食管鳞状细胞癌中超级增强子的研究

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in the world. Although we and others have demonstrated the genomic aberrations in ESCC, little is known regarding the mechanisms controlling oncogenic gene expression program, which hampers the development of novel therapeutic targets for treating this malignancy. Super-enhancers (SE) have recently been discovered as a class of regulatory regions with unusually strong enrichment for the binding of transcriptional factors and co-activators. Interestingly, studies have shown that in several cancer cells, SEs are enriched at genes with known oncogenic function. For example, SEs are found within the MYC locus in several types of cancers. .We hypothesize that characterization of SEs will yield novel insights into the mechanisms controlling aberrant transcription in ESCC. In the preliminary experiments, we annotated 857 and 444 SEs through Chip-seq analysis (using H3K27ac antibody) in two ESCC cells, respectively. To identify SE-driven oncogenes which are highly addicted to continuous transcriptional activation, we performed RNA-seq analysis upon partial inhibition of the transcription-initiation machinery through applying low-dose CDK7 inhibitor, which has been shown to affect particularly SE-driven transcripts. Notably, our complementary profiling approaches revealed many well-known oncogenes in ESCC, such as TP63, SOX2, CTTN, STAT3, etc. More importantly, a number of novel oncogenes have also been uncovered, including RUNX1, YAP1, SREBF2, PAK4 and DNAJB1. Finally, THZ1-mediated blocking of SE-associated oncogenic transcription program potently suppressed the malignancy of ESCC cells in vitro. In this proposal, we will: 1) Comprehensively characterize SEs and their interacting protein complex in primary ESCC specimens and matched normal esophageal epithelial; 2) Functionally explore the biological significance of candidate SE-driven oncogenic transcripts in esophageal cancer cells; 3) Preclinical study of targeting SE-associated oncogenic transcription program using ESCC xenograft models. With very promising preliminary data, we believe that our proposal will provide an important molecular foundation for understanding the transcriptional landscape in ESCC and identifying a catalog of novel oncogenic transcripts. Moreover, our work may establish the therapeutic merit of targeting SE-associated oncogenic transcription program for ESCC treatment.

食管鳞状细胞癌（简称食管鳞癌）是人类常见的恶性肿瘤之一，其发生发展的分子基础包括基因转录调控异常。最新研究发现染色体上存在大量增强子富集的转录调控区域，称为“超级增强子”。超级增强子对很多组织特异性基因，特别是癌基因的转录激活有重要作用。.我们在两株食管鳞癌细胞系中分别鉴定出444和857个超级增强子，注意到超级增强子可驱动食管鳞癌中一些重要癌基因（如TP63、CTTN等）的高表达。进一步研究发现小分子抑制剂THZ1可特异性阻断超级增强子驱动的癌基因表达，并强烈抑制食管鳞癌的恶性表型。结合转录组测序和生物信息学分析，我们还鉴定出一批新的食管鳞癌相关癌基因（如RUNX1、SREBF2等）。本项目中，我们将在食管鳞癌临床标本中深入鉴定新的超级增强子，及与其相互作用的转录因子复合物，并进一步筛选新的超级增强子驱动的癌基因。这些工作将有助于阐明食管鳞癌基因转录机制，为其诊治提供新思路和新靶点。

本项目我们发现，食管鳞癌中，SOX2和TP63作为核心转录因子，可共同调控许多超级增强子相关的基因，包括长非编码RNA CCAT1等的表达。我们通过细胞增殖、克隆形成、裸鼠成瘤等体内外实验，全面评估了受SOX2和TP63共同调控的长非编码RNA CCAT1的肿瘤学功能，并结合ChIP-PCR和萤光素酶报告基因等实验精细阐明了SOX2和TP63调控CCAT1的具体位点。进一步结合RNA-seq、磷酸激酶实验、RIP和ChIRP等实验，我们深入阐明了SOX2和TP63共同调控CCAT1在食管鳞癌中发挥肿瘤学功能的机制：SOX2、TP63及CCAT1共同形成蛋白质/RNA复合物，结合到同为超级增强子驱动的EGFR的超级增强子区域，进而激活MEK/ERK1/2和PI3K/AKT等信号通路，驱动鳞癌的发生发展。通过本项目研究，我们在食管鳞癌中明晰了受超级增强子驱动的基因调控网络；发现了一个建立在超级增强子基础之上的蛋白质/RNA/DNA转录复合体（TP63/SOX2-CCAT1-EGFR）；以该复合体为核心，介导转录失调，促进肿瘤的发生。