CCAR2 as a Target for Prevention of Colorectal Cancer.

CCAR2 作为预防结直肠癌的靶点。

基本信息

批准号：
10358583
负责人：
Roderick H Dashwood
金额：
$ 50.88万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2024-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10358583
关键词：
Acetylation Acute Aftercare Alternative Splicing Antibodies Apoptosis Apoptosis Regulation Gene Attenuated Binding Biopsy Bromodomain CRISPR/Cas technology Cell Cycle Cell Cycle Proteins Cell Death Cell Survival Clinical Colon Colon Carcinoma Colorectal Cancer Complex Cyclin D1 Dietary Factors Dietary Isothiocyanate Dose Down-Regulation Drug Combinations Endoscopy Engineering Excision Familial Adenomatous Polyposis Syndrome Genetic Transcription HDAC3 gene Human Individual Induction of Apoptosis Isothiocyanates Link Modeling Nuclear Nude Mice Null Lymphocytes Patients Polyps Prevention Protocols Preventive Prognosis Protein Family Proteins RNA Binding RNA Processing RNA Splicing Rattus Reader Role Secondary Prevention Signal Pathway Signal Transduction Site Sulforaphane Tertiary Protein Structure Testing Transplantation Tumor Suppression c-myc Genes cancer cell colon cancer patients colon cancer prevention colorectal cancer prevention cruciferous vegetable dietary experimental study in vivo inhibitor member mutant novel overexpression patient derived xenograft model polyposis protein protein interaction tumor

项目摘要

Cell Cycle and Apoptosis Regulator Protein 2 (CCAR2) is overexpressed in human colorectal cancer and is associated with poor prognosis, due in part to its role as a coactivator of -catenin-dependent transcription. The dietary preventive agent sulforaphane (SFN) targets CCAR2 interacting protein histone deacetylase 3 (HDAC3) for inhibition, resulting in acetylation of CCAR2, reduced CCAR2/-catenin nuclear interactions, and attenuated -catenin-dependent transcription. The novel CCAR2 acetylation sites align with a domain linked to S1 RNA binding, providing a new mechanistic link to alternative RNA splicing by SFN and other dietary isothio- cyanates (ITCs). Using novel protein domain arrays, bromodomain-containing proteins were identified that recognized acetylated forms of CCAR2. These acetyl “readers” also were inhibited by JQ1, which binds to bromodomain and extraterminal (BET) family proteins, and synergized with SFN to inhibit cell viability in colon cancer cells. CENTRAL HYPOTHESIS: In the prevention of colon cancer by dietary ITCs and mechanistically- prioritized drug combinations, HDAC3 inhibition leads to acetylation of CCAR2 and changes in protein-protein interactions that enhance apoptosis via the inhibition of -catenin-dependent transcription and via altered RNA splicing. Aim 1a: Test the hypothesis that by inhibiting HDAC3, ITCs alter the acetylation status of CCAR2 in colon cancer cells, with consequences for -catenin signaling and apoptosis induction. Aim 1b: Perform mechanistic studies on the acetyl readers of CCAR2, and test inhibitors of readers that synergize with SFN. Aim 1c: Examine the role of CCAR2 and the DBIRD complex in RNA splicing. Aim 2a: Using the polyposis in rat colon (Pirc) model, extend to working hypothesis in vivo linking HDAC3 inhibition, CCAR2 acetylation, reduced -catenin signaling and apoptosis induction. Single acute doses of SFN, 6-SFN and 9-SFN will be tested alone and in combination with JQ1. The most effective agents from Aim 2a will be assessed for tumor suppression in the Pirc model via primary (Aim 2b) and secondary prevention protocols (Aim 2c). Using sequential endoscopy and polyp resection in live rats, define the precise timing of HDAC3 protein loss, CCAR2 acetylation, reduced nuclear CCAR2/-catenin interactions, and the downregulation of -catenin targets. Aim 2d: Extend mechanistic studies from Aim 1c linking CCAR2 acetylation to alternative RNA splicing after ITC treatment into the Pirc model. Aim 3a: In biopsies from FAP patients, establish the translational relevance of findings in the Pirc model with respect to CCAR2 overexpression and its interactions with -catenin, HDAC3, and ZIRD. Aim 3b: In colonoids from Pirc and FAP patients, test the hypothesis that SFN, 6-SFN and 9-SFN alter the acetylation status of CCAR2 and its protein-protein interactions, with consequences for -catenin signaling and apoptosis induction. Test individual ITCs and their combinations with JQ1. Aim 3c: In nude mice transplanted with FAP patient-derived xenografts or CRISPR/Cas9-engineered CCAR2-null human colon cancer cells transfected with CCAR2 WT and acetylation mutants, examine tumor suppression by ITCs±JQ1.

