Faculty Bibliography

Our current knowledge of the deregulation that occurs during the onset and progression of cancer and other diseases leads us to recognize both genetic and epigenetic alterations as being at the core of the pathological state. The epigenetic landscape includes a variety of covalent modifications that affect the methylation status of DNA but also the post-translational modifications of histones, and determines the structural features of chromatin that ultimately control the transcriptional outcome of the cell to accommodate developmental, proliferative or environmental requirements. MicroRNAs are small non-coding RNAs that regulate the expression of complementary messenger RNAs and function as key controllers in a myriad of cellular processes, including proliferation, differentiation and apoptosis. In the last few years, increasing evidence has indicated that a substantial number of microRNA genes are subjected to epigenetic alterations, resulting in aberrant patterns of expression upon the occurrence of cancer. In this review we discuss microRNA genes that are epigenetically modified in cancer cells, and the role that microRNAs themselves can have as chromatin modifiers.

Two approaches for the evaluation of the relative degree of global DNA methylation through the quantification of 2' deoxynucleosides are described. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA is performed using both high-performance capillary electrophoresis (HPCE) with UV-Vis detection or liquid chromatography with electrospray ionization mass spectrometric detection (LC-ESI/MS). Treatment of genomic DNA with a ribonuclease and generation of nucleosides through enzymatic hydrolysis notably increases the specificity of both techniques. Both approaches have been demonstrated to be highly specific and sensitive, being useful for the rapid quantification of the degree of global DNA methylation and its exploitation for the analysis of poorly purified and/or concentrated DNA samples, such as tumor biopsies.

INTRODUCTION/BACKGROUND:Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer. METHODS:EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome. RESULTS:Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate (P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA re-expression in vitro. Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression (P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation. CONCLUSIONS:EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland.