EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway.

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPß. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

Genetic heterogeneity in supratentorial and infratentorial primitive neuroectodermal tumours of the central nervous system.

AIMS: Medulloblastoma (MB), a kind of infratentorial primitive neuroectodermal tumour (PNET), is the most frequent malignant brain tumour in childhood. In contrast, supratentorial PNET (sPNET) are very infrequent tumours, but they are histologically similar to MB, although they present a worse clinical outcome. We investigated the differences in genetic abnormalities between sPNET and MB. METHODS AND RESULTS: We analysed 20 central PNET (14 MB and six sPNET) by conventional comparative genomic hybridization (CGH) in order to determine whether a different genetic profile for each tumour exists. Isochromosome 17q was detected in four of the 14 MB cases, but not in any sPNET. Gains at 17q and 7 happened more frequently in MB, and those at 1q in sPNET. Losses at chromosome 10 were detected only in MB, while losses at 16p and 19p happened more frequently in sPNET. A new amplification site, on 4q12, was detected in two MB. CONCLUSIONS: Central PNET are a heterogeneous group of tumours from the genetic point of view. The present and previous data, together with further results from larger series, might contribute to the establishment of specific treatments for supratentorial and infratentorial PNET.

Improvement of the methylation specific PCR technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA.

Methylation specific PCR (MSP) is a technique that enables the detection of hypermethylation at a CpG island. The objective of this study is the introduction of small modifications to the MSP technique to make it more suitable for the study of promoter hypermethylation at tumor suppressor genes whenever there is a shortage of material available for study. This commonly happens in the case of using archival material from the Pathology departments. Tumor DNA was extracted from a collection of 40 fresh-frozen soft tissue sarcomas and 19 paraffin-embedded PNETs (primitive neuroectodermal tumors). The MSP technique was performed to detect hypermethylation at the p16 promoter. Also, blood genomic DNA was mixed with herry sperm genomic DNA as a carrier, in nine different combinations, in order to test for the best conditions that could produce MSP bands even when low amounts of genomic tumor DNA is available for study. We demonstrate the benefit of using herry sperm carrier DNA up to 10 microg together with small quantities of tumor DNA. This result will facilitate the incorporation of paraffin-embedded samples for study of promoter hypermethylation at tumor suppressor genes. Other technical conditions for the MSP technique are also studied.

[Molecular genetics of astrocytomas]. / Genética molecular de los astrocitomas.

Astrocytomas are the most frequent group of intracranial tumors. Among them, glioblastoma is the most aggressive one. In this review we will describe the most common genetic abnormalities found out in astrocytomas. We will refer to the epidermal growth factor receptor (EGFR), p53, p16, PTEN and DMBT1 genes. We will also present certain genetic aspects that influence the progression to glioblastoma.