细胞周期和凋亡调节剂蛋白2（CCAR2）在人类结肠癌中过表达，IS 与预后不良有关，部分原因是它是-catenin依赖性转录的共激活因子的作用。饮食预防剂硫烷（SFN）靶向CCAR2相互作用的蛋白组蛋白脱乙酰基酶3 （HDAC3）为抑制作用，导致CCAR2的乙酰化，CCAR2/-catenin核相互作用降低，并且减弱-catenin依赖性转录。新型的CCAR2乙酰化位点与与与 S1 RNA结合，提供了与SFN和其他饮食式Isothio-的替代RNA剪接的新机械链接氰化物（ITC）。使用新型的蛋白质结构域阵列，鉴定出含溴脱域的蛋白公认的CCAR2的乙酰化形式。 JQ1也抑制了这些乙酰“读取器”，该JQ1结合到溴d和外部（BET）家族蛋白，并与SFN协同抑制颜色的细胞活力癌细胞。中央假设：在饮食中预防结肠癌和机械上 - 优先的药物组合，HDAC3抑制会导致CCAR2的乙酰化和蛋白质蛋白质的变化通过抑制-catenin依赖性转录并通过改变的RNA来增强凋亡的相互作用剪接。 AIM 1A：检验以下假设：通过抑制HDAC3，ITC改变了CCAR2的乙酰化状态结肠癌细胞，对-catenin信号传导和凋亡诱导的影响。 AIM 1B：执行关于CCAR2的乙酰读取器的机理研究，以及与SFN协同作用的读取器的抑制剂。 AIM 1C：检查CCAR2和DBIRD复合物在RNA剪接中的作用。 AIM 2A：在大鼠结肠（PIRC）模型，延伸至在体内的工作假设，将HDAC3抑制作用，CCAR2乙酰化，CCAR2乙酰化，降低了-catenin信号传导和凋亡诱导。单一急性剂量的SFN，6-SFN和9-SFN为单独测试并与JQ1结合。 AIM 2A最有效的药物将评估肿瘤通过主要（AIM 2B）和二级预防方案（AIM 2C）在PIRC模型中抑制。使用活大鼠的顺序内窥镜和息肉切除，定义HDAC3蛋白损失的精确时机，CCAR2 乙酰化，核CCAR2/-catenin相互作用减少，以及-catenin靶标的下调。目的 2D：将机械研究从将CCAR2乙酰化连接到ITC后替代RNA剪接的AIM 1C扩展处理PIRC模型。 AIM 3A：在FAP患者的活检中，建立了翻译相关性 PIRC模型中有关CCAR2过表达及其与-Catenin HDAC3的相互作用的发现和Zird。 AIM 3B：在PIRC和FAP患者的结肠体中，检验了SFN，6-SFN和9-SFN的假设改变CCAR2及其蛋白质 - 蛋白质相互作用的乙酰化状态，并影响-catenin 信号传导和凋亡诱导。测试单个ITC及其与JQ1的组合。 AIM 3C：在裸鼠中用FAP患者衍生的Xenographictics或CRISPR/CAS9工程CCAR2-NULL人类结肠移植用CCAR2 WT和乙酰化突变体翻译的癌细胞检查ITCS±JQ1抑制肿瘤。