Defining the function of SETD8 in colorectal cancer

定义 SETD8 在结直肠癌中的功能

基本信息

批准号：
10359781
负责人：
ZVI Cramer
金额：
$ 3.42万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10359781
关键词：
APC mutation Address Binding Biological Assay CRISPR/Cas technology Cancer Etiology Cancer Model Candidate Disease Gene Cell Cycle Progression Cells Cessation of life Chromatin Clinic Co-Immunoprecipitations Collaborations Colonoscopy Colorectal Colorectal Cancer Complex CpG Islands DNA DNA Methylation DNA Repair DNA Sequence Alteration Deposition Disease Dropout Drops Drosophila genus Embryo Engineering Enzymes Epigenetic Process Epithelial Event Gene Expression Gene Targeting Genes Genetic Transcription Guide RNA Histones Human Human Cell Line Hypermethylation Implant In Vitro Injections Internal Ribosome Entry Site Intestines KRAS2 gene Kidney Knowledge LGR5 gene Lysine MADH4 gene Maintenance Malignant Neoplasms Measures Metastatic Carcinoma Metastatic Neoplasm to the Liver Metastatic to Methyltransferase Modeling Modification Mucous Membrane Mus Mutation Neoplasm Metastasis Normal Cell Oncogenes Oncogenic Organoids Patients Play Post-Translational Protein Processing Primary Neoplasm Process Proteins RNA library Reporter Research Retrotransposon Role Shapes TCF Transcription Factor TP53 gene Techniques Testing Therapeutic Therapeutic Index Time Transcriptional Activation Ursidae Family WNT Signaling Pathway Zebrafish base beta catenin cancer stem cell cancer type cell transformation chromatin immunoprecipitation colon cancer cell line colorectal cancer progression combat experimental study genetic regulatory protein histone methyltransferase histone modification implantation in vivo inhibitor knock-down loss of function mortality mutant novel novel therapeutics overexpression pre-clinical prognostic promoter protein function protein protein interaction recruit small hairpin RNA stemness therapeutic development therapeutic target transcription factor transcriptome transcriptome sequencing tumor tumor growth tumorigenesis

项目摘要

Project Summary Colorectal cancer (CRC) is predicted to be the second leading cause of cancer-related deaths in 2018. Greater than 90% of CRC tumors contain a mutation in the canonical Wnt-signaling pathway, which deregulates β-CATENIN-TCF transcriptional activity and drives oncogenic gene expression. Indeed, the canonical Wnt-target LGR5 is proposed mark CRC stem cells, which are required for primary and metastatic tumor growth. In addition to genetic mutations, CRC tumors bear alterations in epigenetic features, including global DNA hypomethylation, CpG island hypermethylation and a variety of changes in histone post-translational modifications. Although these chromatin changes are thought to be important for CRC tumorigenesis, the oncogenic chromatin factors in CRC remain largely undefined, impeding targeting of epigenetic processes in the clinic. To address this gap in knowledge, I conducted an in vitro CRISPR-Cas9 dropout screen using a chromatin-focused pooled lentiviral guide RNA (gRNA) library in a primary murine CRC model. gRNAs targeting Kmt5a (encoding SETD8), the sole histone 4 lysine 20 (H4K20) monomethyltransferase, dropped out significantly during this screen, indicating the importance of this gene in CRC proliferation. SETD8 and H4K20me1 play important roles in regulating transcription, DNA damage repair and cell cycle progression. Particularly relevant for CRC, SETD8 methyltransferase activity is required for β-CATENIN-TCF transcriptional function in human cell lines, zebrafish and Drosophila. Indeed, upon Wnt stimulation, SETD8 directly interacts with β-CATENIN-TCF complexes and deposits H4K20 monomethyl (H4K20me1) modifications at canonical Wnt target genes. Based on these findings, I hypothesize that SETD8 promotes CRC tumorigenesis in part by cooperating with β-CATENIN-TCF to potentiate the canonical Wnt-signaling pathway. To this end, in Aim 1 I will investigate the importance of SETD8 in CRC tumorigenesis in vivo by manipulating SETD8 expression in murine CRC organoids and orthotopically implanting these lines into a syngeneic host. Moreover, I will define an in vitro therapeutic index for the SETD8 inhibitor UNC-0379 in pairs of primary normal and CRC organoids derived from human patients. In Aim 2 I will elucidate the mechanistic role of SETD8 in β-CATENIN-TCF transcriptional activity by manipulating SETD8 levels and measuring canonical Wnt-target activation by transcriptome profiling and functional reporter assays, as well as by quantifying the abundance and activity of Lgr5+ CRC stem cells in vivo. I will also define the protein-protein interactions between β-CATENIN, the TCF factor LEF1 and SETD8 as well as the enrichment of these proteins and H4K20me1 at canonical Wnt-target loci. This study will determine, for the first time, the role of SETD8 in CRC tumorigenesis. The proposed experiments will potentially identify a promising therapeutic avenue to combat this deadly disease.

