title: Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome.
abstract: In light with the view that KEAP1 loss of function may impact tumour behavior and modify response to chemotherapeutical agents, we sought to determine whether KEAP1 gene is epigenetically regulated in malignant gliomas. We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Characteristics (ROC) curve analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman's Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearman's Rho -0.656 P=0.0001) and in a cell line before and after treatment with 5-azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results further suggest that KEAP1 expression is epigenetically regulated. In addition we demonstrated that KEAP1 is frequently methylated in malignant gliomas and a predictor of patient's outcome.
authors.affiliation: Laboratory of Oncology, IRCCS "Casa Sollievo della Sofferenza", San Giovanni Rotondo, Italy.
authors.forename: Lucia Anna
authors.initials: LA
authors.lastname: Muscarella
authors.forename: Raffaela
authors.initials: R
authors.lastname: Barbano
authors.forename: Vincenzo
authors.initials: V
authors.lastname: D'Angelo
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Copetti
authors.forename: Michelina
authors.initials: M
authors.lastname: Coco
authors.forename: Teresa
authors.initials: T
authors.lastname: Balsamo
authors.forename: Annamaria
authors.initials: A
authors.lastname: la Torre
authors.forename: Angelo
authors.initials: A
authors.lastname: Notarangelo
authors.forename: Michele
authors.initials: M
authors.lastname: Troiano
authors.forename: Salvatore
authors.initials: S
authors.lastname: Parisi
authors.forename: Nadia
authors.initials: N
authors.lastname: Icolaro
authors.forename: Domenico
authors.initials: D
authors.lastname: Catapano
authors.forename: Vanna Maria
authors.initials: VM
authors.lastname: Valori
authors.forename: Fabio
authors.initials: F
authors.lastname: Pellegrini
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Merla
authors.forename: Massimo
authors.initials: M
authors.lastname: Carella
authors.forename: Vito Michele
authors.initials: VM
authors.lastname: Fazio
authors.forename: Paola
authors.initials: P
authors.lastname: Parrella
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use; D019175:DNA Methylation; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000188:drug therapy / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000077204:Temozolomide; D016896:Treatment Outcome
chemical_list: D018906:Antineoplastic Agents, Alkylating; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D003606:Dacarbazine; D000077204:Temozolomide