Kazuya Motomura

Mutational landscape and clonal architecture in grade II and III gliomas

Grade II and III gliomas are generally slowly progressing brain cancers, many of which eventually transform into more aggressive tumors. Despite recent findings of frequent mutations in IDH1 and other genes, knowledge about their pathogenesis is still incomplete. Here, combining two large sets of high-throughput sequencing data, we delineate the entire picture of genetic alterations and affected pathways in these glioma types, with sensitive detection of driver genes. Grade II and III gliomas comprise three distinct subtypes characterized by discrete sets of mutations and distinct clinical behaviors. Mutations showed significant positive and negative correlations and a chronological hierarchy, as inferred from different allelic burdens among coexisting mutations, suggesting that there is functional interplay between the mutations that drive clonal selection. Extensive serial and multi-regional sampling analyses further supported this finding and also identified a high degree of temporal and spatial heterogeneity generated during tumor expansion and relapse, which is likely shaped by the complex but ordered processes of multiple clonal selection and evolutionary events.

Efficient delivery of liposome-mediated MGMT-siRNA reinforces the cytotoxity of temozolomide in GBM-initiating cells.

Glioblastoma multiforme (GBM) is one of the most formidable brain tumors with a mean survival period of approximately 12 months. To date, a combination of radiotherapy and chemotherapy with an oral alkylating agent, temozolomide (TMZ), has been used as first-line therapy for glioma. However, the efficacy of chemotherapy for treating GBM is very limited; this is partly because of the high activity levels of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in tumor cells, which creates a resistant phenotype by blunting the therapeutic effect of alkylating agents. Thus, MGMT may be an important determinant of treatment failure and should be considered as a suitable target for intervention, in an effort to improve the therapeutic efficacy of TMZ. In this study, we showed that small-interfering RNA (siRNA)-based downregulation of MGMT could enhance the chemosensitivity of malignant gliomas against TMZ. Notably, TMZ-resistant glioma-initiating cells with increased DNA repair and drug efflux capabilities could be efficiently transduced with MGMT-siRNA by using a novel liposome, LipoTrust. Accordingly, such transduced glioma-initiating cells could be sensitized to TMZ in both in vitro and in vivo tumor models. Taken together, this study provides an experimental basis for the clinical use of such therapeutic combinations.

Benefits of interferon-β and temozolomide combination therapy for newly diagnosed primary glioblastoma with the unmethylated MGMT promoter: A multicenter study.

The aim of the current study was to catalog genomic and epigenomic abnormalities in newly diagnosed glioblastoma patients and determine the correlation among clinical, genetic, and epigenetic profiles and clinical outcome.

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.

Glioma-initiating cells and molecular pathology: implications for therapy

There is now compelling evidence that gliomas harbor a small population of cells, termed glioma-initiating cells (GICs), characterized by their ability to undergo self-renewal and initiate tumorigenesis. The development of therapeutic strategies targeted toward GIC signaling may improve the treatment of malignant gliomas. The characterization of GICs provides a clue to elucidating histological heterogeneity and treatment failure. The role of the stem cell marker CD133 in the initiation and progression of brain tumors is still uncertain. Here, we review some of the signaling mechanisms involved in GIC biology, such as phosphatase and tensin homolog (PTEN), sonic hedgehog, Notch, and WNT signaling pathways, maternal embryonic leucine-zipper kinase (MELK), BMI1, and Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling. In addition, we discuss the role of microRNAs in GICs by focusing on microRNA-21 regulation by type I interferon.

Girdin maintains the stemness of glioblastoma stem cells

This corrects the article DOI: 10.1038/onc.2011.466

Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP+ to NADPH. IDH1 mutations are frequently detected in grades 2–4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to d-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-l-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

Retrovirally engineered T-cell-based immunotherapy targeting type III variant epidermal growth factor receptor, a glioma-associated antigen

