Akira Nakamizo

MiRNA-196 Is Upregulated in Glioblastoma But Not in Anaplastic Astrocytoma and Has Prognostic Significance

Purpose: MicroRNAs (miRNA) are short noncoding RNAs that can play critical roles in diverse biological processes. They are implicated in tumorigenesis and function both as tumor suppressors and as oncogenes. The clinical significance of miRNA expression profiles in malignant gliomas remains unclear. Experimental Design: In this study, we examined the expression levels of 365 mature human miRNAs in 12 malignant gliomas, including 8 glioblastomas and 4 anaplastic astrocytomas, using TaqMan real-time quantitative PCR arrays. A validation study was done to corroborate a subset of the results, including expression levels of miR-196a, -196b, -21, and -15b, by analyzing 92 malignant gliomas by conventional real-time PCR. We modeled the relationship between the expression levels of these miRNAs and the survival rate of 39 glioblastoma patients by Kaplan-Meier method and multivariate analysis. Results: Expression profiles in glioblastomas and anaplastic astrocytomas suggested that 16 miRNAs were candidate markers associated with the malignant progression of gliomas. Among them, miR-196a showed the most significant difference (P = 0.0038), with miR-196b also having a high significance (P = 0.0371). Both miRNAs showed increased expression levels in glioblastomas relative to both anaplastic astrocytomas and normal brains in the validation study. Furthermore, patients with high miR-196 expression levels showed significantly poorer survival by the Kaplan-Meier method (P = 0.0073). Multivariate analysis showed that miR-196 expression levels were an independent predictor of overall survival in all 39 glioblastoma patients (P = 0.021; hazard ratio, 2.81). Conclusions: Our results suggest that miR-196 may play a role in the malignant progression of gliomas and may be a prognostic predictor in glioblastomas. Clin Cancer Res; 16(16); 4289–97. ©2010 AACR.

Associations between microRNA expression and mesenchymal marker gene expression in glioblastoma

The subclassification of glioblastoma (GBM) into clinically relevant subtypes using microRNA (miRNA)- and messenger RNA (mRNA)-based integrated analysis has been attempted. Because miRNAs regulate multiple gene-signaling pathways, understanding miRNA-mRNA interactions is a prerequisite for understanding glioma biology. However, such associations have not been thoroughly examined using high-throughput integrated analysis. To identify significant miRNA-mRNA correlations, we selected and quantified signature miRNAs and mRNAs in 82 gliomas (grade II: 14, III: 16, IV: 52) using real-time reverse-transcriptase polymerase chain reaction. Quantitative expression data were integrated into a single analysis platform that evaluated the expression relationship between miRNAs and mRNAs. The 21 miRNAs include miR-15b, -21, -34a, -105, -124a, -128a, -135b, -184, -196a-b, -200a-c, -203, -302a-d, -363, -367, and -504. In addition, we examined 23 genes, including proneural markers (DLL3, BCAN, and OLIG2), mesenchymal markers (YKL-40, CD44, and Vimentin), cancer stem cell-related markers, and receptor tyrosine kinase genes. Primary GBM was characterized exclusively by upregulation of mesenchymal markers, whereas secondary GBM was characterized by significant downregulation of mesenchymal markers, miR-21, and -34a, and by upregulation of proneural markers and miR-504. Statistical analysis showed that expression of miR-128a, -504, -124a, and -184 each negatively correlated with the expression of mesenchymal markers in GBM. Our functional analysis of miR-128a and -504 as inhibitors demonstrated that suppression of miR-128a and -504 increased the expression of mesenchymal markers in glioblastoma cell lines. Mesenchymal signaling in GBM may be negatively regulated by miR-128a and -504.

Effects of single low dose irradiation on subventricular zone cells in juvenile rat brain.

