Antagonism of the Ah Receptor in Controlling Breast Cancer Growth and Invasion

描述（由申请人提供）：历史上，芳烃受体（AhR）因其对细胞色素P450酶编码基因的转录调控而被研究，细胞色素P450酶将环境和内源性底物代谢为有毒和致突变的中间体。越来越多的研究支持这一假设，即AhR除了在诱变剂的形成和缺乏环境化学物质的情况下，还在恶性上皮细胞的生长和侵袭中发挥重要作用。这一新范式基于以下几个关键观察结果：1)AhR表达在致癌物诱导的大鼠和小鼠乳腺肿瘤以及“自发”的人类乳腺肿瘤系中显著增加。2)在大鼠、小鼠和人类乳腺肿瘤中，AhR的核定位以及在没有环境化学物质的情况下AhR与基因启动子的结合表明AhR的组成性激活。3)组成型活性AhR调节多种基因的表达，包括CYP1B1、CK21和Slug，后者是肿瘤侵袭的主要调控因子。4)最近的研究表明，乳腺肿瘤中AhR活性的增加也有助于细胞迁移和侵袭。5) AhR的分子下调抑制乳腺癌细胞增殖，使细胞恢复到非侵袭性表型。分子和生物学策略已经提供了重要的证据，证明AhR除了突变外，还参与了促进肿瘤形成、生长和侵袭的多种机制。因此，我们可以利用我们的能力来检查组成活性AhR的作用，以确定AhR的化学拮抗如何转化为乳腺癌的预防或抑制肿瘤进展的治疗方法。因此，我们提出了一个新的假设：用天然存在的无毒拮抗剂靶向组成活性AhR代表了一种抑制乳腺肿瘤生长和侵袭的可行治疗方法。提出了三个具体目标：1。通过检查拮抗剂混合物中协同相互作用的潜力，对天然产物提取物库（NCI天然产物库）中新型强效拮抗剂进行高通量筛选，以及研究改善AhR失活的“化学敲除”方法，研究最大化AhR拮抗的策略。2. 通过建立内源性基因表达对AhR反激活的拮抗剂作用，以及检测拮抗剂介导的AhR- dna相互作用的变化，确定乳腺癌模型中组成型活性AhR化学拮抗的分子机制。3. 建立使用最佳AhR拮抗剂化学拮抗本构活性AhR的功能后果。这些研究的转化影响在于已知和新发现的拮抗剂抑制肿瘤生长和侵袭的能力。在这里，潜在的治疗性AhR拮抗剂将被评估其阻断人类乳腺肿瘤细胞系中组成性AhR活性的生物学结果的能力。总的来说，这些研究将为有效的AhR拮抗剂在体内预防和/或治疗乳腺癌的潜力的临床前研究提供基础。公共卫生相关性：我们假设一种叫做芳烃受体的蛋白质的高表达及其与DNA的结合有助于乳腺肿瘤的生长和进展。在这里，我们提出阻碍该受体功能的化学物质（即拮抗剂）将有效下调该蛋白的活性，从而抑制乳腺肿瘤的生长和转移。植物和海洋天然产物库的筛选将提供新的拮抗剂来源，可以测试它们与该受体的相互作用及其干扰肿瘤生长的机制，最终导致治疗乳腺癌的治疗药物的开发。

DESCRIPTION (provided by applicant): Historically, the aryl hydrocarbon receptor (AhR) has been studied for its transcriptional regulation of genes encoding cytochrome P450 enzymes, which metabolize environmental and endogenous substrates into toxic and mutagenic intermediates. Accumulating studies support the hypothesis that the AhR also plays an important role in malignant epithelial cell growth and invasion apart from its role in formation of mutagens and in the absence of environmental chemicals. This new paradigm is based on several key observations: 1) AhR expression is increased dramatically in carcinogen-induced rat and mouse mammary tumors and in "spontaneous" human mammary tumor lines. 2) Constitutive AhR activation is indicated by nuclear AhR localization in rat, mouse, and human mammary tumors and by AhR binding to gene promoters in the absence of environmental chemicals. 3) Constitutively active AhR regulates the expression of multiple genes, including CYP1B1, CK21, and Slug, a master regulator of tumor invasion. 4) Recent studies suggest that increased AhR activity in mammary tumors also contributes to cell migration and invasiveness. 5) Molecular downregulation of the AhR suppresses breast cancer cell proliferation and reverts cells to a non-aggressive phenotype. Molecular and biologic strategies have provided significant evidence that the AhR participates, beyond mutagenesis, in multiple mechanisms that contribute to tumor formation, growth and invasion. Therefore, we can exploit our ability to examine effects of constitutively active AhR to determine how chemical antagonism of the AhR may translate into breast cancer prevention or a therapeutic approach to suppress tumor progression. Thus, we propose a new hypothesis: Targeting the constitutively active AhR with naturally occurring, non-toxic antagonists represents a feasible therapeutic approach to inhibit breast tumor growth and invasion. Three specific aims are proposed: 1. Investigate strategies to maximize antagonism of the AhR by examining the potential for synergistic interaction in mixtures of antagonists, performing a high-throughput screen for novel, potent antagonists from natural product extract libraries (NCI Natural Products Repository) and examining the "chemical knockout" approach for improving AhR inactivation. 2. Define the molecular mechanisms of chemical antagonism of the constitutively active AhR in a breast cancer model by establishing antagonist effects on AhR transactivation of endogenous gene expression and examining antagonist-mediated changes in AhR-DNA interactions. 3. Establish the functional consequences of chemically antagonizing the constitutively active AhR using optimal AhR antagonists. The translational impact of these studies lies in the ability of known and newly identified antagonists to suppress tumor growth and invasion. Here, potentially therapeutic AhR antagonists will be evaluated for their ability to block the biological outcomes of constitutive AhR activity in human mammary tumor cell lines. Collectively, these studies will provide the foundation for preclinical studies on the potential for potent AhR antagonists to prevent and/or treat breast cancer in vivo. PUBLIC HEALTH RELEVANCE: We hypothesize that the hyper-expression of a protein, called the aryl hydrocarbon receptor, and its binding to DNA contributes to the growth and progression of breast tumors. Here we propose that chemicals that impede the function of this receptor (i.e. antagonists) will be effective at downregulating this protein's activity and therefore will suppress breast tumor growth and metastasis. Screening of plant and marine natural product libraries will provide a source of novel antagonists that can be tested for their interaction with this receptor and their mechanism of interference with tumor growth, ultimately resulting in the development of therapeutic agents for the treatment of breast cancer.