The oncogenic role of EPIC1MYC axis in breast cancer

更好地了解导致乳房侵袭性和不良结局的分子事件 癌症对于开发更有效的治疗这种致命疾病的方法是必不可少的。新出现的证据表明 IncRNAs在乳腺癌的发生发展中起着重要作用。然而，乳腺癌中的lncRNAs的研究是 它还处于初级阶段。通过对6475例肿瘤中lncRNA表观遗传格局的综合生物信息学分析 ，我们发现了一种新的基因间lncRNA，EPIC1(表观遗传诱导) InnCRNA1或ENSG00000224271)。EPIC1在B型乳癌中高表达 由于EPIC1基因启动子DNA甲基化缺失。我们继续展示了EPIC1促进 在细胞培养和小鼠异种移植模型中通过直接相互作用形成腔B乳腺癌 致癌转录因子MYC。在这个项目中，我们假设EPIC1是一个致癌的lncRNA。 乳腺癌，尤其是管腔B亚型。机制上，EPIC1增强转录活性 通过直接与MYC蛋白相互作用获得MYC。我们提出了三个具体目标来检验这一假设。在……里面 目的1，我们将用HITS-CLIP分析表征EPIC1-MYC的相互作用，并研究被鉴定的 EPIC1中的MYC结合序列是EPIC1-MYC相互作用的必要条件和充分条件。我们还将 确定负责EPIC1结合的MYC蛋白的功能结构域。在目标2中，我们将确定 EPIC1-MYC轴过表达wt-EPIC1或MYC结合在乳腺癌中的致癌作用 序列缺失突变体EPIC1(Δmyc-epic1)在ZR-75-1和BT-474细胞中的表达然后我们将确定 异种移植小鼠模型中EPIC1‘S致癌作用对myc蛋白的依赖性。最后，我们将调查 8个独立队列中EPIC1‘S与乳腺癌临床特征和预后的关系 其中包括1882例乳腺癌肿瘤。在目标3中，我们将首先确定MYC与其目标的结合模式 基因启动子在wt-或Δ-myc-epic1细胞中的高表达 MYC芯片-后续然后我们将确定EPIC1是否通过EPIC1-MYC结合来调节MYC靶标 用芯片聚合酶链式反应和荧光素酶报告基因分析。最后，我们将构建EPIC1-cMYC调控基因 整合CHIP-SEQ、RNA-SEQ和TCGA乳腺癌数据的乳腺癌网络。我们的研究将 揭示一种新的致癌lncRNA EPIC1在乳腺癌中的作用，通过它与 已确定的癌基因myc。

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.