Abstract 2349: An RNA-based epigenetic network controls the expression of E-cadherin in epithelial normal and cancer cells

Abstract

Abstract Epigenetic mechanisms play important roles in many human diseases. However, what drives epigenetic regulators to specific genomic locations is still an open question. Promoter-associated long non-coding RNAs (paRNAs) have been identified in many genes and have been proposed to act as docking elements for recruitment of epigenetic regulators to gene promoters, although the underlying mechanisms are still poorly characterized. In this study we investigated the role of paRNAs in transcriptional regulation of E-cadherin (CDH1), a trans-membrane glycoprotein and an important determinant of epithelial cell differentiation. Transcriptional silencing of E-cadherin is frequent in epithelial cancers and loss of E-cadherin expression triggers epithelial-to-mesenchymal transition (EMT) and acquisition of tumor-initiating properties. We found that bidirectional transcription occurred from independent initiation sites in the E-cadherin promoter and generated sense (S) and antisense (AS) paRNAs that coordinated the transcriptional activity of the gene. S and AS paRNAs had distinct expression patterns in normal and cancer cell lines and human prostate tumors. Both in cancer cell lines and human tumors the prevalence of S paRNAs and low AS/S paRNA ratio were associated with low E-cadherin expression. We found that the S paRNA bound Argonaute 1 (AGO1) and coordinated silencing of the gene by recruiting, along with AGO1, the histone methyltransferase SUV39H1 to the promoter. Consistently, knockdown of either S paRNA or AGO1 reduced SUV39H1 promoter occupancy and reactivated E-cadherin transcription in low expressing cells. Using promoter reporter and expression constructs we showed that the S paRNA and AGO1 acted in cis to control promoter activity and that the interaction with AGO1 required specific element of the S paRNA. Furthermore, recruitment of AGO1 to the S paRNA and CDH1 promoter depended on an isomiR derived through alternative processing and editing of pre-miR-4534. Accordingly, mutations that disrupted the isomiR binding sequence in the S paRNA reduced AGO1 binding and increased promoter activity, while depletion of the isomiR induced CDH1 expression. Notably, the novel isomiR was more abundant in transformed epithelial cells and cancer cell lines than normal prostate epithelial cells, accumulated preferentially in nuclei and was specifically associated with S paRNA and chromatin-bound AGO1 in low CDH1 expressing cancer cells. This study reveals a complex RNA-based epigenetic network that relies on sequence-specific interactions between a paRNA, a small RNA and AGO1 and coordinates transcriptional silencing of a critical gene involved in tumor development and progression. Our findings give also a new prospective and mechanistic insights on the interplay between epigenetic regulatory factors indentifying paRNAs as relevant elements in these processes and potential targets for gene modulation strategies. Citation Format: Sara Napoli, Giuseppina Pisignano, Ramon Garcia-Escudero, Giuseppina Carbone, Carlo V. Catapano. An RNA-based epigenetic network controls the expression of E-cadherin in epithelial normal and cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2014-2349