate that (89)Zr-transferrin can detect an orthotopic lesion with exceptional contrast. Finally, the tumor-to-brain contrast conferred by (89)Zr-transferrin vastly exceeded that observed with (18)F-FDG, currently the most widely used radiotracer to assess tumor burden in the brain. CONCLUSION The results from this study suggest that (89)Zr-transferrin could be a broadly applicable tool for identifying and monitoring tumors in the brain, with realistic potential for near-term clinical translation. View Publication

The Hedgehog (Hh) pathway regulates the growth of a subset of adult gliomas and better definition of Hh-responsive subtypes could enhance the clinical utility of monitoring and targeting this pathway in patients. Somatic mutations of the isocitrate dehydrogenase (IDH) genes occur frequently in WHO grades II and III gliomas and WHO grade IV secondary glioblastomas. Hh pathway activation in WHO grades II and III gliomas suggests that it might also be operational in glioblastomas that developed from lower-grade lesions. To evaluate this possibility and to better define the molecular and histopathological glioma subtypes that are Hh-responsive, IDH genes were sequenced in adult glioma specimens assayed for an operant Hh pathway. The proportions of grades II-IV specimens with IDH mutations correlated with the proportions that expressed elevated levels of the Hh gene target PTCH1. Indices of an operational Hh pathway were measured in all primary cultures and xenografts derived from IDH-mutant glioma specimens, including IDH-mutant glioblastomas. In contrast, the Hh pathway was not operational in glioblastomas that lacked IDH mutation or history of antecedent lower-grade disease. IDH mutation is not required for an operant pathway however, as significant Hh pathway modulation was also measured in grade III gliomas with wild-type IDH sequences. These results indicate that the Hh pathway is operational in grades II and III gliomas and glioblastomas with molecular or histopathological evidence for evolvement from lower-grade gliomas. Lastly, these findings suggest that gliomas sharing this molecularly defined route of progression arise in Hh-responsive cell types. View Publication

Pediatric high-grade astrocytomas (HGAs) account for 15-20% of all pediatric central nervous system tumors. These neoplasms predominantly involve the supratentorial hemispheres or the pons--diffuse intrinsic pontine gliomas (DIPG). Assumptions that pediatric HGAs are biologically similar to adult HGAs have recently been challenged, and the development of effective therapeutic modalities for DIPG and supratentorial HGA hinges on a better understanding of their biological properties. Here, 20 pediatric HGAs (9 DIPGs and 11 supratentorial HGAs) were subject to gene expression profiling following approval by the research ethics board at our institution. Many of these tumors showed expression signatures composed of genes that promote G1/S and G2/M cell cycle progression. In particular, Aurora kinase B (AURKB) was consistently and highly overexpressed in 6/9 DIPGs and 8/11 HGAs. Array data were validated using quantitative real-time PCR and immunohistochemistry, as well as cross-validation of our data set with previously published series. Inhibition of Aurora B activity in DIPG and in pediatric HGA cell lines resulted in growth arrest accompanied by morphological changes, cell cycle aberrations, nuclear fractionation and polyploidy as well as a reduction in colony formation. Our data highlight Aurora B as a potential therapeutic target in DIPG. View Publication

Large-scale cancer genomics projects are profiling hundreds of tumors at multiple molecular layers, including copy number, mRNA and miRNA expression, but the mechanistic relationships between these layers are often excluded from computational models. We developed a supervised learning framework for integrating molecular profiles with regulatory sequence information to reveal regulatory programs in cancer, including miRNA-mediated regulation. We applied our approach to 320 glioblastoma profiles and identified key miRNAs and transcription factors as common or subtype-specific drivers of expression changes. We confirmed that predicted gene expression signatures for proneural subtype regulators were consistent with in vivo expression changes in a PDGF-driven mouse model. We tested two predicted proneural drivers, miR-124 and miR-132, both underexpressed in proneural tumors, by overexpression in neurospheres and observed a partial reversal of corresponding tumor expression changes. Computationally dissecting the role of miRNAs in cancer may ultimately lead to small RNA therapeutics tailored to subtype or individual. View Publication

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM, a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells, GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However, the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature, which defines aggressiveness and invasion, and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1, which encode a key regulator of selective autophagy, p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs, DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation, thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast, autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally, DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism, in particular reduced ATP and lactate levels. Taken together, these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication

Glioblastoma (GBM) and other malignant gliomas are aggressive primary neoplasms of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed distinct disease subclasses within malignant gliomas whose defining genomic features highlight dysregulated molecular networks as potential targets for therapeutic development. The proneural" designation represents the largest and most heterogeneous of these subclasses� View Publication

The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG, but not (S)-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity, which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications, clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however, the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs, most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification, revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well. View Publication

BACKGROUND: Although both the alkylating agent temozolomide (TMZ) and oncolytic viruses hold promise for treating glioblastoma, which remains uniformly lethal, the effectiveness of combining the two treatments and the mechanism of their interaction on cancer stem cells are unknown. METHODS: We investigated the efficacy of combining TMZ and the oncolytic herpes simplex virus (oHSV) G47Δ in killing glioblastoma stem cells (GSCs), using Chou-Talalay combination index analysis, immunocytochemistry and fluorescence microscopy, and neutral comet assay. The role of treatment-induced DNA double-strand breaks, activation of DNA damage responses, and virus replication in the cytotoxic interaction between G47Δ and TMZ was examined with a panel of pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of DNA repair pathways. Comparisons of cell survival and virus replication were performed using a two-sided t test (unpaired). The survival of athymic mice (n = 6-8 mice per group) bearing GSC-derived glioblastoma tumors treated with the combination of G47Δ and TMZ was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test. RESULTS: The combination of G47Δ and TMZ acted synergistically in killing GSCs but not neurons, with associated robust induction of DNA damage. Pharmacological and shRNA-mediated knockdown studies suggested that activated ataxia telangiectasia mutated (ATM) is a crucial mediator of synergy. Activated ATM relocalized to HSV DNA replication compartments where it likely enhanced oHSV replication and could not participate in repairing TMZ-induced DNA damage. Sensitivity to TMZ and synergy with G47Δ decreased with O(6)-methylguanine-DNA-methyltransferase (MGMT) expression and MSH6 knockdown. Combined G47Δ and TMZ treatment extended survival of mice bearing GSC-derived intracranial tumors, achieving long-term remission in four of eight mice (median survival = 228 days; G47Δ alone vs G47Δ + TMZ, hazard ratio of survival = 7.1, 95% confidence interval = 1.9 to 26.1, P = .003) at TMZ doses attainable in patients. CONCLUSIONS: The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation. View Publication