Miyuki Uno

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas.

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2‐fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT‐PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5‐fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage‐independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3′-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

Galectin-3 as an Immunohistochemical Tool to Distinguish Pilocytic Astrocytomas from Diffuse Astrocytomas, and Glioblastomas from Anaplastic Oligodendrogliomas

The distinction of astrocytomas and oligodendrogliomas, mainly pilocytic astrocytomas (PILOs) from infiltrating astrocytomas and oligodendrogliomas (ODs), and high‐grade oligodendrogliomas from glioblastomas (GBMs), poses a serious clinical problem. There is no useful immunohistochemical (IHC) marker to differentiate these gliomas, and sometimes the differential diagnosis between them is arbitrary. We identified galectin‐3 (Gal‐3) as a possible tool to differentiate them based on gene expression profiles of GBMs. We confirmed the differential expression in 45 surgical samples (thirteen GBMs; seven PILOs; 5 grade II ODs; 5 anaplastic oligodendrogliomas [AODs], including 2 Oligo‐astrocytomas; 8 diffuse astrocytomas [ASTs], and 7 non‐neoplastic samples) by quantification of Gal‐3 gene expression by real‐time quantitative PCR (rt‐PCR). Higher expression of Gal‐3 was observed in GBMs and PILOs than in OD, AODs and ASTs. The IHC expression of Gal‐3 was evaluated in 90 specimens (fifteen PILOs, fourteen ASTs, 10 anaplastic astrocytomas, fifteen GBMs, eleven ODs, fifteen AODs, and 10 dysembryoplastic neuroepithelial tumors). The mean labeling score for Gal‐3 determined according to the percentage of labeled cells in the tumor bulk was significantly different in GBMs versus AODs and in PILOs versus ASTs. Hence, Gal‐3 is differentially expressed in central nervous system tumors, making IHC detection of Gal‐3 a useful tool in distinguishing between these gliomas.

Proteomic analysis of low‐ to high‐grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non‐neoplastic brain tissue as control using 2‐DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up‐regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down‐regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real‐time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Angiogenesis and expression of PDGF‐C, VEGF, CD105 and HIF‐1α in human glioblastoma

Glioblastoma (GBM), the most frequent and aggressive brain tumor, is characterized by marked angiogenesis directly related to invasiveness and poor prognosis. Hypoxia is considered to be an important stimulus for angiogenesis by inducing hypoxia‐inducible factor 1‐alpha (HIF‐1α) overexpression that activates platelet‐derived growth factor (PDGF) and VEGF. The aim of this study is to analyze the expression of PDGF‐C, VEGF in endothelial and tumor cells of GBM and their relation to HIF‐1α expression. Two hundred and eight GBM cases were studied by tissue microarray immunohistochemical preparation. Expression of HIF‐1α, VEGF and PDGF‐C was observed in 184 (88.5%), 131 (63%) and 160 (76.9%) tumor cases, respectively. The numbers of vessels were quantified by CD34, PDGF‐C, VEGF and CD105 staining, and were in median 20, 16, 5 and 6, respectively. The GBMs that showed positive or negative expression for HIF‐1α showed a median vascular density of 30 and 14, respectively, for CD34 (P < 0.015). Positive expression for HIF‐1α was correlated with VEGF and PDGF‐C expression in tumors (P < 0.001). There was a significant correlation between VEGF and PDGF‐C expression in the cytoplasm of GBM tumor cells (P < 0.0001). We showed that VEGF expression in tumor cells was correlated with its expression in blood vessels (P < 0.0001). Endothelial cells with PDGF‐C and VEGF positive expression were also positive for CD105 and their nuclei for Ki‐67, confirming the neoangiogenic and proliferative influence of VEGF and PDGF‐C. VEGF nuclear staining in tumor cells (P = 0.002) as well as nuclear staining for HIF‐1α and VEGF (P = 0.005) correlated with survival. In summary, our present findings of the concomitant upregulation of PDGF‐C with VEGF in GBM tumor cells and vessels further reinforce the benefit of using combined anti‐angiogenic approaches to potentially improve the therapeutic response for GBM.

Lifestyle factors associated with atrophic gastritis among Helicobacter pylori-seropositive Japanese-Brazilians in SÃo Paulo

BackgroundStudies of lifestyle factors related to gastric atrophy development in Helicobacter pylori-infected individuals are limited. The present cross-sectional study aimed to examine the associations between lifestyle factors and serum pepsinogens (PGs) among anti-H. pylori antibody-seropositive Japanese in Brazil, where gastric cancer mortality was reported to be as high as in Japanese in Japan, and seropositive individuals were still frequently detected.MethodsThe subjects were 291 seropositive individuals (129 males and 162 females; age, 30 to 69 years) out of 656 Japanese-Brazilian volunteers in SÃo Paulo city. Information on lifestyle factors was obtained using a self-administered questionnaire. Atrophic gastritis was defined as a PG1 serum level less than 70 ng/ml and PG1/PG2 ratio less than 3.ResultsThe prevalence of atrophic gastritis was 31.9% (95% confidence intervals, 26.6%–37.6%). The proportion of subjects with atrophic gastritis increased with age, but there were no significantly marked differences in the proportions of subjects with atrophic gastritis among the three generations studied (first generation [Issei], second generation [Nisei], and third generation [Sansei]) for any 10-year age group. The associations with smoking and alcohol drinking were not significant. Length of education was inversely associated with gastric atrophy, while infrequent rice intake was preventive; the odds ratio relative to everyday rice intake was 0.13 (95% confidence intervals, 0.39–0.46) on multivariate analysis.ConclusionsThe present study demonstrated that frequent rice intake was a risk factor for atrophic gastritis among the H. pylori-infected Japanese-Brazilians, suggesting that diet including rice plays a role in the step from H. pylori infection to gastric atrophy.

