Takashi Sasayama

MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC

MicroRNAs (miRNAs) are effective post‐transcriptional regulators of gene expression and are important in many biological processes. Although the oncogenic and tumor suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumor invasion and migration remains largely unexplored. Recently, miR‐10b was identified as an miRNA highly expressed in metastatic breast cancer, promoting cell migration and invasion. Here, we performed real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) assays on 43 glioma samples (17 glioblastoma, 6 anaplastic astrocytoma, 10 low‐grade astrocytoma, 6 oligodendroglioma and 4 ependymoma) and 6 glioma cell lines. We found that miR‐10b expression was upregulated in all glioma samples compared to non‐neoplastic brain tissues. The expression levels of miR‐10b were associated with higher grade glioma. In addition, mRNA expressions of RhoC and urokinase‐type plasminogen activator receptor (uPAR), which were thought to be regulated by miR‐10b via HOXD10, were statistically significantly correlated with the expression of miR‐10b (p < 0.001, p = 0.001, respectively). Also, protein expression levels of RhoC and uPAR were associated with expression levels of miR‐10b (p = 0.009, p = 0.014, respectively). Finally, multifocal lesions on enhanced MRI of 7 malignant gliomas were associated with higher expression levels of miR‐10b (p = 0.02). Our data indicated that miR‐10b might play some role in the invasion of glioma cells.

Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells

Background: Various mitotic events are controlled by Cdc2‐cyclin B and other mitotic kinases. Aurora/Ipl1‐related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora‐A is a mammalian counterpart of aurora/Ipl1‐related kinases and is thought to be a potential oncogene. However, the regulation of aurora‐A activation and the commitment of aurora‐A in the progression of G2‐M phase are largely unknown in mammalian cells.

PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas

Gene rearrangement in the form of an intragenic deletion is the primary mechanism of oncogenic mutation of the epidermal growth factor receptor (EGFR) gene in gliomas. However, the incidence of platelet-derived growth factor receptor-α (PDGFRA) gene rearrangement in these tumors is unknown. We investigated the PDGFRA locus in PDGFRA-amplified gliomas and identified two rearrangements, including the first case of a gene fusion between kinase insert domain receptor (KDR) (VEGFRII) and the PDGFRA gene, and six cases of PDGFRA(Δ8, 9), an intragenic deletion rearrangement. The PDGFRA(Δ8, 9) mutant was common, being present in 40% of the glioblastoma multiformes (GBMs) with PDGFRA amplification. Tumors with these two types of PDGFRA rearrangement displayed histologic features of oligodendroglioma, and the gene products of both rearrangements showed constitutively elevated tyrosine kinase activity and transforming potential that was reversed by PDGFR blockade. These results suggest the possibility that these PDGFRA mutants behave as oncogenes in this subset of gliomas, and that the prevalence of such rearrangements may have been considerably underestimated.

Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment

The mechanistic target of rapamycin (mTOR) is hyperactivated in many types of cancer, rendering it a compelling drug target; however, the impact of mTOR inhibition on metabolic reprogramming in cancer is incompletely understood. Here, by integrating metabolic and functional studies in glioblastoma multiforme (GBM) cell lines, preclinical models, and clinical samples, we demonstrate that the compensatory upregulation of glutamine metabolism promotes resistance to mTOR kinase inhibitors. Metabolomic studies in GBM cells revealed that glutaminase (GLS) and glutamate levels are elevated following mTOR kinase inhibitor treatment. Moreover, these mTOR inhibitor-dependent metabolic alterations were confirmed in a GBM xenograft model. Expression of GLS following mTOR inhibitor treatment promoted GBM survival in an α-ketoglutarate-dependent (αKG-dependent) manner. Combined genetic and/or pharmacological inhibition of mTOR kinase and GLS resulted in massive synergistic tumor cell death and growth inhibition in tumor-bearing mice. These results highlight a critical role for compensatory glutamine metabolism in promoting mTOR inhibitor resistance and suggest that rational combination therapy has the potential to suppress resistance.

