Expression of ncRNAs on the DLK1-DIO3 Locus Is Associated With Basal and Mesenchymal Phenotype in Breast Epithelial Progenitor Cells.

Zuzana Budkova,Snævar Sigurdsson,Bylgja Hilmarsdottir,Magnus Karl Magnusson,Thorarinn Gudjonsson,Anna Karen Sigurdardottir,Jon Thor Bergthorsson,Eirikur Briem,Gunnhildur Asta Traustadottir

doi:10.3389/fcell.2020.00461

Abstract

Epithelial-to-mesenchymal transition (EMT) and its reversed process mesenchymal-to-epithelial transition (MET) play a critical role in epithelial plasticity during development and cancer progression. Among important regulators of these cellular processes are non-coding RNAs (ncRNAs). The imprinted DLK1-DIO3 locus, containing numerous maternally expressed ncRNAs including the lncRNA maternally expressed gene 3 (MEG3) and a cluster of over 50 miRNAs, has been shown to be a modulator of stemness in embryonic stem cells and in cancer progression, potentially through the tumor suppressor role of MEG3. In this study we analyzed the expression pattern and functional role of ncRNAs from the DLK1-DIO3 locus in epithelial plasticity of the breast. We studied their expression in various cell types of breast tissue and revisit the role of the locus in EMT/MET using a breast epithelial progenitor cell line (D492) and its isogenic mesenchymal derivative (D492M). Marked upregulation of ncRNAs from the DLK1-DIO3 locus was seen after EMT induction in two cell line models of EMT. In addition, the expression of MEG3 and the maternally expressed ncRNAs was higher in stromal cells compared to epithelial cell types in primary breast tissue. We also show that expression of MEG3 is concomitant with the expression of the ncRNAs from the DLK1-DIO3 locus and its expression is therefore likely indicative of activation of all ncRNAs at the locus. MEG3 expression is correlated with stromal markers in normal tissue and breast cancer tissue and negatively correlated with the survival of breast cancer patients in two different cohorts. Overexpression of MEG3 using CRISPR activation in a breast epithelial cell line induced partial EMT and enriched for a basal-like phenotype. Conversely, knock down of MEG3 using CRISPR inhibition in a mesenchymal cell line reduced the mesenchymal and basal-like phenotype of the cell line. In summary our study shows that maternally expressed ncRNAs are markers of EMT and suggests that MEG3 is a novel regulator of EMT/MET in breast tissue. Nevertheless, further studies are needed to fully dissect the molecular pathways influenced by non-coding RNAs at the DLK1-DIO3 locus in breast tissue.

Highlights

Breast cancer is the most common cancer in women and the second most common cancer overall (Ghoncheh et al, 2016)
We show that ncRNAs from the DLK1-DIO3 locus are highly expressed in stromal/mesenchymal cells in the breast and positively correlate with the expression of Epithelial-to-mesenchymal transition (EMT) genes in breast tissue
We demonstrate that enhanced maternally expressed gene 3 (MEG3) expression accompanied by increased expression of the ncRNAs at the DLK1-DIO3 locus, contributes to partial EMT more correctly referred to as epithelial plasticity, seen by increased expression of EMT related TFs, increase of basal/mesenchymal markers and enhanced properties such as migration, resistance to apoptosis and clonogenic capacity

Summary

Introduction

Breast cancer is the most common cancer in women and the second most common cancer overall (Ghoncheh et al, 2016). Cells undergoing EMT, acquire increased migration and invasive properties and show increased resistance to apoptosis (Robson et al, 2006; Cao et al, 2016). Through these processes, EMT is considered a major mediator of phenotypic plasticity in cancer cells, metastatic formation and drug resistance (Mani et al, 2008; Scheel and Weinberg, 2012; Ansieau, 2013; Nieto et al, 2016; Lu and Kang, 2019). This, is debated and further studies will increase our knowledge of the role of EMT/MET in cancer progression and metastasis

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