CCAR2 as a Target for Prevention of Colorectal Cancer.

细胞周期和凋亡调节蛋白2（CCAR 2）在人结直肠癌中过表达， 与不良预后相关，部分原因是其作为β-连环蛋白依赖性转录的共激活因子的作用。 膳食预防剂萝卜硫素（SFN）靶向CCAR 2相互作用蛋白组蛋白脱乙酰酶3 （HDAC 3）用于抑制，导致CCAR 2的乙酰化，减少CCAR 2/β-连环蛋白核相互作用，以及 减弱的β-连环蛋白依赖性转录。新的CCAR 2乙酰化位点与连接到 S1 RNA结合，提供了一个新的机制连接到选择性RNA剪接的SFN和其他饮食异硫代- 氰酸酯（ITC）。使用新的蛋白质结构域阵列，鉴定了含溴结构域蛋白， CCAR 2的乙酰化形式。这些乙酰基“阅读器”也被JQ 1抑制，JQ 1结合到 溴结构域和末端外（BET）家族蛋白，并与SFN协同抑制结肠中的细胞活力 癌细胞中枢假设：在通过饮食ITC和机制预防结肠癌中- 优先的药物组合，HDAC 3抑制导致CCAR 2的乙酰化和蛋白质-蛋白质 通过抑制β-连环蛋白依赖性转录和通过改变RNA的相互作用， 拼接目的1a：检验通过抑制HDAC 3，ITC改变了CCAR 2的乙酰化状态的假设。 结肠癌细胞，导致β-连环蛋白信号传导和凋亡诱导。目标1b：执行 对CCAR 2的乙酰基阅读器的机制研究，以及与SFN协同的阅读器的测试抑制剂。 目的1c：检测CCAR 2和DBIRD复合物在RNA剪接中的作用。目标2a：使用息肉 大鼠结肠（Pirc）模型，扩展到体内工作假设，其将HDAC 3抑制，CCAR 2乙酰化， 降低β-连环蛋白信号传导和凋亡诱导。单次急性剂量的SFN、6-SFN和9-SFN将被 单独测试和与JQ 1组合测试。将对目标2a中最有效的药物进行肿瘤评估 通过一级预防方案（Aim 2b）和二级预防方案（Aim 2c）在Pirc模型中进行抑制。使用 在活体大鼠中连续的内窥镜检查和息肉切除术，确定了HDAC 3蛋白丢失、CCAR 2 乙酰化，减少核CCAR 2/β-连环蛋白相互作用，以及β-连环蛋白靶点的下调。目的 2d：从Aim 1c扩展ITC后将CCAR 2乙酰化连接到可变RNA剪接的机制研究 治疗进入Pirc模式。目的3a：在FAP患者的活检中，确定 Pirc模型中关于CCAR 2过表达及其与β-连环蛋白，HDAC 3， 和ZIRD。目的3b：在来自Pirc和FAP患者的结肠样物质中，检验SFN、6-SFN和9-SFN与Pirc和FAP患者的结肠样物质之间的相互作用的假设。 改变CCAR 2的乙酰化状态及其蛋白质-蛋白质相互作用， 信号传导和凋亡诱导。测试单个ITC及其与JQ 1的组合。目的3c：裸鼠 移植有FAP患者来源的异种移植物或CRISPR/Cas9工程化的CCAR 2缺失的人结肠 用CCAR 2 WT和乙酰化突变体转染的癌细胞检查ITC ± JQ 1的肿瘤抑制。

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.