Abstract B08: Expression of noncoding RNAs on the DLK1-DIO1 locus in EMT and breast cancer
Authors: Hilmarsdottir Bylgja , Briem Eirikur , Bergthorsson Jon Thor , Gudjonsson Thorarinn , Magnusson Magnus Karl 
Published: May 2016

Abstract
The DLK1-DIO3 locus contains three paternally expressed protein-coding genes and maternally expressed non-protein coding genes, including the lncRNA MEG3 and a cluster of 54 miRNA, making it one of the largest miRNA clusters in the genome. A recent study showed that the degree of activation of the maternally inherited homolog of this region was correlated with the pluripotency of stem cells. Also, the maternal ncRNAs from the DLK1-DIO3 locus are amongst the few regions in the transcriptome able to discriminate between induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), thereby linking this locus to the maintenance of stem cell character. In humans DLK1-DIO3-derived ncRNAs have been linked to both progression and inhibition of cancer.
The objective of this study was to analyze the expression profile of the DLK1-DIO3-derived ncRNAs and in particular the lncRNA MEG3 in breast epithelial progenitor cell lines (D492 and HMLE) with high degree of plasticity towards epithelial to mesenchymal transition (EMT) and cancer progression. D492M and HMLEmes are mesenchymal derivatives of D492 and HMLE, respectively. Total RNA and small RNA sequencing revealed marked upregulation of the maternally expressed lncRNAs and the miRNAs on the DLK1-DIO3 locus in D492M compared to D492. This miRNA-cluster along with the lncRNA MEG3 is expressed from the maternal inherited homolog. Allele specific expression analysis shows that the increased expression of MEG3 in D492M is not caused by loss of imprinting but increased expression of the already expressed allele. These ncRNAs are also upregulated in HMLEmes compared to HMLE, showing that these effects are not cell line specific. qPCR analysis of primary tissue shows that MEG3 expression is 8-15 fold higher in stromal cells than epithelial cells of the breast. These results indicate that the MEG3 upregulation and/or the maternally expressed miRNA cluster could be critical regulators/effectors in the EMT/mesenchymal phenotype. Data from the The Cancer Genome Atlas consistently shows positive correlation between the expression of Meg3 and the miRNAs located at the DLK1-DIO3 genomic region. We have characterized the main splice-forms of the MEG3 gene, expressed in D492 and D492M. Out of the 12 different splice-forms, splice-form 1 (NR_002766) was mostly responsible for increased expression in D492M. Since EMT has been linked to metastatic behavior of epithelial-originating cancers we measured the expression of MEG3 in a series of clinically well-defined breast tumors (N= 125). Survival analysis showed lower overall survival in patients with high MEG3 expression (P=0.01). Comparison with expression and survival data from the online GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database confirmed our observed negative relationship between MEG3 expression and survival which was particularly evident in luminal B type tumors. MEG3 is also negatively correlated with distant metastasis-free survival of poorly differentiated (grade 3) tumors, suggesting that MEG3 expression may be a valid marker of EMT during tumorigenesis.
Conclusions: ncRNAs are increasingly seen as intrinsic regulators of stemness, tissue morphogenesis and cancer progression. Our data indicates that the maternally expressed ncRNAs at the DLK1-DIO3 region could be regulators of EMT in breast cancer. Future works will aim at understanding the biological role of MEG3 and the miRNA-cluster in these processes.
Citation Format: Bylgja Hilmarsdottir, Eirikur Briem, Jon Thor Bergthorsson, Thorarinn Gudjonsson, Magnus Karl Magnusson. Expression of noncoding RNAs on the DLK1-DIO1 locus in EMT and breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B08.