Bioassay for Breast Cancer Prevention

描述（由申请人提供）：本申请涉及广泛的挑战领域（01）行为，行为改变和预防，以及特定的挑战主题01- ca -102，营养在癌症生物学中的作用。我们的长期目标是描述基因组稳定性在饮食预防癌症中的作用，并开发一种DNA损伤的中间生物测定法，以测试饮食制剂的功效。这些研究将为这一概念的原理提供证明，并通过研究维生素D和基因组稳定性建立生物测定方法。本项目针对“预防”和“营养在癌症生物学中的作用”这两个挑战，从以下几个方面进行了研究：关注正常乳腺细胞和癌症预防。我的实验室广泛研究了维生素D对乳腺癌细胞的影响，并提出了针对维生素D受体（VDR）进行癌症治疗的想法。不幸的是，在临床试验中，患者患有晚期癌症，并且以维生素D为基础的治疗效果甚微。许多其他营养素和营养衍生物（如维生素A、维生素E等）也报道了类似的结果。因此，我们开始研究维生素D对正常乳腺的影响，并认为维生素D可以有效预防癌症。事实上，我们发现VDR无效小鼠对化学和致癌基因诱导的癌变表现出增强的敏感性。该提案直接针对预防的广泛挑战领域，计划在体外和体内全面研究维生素D治疗如何影响正常人和小鼠乳腺细胞的DNA损伤反应途径。2. 在体内测试膳食维生素D的补充。饮食中维生素D的需求最初是为了预防骨病佝偻病而设定的。当时，人们还没有认识到维生素D在预防癌症方面的作用。因此，我们知道最佳骨骼生长需要多少维生素D，然而，我们不知道表达VDR的其他组织（如乳房）需要多少维生素D才能达到最佳健康状态。我们方法的一项创新是，我们将在“标准啮齿动物需求”的基础上，将膳食维生素D增加5倍、10倍和20倍。这是基于这样一个事实，即我们只是不知道多少才足够（我们从之前对钙代谢的研究中知道，这些水平是无毒的）。3. 化学预防剂对DNA损伤反应的中间生物测定方法的建立。尽管基于细胞的研究已经开始证明营养物质对基因组稳定性的影响，但将体外数据扩展到更复杂的组织环境中，以模仿体内癌症的发展，显然是一个挑战。关于监测由双链断裂（DSB）引起的DNA损伤，体内研究尤其困难，因为基因毒性药物在多个组织的随机位点诱导DSB，并且这些断裂的易出错修复导致整个基因组突变。一旦癌症最终发展，DNA最初的损伤就无法辨认了。因此，我们建议使用暴露于遗传毒性物质的器官培养中的乳腺来验证生物测定（我们将使用辐射和阿霉素，但该测定可以适用于其他感兴趣的遗传毒性物质）。这将允许精确控制暴露于乳腺内的遗传毒性物质，并监测即时损害（DSBs）。此外，我们相信利用完整组织的生物测定将更紧密地模拟与癌症预防相关的体内生物学。我们设计的一个主要优势将是能够在去除腺体进行培养之前操纵小鼠的饮食，因此可以在饮食/血清营养水平与生物测定中的有效性之间建立相关性。我们这项为期两年的挑战拨款的目标是通过研究这种方法补充膳食维生素D的效果来提供原理证明。如果得到验证，这种生物测定法将被证明是一种有用的中间方法，用于筛选优化DNA损伤反应途径的膳食制剂。当然，也可以使用基因敲除或转基因小鼠作为供体来探测乳腺中DNA损伤反应通路调节中的特定基因-营养相互作用。4. 启动预防基因组数据库。我们方法的另一个吸引人的方面是，人们可以从同一动物身上收集多个腺体，同时比较化学预防剂在生物测定中的基因组或蛋白质组学特征的有效性。与细胞培养研究相反，生物测定将允许在所有乳腺细胞群（上皮细胞、基质细胞、免疫细胞、内皮细胞等）的背景下分析基因表达变化。从长远来看，将开发一个由这种生物测定有效的预防药物诱导的基因组图谱数据库。最终，该数据库有望产生一组与维持基因组稳定性的癌症预防剂相关的共同生物标志物。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic 01-CA-102, The Role of Nutrition in Cancer Biology. Our long term goals are to characterize the role of genomic stability in dietary prevention of cancer and to develop an intermediate bioassay of DNA damage for testing efficacy of dietary agents. These studies will provide proof of principle of this concept and establish the bioassay through study of vitamin D and genomic stability. This project specifically targets the Challenges "Prevention" and "Role of Nutrition in Cancer Biology" in the following ways: 1. Focus on normal mammary cells and cancer prevention. My lab has extensively studied the effects of vitamin D on breast cancer cells with the idea of targeting the vitamin D receptor (VDR) for cancer therapy. Unfortunately, in clinical trials, patients had late stage cancer and exhibited minimal improvement with vitamin D based therapies. Similar results have been reported with many other nutrients and nutrient derivatives (ie, vitamin A, vitamin E, etc). Thus, we began studying the effects of vitamin D on normal mammary gland with the idea that vitamin D would be effective in prevention of cancer. Indeed, we showed that VDR null mice display enhanced sensitivity to chemical and oncogene induced carcinogenesis. This proposal directly targets the Broad Challenge Area of Prevention with the plan to comprehensively study how vitamin D treatment impacts on DNA damage response pathways in normal human and mouse mammary cells in vitro and in vivo. 2. Testing dietary vitamin D supplementation in vivo. Dietary requirements for vitamin D were originally set for prevention of the bone disease rickets. At the time, a role for vitamin D in cancer prevention was not recognized. Thus, we know how much vitamin D is required for optimal bone growth, however, we do not know how much vitamin D is required for optimal health of other tissues that express the VDR such as the breast. One innovation of our approach is that we will increase dietary vitamin D 5, 10 and 20 fold above the "standard rodent requirement". This is based on the fact that we just don't know how much is enough (we do know from our previous studies on calcium metabolism that these levels are not toxic). 