Elucidating the role of acetylation of APE1 in tumor growth and chemoresistance

阐明 APE1 乙酰化在肿瘤生长和化疗耐药中的作用

基本信息

批准号：
9813757
负责人：
Kishor K Bhakat
金额：
$ 8.04万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9813757
关键词：
ABCB1 gene Acetylation Adenomatous Polyposis Coli Alkaline Single-Cell Gel Electrophoresis Assay ApcMin/+ mice Base Excision Repairs Biological Assay CRISPR/Cas technology Cancer Biology Cancer Model Cancer Patient Carcinogens Carrier Proteins Cell Line Cells Chromatin Colon Carcinoma Colorectal Cancer DNA DNA Damage DNA Repair DNA Repair Enzymes DNA lesion DNA-(apurinic or apyrimidinic site) lyase Down-Regulation Drug Efflux EP300 gene Exposure to Fluorouracil Future Genes Genetic Genome HCT116 Cells Human Human Genome In Vitro Knock-in Knock-in Mouse Lysine Malignant Neoplasms Measures Modeling Monitor Mus N-terminal Pathway interactions Patients Pharmaceutical Preparations Pharmacotherapy Play Primary Neoplasm Proteins Radiation Radiation therapy Research Research Personnel Resistance Role Short Interspersed Nucleotide Elements Site System Testing Tissues Transcriptional Regulation Tumor Tissue Xenograft Model base cancer type chemotherapeutic agent chemotherapy colon cancer cell line colon cancer patients colorectal cancer treatment cytotoxic drug efficacy endonuclease genetic approach in vitro Assay in vivo in vivo Model mouse model mutant neoplastic cell new therapeutic target novel outcome forecast overexpression pre-clinical preclinical study repair enzyme repaired stable cell line tumor tumor growth

项目摘要

Project Summary Common forms of DNA damage in the genome are Apurinic/apyrimidinic (AP) sites, which can be generated either spontaneously or following treatment with chemotherapeutic drugs or radiation. Human AP-endonuclease (APE1) is the primary enzyme that repairs AP sites through the Base Excision Repair (BER) pathway. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the direct evidence that APE1 contributes in promoting chemoresistance in a tumor model in vivo has not been established. We have been elucidating the regulatory roles of acetylation of multiple lysine residues in N-terminal domain of APE1 in DNA damage repair and transcriptional regulation. We discovered earlier that multiple Lys residues (Lys 6,7, 27, 31 and 32) are acetylated in cells by the histone acetyltransferase p300. We have recently shown that that these lysine residues in APE1 are acetylated (AcAPE1) at AP site damage in chromatin and that acetylation enhances damage repair activity of APE1. We further showed elevated levels of AcAPE1 in diverse cancer types and demonstrated that loss of APE1 or its acetylation results in accumulation of AP sites in the genome and increased sensitivity of cells to many chemotherapeutic drugs. Moreover, our earlier studies showed that AcAPE1 regulates expression of hundreds of genes including a key drug efflux transporter protein, MDR1. To determine the role of APE1 acetylation in inducing chemo resistance in vivo, using CRISPR/cas9 system we have established a APE1 knock-in mouse line defective in APE1 acetylation at Lys 6, 7 residues (Ape1A/A). Using this mouse model, we will test the hypothesis that acetylation of APE1 promotes 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC) in vivo. In Aim 1, using two 5-FU-resistant CRC cell lines (HCT116 and DLD-1) expressing WT or acetylation-defective APE1 mutant, we will monitor 5-FU induced damage and repair using alkaline comet assay. We will measure sensitivity to 5-FU by colony formation assay in vitro. We will examine tumor growth and the efficacy of 5-FU on tumor growth in xenograft model in vivo. In AIM2, we will use acetylation-defective APE1 knock-in (Ape1A/A) mouse model to probe the potential role of AcAPE1 in promoting 5-FU resistance in the context of (a) carcinogen (AOM/DSS)-induced and (b) genetic APCMin/+ (Adenomatous Polyposis Coli) colon cancer model. Our project will establish the role of APE1 acetylation in promoting 5-FU resistance in colon cancer in vivo. Furthermore, our APE1-acetylation defective knock-in genetic mouse model will be instrumental for future preclinical studies in examining whether acetylation of APE1 could be used as a novel target for sensitization of tumor cells to many chemotherapeutic drugs.

项目概要基因组中 DNA 损伤的常见形式是无嘌呤/无嘧啶 (AP) 位点，可生成自发地或在化疗药物或放射治疗后。人AP核酸内切酶 (APE1) 是通过碱基切除修复 (BER) 途径修复 AP 位点的主要酶。虽然 APE1 在多种癌症类型中的过度表达以及 APE1 表达与化疗的关联耐药性和不良预后都有据可查，APE1 有助于促进体内肿瘤模型中的化疗耐药性尚未建立。我们一直在阐明 N 末端结构域中多个赖氨酸残基乙酰化的调节作用 APE1 在 DNA 损伤修复和转录调控中的作用。我们之前发现多个Lys残基（Lys 6,7,27,31 和 32) 在细胞中被组蛋白乙酰转移酶 p300 乙酰化。我们最近表明 APE1 中的这些赖氨酸残基在染色质的 AP 位点损伤处被乙酰化 (AcAPE1)，并且乙酰化增强 APE1 的损伤修复活性。我们进一步发现不同癌症类型中 AcAPE1 水平升高并证明 APE1 或其乙酰化的缺失会导致基因组中 AP 位点的积累，细胞对许多化疗药物的敏感性增加。此外，我们早期的研究表明 AcAPE1 调节数百个基因的表达，包括关键的药物外排转运蛋白 MDR1。到使用 CRISPR/cas9 系统确定 APE1 乙酰化在诱导体内化疗耐药性中的作用已经建立了 APE1 敲入小鼠品系，该小鼠品系在 Lys 6、7 残基处存在 APE1 乙酰化缺陷（Ape1A/A）。使用在这个小鼠模型中，我们将测试 APE1 乙酰化促进 5-氟尿嘧啶 (5-FU) 的假设体内结直肠癌（CRC）的耐药性。在目标 1 中，使用两种 5-FU 耐药 CRC 细胞系 (HCT116 和 DLD-1) 表达 WT 或乙酰化缺陷的 APE1 突变体，我们将监测 5-FU 诱导的损伤和使用碱性彗星试验进行修复。我们将通过体外集落形成测定来测量对 5-FU 的敏感性。我们将在体内异种移植模型中检查肿瘤生长以及 5-FU 对肿瘤生长的功效。在AIM2中，我们将使用乙酰化缺陷型 APE1 敲入 (Ape1A/A) 小鼠模型来探讨 AcAPE1 在在 (a) 致癌物 (AOM/DSS) 诱导和 (b) 遗传 APCMin/+ 的背景下促进 5-FU 耐药性（腺瘤性结肠息肉病）结肠癌模型。我们的项目将确定 APE1 乙酰化在体内促进结肠癌 5-FU 耐药性中的作用。此外，我们的 APE1 乙酰化缺陷敲入基因小鼠模型将有助于未来检验 APE1 乙酰化是否可以作为致敏新靶点的临床前研究肿瘤细胞对许多化疗药物的作用。