Hiroyuki Momota

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.

Perifosine Inhibits Multiple Signaling Pathways in Glial Progenitors and Cooperates With Temozolomide to Arrest Cell Proliferation in Gliomas In vivo

Perifosine is an oral Akt inhibitor which exerts a marked cytotoxic effect on human tumor cell lines, and is currently being tested in several phase II trials for treatment of major human cancers. However, the efficacy of perifosine in human gliomas has not been established. As Akt is activated in approximately 70% of human glioblastomas, we investigated the impact of perifosine on glia in culture and on a mouse glioma model in vivo. Here we show that perifosine strongly reduces phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk) 1/2, induces cell cycle arrest in G1 and G2, and causes dose-dependent growth inhibition of mouse glial progenitors in which Akt and/or Ras-Erk 1/2 pathways are activated. Furthermore, because temozolomide is a common oral alkylating agent used in the treatment of gliomas, we investigated the effect of perifosine in combination with temozolomide. We observed an enhanced effect when both were used in culture. With these results, we combined perifosine and temozolomide as treatment of platelet-derived growth factor B-driven gliomas in mice. Animal studies showed that perifosine and temozolomide combination therapy was more effective than temozolomide treatment alone (P < 0.01). These results indicate that perifosine is an effective drug in gliomas in which Akt and Ras-Erk 1/2 pathways are frequently activated, and may be a new candidate for glioma treatment in the clinic.

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.

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma.

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O 6-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.

Immunohistochemical analysis‐based proteomic subclassification of newly diagnosed glioblastomas

Recent gene expression and copy number profilings of glioblastoma multiforme (GBM) by The Cancer Genome Atlas (TCGA) Research Network suggest the existence of distinct subtypes of this tumor. However, these approaches might not be easily applicable in routine clinical practice. In the current study, we aimed to establish a proteomics‐based subclassification of GBM by integrating their genomic and epigenomic profiles. We subclassified 79 newly diagnosed GBM based on expression patterns determined by comprehensive immunohistochemical observation in combination with their DNA copy number and DNA methylation patterns. The clinical relevance of our classification was independently validated in TCGA datasets. Consensus clustering identified the four distinct GBM subtypes: Oligodendrocyte Precursor (OPC) type, Differentiated Oligodendrocyte (DOC) type, Astrocytic Mesenchymal (AsMes) type and Mixed type. The OPC type was characterized by highly positive scores of Olig2, PDGFRA, p16, p53 and synaptophysin. In contrast, the AsMes type was strongly associated with strong expressions of nestin, CD44 and podoplanin, with a high glial fibrillary acidic protein score. The median overall survival of OPC‐type patients was significantly longer than that of the AsMes‐type patients (19.9 vs 12.8 months). This finding was in agreement with the Oncomine analysis of TCGA datasets, which revealed that PDGFRA and Olig2 were favorable prognostic factors and podoplanin and CD44 were associated with a poor clinical outcome. This is the first study to establish a subclassification of GBM on the basis of immunohistochemical analysis. Our study will shed light on personalized therapies that might be feasible in daily neuropathological practice. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02377.x, 2012)

Glioblastoma simultaneously present with adjacent meningioma: case report and review of the literature

The simultaneous occurrence of multiple primary intracranial tumors has been reported previously. However, most of these tumors arise after cranial radiotherapy or in association with familial tumor syndromes. Double tumors of different histologies that are unrelated to radiotherapy or genetic disorders are very rare. We present a case of two primary intracranial tumors occurring simultaneously at adjacent sites. Preoperative gadolinium-enhanced magnetic resonance imaging of these tumors revealed a single continuous lesion. Postoperative histological examination revealed the presence of two distinct tumors, meningioma and glioblastoma multiforme. To elucidate the mechanism of synchronous tumor formation, we performed immunohistochemical analysis of the proteins involved in the receptor tyrosine kinase, Wnt, and Notch signaling pathways. These analyses showed that platelet-derived growth factor (PDGF) receptors-α and β were overexpressed in both tumors, thereby indicating the oncogenic effects of activated signaling of these receptors. The PDGF-mediated paracrine system may induce one tumor from another.

Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase

Background: Roles of GD3 in gliomas are not well understood. Results: PDGF receptor α was identified as a GD3-associated molecule by enzyme-mediated activation of radical sources and mass spectrometry, and its association with GD3 and Yes leads to increased invasiveness. Conclusion: GD3 enhances invasiveness by forming a molecular complex. Significance: GD3/PDGF receptor α·Yes complex is a potential target for glioma therapy. There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.

p53 abnormality and tumor invasion in patients with malignant astrocytoma

Malignant astrocytomas are characterized by diffusely infiltrating nature, and the abnormality of p53 is a cytogenetic hallmark of astrocytic tumors. To elucidate the relationship between p53 abnormality and invasiveness of the tumors, we studied mutation and protein expression of p53 in 48 consecutive patients with malignant astrocytoma (14 anaplastic astrocytomas and 34 glioblastoma multiformes). The tumors were classified into three categories according to the features of magnetic resonance imaging, and 5, 7, and 36 tumors were classified into diffuse, multiple, and single type, respectively. We then examined how these tumor types correlate with MIB-1 staining index, TP53 gene mutation, and p53 protein expression. We found that p53 immunopositivity or TP53 mutation was frequently observed in diffuse and multiple types. These abnormalities of p53 were also associated with high MIB-1 staining index and strong expression of vascular endothelial growth factor. Furthermore, diffuse- and multiple-type tumors were significantly correlated with poor progression-free survival, whereas only multiple-type tumors were significantly correlated with poor overall survival. As diffuse and multiple features on imaging modalities represent invasive characteristics of the tumors, p53 abnormalities may affect the invasive and aggressive nature of malignant astrocytomas.

Neural stem cell-based dual suicide gene delivery for metastatic brain tumors.

In our previous works, we demonstrated that human neural stem cells (NSCs) transduced with the cytosine deaminase (CD) gene showed remarkable ‘bystander killer effect’ on glioma and medulloblastoma cells after administration of the prodrug 5-fluorocytosine (5-FC). In addition, herpes simplex virus thymidine kinase (TK) is a widely studied enzyme used for suicide gene strategies, for which the prodrug is ganciclovir (GCV). To apply this strategy to brain metastasis treatment, we established here a human NSC line (F3.CD-TK) expressing the dual suicide genes CD and TK. We examined whether F3.CD-TK cells intensified the antitumor effect on lung cancer brain metastases. In vitro studies showed that F3.CD-TK cells exerted a marked bystander effect on human lung cancer cells after treatment with 5-FC and GCV. In a novel experimental brain metastases model, intravenously administered F3 cells migrated near lung cancer metastatic lesions, which were induced by the injection of lung cancer cells via the intracarotid artery. More importantly, F3.CD-TK cells in the presence of prodrugs 5-FC and GCV decreased tumor size and considerably prolonged animal survival. The results of the present study indicate that the dual suicide gene–engineered, NSC-based treatment strategy might offer a new promising therapeutic modality for brain metastases.

Acute lymphoblastic leukemia after temozolomide treatment for anaplastic astrocytoma in a child with a germline TP53 mutation

We present a case of a 12‐year‐old female with a germline TP53 mutation who presented with anaplastic astrocytoma and subsequent acute lymphoblastic leukemia (ALL) 13 months after starting treatment with temozolomide (TMZ). The patient had no family history of malignancy except her grand father and his siblings. Although alkylating agents such as TMZ are known to induce secondary hematologic malignancy, only several cases of treatment‐related acute leukemia have been reported after TMZ‐alone chemotherapy for malignant gliomas. We demonstrate a rare case of TMZ‐related ALL in a child with glioma possibly associated with a germline TP53 mutation. Pediatr Blood Cancer. 2010;55:577–579.