CXCR4 as a target for colon cancer chemoprevention

项目摘要 结直肠癌（CRC）的风险在食用西方饮食（WD）的人群中显著较高， 动物模型证实了这种关联。WD诱导的肿瘤促进需要表皮生长因子受体 （EGFR）信号由增加的EGFR配体驱动。EGFR配体释放受蛋白酶控制 ADAM 17.结肠细胞缺失或药物阻断ADAM 17抑制肿瘤发生。化因子 受体CXCR 4在结肠细胞上表达，随着癌症进展而上调，并且可以激活ADAM 17。 CXCR 4在饮食促进的肿瘤发生中的作用尚未阐明。我们发现WD上调了 CXCR 4及其配体Sdf 1 α（又名CXCL 12）在结肠粘膜中的表达。总之，这些数据表明，CXCR 4 是WD通过信号转导反式激活结肠促进肿瘤发展的靶向上游调节因子 癌细胞EGFR通过ADAM 17依赖性机制。虽然CXCR 4在癌症中很少突变，但其 转录因子（TF）和表观遗传变化上调表达，进一步证明了因子 如饮食调节其活性。在初步研究中，MSX-122，一种在临床中耐受良好的CXCR 4抑制剂， 试验中，在氧化偶氮甲烷（AOM）处理的Apc+/Min小鼠中，结肠肿瘤发病率降低了近70%。此外，委员会认为， 缺失结肠细胞CXCR 4的小鼠比缺失APC的CXCR 4 +/+小鼠发生更少的肿瘤。 背景我们假设CXCR 4是WD促进肿瘤发生所必需的，而天然的CXCR 4是WD促进肿瘤发生所必需的。 CXCR 4的NP产物抑制是一种有前途的化学预防策略。研究CXCR 4 调节和效应信号，并确定新的NP抑制剂可以收敛，以发现新的CRC靶点 预防为了研究CXCR 4在WD促进肿瘤中的作用并鉴定新的抑制剂，我们建议： 三个具体目的：目的1确定WD促进的肿瘤发生是否需要CXCR 4活性。1a） 评估CXCR 4抑制剂MSX-122抑制AOM肿瘤发生的能力。我们将比较MSX效果 在Std饮食和WD中，在癌前和恶性阶段，并通过RNAseq和EGFR评估效应信号 activation. 1b）为了从遗传学上评估CXCR 4在CRC中的作用，将结肠细胞缺失CXCR 4 Δ/Δ小鼠与 CXCR 4 +/Δ和CXCR 4 +/+小鼠（均在Apc缺陷背景下），并测量目标1a中描述的终点。 目的2a：确定WD和肿瘤进展对调节Sdf 1 α的TF和组蛋白修饰的影响 和CXCR 4局部使用ChIP测定，并使用染色质评估饮食和肿瘤发生的总体影响 ATAC-seq和使用来自条件Apc-CXCR 4模型的类器官通过纳米-5 hmC-seal进行的DNA甲基化。5mC 例如，全球发现的5 hmC信号调节局部组蛋白修饰。2b）评估Sdf 1 α- 使用如2a中的类器官的人结肠肿瘤发生中的CXCR 4基因调控和效应信号。 目的3a筛选具有CXCR 4抑制活性的NP文库。CXCR 4报告基因测定（Ca 2+瞬变，迁移 分析和配体结合）将用于确认计算预测的NP命中3b 在AIM 3A中鉴定的将被优先化并在小鼠模型中测试化学预防功效。

PROJECT SUMMARY The risk of colorectal cancer (CRC) is substantially higher in populations consuming Western diets (WDs) and animal models confirm this association. WD-induced tumor promotion requires epidermal growth factor receptor (EGFR) signals that are driven by increased EGFR ligands. EGFR ligand release is controlled by proteinase ADAM17. Colonocyte deletion or pharmacological blockade of ADAM17 suppressed tumorigenesis. Chemokine receptor CXCR4 is expressed on colonocytes up-regulated with cancer progression and can activate ADAM17. The role of CXCR4 in diet-promoted tumorigenesis has not been elucidated. We found that WD up-regulated both CXCR4 and its ligand Sdf1α (aka CXCL12) in colonic mucosa. Together, these data suggest that CXCR4 is a targetable upstream regulator of WD-promoted tumor development by signaling transactivated colon cancer cell EGFR via an ADAM17-dependent mechanism. While CXCR4 is rarely mutated in cancer, its expression is up-regulated by transcription factors (TFs) and epigenetic changes, further evidence that factors such as diet regulate its activity. In preliminary studies, MSX-122, a CXCR4 inhibitor well tolerated in clinical trials, reduced colon tumor incidence by nearly 70% in azoxymethane (AOM)-treated Apc+/Min mice. Moreover, mice deleted of colonocyte CXCR4 developed fewer tumors than CXCR4+/+ mice on an Apc-deficient background. We hypothesize that CXCR4 is required for WD-promoted tumorigenesis, and that natural product (NP) inhibition of CXCR4 is a promising chemopreventative strategy. Studies that dissect CXCR4 regulation and effector signals and identify novel NP inhibitors could converge to uncover new targets for CRC prevention. To study the role of CXCR4 in tumor promotion by WDs and identify novel inhibitors, we propose three specific aims: Aim 1 To determine if CXCR4 activity is required for WD-promoted tumorigenesis. 1a)To assess the ability of CXCR4 inhibitor MSX-122 to suppress AOM tumorigenesis. We will compare MSX effects in Std diet and WD in premalignant and malignant phases and assess effector signals by RNAseq and EGFR activation. 1b) To genetically assess the role of CXCR4 in CRC, comparing colonocyte null CXCR4Δ/Δ mice to CXCR4+/Δ and CXCR4+/+ mice (all on Apc deficient background) and measure end points described in aim 1a. Aim 2a: To determine effects WD and neoplastic progression on TFs and histone modifications regulating Sdf1α and CXCR4 locally using ChIP assays and assess global effects of diet and tumorigenesis using chromatin ATAC-seq and DNA methylation by nano-5hmC-seal using organoids from conditional Apc-CXCR4 model. 5mC and 5hmC signals discovered globally, for example, regulate local histone modifications. 2b) To assess Sdf1α- CXCR4 gene regulation and effector signals in human colonic tumorigenesis using organoids as in 2a. Aim 3a To screen NP libraries for CXCR4 inhibitory activity. CXCR4 reporter assays (Ca2+ transients, migration assays and ligand-binding) will be used to confirm computationally predicted NP hits 3b Promising agents identified in aim3a will be prioritized and tested for chemopreventive efficacy in mouse models.