The oncogenic role of EPIC1MYC axis in breast cancer

更好地了解导致乳腺癌侵袭性和不良结局的分子事件 癌症对于开发针对这种致命疾病的更有效疗法至关重要。新出现的证据表明 lncRNA在乳腺癌的发生发展中起重要作用。然而，lncRNAs在乳腺癌中的研究 还处于初期阶段通过对6475肿瘤中lncRNA表观遗传景观的综合生物信息学分析， 通过对781个癌细胞株和100个癌细胞株的研究，我们发现了一种新的基因间lncRNA，EPIC 1（EPigenetically Induced InCRNA 1或ENSG 00000224271）。EPIC 1在管腔型B乳腺癌亚型中显著过表达 这是由于EPIC 1基因启动子处的DNA甲基化缺失所致。我们继续证明EPIC 1促进了 在细胞培养物和小鼠异种移植物模型中通过与 致癌转录因子MYC。在这个项目中，我们假设EPIC 1是一个致癌的lncRNA， 乳腺癌，特别是管腔型B亚型。EPIC 1在机制上增强了转录活性， 通过与MYC蛋白直接相互作用来抑制MYC。我们提出了三个具体目标来检验这一假设。在 目的1，我们将使用HITS-CLIP测定来表征EPIC 1-MYC相互作用，并研究是否鉴定出了EPIC 1-MYC相互作用。 EPIC 1中的MYC结合序列对于EPIC 1-MYC相互作用是必需的且充分的。我们还将 确定负责EPIC 1结合的MYC蛋白的功能结构域。在目标2中，我们将确定 EPIC 1-MYC轴在乳腺癌中的致癌作用通过过度表达wt-EPIC 1或MYC结合 序列缺失突变体EPIC 1（Δ MYC-EPIC 1）在ZR-75-1和BT-474细胞中的表达。然后我们将确定 在异种移植小鼠模型中EPIC 1的致癌作用对MYC蛋白的依赖性。最后，我们将调查 EPIC 1与8个独立患者队列中乳腺癌临床特征和结局的相关性 包括1882例乳腺癌肿瘤。在目标3中，我们将首先确定MYC与其靶标结合的模式 在野生型或Δ MYC-EPIC 1过表达ZR-75-1和BT-474细胞中的基因启动子， MYC ChIP序列然后，我们将确定EPIC 1是否通过EPIC 1-MYC结合调节MYC靶点 使用ChIP-PCR和荧光素酶报告基因分析的位点。最后，我们将构建EPIC 1-cMYC调控基因， 通过整合ChIP-seq，RNA-seq和TCGA乳腺癌数据，在乳腺癌中建立网络。我们的研究将 揭示了一种新的致癌lncRNA，EPIC 1，在乳腺癌中的作用，通过其功能的串扰与乳腺癌。 明确的致癌基因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.