P2.01-036 Identification of a Novel Oncogenic Ubiquitin Ligase from a Lung Cancer Epigenome-Wide Association Study (EWAS)

Abstract

Lung cancer (LC) is the leading cancer-related cause of death worldwide with a 5-year survival rate of only 8%. Smoking is the main risk factor for lung malignancies, but not every smoker develops LC. DNA methylation alterations in tumor tissue lead to genome instability and thus can contribute to malignant cell transformation. Smoking-associated blood DNA methylation changes have been shown to indicate LC risk. We aimed to investigate differential blood DNA methylation in healthy smokers for the identification of novel oncogenes. We performed Illumina 450K epigenome-wide DNA methylation arrays for 66 smoking prediagnostic lung cancer case-control pairs. The blood DNA samples for this nested case-control design came from the European Prospective Investigation into Cancer and Nutrition (EPIC) Heidelberg cohort. Differentially methylated candidate CpGs were then tested in lung tumor versus adjacent normal tissue for differential methylation in multiple patient sample sets. Additionally, we tested whether differential methylation leads to differential gene expression in lung tumor versus adjacent normal tissue. The top candidate CpG proximal gene was evaluated in functional LC cell based assays to investigate the consequences of its elevated expression in LC. The top differentially methylated candidate CpGs from EPIC were also found differentially methylated in lung tumor versus adjacent normal tissue. The two top hypermethylated CpGs were located within differentially methylated regions (DMRs) and the proximal or associated genes were differentially expressed in lung tumors. The top DMR revealed the regulation of gene expression by DNA methylation of a downstream ubiquitin E3 ligase. Deregulated expression of that gene was associated with LC cell proliferation, migration and glucose uptake in vitro. The gene was found to be involved in the activation of AKT by mTORC2. When the gene was knocked down, apoptotic genes which are suppressed by activated AKT in LC cells were re-expressed. Expression of a cell cycle promoting regulator stimulated by activated AKT was found repressed in LC knock down cells. We identified differential DNA methylation in blood from healthy smokers who later developed LC. The top differentially methylated CpGs were also differentially methylated in lung tumor versus adjacent normal tissue samples. The top DMR lead to expression of a proximal ubiquitin E3 ligase. We showed, that this gene is crucial for the mTORC2-mediated activation of AKT. This ubiquitin E3 ligase has not previously been associated with cancer but our findings identify it as a novel epigenetically deregulated LC oncogene.