A better understanding of the molecular events that contribute to the aggressiveness and poor outcome of breast cancer is essential to develop more effective therapies for this deadly disease. Emerging evidence suggests that lncRNAs play important roles in breast cancer tumorigenesis. However, research of lncRNAs in breast cancer is still in its infancy. Through an integrated bioinformatic analysis of the lncRNA epigenetic landscape in 6475 tumor samples and 781 cancer cell lines, we discovered a novel intergenic lncRNA, EPIC1 (EPigenetically Induced lnCRNA1, or ENSG00000224271). EPIC1 is significantly overexpressed in the luminal B breast cancer subtype due to the loss of DNA methylation at the EPIC1 gene promoter. We went on to show that EPIC1 promotes luminal B breast cancer tumorigenesis in cell cultures and in mouse xenograft models by directly interacting with the oncogenic transcription factor MYC. In this project, we hypothesize that EPIC1 is an oncogenic lncRNA in breast cancer, particularly in the luminal B subtype. Mechanistically, EPIC1 enhances the transcriptional activity of MYC by directly interacting with the MYC protein. We propose three specific aims to test this hypothesis. In Aim 1, we will characterize the EPIC1-MYC interaction using HITS-CLIP assay and investigate if the identified MYC binding sequences in EPIC1 are necessary and sufficient for EPIC1-MYC interaction. We will also determine the functional domain of MYC protein responsible for the EPIC1 binding. In Aim 2, we will determine the oncogenic role of the EPIC1-MYC axis in breast cancer by overexpressing wt-EPIC1 or MYC binding sequence deletion mutant EPIC1 (ΔMYC-EPIC1) in ZR-75-1 and BT-474 cells. We will then determine the dependency of EPIC1’s oncogenic role on MYC protein in xenograft mouse models. Finally, we will investigate EPIC1’s association with breast cancer clinical characteristics and outcomes in eight independent patient cohorts including 1882 breast cancer tumors. In Aim 3, we will first determine the pattern of MYC binding to its target gene promoters in wt- or ΔMYC-EPIC1 overexpressing ZR-75-1 and BT-474 cells by performing RNA-seq and MYC ChIP-seq. We will then determine whether EPIC1 regulates the MYC targets through EPIC1-MYC binding sites using ChIP-PCR and luciferase reporter analyses. Finally, we will construct the EPIC1-cMYC regulatory network in breast cancer by integrating ChIP-seq, RNA-seq, and TCGA breast cancer data. Our study will uncover the role of a novel oncogenic lncRNA, EPIC1, in breast cancer through its functional crosstalk with the well-established oncogene MYC.

Project narrative The goal of this project is to understand the mechanism by which EPIC1-MYC axis regulates the aggressiveness and poor outcomes of breast cancer. Our preliminary data has shown that in breast cancer cells and mouse model, EPIC1 promotes breast cancer tumorigenesis by directly interacting with the oncogenic transcriptional factor MYC. In this project, we hypothesize that EPIC1 is an oncogenic lncRNA in breast cancer. Mechanistically, EPIC1 enhances the transcriptional activity of MYC by directly interacting with MYC protein. We propose three specific aims to test this hypothesis: (1) we will characterize the EPIC1-MYC interaction; (2) we will determine the oncogenic role of the EPIC1-MYC axis in breast cancer in vitro, in vivo, and in patients; (3) we will determine the mechanism by which EPIC1 dictates MYC-mediated gene regulation. This project will uncover a novel mechanism by which MYC regulates specific gene expression and will pave the way for developing new cancer therapies that target MYC through its interaction with EPIC1.