Abstract
The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p < 0.001). The CNV assay was validated investigating HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.
Highlights
Glioblastoma multiforme (GBM) is the highest grade glioma, according to World Health Organization classification, and has an annual incidence of 5 cases per 100,000 people [1,2]
To differentiate epidermal growth factor receptor gene (EGFR) amplification from polisomy of chromosome 7 we considered the ratio between copy number variation (CNV) of EGFR and HGF, both located on chromosome 7, normalised to the reference gene TERT
Functional validation of NFKBIA heterozygous deletion To validate further CNV data, we investigated the functional consequences of NFKBIA deletion
Summary
Glioblastoma multiforme (GBM) is the highest grade glioma, according to World Health Organization classification, and has an annual incidence of 5 cases per 100,000 people [1,2]. A huge effort was made to achieve a more thorough characterization of genetic and molecular signatures of GBM, facilitating the identification of new molecular targets and leading to a classification in four molecular subtypes: classical, mesenchymal, proneural and neural [3,4]. The classical subtype is mostly characterized by loss of chromosome 10 and amplification of the epidermal growth factor receptor gene (EGFR). NF-kB complexes are maintained inactive in the cytoplasm through interaction with their inhibitor IkBα, encoded by the NFKBIA gene located on chromosome 14q13.2. Most stimuli activate this pathway through phosphorylation of the IKK complex, which in turn phosphorylates IkBα, leading the inhibitor to degradation and allowing nuclear translocation of NF-kB. Two pathways of NF-kB activation have been described: canonical and non-canonical, involving different types of kinases (STAT3, PI3K/Akt, MAPK) and distinct heterodimers (p65/p50; p100/RelB) [10]
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