Satoko Nakada

Molecular targets of glioma invasion

Abstract.Glioblastoma multiforme is the most common and lethal primary malignant brain tumor. Although considerable progress has been made in technical proficiencies of surgical and radiation treatment for brain tumor patients, the impact of these advances on clinical outcome has been disappointing, with median survival time not exceeding 15 months. Over the last 30 years, no significant increase in survival of patients suffering from this disease has been achieved. A fundamental source of the management challenge presented in glioma patients is the insidious propensity of tumor invasion into distant brain tissue. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure and chemotherapy. Recent improved understanding of biochemical and molecular determinants of glioma cell invasion provide valuable insight into the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. These findings are moving forward to translational research and clinical trials as novel antiglioma therapies.

Ephrin-B3 ligand promotes glioma invasion through activation of Rac1

Eph receptor tyrosine kinases are involved in nervous system development. Eph ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in glioma cells increases cell invasion. Here, expression profiles of ephrin-B family members were determined in four glioma cell lines and in invading glioblastoma cells collected by laser capture microdissection. Ephrin-B3 mRNA was up-regulated in migrating cells of four of four glioma cell lines (1.3- to 1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251, SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA negated the increased invasion by addition of EphB2/Fc. In human glioma specimens, ephrin-B3 expression and phosphorylation correlated with increasing tumor grade. Immunohistochemistry revealed robust staining for phosphorylated ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that ephrin-B3 expression and signaling through Rac1 are critically important to glioma invasion.

MAP-ing glioma invasion: Mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival

Although astrocytic brain tumors do not metastasize systemically, during tumorigenesis glioma cells adopt an invasive phenotype that is poorly targeted by conventional therapies; hence, glioma patients die of recurrence from the locally invasive tumor population. Our work is aimed at identifying and validating novel therapeutic targets and biomarkers in invasive human gliomas. Transcriptomes of invasive glioma cells relative to stationary cognates were produced from a three-dimensional spheroid in vitro invasion assay by laser capture microdissection and whole human genome expression microarrays. Qualitative differential expression of candidate invasion genes was confirmed by quantitative reverse transcription-PCR, clinically by immunohistochemistry on tissue microarray, by immunoblotting on surgical specimens, and on two independent gene expression data sets of glial tumors. Cell-based assays and ex vivo brain slice invasion studies were used for functional validation. We identify mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) as a key activator of p38 MAPK in glioma; MKK3 activation is strongly correlated with p38 activation in vitro and in vivo. We further report that these members of the MAPK family are strong promoters of tumor invasion, progression, and poor patient survival. Inhibition of either candidate leads to significantly reduced glioma invasiveness in vitro. Consistent with the concept of synthetic lethality, we show that inhibition of invasion by interference with these genes greatly sensitizes arrested glioma cells to cytotoxic therapies. Our findings therefore argue that interference with MKK3 signaling through a novel treatment combination of p38 inhibitor plus temozolomide heightens the vulnerability of glioma to chemotherapy. [Mol Cancer Ther 2007;6(4):1212–22]

The Phosphorylation of Ephrin-B2 Ligand Promotes Glioma Cell Migration and Invasion

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin‐B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin‐B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin‐B1 and ‐B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan–Meier analysis demonstrated ephrin‐B2, but not ephrin‐B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin‐B2 were high in GBM. Immunohistochemistry demonstrated ephrin‐B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin‐B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin‐B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin‐B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin‐B2. These results demonstrate that high expression of ephrin‐B2 is a strong predictor of short‐term survival and that ephrin‐B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.

Glioma cells on the run – the migratory transcriptome of 10 human glioma cell lines

BackgroundGlioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death.To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis.ResultsGene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells.ConclusionGene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

Narrow band imaging-guided endoscopic biopsy for intraventricular and paraventricular brain tumors: clinical experience with 14 cases

BackgroundNarrow-band imaging (NBI) has been confirmed as a useful endoscopic technique to distinguish neoplasm from normal tissue, on the basis of the enhanced neovascularity of tumor tissue. NBI-guided tissue biopsy for laryngopharyngeal and digestive lesions is a novel methodology, but the feasibility for central nervous system tumors remains unclear. The aim of our study was to evaluate the feasibility of NBI-guided biopsy for intraventricular and paraventricular tumor.MethodsFourteen patients with intraventricular or paraventricular tumors underwent neuroendoscopic biopsy using a videoscope with NBI. Ventricular walls and tumors were observed using conventional imaging, followed by NBI. Colors of ventricle walls and tumors visualized using NBI were compared to those visualized under conventional imaging. Extracted specimens were stained using CD31 antibody and numbers of microvessels in each specimen were counted for analyzing vascular density.ResultsNormal ventricle walls were a similar color under conventional imaging and NBI. Tumor surfaces appeared to be cyan in color under NBI. Vessels on the tumor were more clearly visualized with NBI than with conventional imaging. NBI was able to identify tumor surfaces that were not perceptible on conventional imaging. All specimens in the lesion surfaces from cyan-colored areas under NBI contained tumor cells. Specimens extracted from regions that appeared cyan in color under NBI (51.0 vessels/mm2) had significantly greater vascular density than regions that appeared a normal color (17.4 vessels/mm2; p = 0.039).ConclusionNBI-guided biopsy of intraventricular and paraventricular tumors is feasible for visualizing tumor surface-enhancing neovascularities. NBI would contribute to accurate histological diagnosis while minimizing injury to surrounding structures.

