Abstract
Simple SummaryIn the present study, we define the expression of 15 selected circular RNAs, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of tumors and consisting of three distinct groups according to the mesenchymal/epithelial phenotype. We identify circDOCK1-1 generated from the DOCK1 gene as an important regulator of the epithelial differentiation of mammary tumour cells. In addition, our high-throughput transcriptomic studies provide information on the genes that are regulated by circDOCK1-1, casting new light on the gene networks involved in the processes underlying the development of mammary tumours. Our results further indicate that circDOCK1-1 controls cell–cell adhesion and consequently random as well as directional motility. Besides their interest from a basic point of view, the data generated may be of translational relevance, as they suggest that strategies aimed at increasing the amounts or at stimulating the activity of circDOCK1-1 may be of significance for the treatment of a subset of triple-negative breast cancer patients.Circular RNAs are regulatory molecules involved in numerous cellular processes and may be involved in tumour growth and diffusion. Here, we define the expression of 15 selected circular RNAs, which may control the process of epithelial-to-mesenchymal transition, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of these tumours and consisting of three groups according to the mesenchymal/epithelial phenotype. A circular RNA from the DOCK1 gene (hsa_circ_0020397) shows low/undetectable levels in triple-negative mesenchymal cell lines, while its content is high in epithelial cell lines, independent of estrogen receptor or HER2 positivity. RNA-sequencing experiments performed on the triple-negative/mesenchymal MDA-MB-231 and MDA-MB-157 cell lines engineered to overexpress hsa_circ_0020397 demonstrate that the circRNA influences the expression of 110 common genes. Pathway analysis of these genes indicates that overexpression of the circular RNA differentiates the two mesenchymal cell lines along the epithelial pathway and increases cell-to-cell adhesion. This is accompanied by growth inhibition and a reduction in the random/directional motility of the cell lines. The upregulated AGR2, ENPP1, and PPP1R9A genes as well as the downregulated APOE, AQP3, CD99L2, and IGFBP4 genes show an opposite regulation by hsa_circ_0020397 silencing in luminal CAMA1 cells. The results provide novel insights into the role played by specific circular RNAs in the generation/progression of breast cancer.
Highlights
Circular RNAs are covalent circles of single-stranded RNA that are generated from non-canonical back-splicing of pre-mRNAs and non-coding RNAs [1,2,3]
We focus our interest on a small set of circular RNAs that may be involved in the process of epithelial-to-mesenchymal transition (EMT) [22] and we define their profile of expression in a relatively large panel of cell lines recapitulating the heterogeneity of the disease [23]
We investigated the epithelial/mesenchymal nature of the gene products commonly regulated by circDOCK1-1 in MDA-MB-231 and MDA-MB-157 cells, using the EMT gene signatures determined in breast cancer [30]. circDOCK1-1 caused a significant induction (Figure 3D) of various mRNAs coding for proteins annotated as epithelial (AGR2, ARAP2, CLDN4, KRT7, RAB11FIP2, and RBM47; see Supplementary Materials for the full name of the genes), whereas it downregulated different transcripts whose protein products are annotated as mesenchymal (CHRDL1, SDC2, CRISPLD2, and SPOCK1) (Figure 3D)
Summary
Circular RNAs are covalent circles of single-stranded RNA that are generated from non-canonical back-splicing of pre-mRNAs and non-coding RNAs [1,2,3]. There is emerging evidence indicating that one of the main mechanisms underlying the regulatory action of circular RNAs is related to the ability of these molecules to act as sponges and to sequester microRNAs [15]. Despite this background, there are relatively few studies focussing on the involvement of circular RNAs in the development and progression of solid tumours, with particular reference to breast cancer [15,16,17], a very heterogeneous type of tumour consisting of a large collection of different diseases [18,19,20,21]
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