Abstract Although the juvenile human brain is relatively radioresistant, irradiation can result in brain growth retardation, progressive mental disturbance, and neurologic abnormalities. As neural stem cells or progenitor cells may be a target of radiation injury and may play an important role in the brains functional recovery, we examined the effects of whole brain irradiation on these cells in juvenile rat. Six-week-old Wistar rats, where the brain is still growing, were irradiated with single doses of 1, 2, or 3 Gy X-ray. We measured their body and brain weights at 30 or 60 days after irradiation. The chronological changes of the subventricular zone (SVZ) were examined at 6 h, 2, 7, 14, 30, or 60 days after irradiation by immunohistochemistry, specifically looking at the neural stem cells or progenitor cells using anti-nestin antibodies specific for these cells. The rate of brain weight gain of irradiated rats significantly decreased in comparison to controls, although that of body weight gain was similar among them. Multiple apoptotic cells appeared in the SVZ at 6 h after irradiation with simultaneous reduction in nestin-positive cells (69% of the control). The cell levels recovered within a week, with the nestin-positive cells reaching maximal numbers (182%) on Day 14. Nestin-positive cells returned to baseline levels within 30 days (96%) and remained unchanged for the subsequent 60 days. The X-ray dosage did not affect these findings. Our findings revealed that single low dose X-ray administration reversibly affected the levels of neural stem and progenitor cells in the SVZ region. These results suggest that continuous multiple administrations of X-rays in clinical treatment may affect irreversible changes on neural stem or progenitor cells, causing brain growth retardation, or dysfunction.

Clinical implications of microRNAs in human glioblastoma

Glioblastoma (GBM) is one of the most common and dismal brain tumors in adults. Further elucidation of the molecular pathogenesis of GBM is mandatory to improve the overall survival of patients. A novel small non-coding RNA molecule, microRNA (miRNA), appears to represent one of the most attractive target molecules contributing to the pathogenesis of various types of tumors. Recent global analyses have revealed that several miRNAs are clinically implicated in GBM, with some reports indicating the association of miRNA dysregulation with acquired temozolomide (TMZ) resistance. More recent studies have revealed that miRNAs could play a role in cancer stem cell (CSC) properties, contributing to treatment resistance. In addition, greater impact might be expected from miRNA-targeted therapies based on tumor-derived exosomes that contain numerous functional miRNAs, which could be transferred between tumor cells and surrounding structures. Tumor-derived miRNAs are now considered to be a novel molecular mechanism promoting the progression of GBM. Establishment of miRNA-targeted therapies based on miRNA dysregulation of CSCs could provide effective therapeutic strategies for TMZ-resistant GBM. Recent progress has revealed that miRNAs are not only putative biological markers for diagnosis, but also one of the most promising targets for GBM treatment. Here in, we summarize the translational aspects of miRNAs in the diagnosis and treatment of GBM.

Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ) is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma (GBM). Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been described as the main modulator to determine the sensitivity of GBM to TMZ, a subset of GBM does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR), and the base excision repair (BER) pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break repair and double-strand break repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.

Expression of VEGF and its receptor genes in intracranial schwannomas

Vascular endothelial growth factor (VEGF) is considered to be a major regulator of angiogenesis in various brain tumors. In this study, we determined the expression levels of VEGF, and vascular endothelial growth factor receptor (VEGFR)-1 and -2 mRNA in 46 intracranial schwannomas by quantitative real-time PCR, and correlated these with various clinical factors or other molecular markers. We found that these tumors expressed significant amounts of VEGF mRNA in comparison with other brain tumors, including malignant gliomas and meningiomas. In addition, we performed immunohistochemical studies for VEGF and VEGFR-1, and confirmed that these tumors prominently express these proteins. The expression levels of VEGF and VEGFR-1 mRNA in recurrent tumors were higher than those in primary tumors. When we divided patients into two groups according to VEGF mRNA expression in the tumor, there was no significant difference in patient age, gender, or cranial nerves of origin between groups; however, the tumor volume tended to be larger in the high VEGF group than in the low VEGF group. The levels of VEGFR-1 mRNA and neurofibromatosis-2 mRNA in the high VEGF group were significantly greater than those in the low VEGF group. Levels of VEGFR-2 mRNA and DNA topoisomerase IIα mRNA, and the MIB-1 labeling index in the high VEGF group were slightly higher than those in the low VEGF group; however, the difference was not statistically significant. Based on these observations, the significance of VEGF and its receptor genes in intracranial schwannomas is discussed.

Type V phosphodiesterase expression in cerebral arteries with vasospasm after subarachnoid hemorrhage in a canine model

