Breast Cancer Prevention by Ayurvedic Medicine Constituents

描述（由申请人提供）： 背景：乳腺癌仍然是全球女性癌症相关死亡的主要原因，选择性雌激素受体 (ER) 调节剂 (SERM) 和芳香酶抑制剂的临床成功证明，乳腺癌的化学预防是可行的。然而，SERM 对 ER 阴性乳腺癌无效，并且具有严重的副作用。芳香酶抑制剂似乎有望预防高危绝经后女性的乳腺癌。尽管芳香酶抑制剂在预防性环境中的安全性仍在研究中，但其使用引起了人们对心血管事件和骨折风险增加的担忧。因此，非常需要一种对 ER 阳性和 ER 阴性乳腺癌均有效的无毒预防干预措施。在之前的资助期间，我们证明了一种源自阿育吠陀药用植物（Withania somnifera）的小分子（withaferin A；以下简称WA）不仅可以在临床相关转基因小鼠模型（MMTV-neu）中预防ER阴性乳腺癌的发展，且没有任何毒性，而且还能在药理学剂量下抑制培养的人乳腺癌细胞中ER-�的表达和活性。在分子水平上，我们已经确定了WA的新机制靶标，包括微管蛋白（β-和β-微管蛋白的下调以及MCF-7细胞中β-微管蛋白的Cys-303的共价修饰）和线粒体电子传递链的复合物III（活性抑制），可能通过引发有丝分裂停滞和活性氧（ROS）依赖性细胞凋亡来促进其化学预防活性。 WA 的这些作用在代表主要亚型的乳腺癌细胞中观察到，包括 ER 阳性、ER 阴性和三阴性乳腺癌。更重要的是，与乳腺癌细胞相比，正常乳腺上皮细胞对 WA 靶向的微管蛋白和复合物 III 具有更强的抵抗力。尽管有这些令人兴奋的机制发现，微管蛋白下调或 Cys-303 修饰在 WA 生长停滞中的功能意义仍不清楚。同样，WA 介导的复合物 III 活性抑制的分子基础仍然难以捉摸。假设：目前的更新申请在逻辑上建立在这些新颖且大量发表的观察结果的基础上，以测试一个令人兴奋的假设，即WA给药可预防相关动物模型中的ER阳性和ER阴性（已在之前的资助期中显示）乳腺癌，与微管蛋白和复合物III亚基的翻译后修饰相关，导致癌细胞中有丝分裂停滞并最终选择性地凋亡细胞死亡。具体目标：本研究利用相关的乳腺癌细胞和体内动物模型以及尖端技术来：（1）在成熟的化学诱导（N-甲基-N-亚硝基脲）乳腺癌大鼠模型中确定膳食WA给药预防ER阳性乳腺癌的功效； (2) 研究微管蛋白下调和翻译后修饰在WA有丝分裂阻滞中的功能意义； (3)确定WA抑制复合物III活性的分子机制。转化影响：上一个资助期的进展超出了我们自己的预期，拟议工作的完成和出版以及西澳新机制目标的确定就证明了这一点。本次更新申请中提出的研究的转化影响最终通过以下方式实现：(a) 在新辅助窗口设置中合理设计生物标志物驱动的试点试验，这超出了本申请的范围，因为在没有充分了解 WA 分子药理学的情况下进行临床试验设计还为时过早； (b) 鉴定预测 WA 暴露和可能反应的机械生物标志物，这对其临床开发至关重要，因为原发性癌症发病率对终点要求太高； (c) 确定一种无毒方案，用于选择性地靶向癌细胞中的微管蛋白/微管网络，因为目前可用的抗有丝分裂药物（例如紫杉烷）具有副作用。在这个靶向治疗和个性化医疗的时代，微管蛋白/微管网络仍然是乳腺癌和其他癌症的有吸引力的治疗靶点。

DESCRIPTION (provided by applicant): Background: Chemoprevention of breast cancer, which continues to be a leading cause of cancer-related mortality among women globally, is feasible as exemplified by clinical success of selective estrogen receptor (ER) modulators (SERM) and aromatase inhibitors. However, the SERMs are ineffective against ER- negative breast cancers and have serious side effects. The aromatase inhibitors appear promising for prevention of breast cancer in high-risk postmenopausal women. Even though the safety of aromatase inhibitors in a preventative setting is still under study, their use raises concerns for increased risk of cardiovascular events and bone fracture. Therefore, a non-toxic preventive intervention effective against both ER-positive and ER-negative breast cancers is highly desirable. In the previous funding period, we demonstrated that a small-molecule (withaferin A; hereafter abbreviated as WA) derived from an Ayurvedic medicine plant (Withania somnifera) not only prevents ER-negative mammary cancer development in a clinically-relevant transgenic mouse model (MMTV-neu) without any toxicity, but also inhibits ER- � expression and activity at pharmacologic doses in cultured human breast cancer cells. At the molecular level, we have identified novel mechanistic targets of WA, including tubulin (downregulation of �- and �- tubulin as well as covalent modification of Cys-303 of � -tubulin in MCF-7 cells) and complex III of the mitochondrial electron transport chain (activity inhibition), potentially contributing to its chemopreventive activity by eliciting mitotic arrest and reactive oxygen species (ROS)-dependent apoptotic cell death. These effects of WA are observed in mammary cancer cells representing major subtypes, including ER-positive, ER-negative, and triple-negative breast cancer. More importantly, normal mammary epithelial cells are significantly more resistant to tubulin and complex III targeting by WA compared with breast cancer cells. Despite these exciting mechanistic findings, the functional significance of down-regulation or Cys-303 modification of tubulin in growth arrest by WA is still unclear. Likewise, the molecular basis for WA-mediated inhibition of complex III activity remains elusive. Hypothesis: The present renewal application logically builds upon these novel and largely published observations to test an exciting hypothesis that WA administration prevents both ER-positive and ER-negative (already shown in the previous funding period) breast cancers in relevant animal models in association with post-translational modifications of tubulin(s) and complex III subunit(s) leading to mitotic arrest and ultimately apoptotic cell death selectively in cancerous cells. Specific Aims: The proposed research utilizes relevant cellular and in vivo animal models of breast cancer and cutting-edge technologies to: (1) determine the efficacy of dietary WA administration for prevention of ER-positive mammary cancer in a well-established rat model of chemically-induced (N-methyl- N-nitrosourea) breast cancer; (2) study the functional significance of downregulation and post-translational modification of tubulins in mitotic arrest by WA; and (3) determine the molecular mechanism by which WA inhibits complex III activity. Translational Impact: The progress in the previous funding period exceeded our own expectations as evidenced by completion and publication of the proposed work as well as identification of novel mechanistic targets of WA. The translational impact of the studies proposed in this renewal application is ultimately realized by: (a) rational design of a pilot biomarker-driven trial in a neoadjuvant window setting, which is beyond the scope of this application because clinical trial design without a full appreciation of the molecular pharmacology of WA is premature; (b) identification of mechanistic biomarker(s) predictive of WA exposure, and possibly response, which is critical for its clinical development because primary cancer incidence is too demanding of an end point; and (c) identification of a non-toxic regimen for targeting tubulin/microtubule network selectively in cancer cells as currently available anti-mitotics (e.g., taxanes) have side effects. In this era of targeted therapies and personalized medicine, the tubulin/microtubule network still remains an attractive therapeutic target for breast and other cancers.