Roseli da Silva

LOX Expression and Functional Analysis in Astrocytomas and Impact of IDH1 Mutation

Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

IDH1 mutations in a Brazilian series of Glioblastoma

GBM may manifest rapidly de novo(primary GBM), or may develop slowly from grade II orgrade III astrocytomas (secondary GBM), suggesting thatthey are distinct disease entities that evolve throughdifferent genetic pathways.In recent genome-wide analyses, high rates of sponta-neous mutations in the gene encoding cytosolic NADP-dependent isocitrate dehydrogenase 1 (IDH1) have beenreported in diffuse gliomas including WHO grades II andIII astroglial and oligodendroglial lineages.

Methylenetetrahydrofolate reductase gene polymorphism is not related to the risk of ischemic cerebrovascular disease in a Brazilian population

PURPOSE Data are conflicting concerning the risk for ischemic stroke associated with a common polymorphism in the gene encoding 5,10-methylenetetrahydrofolate reductase C677T, which predisposes carriers to hyperhomocysteinemia. A meta-analysis study suggested that the 5,10-methylenetetrahydrofolate reductase 677TT genotype might have a small influence in determining susceptibility to ischemic stroke. METHODS We analyzed the 5,10-methylenetetrahydrofolate reductase 677TT genotype polymorphism in Brazilian subjects with ischemic stroke, using a case-control design. RESULTS We compared 5,10-methylenetetrahydrofolate reductase genotypes in groups of subjects presenting ischemic stroke (n = 127) and normal control (n = 126) and found an odds ratio of 1.97 (95% CI, 0.84-4.64) in a multivariate analysis in which results were adjusted to baseline clinical characteristics of study participants. CONCLUSION We found that the homozygous 5,10-methylenetetrahydrofolate reductase C677T genotype was not a risk factor for ischemic stroke in these Brazilian subjects.

Xenograft transplantation of human malignant astrocytoma cells into immunodeficient rats: an experimental model of glioblastoma

INTRODUCTION: Astrocytic gliomas are the most common intracranial central nervous system neoplasias, accounting for about 60% of all primary central nervous system tumors. Despite advances in the treatment of gliomas, no effective therapeutic approach is yet available; hence, the search for a more realistic model to generate more effective therapies is essential. OBJECTIVE: To develop an experimental malignant astrocytoma model with the characteristics of the human tumor. METHOD: Primary cells from subcutaneous xenograft tumors produced with malignant astrocytoma U87MG cells were inoculated intracerebrally by stereotaxis into immunosuppressed (athymic) Rowett rats. RESULTS: All four injected animals developed non-infiltrative tumors, although other glioblastoma characteristics, such as necrosis, pseudopalisading cells and intense mitotic activity, were observed. CONCLUSION: A malignant astrocytoma intracerebral xenograft model with poorly invasive behavior was achieved in athymic Rowett rats. Tumor invasiveness in an experimental animal model may depend on a combination of several factors, including the cell line used to induce tumor formation, the rat strains and the status of the animal’s immune system.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1‐siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ‐treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

CD99 is upregulated in placenta and astrocytomas with a differential subcellular distribution according to the malignancy stage

In the present study, we searched for genes highly expressed in placenta and that could contribute to the establishment and maintenance of a malignant phenotype in different types of tumours, and in astrocytomas in particular. We employed a strategy based on the integration of in silico data from previously generated massively parallel signature sequencing and public serial analysis of gene expression databases. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both cell lines. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

Abstract 66: CD99 functional analysis in glioblastoma by RNAseq

CD99 is a membrane protein expressed in a wide variety of normal tissues and implicated in several cellular physiologic processes, including haematopoietic cell differentiation, proliferation, diapedesis, cell-cell adhesion, migration, apoptosis induction, cellular architecture maintenance and transmembrane protein transport. Additionally, aberrant CD99 expression has been associated with numerous malignancies, including astrocytomas. Amongst astrocytomas of different malignant grades, glioblastoma (GBM) is the most aggressive and common of central nervous system tumors. Despite current multimodal therapies, including surgery, chemotherapy and radiotherapy, the median survival duration of patients is 17 months. We have previously demonstrated increased CD99 expression at mRNA and protein levels in astroctyoma of all malignant grades, with GBM showing the highest levels. Knocking down CD99 expression by siRNA efficiently decreased CD99 at mRNA and protein levels and concurrently decreased the migratory property of two GBM cell lines, U87MG and A172. We subsequently analysed the transcriptoma of both cell lines by RNA-seq in Illumina platform after transfection with siRNA for CD99 and compared to non-target control (NTC). CD99 gene and protein silencing was confirmed in both cell lines. A total of 413 and 340 genes for U87MG and A172, respectively, with expression ≤1.3 (p Citation Format: Sueli M. Oba-Shinjo, Lais C. Cardoso, Roseli da Silva, Antonio M. Lerario, Miyuki Uno, Suely S.K. Marie. CD99 functional analysis in glioblastoma by RNAseq. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2015-66