The epigenetic landscape of miR-126 in breast cancer: implications of EBF1/ETS2/KLF2 transcription factor dysregulation

MicroRNAs (miRNAs) are short and single-stranded non-coding RNAs that act as post-transcriptional repressors of gene expression, thus playing a crucial role in both physiological and pathological conditions. miR-126 is a master regulator of angiogenesis and vascular integrity, whose deregulation has been observed in angiogenesis-related disorders, as well as in several other diseases. An oncosuppressive role of miR-126 has also been postulated since this miRNA is frequently lost in cancer and restoration of physiological levels represents an alluring perspective with therapeutic implications. In our laboratory, we have previously demonstrated that activated platelets release a huge amount of miR-126-enriched microvesicles, efficiently internalized by breast cancer (BC) cells, with notable anti-cancer effects. Based on this background, the first part of this PhD study focused on potential mechanisms underlying the positive effects of delivered miR-126. Obtained results suggested that platelets may represent a valid platform for the horizontal transfer of miRNA into BC cells, by specifically targeting components of the PI3K/AKT signalling cascade. The second part of the project concerned the deciphering of molecular mechanism(s) responsible for low miR-126 levels in BC. Ex-vivo studies on six human luminal BC biopsies and matched normal tissues confirmed miR-126 downregulation in tumour specimens; we also found a similar trend for its precursor pre-miR-126, as well as for the host gene EGFL7. These data, supported by bioinformatics, led to investigate potential transcriptional control of miR-126 expression. To this aim, we looked at the epigenetic profile of BC and normal cells, by examining genome-browser traces of two active (H3K4me3, H3K27ac) and repressive (H3K9me3 and H3K27me3) H3 modification ChIP-Seq data, encompassing the whole EGFL7/miR-126 gene. No evident changes were detectable in four identified promoters, indicating that deregulation of specific transcription factors may impact EGFL7/miR-126 expression. Therefore, we analysed genome binding/occupancy profiling of KLF2, EBF1, and ETS2, and we found two binding sites for KLF2 and EBF1 and four binding sites for ETS2. Silencing experiments carried out on primary endothelial HUVEC cells, which displayed complete open promoter configuration and high miR-126 expression, demonstrated the involvement of these three transcription factors in EGFL7/miR-126 expression. This finding was also confirmed, at least partially, by positive correlation between EBF1/ETS2 and EGFL7/miR-126 expression in our BC biopsies. Whole epigenetic profile (histone modifications, DNA methylation and chromatin state) of the three transcription factors effectively showed that EBF1 and ETS2 were in a closed repressive state in BC, while KLF2 showed an open chromatin state both in normal and tumour tissues, even if H3K36me3 (indicative of active transcription) was more widespread in healthy samples. In conclusion, this PhD investigation demonstrated that miR-126 was downregulated in BC, because of EBF1 and ETS2 (maybe also KLF2) epigenetic dysregulation. To the best of our knowledge, we demonstrated for the first time upstream event(s) responsible for miR-126 downregulation in cancer. Highlights • miR-126 is transcriptionally downregulated in breast cancer • EBF1, ETS2 and KLF2 control miR-126 expression • These transcription factors are epigenetically dysregulated in breast cancer • miR-126 levels can be restored by either epigenetic therapy or platelet-mediated delivery.