The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down-regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n 5 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12a promoter but not of the AKAP12b promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5-aza-2 0 deoxycytidine drastically increased AKAP12a mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12a promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA-mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR-183 and miR-186 are up-regulated in CL and DN and are able to target AKAP12. Conclusion: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis. (HEPATOLOGY 2010;52:2023-2034 A kinase anchor proteins (AKAPs) are a diverse group of functionally related scaffolding proteins that target protein kinase A (PKA) and other enzymes, thereby coordinating a range of signaling events.1 Human AKAP12 (synonymous: Gravin/ AKAP250) is a large protein up-regulated in contactinhibited cells and down-regulated by Src, Ras, and PKC.2 Interestingly, AKAP12 is able to modulate both protein kinase A and C, indicating that this protein is involved in the regulation of several signaling pathways. Other effects of AKAP12 are direct sequestration of cyclin D1, inhibition of ERK2 activation, and actin cytoskeleton interaction.3 Cyclin D1 overexpression is a frequent event in hepatocarcinogenesis and has been Abbreviations: aCGH, array-based comparative genomic hybridization; AKAP12, a kinase anchor protein 12; 5-aza-dC, 5-aza-2 0

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change 6 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n 5 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p 5 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n 5 89), and was independent of gender, performance (KPS) and MGMT status (p 5 0.0485, HR 5 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs.Glioblastoma multiforme (GBM) still constitutes one of the most aggressive and deadly human cancers with a median overall survival (OS) of about 15 months.1,2 Despite this unfavorable prognosis, 16% of GBM patients survive for >36 months.3 These long-term survivors (LTS) usually are younger in age and were shown to have a higher preoperative Karnofsky performance score (KPS). [4][5][6] Recently, a considerable fraction of GBM LTS (34%) were found to harbor IDH1 mutations. 7 Concerted efforts in deciphering the biology of IDH1-mutant GBM have led to the conclusion that these tumors constitute a subgroup differing significantly from IDH1 wild-type GBMs. [8][9][10][11] Gain-of-function mutations