Annie-Claire Diserens

Frequent Co-Alterations of TP53, p16/CDKN2A, p14ARF, PTEN Tumor Suppressor Genes in Human Glioma Cell Lines.

In this study we established the simultaneous status of TP53, p16, p14ARF and PTEN tumor suppressor genes in 34 randomly chosen human glioma cell lines. Nine cell lines (26.4%) harbored mutations or deletions in all four tumor suppressor genes and 22 cell lines (64%) had alterations in at least three. Mutations/deletions were found at the following frequencies: TP53 (76.5%), p14ARF (64.7%), p16 (64,7%), PTEN (73.5%). Thus, there was a high incidence of alterations in the cellular pathways involving the p53 transcription factor (94.1%), the retinoblastoma protein (64.7%) and the PTEN phosphatase (73.5%) and 91% of cell lines carried mutations in two or more pathways. This provides the first clear genetic evidence that these tumor suppressors participate in biological pathways which are functioning separately/independently in glioma cells. The status of the gene alterations did not correlate with tumorigenicity in immunocompromized mice or any clinical parameters. Although the mutation rate was higher in glioma cell lines than that reported for glioma tissues, the alterations were molecularly representative of those found in adult de novo glioblastoma. This study highlights the importance of developing therapeutic approaches applicable to tumors with a broad range of genetic alterations and also provides an invaluable panel of glioma cell lines to make this possible.

Anti-O6-methylguanine-methyltransferase (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as clinical biomarker.

Silencing of O6‐methylguanine‐DNA methyltransferase (MGMT) protein expression because of MGMT gene promoter hypermethylation is considered to be associated with postoperative chemoradiotherapy benefits in glioblastoma multiforme (GBM) patients. The objective of this study was to clarify the usability of MGMT immunohistochemistry (IHC) as a clinical biomarker.

Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohens kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

Regulation of interleukin-8 expression by reduced oxygen pressure in human glioblastoma.

Oxygen deprivation is an important biological feature of tumor growth. We previously showed that in glioma, anoxia increases expression of IL-8, a chemokine and angiogenic factor. Here, we analysed for the first time the biochemical mechanisms inducing the IL-8 gene upon anoxia in glioma cells, and showed that they differ from those inducing the VEGF gene. Both genes are induced in biologically and genetically heterogenous glioblastoma cell lines (LN-229, LN-Z308, U87MG, T98G), whereas, in gliosarcoma cells (D247MG), only the VEGF gene is induced. The kinetics of IL-8 and VEGF mRNA inductions differ in these cells and reoxygenation experiments showed that the induction is due to the anoxic stress per se. Furthermore, in LN-229 and LN-Z308 cell lines actinomycin D, DRB and nuclear run-on experiments showed that anoxia stimulates increased transcription of both genes. Electromobility shift assays show increased protein binding to the AP-1 site on the IL-8 promoter following anoxia treatment. Finally, in situ hybridization on glioblastoma sections shows that the in vivo expression patterns of IL-8 and VEGF genes overlap, but are not identical. Since intratumoral augmentation of IL-8 and VEGF secretion, following microenvironmental decreases in oxygen pressure, may promote angiogenesis, further definition of these pathways is essential to appropriately target them for antitumoral therapy.

Pathway Analysis of Glioblastoma Tissue after Preoperative Treatment with the EGFR Tyrosine Kinase Inhibitor Gefitinib—A Phase II Trial

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation. Mol Cancer Ther; 10(6); 1102–12. ©2011 AACR.

The Endothelin System in Human Glioblastoma

Endothelin-1 (ET-1) is a powerful mitogenic and/or anti-apoptotic peptide produced by many cancer cells. To evaluate the potential role of the endothelin system in glioblastoma we first determined the cellular distribution of the mRNA and proteins of the components of the endothelin system, preproendothelin-1 (PPET-1), endothelin-converting enzyme-1 (ECE-1), and ETA and ETB receptors in human glioblastoma tissue and glioblastoma cell lines. PPET-1, ECE-1, and ETA receptor were highly expressed in glioblastoma vessels and in some scattered glioblastoma areas whereas ETB receptor was mainly found in cancer cells. This suggests that glioblastoma vessels constitute an important source of ET-1 that acts on cancer cells via the ETB receptor. Four human glioblastoma cell lines expressed mRNA for all of the components of the ET-1 pathway. Bosentan, a mixed ETA and ETB receptor antagonist, induced apoptosis in these cell lines in a dose-dependent manner. Apoptosis was potentiated by Fas Ligand (APO-1L, CD95L), a pro-apoptotic peptide, only in LNZ308 cells, corresponding to the known functional Fas expression in these cell lines. LNZ308 cells also expressed the long and short forms of the cellular FLICE/caspase-8 inhibitory protein (FLIP). Bosentan and a protein kinase C inhibitor down-regulated short FLIP in these cells. ET-1 induced transient phosphorylation of extracellular signal-regulated kinase but did not induce long-term thymidine incorporation in LNZ308 glioblastoma cells. These results suggest that, in glioblastoma cells, ET-1, mainly acting via the ETB receptor, is a survival/anti-apoptotic factor produced by tumor vasculature, but not a proliferation factor, involving protein kinase C and extracellular signal-regulated kinase pathways, and stabilization of the short form of FLIP.

Characterization of an established human malignant glioma cell line: LN-18

SummaryA human malignant glioma cell line, LN-18, has been established in monolayer culture and subcultured for more than 115 passages. LN-18 cells grow in vitro as bipolar or stellate cells with pleomorphic nuclei, have a doubling time of about 72 h and a plating efficiency of 3%. The glial nature of these cells has been assessed by ultrastructural examination. The synthesis of glial fibrillary acidic and S-100 proteins could not be demonstrated, although the initial biopsy tissue and the early cultures were positive for the former. The presence of Ia-like antigens on the surface of these cells was demonstrated using allo and xeno antisera. LN-18 cells were also shown to synthesize large quantities of fibronectin. The injection of LN-18 cells into nude mice induced the formation of solid tumor masses that could be retransplanted every 3 weeks and showed a morphology comparable to that of the initial biopsy. Karyotype analysis revealed the presence of three marker chromosomes, constantly present before and after hetero-transplantation.

Extent and Patterns of MGMT Promoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.

In vitro prostaglandin E2 production by glioblastoma cells and its effect on interleukin-2 activation of oncolytic lymphocytes.

SummaryGlioblastoma cells constitutively produce various amounts of PGEZ (prostaglandin E2)in vitro. The amounts of PGE2 found in the conditioned medium of the glioblastoma cultures (less than 5 ng/ml) were not enough to inhibit the IL-2 (interleukin-2) activation of peripheral blood lymphocytes. However the amount of PGE2 produced by approximately 1 x 107 of the glioblastoma cells can be assumed to suppress the generation of IL-2-induced killing activity against glioblastoma cells.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.