Association between epidermal growth factor receptor amplification and ADP-ribosylation factor 1 methylation in human glioblastoma.

PURPOSE: Glioblastoma (GB) is the most frequent and most malignant primary brain tumor in adults. Previously, it has been found that both genetic and epigenetic factors may play critical roles in its etiology and prognosis. In addition, it has been found that the epidermal growth factor receptor gene (EGFR) is frequently over-expressed and amplified in primary GBs. Here, we assessed the promoter methylation status of 10 genes relevant to GB and explored associations between these findings and the EGFR gene amplification status.

METHODS: Tumor samples were obtained from 36 patients with primary GBs. In addition, 6 control specimens were included from patients who were operated for diseases other than brain tumors. The amplification status of the EGFR gene, and its deletion mutant EGFRvIII, were evaluated using FISH and MLPA, respectively. The IDH1/2 gene mutation status was verified using Sanger sequencing. A commercial DNA methylation kit was used to assess the promoter methylation status of 10 pre-selected genes. Metabolic profiles were measured using HR-MAS NMR spectroscopy. The EGFR and ARF1 mRNA expression levels were quantified using qRT-PCR.

RESULTS: Of the 10 genes analyzed, we found that only ARF1 promoter hypermethylation was significantly associated with EGFR gene amplification. ARF1 is a GTPase that is involved in vesicle trafficking and the Golgi apparatus. Subsequent tumor metabolism measurements revealed a positive association between EGFR amplification and different membrane precursors and methyl-donor metabolites. Finally, we found that EGFR gene amplifications were associated with distinct tumor infiltration patterns, thus representing a putative novel functional association between EGFR gene amplification and ARF1 gene promoter methylation in GB.

CONCLUSIONS: The results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism(s) underlying these connections and their functional consequences remain to be established.