Epithelioid glioblastoma changed to typical glioblastoma: the methylation status of MGMT promoter and 5-ALA fluorescence

A 55-year-old man was admitted to our hospital complaining of left hemiparesis. Magnetic resonance imaging (MRI) showed a smooth ring-like enhanced cystic tumor in the right parietal lobe. He underwent gross total resection of the tumor under neuronavigation and 5-aminolevulinic acid (5-ALA) fluorescence guiding method. Histopathological examination of the tumor showed small cells formed epithelioid solid nests with some focus of duct-like structure. On the basis of the MRI and operative and histological findings, this tumor was diagnosed as a metastatic poorly differentiated carcinoma, although the primary cancer could not be detected by metastatic work-ups. Afterward, this tumor recurred repeatedly. Histopathological examination of specimen from the fourth surgery indicated that the tumor was a glioblastoma (GBM). In the review of the histology and immunohistochemistry of the first tumor, atypical fibrillary cells were seen between solid nests and positive for glial fibrillary acidic protein, therefore the tumor was retrospectively diagnosed as epithelioid GBM. We assessed whether the changes in histopathology were accompanied by changes in the methylation status of O6-methylguanine methyltransferase (MGMT) promoter and the status of 5-ALA fluorescence. The methylation status of the MGMT promoter was found to have changed from methylated to unmethylated and 5-ALA fluorescence became positive along with the histological change.

Benign mesothelial nodules reflux within acquired cutaneous lymphangiectasia associated with huge ovarian clear cell carcinoma: Letter to the Editor

To the Editor: There have been some interesting reports describing a unique case of benign mesothelial nodule reflux (BMNR) in the cutaneous lymph vessels (the benign mesothelial nodules are composed of polygonal mesothelial cells admixed with a variable number of inflammatory cells, especially the histiocytes) including: (i) ‘Embolization of mesothelial cells’ in lymphatics of a 60-year-old male on the abdominal skin with hernia, accompanied by alcoholic cirrhosis and severe ascites; (ii) ‘Mesothelial cells reflux’ within acquired cutaneous lymphangiectasia of a 56-year-old male on the abdominal skin, accompanied by hepatitis C virus-induced cirrhosis and severe ascites; and (iii) ‘Benign mesothelial nodules’ in lymphatics of a 55-year-old female on the umbilical skin with huge hernia, associated with massive ovarian fibroma. Although mesothelial cell inclusions within the mediastinal or abdominal lymph nodes are a well known phenomenon, BMNR in the cutaneous lymphatics is rarely reported and potentially represents a new morphological and clinicopathological entity. To the best of our knowledge, BMNR was first described as the lymphatic dissemination and embolization of mesothelial cells by Rossi Su arez-Viela and Izquierdo-Garcia in the late 1990s, and to date, only four cases of BMNR (including our own) have been reported in the English literature. BMNR in the cutaneous lymph vessels is an extremely uncommon and unestablished entity; however, we should be aware that pathologists might misinterpret BMNR as the lymphatic dissemination of malignant cells. We showed the first case of BMNR within acquired cutaneous lymphangiectasia, which was associated with huge ovarian clear cell carcinoma and ascites in an obese patient. The patient, who was a woman in her late forties with marked obesity (BMI: 45 kg/m) and an unremarkable previous medical history, presented with abnormal vaginal/ uterine bleeding due to a huge ovarian carcinoma, accompanied by a subsequent and diffuse erythema with local heat. The erythema was approximately 25 cm in diameter and covered the whole abdominal skin (Fig. 1a). Dermatologists first interpreted it as cellulitis, and antibiotics were administered but were not effective. Scanning magnification of a biopsy specimen of the umbilical lesion (Fig. 1b) revealed that the deep dermis and superficial subcutis demonstrated a collection of variably dilated lymphatic vessels; microscopy revealed a mildly thickened wall; the endothelium was positive for D2-40 and CD31, and was frequently filled with small glomeruloid nodules (Fig. 1c). On a high-power view, the cellular nodules consisted of a substantial number of CD68-positive histiocytes and siderophages, admixed with lymphocytes/neutrophils, CD31positive microvessels and a-SMA-positive myofibroblast-like cells The nodules were characteristically lined by mediumsized to large polygonal or cuboidal epithelioid cells (Fig. 1c). These lining cells contained relatively abundant eosinophilic cytoplasm and likely centrally-located nuclei (Fig. 1c), and were immunohistochemically positive for calretinin (Fig. 1d), D2-40, WT1 and cytokeratins (AE1/AE3 and CK5/6) and negative for IMP3, GLUT1, HNF-1b and Napsin A. In addition, the deletion of p16 or BAP1 was not detected by fluorescence in situ hybridization (FISH) testing or immunohistochemistry, respectively. Based on all of these features, we concluded that the lining epithelioid cells were benign mesothelial cells. In contrast, on gross examination, the cut surface of the ovarian multilocular tumor showed a solid firm and lobulated mass, measuring more than 30 18 13 cm in size, which appeared yellow-whitish to gray-whitish in color, accompanied by focal necrosis and hemorrhage. The typical microscopic findings (Fig. 1e) included a proliferation of medium-sized to large highly atypical epithelial cells with hyperchromatic pleomorphic nuclei and abundant clear to eosinophilic cytoplasm, arranged in a predominantly papillary/micropapillary or microcystic growth fashion that frequently shows a hobnail appearance. Those carcinoma cells were immunohistochemically positive for HNF-1b and Napsin A. Neither distant metastasis nor the peritoneal dissemination of clear cell carcinoma were observed. A gross examination revealed light hemorrhagic ascites, while a histopathological examination revealed numerous inflammatory foci on the surface of omentum, very similar to the abovementioned small intralymphatic glomeruloid nodules, consisting of granulation-like tissue, lined by variably hyperplastic mesothelial cells without any atypia (Fig. 1f). We ultimately made a diagnosis of cutaneous BMNR within acquired lymphangiectasia due to the peritoneal mesothelial and inflammatory nodule drainage, which was potentially induced by the gradient of abdominal pressure secondary to huge ovarian clear cell carcinoma and ascites. To date, the patient has been followed for approximately one year since surgery, and remains well without any sign of recurrence.