Abstract Cyclic GMP (cGMP) mediates smooth muscle relaxation in the central nervous system. In subarachnoid hemorrhage (SAH), decreases in intrinsic nitric oxide (NO) cause cerebral vasospasms due to the regulation of cGMP formation by NO-mediated pathways. As phosphodiesterase type V (PDE V) selectively hydrolyzes cGMP, we hypothesized that PDE V may function in the initiation of vasospasm. This study sought to identify the altered PDE V expression and activity in the vasospastic artery in a canine SAH model. We also used this system to examine possible therapeutic strategies to prevent vasospasm. Using a canine model of SAH, we induced cerebral vasospasm in the basilar artery (BA). Following angiographic confirmation of vasospasm on day 7, PDE V expression was immunohistochemically identified in smooth muscle cells of the vasospastic BA but not in cells of a control artery. The isolation of PDE enzymes using a sepharose column confirmed increased PDE V activity in the vasospastic artery only through both inhibition studies, using the highly selective PDE V inhibitor, sildenafil citrate, and Western blotting. Preliminary in vivo experiment using an oral PDE V inhibitor at 0.83 mg kg-1 demonstrated partial relaxation of the spastic BA. PDE V activity was increased from control levels within the BA seven days after SAH. PDE V expression was most prominent in smooth muscle cells following SAH. These results suggest that clinical administration of a PDE V inhibitor may be a useful therapeutic tool in the prevention of vasospasm following SAH. [Neurol Res 2002; 24: 607-612]

Preliminary Study on the Clinical Application of Augmented Reality Neuronavigation

OBJECTIVE To develop an augmented reality (AR) neuronavigation system with Web cameras and examine its clinical utility. METHODS The utility of the system was evaluated in three patients with brain tumors. One patient had a glioblastoma and two patients had convexity meningiomas. Our navigation system comprised the open-source software 3D Slicer (Brigham and Womens Hospital, Boston, Massachusetts, USA), the infrared optical tracking sensor Polaris (Northern Digital Inc., Waterloo, Canada), and Web cameras. We prepared two different types of Web cameras: a handheld type and a headband type. Optical markers were attached to each Web camera. We used this system for skin incision planning before the operation, during craniotomy, and after dural incision. RESULTS We were able to overlay these images in all cases. In Case 1, accuracy could not be evaluated because the tumor was not on the surface, though it was generally suitable for the outline of the external ear and the skin. In Cases 2 and 3, the augmented reality error was ∼2 to 3 mm. CONCLUSION AR technology was examined with Web cameras in neurosurgical operations. Our results suggest that this technology is clinically useful in neurosurgical procedures, particularly for brain tumors close to the brain surface.

Expression of stem cell marker and receptor kinase genes in glioblastoma tissue quantified by real-time RT-PCR

Glioblastoma is dependent on a specific signaling pathway to maintain its tumor phenotype. The receptor tyrosine kinase (RTK) family mediates the multiple oncogenic growth factor receptor signaling and contributes to the pathogenesis of glioblastoma. Recently, many studies have shown that the expression of stem cell marker in glioblastoma tissue has prognostic significance, which indicates that the quantification of stem cell markers and RTK genes yields biological information about glioblastoma. In this study, we quantified RNA expression levels of stem cell markers [CD133, Nestin, BMI-1, maternal embryonic leucine zipper kinase (MELK), and Notch1–4] as well as RTKs (EGFR, ErbB4, VEGFR1-3, FGFR1, -2, PDGFRΑ, and PDGFRΒ) in 42 clinical samples of glioblastoma by the real-time RT-PCR method. We demonstrated that the expression of MELK is exclusively upregulated in glioblastoma tissue. Notch receptor expression is moderately upregulated and is correlated with that of VEGFR2, VEGFR3, and PDGFRβ. Unsupervised clustering identified one unique sample group that showed high expression of most of the genes analyzed. Our results suggest that quantification of these stem cell markers and RTK genes can stratify patients based on the expression profile, which might provide insight into the glioma biology in each cluster.

MicroRNAs in Human Malignant Gliomas

MicroRNA (miRNA) is a new class of small noncoding RNA molecules that regulate a wide spectrum of gene expression in a posttranscriptional manner. MiRNAs play crucial roles in tumorigenesis, angiogenesis, invasion, and apoptosis for various types of tumor. Recent studies have identified dysregulation of specific miRNAs in malignant gliomas. Global expression profiling of miRNAs has revealed several miRNAs clinically implicated in human glioblastomas. Some miRNAs are clearly associated with clinical outcome and chemo- and radio-therapy resistance in these tumors. Furthermore, miRNAs also regulate specific signaling pathways, including the critical core pathways in glioblastoma. As a result, miRNAs have the potential to affect the responses to molecular-targeted therapies. More recent studies have revealed that miRNAs might be associated with cancer stem cell properties, affecting tumor maintenance and progression. Recent investigation have revealed that miRNAs are not only biological markers with diagnostic implications, but also one of the most promising treatment targets in human glioblastoma. Herein, we summarized the novel insights of miRNAs into human malignant gliomas.