Cerebrospinal fluid interleukin-10 is a potentially useful biomarker in immunocompetent primary central nervous system lymphoma (PCNSL)

The diagnosis of primary central nervous system lymphoma (PCNSL) by radiographical examination is often difficult because of its similarity to other brain tumors. To test whether interleukin-10 (IL-10) and IL-6 can be used to distinguish PCNSL from other brain tumors that are radiographically similar, cerebrospinal fluid (CSF) levels of IL-10 and IL-6 were measured in 66 patients with intracranial tumors (PCNSLs: 26 cases; other brain tumors: 40 cases). In the patients with PCNSLs, the median CSF levels of IL-10 and IL-6 were 27 pg/mL and 5.4 pg/mL, respectively. The CSF IL-10 and IL-6 levels were significantly higher in PCNSLs than in the other brain tumors. To validate the diagnostic value of CSF IL-10 in PCNSL, we prospectively examined 24 patients with brain lesions that were suspected to be PCNSL. We observed that the CSF IL-10 levels were significantly higher in PCNSLs than in other brain tumors. At an IL-10 cutoff level of 9.5 pg/mL, the sensitivity and specificity were 71.0% and 100%, respectively. After therapy, the CSF IL-10 levels were decreased in all patients and were increased at relapse in most of these patients. Immunohistochemically, all PCNSLs, except for 1 unclassified PCNSL, expressed both IL-10 and IL-10 receptor-A. In the patients with high CSF IL-10, IL-10 expression levels in tumor were relatively higher, compared with low CSF IL-10; however, there was no significant difference between these groups. In addition, elevated CSF level of IL-10 was significantly associated with having a shorter progression-free survival (hazard ratio, 3.37; 95% confidence interval, 0.985-11.528; log-rank, P= .038). These results indicate that the CSF level of IL-10 may be a useful diagnostic and prognostic biomarker in patients with PCNSLs.

MicroRNA-183 upregulates HIF-1α by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells

MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression and contribute to cell proliferation, differentiation and metabolism. Our previous study revealed the extensive modulation of a set of miRs in malignant glioma. In that study, miR microarray analysis demonstrated the upregulation of microRNA-183 (miR-183) in glioblastomas. Therefore, we examined the expression levels of miR-183 in various types of gliomas and the association of miR-183 with isocitrate dehydrogenase 2 (IDH2), which has complementary sequences to miR-183 in its 3′-untranslated region (3′UTR). In present study, we used real-time PCR analysis to demonstrate that miR-183 is upregulated in the majority of high-grade gliomas and glioma cell lines compared with peripheral, non-tumorous brain tissue. The mRNA and protein expression levels of IDH2 are downregulated via the overexpression of miR-183 mimic RNA in glioma cells. Additionally, IDH2 mRNA expression is upregulated in glioma cells expressing anti-miR-183. We verified that miR-183 directly affects IDH2 mRNA levels in glioma cells using luciferase assays. In malignant glioma specimens, the expression levels of IDH2 were lower in tumors than in the peripheral, non-tumorous brain tissues. HIF-1α levels were upregulated in glioma cells following transfection with miR-183 mimic RNA or IDH2 siRNA. Moreover, vascular endothelial growth factor and glucose transporter 1, which are downstream molecules of HIF-1α, were upregulated in cells transfected with miR-183 mimic RNA. These results suggest that miR-183 upregulation in malignant gliomas induces HIF-1α expression by targeting IDH2 and may play a role in glioma biology.

Recurrent mutations of CD79B and MYD88 are the hallmark of primary central nervous system lymphomas

Primary central nervous system lymphoma (PCNSL) manifest aggressive clinical behaviour and have poor prognosis. Although constitutive activation of the nuclear factor‐κB (NF‐κB) pathway has been documented, knowledge about the genetic alterations leading to the impairment of the NF‐κB pathway in PCNSLs is still limited. This study was aimed to unravel the underlying genetic profiles of PCNSL.

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1.