3. Development of an intermediate bioassay for testing effects of chemopreventive agents on the DNA damage response. Although cell based studies have begun to demonstrate effects of nutrients on genomic stability, extension of in vitro data to a more complex tissue environment that mimics cancer development in vivo is obviously a challenge. With respect to monitoring DNA damage resulting from double strand breaks (DSB), in vivo studies are especially difficult because genotoxic agents induce DSBs at random sites in multiple tissues, and it is the error-prone repair of these breaks that cause mutations throughout the genome. The initial damage to the DNA is no longer identifiable once cancer eventually develops. We therefore propose to validate a bioassay using mammary gland in organ culture exposed to genotoxic agents (we will use radiation and doxorubicin, but this assay could be adapted for other genotoxic agents of interest). This will allow exact control over exposure to genotoxic agents within the mammary gland, and monitoring of the immediate damage (DSBs). In addition, we believe that a bioassay that utilizes intact tissue will more closely model the in vivo biology relevant to cancer prevention. A major strength of our design will be the ability to manipulate the diets of mice prior to removal of the glands for culture and thus correlation can be made between dietary/serum levels of nutrients and effectiveness in the bioassay. Our aim for this two year challenge grant is to provide proof of principle by studying the effects of supplemental dietary vitamin D with this approach. If validated, this bioassay could prove useful as an intermediate approach for screening dietary agents that optimize DNA damage response pathways. Alternatively, of course, one could use knockout or transgenic mice as donors to probe specific gene-nutrient interactions in modulation of DNA damage response pathways in the mammary gland. 4. Initiation of a prevention genomic database. Another attractive aspect of our approach is that one can collect multiple glands from the same animal and simultaneously compare effectiveness of chemopreventive agents in the bioassay with genomic or proteomic profiles. In contrast to cell culture studies, the bioassay will allow profiling of gene expression changes in the context of all mammary cell populations (epithelial, stromal, immune, endothelial, etc). In the long term, a database of genomic profiles induced by preventive agents effective in this bioassay will be developed. Ultimately, this database is expected to generate a common set of biomarkers associated with cancer preventive agents that maintain genomic stability. PUBLIC HEALTH RELEVENCE: People are continuously exposed to environmental agents that can cause genetic mutations and increase the risk of cancer development. Fortunately, healthy tissues can repair the majority of these mutations, some cells do eventually accumulate enough damage over time that they become cancerous. Based on this paradigm of age-related accumulation of damage, it stands to reason that dietary factors might delay cancer development by maximizing tissue repair processes. In this project we will develop methods to study damage and repair processes in mammary gland, and will perform initial experiments to test whether vitamin D acts to prevent breast cancer via enhancement of cellular protective responses.