PO-388 Epigenetic silencing of ZNF177 by MIR-877–3 p could be involved in cervical cancer progression

Abstract

Introduction Breast cancer (BC) is the most frequent neoplasia among women and one of the leading causes of cancer-related death worldwide. It is a clinically heterogeneous disease, with the triple negative (TN) as the most aggressive subtype. Epigenetic alterations are common molecular abnormalities in cancer, including changes in DNA methylation, microRNA profiling and post-translational modifications of histones. Aberrant DNA methylation has been recognised as one of the most common epigenetic modifications in BC. Methylation of certain genes is associated with pathological features, differential response to therapy or metastases during follow-up, being also a predictor of survival in BC; but little is known about its involvement in TN BC. Our objective was to identify the methylome of TN BC compared to that of non-neoplastic mammary tissue. Material and methods A 450k-methylation array was performed to identify differentially methylated genes in 8 TN BC samples (T) and 5 non-neoplastic (N) tissues from reduction mammoplasties. For that, DNA was extracted, bisulphite-converted, whole-genome amplified and hybridised to Infinium HumanMethylation450 BeadChip array. The level of DNA methylation at each CpG locus (b-value) was calculated as M/(M+U), ranging from 0 (100% unmethylated, U) to 1 (100% methylated, M). Differentially expressed probes were obtained by limma t-test (False Discovery Rate<0.05). Those probes with delta b-value (Db) >0.2 and located in CpG islands in the 5´UTR, 1500-200 bp upstream the transcription start site or in the first exon, were considered. ADAM12, VWCE and TSPAN9 genes were selected for further validation by pyrosequencing in a series of 30 T and 20 n, using a PyroMark Q24 after PCR amplification.Protein expression was explored by immunohistochemistry. Results and discussions A total of 700 hypermethylated and 349 hypomethylated probes in T compared to N, corresponding to 15 and 7 hyper-and hypomethylated genes, respectively, were found to fulfil our criteria. ADAM12, VWCE and TSPAN9 genes showed lower methylation levels in T than in N (p<0.05). The increased protein expression in T compared to N was confirmed, highlighting the causal role of the hypomethylation in the regulation of the expression of these genes. No association was found between these alterations and clinicopathological variables. Conclusion This is the first description of ADAM12, VWCE and TSPAN9 hypomethylation and overexpression in TN BC compared to non-neoplastic mammary tissues.