Aurora‐A is a centrosomal serine‐threonine kinase that regulates mitosis. Over‐expression of Aurora‐A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora‐A over‐expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over‐expressed Aurora‐A is not restricted to the centrosomes but is also found in the cytoplasm. This over‐expressed Aurora‐A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora‐As role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over‐expressed Aurora‐A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h‐CPEB. In vitro experiments revealed that Aurora‐A binds directly to, and phosphorylates, h‐CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora‐A and h‐CPEB were over‐expressed, and they were further promoted in the presence of an Aurora‐A activator Ajuba. Our results suggest a function of ectopically over‐expressed Aurora‐A that might be relevant for carcinogenesis.

GC/MS-based metabolomic analysis of cerebrospinal fluid (CSF) from glioma patients

Metabolomics has recently undergone rapid development; however, metabolomic analysis in cerebrospinal fluid (CSF) is not a common practice. We analyzed the metabolite profiles of preoperative CSF samples from 32 patients with histologically confirmed glioma using gas chromatography/mass spectrometry (GC/MS). We assessed how alterations in the metabolite levels were related to the World Health Organization (WHO) tumor grades, tumor location, gadolinium enhancement on magnetic resonance imaging (MRI), and the isocitrate dehydrogenase (IDH) mutation status. Sixty-one metabolites were identified in the CSF from glioma patients using targeted, quantitative and non-targeted, semi-quantitative analysis. The citric and isocitric acid levels were significantly higher in the glioblastoma (GBM) samples than in the grades I–II and grade III glioma samples. In addition, the lactic and 2-aminopimelic acid levels were relatively higher in the GBM samples than in the grades I–II glioma samples. The CSF levels of the citric, isocitric, and lactic acids were significantly higher in grade I–III gliomas with mutant IDH than in those with wild-type IDH. The tumor location and enhancement obtained using MRI did not significantly affect the metabolite profiles. Higher CSF levels of lactic acid were statistically associated with a poorer prognosis in grades III–IV malignant gliomas. Our study suggests that the metabolomic analysis of CSF from glioma patients may be useful for predicting the glioma grade, metabolic state, and prognosis of gliomas.

Combined IDH1 mutation and MGMT methylation status on long-term survival of patients with cerebral low-grade glioma

OBJECTIVE The management of low-grade glioma (LGG) still remains controversial because the effectiveness of early and extensive resection is unclear, and the use of radiation therapy or chemotherapy is not well-defined. In particular, the importance of prognostic factors for survival remains a matter of discussion. The purpose of this study was to validate prognostic factors for survival in patients with LGG. MATERIALS AND METHODS A consecutive series of 55 patients with WHO grade II LGG treated in our institute between 1983 and 2013 were retrospectively reviewed to determine the prognostic factors for survival. All data were retrospectively analyzed from the aspect of baseline characteristics, pathological findings, genetic change, surgical treatments, adjuvant therapies, and survival time. Cox multivariate analysis was performed to determine the prognostic factors for survival. RESULTS There were 28 patients with diffuse astrocytoma (DA), 21 patients with oligodendroglioma (OG), and 6 patients with oligoastrocytoma (OA) diagnosed on initial surgery. The median overall survival was 193 months and fifteen patients (27.3%) died. A mutation in isocitrate dehydrogenase-1 (IDH1) was found in 72.9% of LGG, and this mutation was positively correlated with methylation of O6-methylguanine-DNA methyltransferase (MGMT) (p=0.02). A better prognosis was significantly associated with combined IDH1 mutation and MGMT methylation status (both positive vs both negative, HR 0.079 [95% CI 0.008-0.579], p=0.012), as well as histology (OG vs DA and OA, HR 0.158 [95% CI 0.022-0.674], p=0.011) and tumor size (<6 cm vs ≥6 cm, HR 0.120 [95% CI 0.017-0.595], p=0.008). CONCLUSIONS Tumor histology, size and IDH-mutation status are important predictors for prolonged overall survival in patients with LGG and may provide a reliable tool for standardizing future treatment strategies.