BIOLOGY

Abstract

PURPOSE: To investigate whether tissue factor (TF) regulates fibroblast growth factor (FGF)-2-induced angiogenesis in retinoblastoma. METHODS: In an orthotopic transplantation mouse model of retinoblastoma, immunofluorescence staining for TF and CD31 (an endothelial cell maker) was performed. With treatment of FGF-2 (10 ng/ml), TF expression in human umbilical vein endothelial cells (HUVECs) was measured by Western blotting. To confirm the role of TF in tumor angiogenesis in retinoblastoma, anti-angiogenic activity of TF pathway inhibitor (TFPI) was investigated by treating TFPI on FGF-2-induced proliferation, migration and in vitro tube formation of HUVECs. In addition, inhibition of ERK1/2 phosphorylation by TFPI was measured by Western blot analysis. RESULTS: TF was highly expressed on vascular endothelial cells of retinoblastoma, co-localized with CD31. With FGF-2-induced proliferation of HUVECs, TF expression was significantly up-regulated. Interestingly, TFPI effectively inhibited FGF-2-induced proliferation, migration and in vitro tube formation of HUVECs, which was accompanied by inhibition of ERK1/2 phosphorylation. CONCLUSIONS: TF is involved in tumor angiogenesis of retinoblastoma via extracellular signal-regulated kinase pathway. PURPOSE: Nuclear exclusion of p53 has been suggested to be an important mechanism of tumor suppressor gene inactivation in the retinoblastoma. Here, we investigated whether the mature tumor vascular endothelium of human retinoblastoma also shows the cytogenetic abnormality of tumor cell by verifying the phenomenon of p53 nuclear exclusion in the tumor vascular endothelium of human retinoblastoma. METHODS: The formalin-fixed paraffin-embedded specimens of primarily enucleated eyes of retinoblastoma (7 eyes from 7 patients) were sectioned for immunofluorescent staining. Double immunofluorescence stain with anti-von Willebrand factor and anti-p53 antibodies and DAPI nuclear counterstain were performed to identify the presence of mature tumor vascular endothelium which shows nuclear exclusion of p53 protein. To determine the proportion of p53 nuclear exclusion among mature tumor vascular endothelium, two blinded investigators counted at least 10 randomly selected high power fields. RESULTS: Most of tumor cells from human retinoblastoma specimens showed nuclear exclusion of p53 protein. Overall, 45.1% of mature tumor vascular endothelium showed nuclear exclusion of p53, whereas the endothelium of pre-existing retinal vessels which supply the relatively preserved retina consistently showed negative stain for anti-p53 antibody. CONCLUSIONS: Some proportion of mature tumor vascular endothelium shares the same cytogenetic abnormality with retinoblastoma cells. These data suggest that an endothelial differentiation of retinoblastoma cell might partially contribute to the tumor vascularization in retinoblastoma. Embryonal Tumors with Abundant Neuropil and True Rosettes (ETANTR) is a rare CNS tumor in children. Due to its rarity molecular pathways and treatment guidelines remain ill-defined. To our knowledge this is the second case report of this tumor presenting as an isolated spinal lesion and the first report of a personalized therapy approach in ETANTR. A four year male presented with headache, gluteal area discomfort and lower extremity weakness. MRI spine showed a lumbar mass. Surgical resection was performed and pathology was consistent with ETANTR. He was treated with chemoradiation followed by adjuvant chemotherapy with cisplatin, vincristine and cyclophosphamide. He is currently receiving maintenance therapy with cis-retinoic acid, melatonin, metformin and is clinically doing well. Our previous submission demonstrated an innovative approach of molecular tumor profiling using morphoproteomic (MP) analysis and quantifying immunohistochemistry (IHC) signaling intensity of major pathways identifying activation of ras/Raf kinase/ERK and Akt/mTOR pathway in this tumor. Use of melatonin, metformin and cisretinoic acid in maintenance chemotherapy is based on our MP data of ETANTR. Metformin inhibits components of the mTORC2 pathway and thereby down-regulating the expression of p-mTOR and p-Akt. We identified MTORC2 as a dominant complex in these tumors supported by nuclear compartmentalization of p-mTOR(Ser 2448) and correlative expression of its putative downstream effector phosphorylated Akt (Ser 473). Expression of melatonin 1a melatonin receptor known to have cystostatic and cytodifferentiating properties was negative in these tumors validating the supplement of melatonin. Melatonin is known to potentiate all-trans retinoic acid effect on retinoic acid receptor and their sequential use induces apoptosis in tumor cells. These preliminary data suggests the potential use of metformin, melatonin and cis-retinoic acid as a maintenance therapy in ETANTR. Larger studies are needed to evaluate and validate this innovative method of target identification and personalized