The isotype of epidermal growth factor receptor variant III (EGFRvIII) is often identified in glioblastomas. Previously, we created a mouse monoclonal antibody, 3C10 (IgG2b), that specifically recognized EGFRvIII, and a recombinant single‐chain variable fragment of 3C10. The aim of the current study was to develop genetically engineered T cells, termed T‐bodies, that express a chimeric receptor consisting of the 3C10 single‐chain variable fragment coupled to signaling modules such as the CD3zeta (ζ) chain, for the treatment of tumors expressing mutant EGFR. After successful construction of the chimeric 3C10/CD3ζ T‐cell receptor, its expression on the T‐body was observed using western blotting and flow cytometry. The specificity of the T‐body for EGFRvIII was evaluated using an interferon‐gamma Elispot assay and a standard 51Cr‐release cytotoxicity assay. Furthermore, we demonstrated that the systemically delivered T‐body infiltrated the intrabrain tumor and significantly delayed tumor growth. These results indicate that the T‐body expressing the chimeric 3C10/CD3ζ T‐cell receptor specifically recognized glioma cells expressing EGFRvIII. In conclusion, T‐body‐based immunotherapy appears to be a promising approach for the treatment of glioma. (Cancer Sci 2010; 101: 2518–2524)

The Modulation of MicroRNAs by Type I IFN through the Activation of Signal Transducers and Activators of Transcription 3 in Human Glioma

Type I IFNs are involved in double-stranded RNA responses. Here, we investigated the possibility that IFN-β may induce or downregulate cellular microRNAs (miRNA) in human neoplasms and thereby use the RNA interference system to show antitumor effects. Because of its known connection to glioma biology, we focused on miR-21 among seven miRNAs influenced by IFN-β. We analyzed the effect of IFN-β treatment on miR-21 expression in glioma cells and intracranial glioma xenografts. IFN-β treatment reduced miR-21 expression in glioma cells markedly, and IFN-β administration suppressed the growth of glioma-initiating cell–derived intracranial tumors. The levels of primary miR-21 gene transcripts, precursor miR-21, and mature miR-21 decreased 6 hours after the addition of IFN-β, indicating that the reduction in miR-21 levels was due to transcriptional suppression. We did reporter assays to elucidate the IFN-β–mediated suppression of miR-21; the addition of signal transducers and activators of transcription 3 (STAT3)–expressing vectors induced the IFN-β–mediated suppression of miR-21, whereas STAT3-inhibiting agents inhibited the miR-21 suppression. Thus, the results of our study show that the downregulation of miR-21 contributes to the antitumor effects of IFN-β and that miR-21 expression is negatively regulated by STAT3 activation. These results highlight the importance of understanding the transcriptional regulation of the miRNAs involved in oncogenesis. (Mol Cancer Res 2009;7(12):2022–30)

Intraoperative subcortical mapping of a language-associated deep frontal tract connecting the superior frontal gyrus to Broca's area in the dominant hemisphere of patients with glioma

OBJECT The deep frontal pathway connecting the superior frontal gyrus to Brocas area, recently named the frontal aslant tract (FAT), is assumed to be associated with language functions, especially speech initiation and spontaneity. Injury to the deep frontal lobe is known to cause aphasia that mimics the aphasia caused by damage to the supplementary motor area. Although fiber dissection and tractography have revealed the existence of the tract, little is known about its function. The aim of this study was to determine the function of the FAT via electrical stimulation in patients with glioma who underwent awake surgery. METHODS The authors analyzed the data from subcortical mapping with electrical stimulation in 5 consecutive cases (3 males and 2 females, age range 40-54 years) with gliomas in the left frontal lobe. Diffusion tensor imaging (DTI) and tractography of the FAT were performed in all cases. A navigation system and intraoperative MRI were used in all cases. During the awake phase of the surgery, cortical mapping was performed to find the precentral gyrus and Brocas area, followed by tumor resection. After the cortical layer was removed, subcortical mapping was performed to assess language-associated fibers in the white matter. RESULTS In all 5 cases, positive responses were obtained at the stimulation sites in the subcortical area adjacent to the FAT, which was visualized by the navigation system. Speech arrest was observed in 4 cases, and remarkably slow speech and conversation was observed in 1 case. The location of these sites was also determined on intraoperative MR images and estimated on preoperative MR images with DTI tractography, confirming the spatial relationships among the stimulation sites and white matter tracts. Tumor removal was successfully performed without damage to this tract, and language function did not deteriorate in any of the cases postoperatively. CONCLUSIONS The authors identified the left FAT and confirmed that it was associated with language functions. This tract should be recognized by clinicians to preserve language function during brain tumor surgery, especially for tumors located in the deep frontal lobe on the language-dominant side.