项目概要结直肠癌 (CRC) 预计将成为 2018 年癌症相关死亡的第二大原因。超过 90% 的 CRC 肿瘤在经典 Wnt 信号通路中含有突变，从而导致失调 β-CATENIN-TCF 转录活性并驱动致癌基因表达。事实上，这是典型的 Wnt 靶标。 LGR5被提议标记CRC干细胞，这是原发性和转移性肿瘤生长所必需的。由于基因突变，结直肠癌肿瘤具有表观遗传特征的改变，包括整体 DNA 低甲基化， CpG 岛高甲基化和组蛋白翻译后修饰的多种变化。染色质变化被认为对结直肠癌肿瘤发生很重要，结直肠癌中的致癌染色质因子仍然很大程度上未定义，阻碍了临床上表观遗传过程的目标。为了解决这一知识差距，我使用原代小鼠 CRC 模型中以染色质为中心的混合慢病毒指导 RNA (gRNA) 文库。 Kmt5a（编码 SETD8），唯一的组蛋白 4 赖氨酸 20 (H4K20) 单甲基转移酶，脱落在此筛选过程中显着，表明该基因在 CRC 增殖中的重要性。 H4K20me1 在调节转录、DNA 损伤修复和细胞周期进程中发挥重要作用。与 CRC 特别相关，β-CATENIN-TCF 转录需要 SETD8 甲基转移酶活性事实上，在 Wnt 刺激下，SETD8 直接相互作用。具有 β-CATENIN-TCF 复合物并在规范 Wnt 处沉积 H4K20 单甲基 (H4K20me1) 修饰基于这些发现，我敢说 SETD8 促进 CRC 肿瘤发生的部分原因是与 β-CATENIN-TCF 合作增强经典的 Wnt 信号通路为此，我将在目标 1 中进行。通过操纵小鼠 SETD8 表达来研究 SETD8 在体内 CRC 肿瘤发生中的重要性 CRC 类器官并将这些细胞系原位植入同基因宿主中此外，我将定义体外。 SETD8 抑制剂 UNC-0379 在原发性正常和 CRC 类器官中的治疗指数在目标 2 中，我将阐明 SETD8 在 β-CATENIN-TCF 转录中的机制作用。通过操纵 SETD8 水平并通过转录组分析测量典型 Wnt 靶标激活来确定活性和功能报告分析，以及通过量化 Lgr5+ CRC 干细胞的丰度和活性我还将 β-CATENIN、TCF 因子 LEF1 和 SETD8 之间的蛋白质-蛋白质相互作用定义为以及这些蛋白质和 H4K20me1 在典型 Wnt 靶位点的富集。这项研究将首次确定 SETD8 在 CRC 肿瘤发生中的作用。实验将有可能找到一种有前途的治疗途径来对抗这种致命的疾病。