The Combination Of Weak Expression Of PRDX4 And Very High MIB-1 Labelling Index Independently Predicts Shorter Disease-free Survival In Stage I Lung Adenocarcinoma

Background:Oxidative stress plays pivotal roles in the progression of lung adenocarcinoma (LUAD) through cell signaling related closely to cancer growth. We previously reported that peroxiredoxin 4 (PRDX4), a secretory-type antioxidant enzyme, can protect against the development of various diseases, including potential malignancies. Since many patients with early-stage LUAD develop recurrence, even after curative complete resection, we investigated the association of the PRDX4 expression with the clinicopathological features and recurrence/prognosis using post-surgical samples of stage I-LUAD. Methods: The expression of PRDX4 and MIB-1, a widely accepted Ki67 protein, was immunohistochemically analysed in 206 paraffin-embedded tumour specimens of patients with stage I-LUAD. The PRDX4 expression was considered to be weak when less than 25% of the adenocarcinoma cells showed positive staining. Results: A weak PRDX4+ expression demonstrated a significantly close relationship with pathologically poor differentiation, highly invasive characteristics and recurrence. The decrease in PRDX4-positivity potentially induced cell growth in LUAD, which was correlated significantly with a very high MIB-1 labelling index (≥17.3%). Univariate/multivariate analyses revealed that the subjects with both weak PRDX4+ expression and a very high MIB-1 index had significantly worse disease-free survival rates than other subjects. Conclusions: The combination of weak PRDX4 expression and a very high MIB-1 index can predict high proliferating activity and recurrence with a potential poor prognosis, especially in post-operative stage I-LUAD patients.

The clinicopathological comparison among nodal cases of idiopathic multicentric Castleman disease with and without TAFRO syndrome

Multicentric Castleman disease (MCD) is a systemic inflammatory disease potentially caused by an increase in the serum interleukin-6 (IL-6) level. Idiopathic MCD (iMCD) is histopathologically classified into three types: plasmacytic (PC), mixed, and hypervascular (hyperV) types. Recently, a unique clinical phenotype with a poor prognosis overlap with iMCD, thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly (TAFRO syndrome), has been reported from Japan, but its detailed clinicopathological features remain unclear. In this study, we performed a clinicopathological analysis of 70 nodal cases of iMCD with and without TAFRO syndrome (n = 37 versus n = 33). Compared with iMCD without TAFRO, iMCD with TAFRO showed more atrophic lymphoid follicles (LF), greater distances between follicles, increased glomeruloid vascular proliferation within the germinal center, and increased follicular dendritic cells. In addition, the hyperV type in particular demonstrated severe atrophic LF and interfollicular vascular proliferation. Among the mixed-type cases, the serum IL-6 levels in iMCD with TAFRO were significantly higher than those in iMCD without TAFRO. Furthermore, compared to iMCD without TAFRO, the numbers of immunoglobulin G4 (IgG4)-positive and CD38-positive plasma cells were significantly decreased in iMCD with TAFRO.