CXCR4 as a target for colon cancer chemoprevention

项目总结 在食用西式饮食(WDS)的人群中，患结直肠癌(CRC)的风险大大高于 动物模型证实了这种联系。WD诱导的肿瘤促进需要表皮生长因子受体 (EGFR)由增加的EGFR配体驱动的信号。蛋白水解酶控制EGFR配体的释放 ADAM17.ADAM17的克隆细胞缺失或药物阻断可抑制肿瘤的发生。趋化因子 CXCR4受体在肿瘤进展过程中上调表达的结肠细胞上表达，并可激活ADAM17。 CXCR4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 CXCR4及其配体SDF1α(又名CXCL12)在结肠粘膜中均有表达。总之，这些数据表明，CXCR4 是通过信号转导反式激活的结肠促进WD促进肿瘤发展的靶向上游调节因子 癌细胞EGFR通过ADAM17依赖的机制。虽然CXCR4在癌症中很少发生突变，但它的 转录因子(TF)和表观遗传学变化上调了表达，进一步证明这些因子 如饮食调节其活动。在初步研究中，临床耐受性良好的CXCR4抑制剂MSX-122 试验表明，偶氮甲烷(AOM)治疗的APC+/Min小鼠的结肠癌发病率降低了近70%。此外， 在APC缺陷的情况下，CXCR4缺失的小鼠比CXCR4+/+小鼠患肿瘤更少 背景资料。我们假设CXCR4在WD促进的肿瘤发生中是必需的，并且自然地 抑制CXCR4的产物(NP)是一种很有前途的化学预防策略。剖析CXCR4的研究 调控和效应信号以及识别新的NP抑制剂可能融合在一起，以发现结直肠癌的新靶点 预防。为了研究CXCR4在wds促肿瘤中的作用，并寻找新的抑制物，我们建议 三个具体目标：目标1确定CXCR4活性是否是WD促进的肿瘤发生所必需的。1a)至 评估CXCR4抑制剂MSX-122抑制AOM发生的能力。我们将比较MSX的影响 在STD饮食和WD癌前和癌变阶段，用RNAseq和EGFR评估效应信号 激活。1b)从遗传学角度评估CXCR4在结直肠癌中的作用，将克隆细胞缺失的CXCR4Δ/Δ小鼠与 CXCR4+/Δ和CXCR4+/+小鼠(均为APC缺陷背景)，并测量目标1a所述的终点。 目的2a：确定WD和肿瘤进展对转录因子和组蛋白修饰调节SDF1α的影响 和CXCR4在当地使用芯片分析，并使用染色质评估饮食和肿瘤形成的全球影响 使用条件APC-CXCR4模型中的有机化合物进行纳米5HmC-Seal的ATAC-SEQ和DNA甲基化。5mC 例如，全球发现的5hmC信号调节局部的组蛋白修饰。2B)评估SDF1α- CXCR4基因调控和效应信号在使用有机化合物的人结肠肿瘤发生中的作用。 目的3a筛选CXCR4抑制活性的NP文库。CXCR4报告分析(钙瞬变、迁移 分析和配基结合)将用于确认计算预测的NP HITS 3b有希望的试剂 将对aim3a中确定的药物进行优先排序，并在小鼠模型中测试其化学预防效果。

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.