therapy in these rare tumors. MicroRNA-9,(miR-9), which mediates neuronal differentiation is found to be dysexpressed in brain tumors, nevertheless the functional implications of altered miR-9 expression in medulloblastoma MB is yet unclear. In this study, we investigated the expression level and the functional role of miR-9 in MB. Compared to normal human cerebellum, miR-9 was found to be under-expressed in 22 out of 29 MB primary samples (2-90 fold) as well as in 6/6 MB cell lines tested (3-13 fold). Methylation-specific PCR analysis for miR-9-1 promoter showed methylation in 5/6 MB cell lines, but not in normal cerebellum, whilst ethylation-mediated silencing could be reverted through treatment with the demethylating agent 5-aza-2'-deoxycytidine. An in silico search revealed that the Hes1 oncogene, which is the principal effector of the Notch pathway and known to be elevated in MB with unfavourable prognosis, is a direct target of miR-9. Consistently, a Hes1 3' UTR luciferase reporter assay experiment confirmed the inhibitory effect of miR-9 on Hes1 expression. Further analysis showed that increasing the expression of miR-9 in four MB cell lines down-regulates Hes1 protein and promotes mRNAs expression of its main responsive genes, P21 and Mash1. The resulting up-regulation of P21 increased the number of MB cells in the G1 phase of cell cycle and decreased cells in S phase, whereas the elevation of Mash1 increased protein expression of the neuronal differentiation markers Tuj1 and Nestin. In summary, our results suggest that miR-9 may act as a tumor suppressor and the failure of MB cells to express it may contribute to the disease through the over-expression of the Hes1. Moreover, the finding that miRNA-9 re-expression in MB cells results in the withdrawal from the cell cycle and in increased expression of neural-specific differentiation markers, qualifies miRNA-9 as a potential therapeutically efficacious remedy for the treatment of this disease. PURPOSE: Isocitrate dehydrogenase 1 (IDH1) mutations are genetic alteration with a high prevalence (50-70% of cases) in WHO grade II or III gliomas and secondary glioblastoma. The acquisition of IDH1 mutations occurs early in the development of a glioma from a glial progenitor cell that can give rise to both astrocytes and oligodendrocytes. These results, however, are based on studies of adult gliomas, but not pediatric gliomas. In this study, we examined IDH1 mutations in pediatric glioma patients with the following goals: (1) to compare the prevalence of IDH1 mutations in pediatric gliomas with those of adult ones; (2) to evaluate the relationship between IDH1 mutations and loss of 1p/19q or O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. MATERIALS & METHODS: We analyzed the mutational hot spot, codon 132 of IDH1 in 19 newly diagnosed pediatric gliomas treated in our hospital. Real-time PCR system followed by high-resolution melting (HRM) analysis were used to detect IDH1 mutaions. We estimated loss of 1p/19q and MGMT methylation status by fluorescence in situ hybridization and methylation specific HRM, respectively. RESULTS: Of the 19 cases, IDH1 was mutated in only one glioma (anaplastic astrocytoma). This case was accompanied with methylated MGMT. Four pediatric oligodendrogliomas with co-deletion of 1p/19q had no IDH1 mutations and no methylated MGMT. CONCLUSIONS: The frequency of IDH1 mutations in pediatric gliomas was significantly low irrespective of tumor grades or histology. All 1p/19q co-deleted adult oligodendrogliomas showed IDH1 mutations, whereas pediatric oligodendrogliomas did not. These results suggest that there is a difference between pediatric gliomas and adult ones in genetic significance of IDH1 mutataions in gliomagenesis. {beta}-adrenergic receptors ({beta}-ARs) are G-protein-coupled molecules that activate the protein kinase A pathway by accumulation of the second messenger cAMP. This signal transduction pathway seems to increase VEGF gene expression, resulting in enhanced tumor vascularization and more aggressive growth. Moreover, several epidemiologic studies documented a significantly lower risk of cancer development or recurrence in humans treated with {beta}-blocking agents. In malignant brain tumors, such as medulloblastoma, glioblastoma and anaplastic ependymoma, hypervascularization might result also from an enhancement of the {beta}-adrenergic signaling pathway. In this study we addressed the issue of the expression of {beta}-ARs in a population of 12 children affected by malignant primary brain tumors. We found a significant expression of {beta}1, {beta}2 -ARs in all of 12 samples as well as the 3 cell lines tested, U87MG, T98G and DAOY. Median absolute {beta}1-AR mRNA level standardized for GAPDH was 5.81 (range -7.91 to 11.20) for brain tumors and 8.50 (range 6.046 to 12.59) for cell lines (U87MG, DAOY and T98G) respectively; Median absolute {beta}2-AR mRNA level was 4.74 (range -9.30 to 8.45) for tumor specimens an