Mitochondrial DNA depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human diffusely infiltrating astrocytomas.

Mitochondrial DNA (mtDNA) alterations and their clinical and pathological implications have been analyzed in several human malignancies. A marked decrease in mtDNA copy number along with the increase in malignancy was observed in diffusely infiltrating astrocytomas (24 WHO grade II, 18 grade III, and 78 grade IV or GBM) compared to non-neoplastic brain tissues, being mostly depleted in GBM. Although high relative gene expression levels of mtDNA replication regulators (mitochondrial polymerase catalytic subunit (POLG), transcription factors A (TFAM), B1 (TFB1M) and B2 (TFB2M)) were detected, it cannot successfully revert the mtDNA depletion observed in our samples. On the other hand, a strong correlation among the expression levels of mitochondrial transcription factors corroborates with the TFAM role in the direct control of TFB1M and TFB2M during initiation of mtDNA replication. POLG expression was related to decreased mtDNA copy number, and its overexpression associated with TFAM expression levels also have an impact on long-term survival among GBM patients, interpreted as a potential predictive factor for better prognosis.

A comparison of the prevalence of the metabolic syndrome and its components among native Japanese and Japanese Brazilians residing in Japan and Brazil.

Background This study investigated the prevalence of risk factors associated with the metabolic syndrome (MetSyn) among individuals of Japanese descent exposed to different cultural environments. Design A cross-sectional study to assess component risk factors for the diagnosis of MetSyn was undertaken in urban areas in Japan and Brazil. A total of 773 men and women aged 35 years or over were included in three groups: 249 native Japanese, 269 Brazilian individuals of Japanese ancestry residing in Japan, and 255 Brazilian individuals of Japanese ancestry residing in Brazil. Results Higher rates of metabolic abnormalities with respect to central obesity and serum lipid profiles were observed among Brazilian individuals of Japanese ancestry residing in Brazil compared with those residing in Japan and native Japanese. Likewise, an increased risk of hypertension was observed among Japanese Brazilian individuals residing in Japan. The prevalence of MetSyn in men was significantly higher among Brazilians of Japanese ancestry residing in Brazil (37.5%) compared with those residing in Japan (25.3%) or native Japanese (21.4%), whereas no significant difference was observed among women. In the logistic model, Brazilian individuals of Japanese ancestry residinginBrazil weretwice as likely to develop MetSyn compared with native Japanese, whereas no significant differences were found among those residing in Japan. Conclusions These findings underscore the significant heterogeneity in risk factors among communities of Japanese ancestry residing in Brazil and Japan, and suggest that immigrants exposed to the Brazilian cultural environment are more susceptible to the development of risk factors associated with MetSyn than native Japanese.

Helicobacter pylori Seropositivity among 963 Japanese Brazilians According to Sex, Age, Generation, and Lifestyle Factors

Seropositivity of anti–Helicobacter pylori antibody (HP+) was examined among Japanese Brazilians. The study was announced through 18 Japanese community culture associations in São Paulo, Curitiba, Mogi das Cruzes, and Mirandopolis in 2001. Among 969 participants, 963 individuals aged 33–69 years were analyzed. The overall HP+% was 48.1% (95% confidence interval, 44.9–51.3%). There was no difference in HP+% between 399 males and 564 females (49.6% and 47.0%, respectively). The HP+% increased with age; 35.3% for those aged 33–39 years, 46.2% for those aged 40–49 years, 46.5% for those aged 50–59 years, and 56.9% for those aged 60–69 years, but no differences were observed among the generations (Issei, Nisei, and Sansei) for each 10–year age group. Mogi das Cruzes, a rural area, showed a higher HP+%. Length of education was inversely associated with the positivity; the odds ratio (OR) relative to those with eight years or less of schooling was 0.61 (0.42–0.89) for those with 12 years or more. The associations with smoking and alcohol drinking were not significant. Fruit intake was associated with the HP+%; the OR relative to everyday intake was 1.38 (1.05–1.83) for less frequent intake, while intake frequencies of green tea, miso soup, and pickled vegetables (tsukemono) were not. Multivariate analysis including sex, 10–year age group, residence, education, and fruit intake showed that all factors except sex were significant. This is the largest study of HP infection among Japanese Brazilians, and the results indicated a similar pattern of age–specific infection rate to that for Japanese in Japan.