Dissecting the Mechanisms of Tamoxifen Action

描述（由申请人提供）：他莫昔芬是治疗雌激素受体（ER）阳性乳腺癌的主要药物。然而，对乳腺癌高危女性的长期治疗仅使侵袭性和非侵袭性ER+乳腺癌的发病率降低约50%，并适度增加子宫内膜癌的风险。乳腺肿瘤也可能依赖于药物生长。因此，与他莫昔芬相比有相当大的改进空间。他莫昔芬的临床局限性反映在乳腺和子宫内膜肿瘤异种移植模型中。了解他莫昔芬不同的分子机制方面是必要的，以改善这种药物在降低乳腺肿瘤的发病率和复发。雌激素（E2）作用的一个显著不足的方面是基因抑制，它经常被他莫昔芬（去抑制）阻止。我们已经建立了一种新的非经典的机制，直接基因阻遏E2在ER形成一个TAFII30相关的共阻遏复合物;他莫昔芬防止这种镇压简单地解离复合物。我们已经确定了合成的ER配体，模拟他莫昔芬在各种模型启动子和基因，然而，这些化合物不同于他莫昔芬，因为它们的行为就像E2的上述机制的直接基因抑制。值得注意的是，新化合物不仅阻断了E2刺激的MCF-7乳腺肿瘤细胞增殖，而且在体内开发的他莫昔芬耐药MCF-7细胞中也具有抗增殖作用;与他莫昔芬相比，它们还阻断了E2刺激的石川子宫内膜癌细胞的生长。化合物不影响ER阴性细胞的生长。这些发现提出了一个问题，即他莫昔芬对基因去抑制的不同机制的生理相关性可能是什么，特别是他莫昔芬对乳腺和子宫内膜肿瘤的发病率和生长的影响。我们假设，他莫昔芬的基因去抑制提供了独特的机制，可以提供识别的机制类别的ER拮抗剂在乳腺癌的上级治疗效果的基础。换句话说，他莫昔芬去抑制某些基因的特性本身或通过与其他效应的关联，可以帮助创造允许不同存活/增殖机制的开始或成功的条件，从而限制药物在某些细胞环境中的有益作用;在某些ER拮抗剂中消除这种机制可能具有间接抑制细胞存活/增殖的净效应，从而增加肿瘤抑制的持续时间。我们已经鉴定的ER拮抗剂（以及我们预期将来鉴定的那些）是解决该假设的极好工具，因为它们具有与基因阻遏/去阻遏相关的共同机制差异。由于化合物与他莫昔芬的基因去阻遏谱的预期差异相对较小，因此使用生物统计分析结合多基因敲低方法来最终将一组被他莫昔芬去阻遏的E2靶标与药物的特定生理效应相关联甚至是可行的。无论结果如何，在这个时候启动系统性研究以阐明他莫昔芬基因组作用的新的和未调查的方面的可能影响的性质和程度是必要的和可行的。这里提出的机制为基础的化学生物学方法是及时的，因为大量收集的部分特征的ER配体，其分类一直是有限的，主要是经验的可用性。目的1：进一步研究三苯氧胺对基因去阻遏作用的分子机制，并确定代表每种机制的其他模型基因。目标二：继续确定新的机制类的ER拮抗剂;比较他莫昔芬和新化合物在ER+乳腺癌和子宫内膜癌细胞系的细胞效应在体外;确定差异基因抑制模式，并试图确定关键的基因靶点。目标3：在异种移植模型中使用ER+人肿瘤细胞，对他莫昔芬和选定的新试剂在乳腺和子宫内膜肿瘤抑制和潜伏期方面的作用进行比较评价，这些模型先前提供了他莫昔芬最具预测性的临床信息，并检查了促子宫效应。公共卫生相关性：药物他莫昔芬是抑制乳腺癌生长和降低乳腺癌发病率/复发的主要药物。为了改善这种药物，有必要了解其作用机制的各个方面。我们已经发现了他莫昔芬分子作用的一个新方面，在适当的条件下，它可以通过抵消其自身抑制肿瘤生长的能力来限制药物的有效性。我们建议进一步研究这一点和其他机制的他莫昔芬行动和在这个过程中，以确定领先的化合物，其性能是上级的他莫昔芬。

DESCRIPTION (provided by applicant): Tamoxifen is a mainstay in the treatment of estrogen receptor (ER)-positive breast cancer. However, long-term treatment of women at high risk for breast cancer reduces the incidence of both invasive and non-invasive ER+ breast cancer by only about 50 percent and also modestly increases the risk of endometrial cancer. Breast tumors could also become dependent on the drug for growth. Therefore, there is considerable room for improvement over tamoxifen. The clinical limitation of tamoxifen is reflected in breast and endometrial tumor xenograft models. Understanding different molecular mechanistic aspects of tamoxifen is necessary for improving upon this drug in decreasing the incidence and recurrence of breast tumors. A remarkably under-investigated aspect of estrogen (E2) action is gene repression which is frequently prevented by tamoxifen (de-repression). We have established a novel non-classical mechanism for direct gene repression by E2 in which ER forms a TAFII30-associated co-repressor complex; tamoxifen prevents this repression by simply dissociating the complex. We have identified synthetic ER ligands that mimic tamoxifen in a variety of model promoters and genes; however, these compounds differ from tamoxifen in that they behave like E2 with respect to the above mechanism of direct gene repression. Remarkably, the new compounds not only blocked E2-stimulated MCF-7 breast tumor cell proliferation but were also antiproliferative in tamoxifen-resistant MCF-7 cells developed in vivo; they also blocked E2-stimulated growth of Ishikawa uterine endometrial cancer cells in contrast to tamoxifen. The compounds did not affect the growth of ER-negative cells. The findings beg the question of what the physiological correlates of different mechanisms of gene de- repression by tamoxifen might be, particularly in relation to the effects of tamoxifen on the incidence and growth of breast and endometrial tumors. We hypothesize that gene de-repression by tamoxifen offers distinctive mechanisms that can provide the basis for identification of mechanistic classes of ER antagonists with superior therapeutic effects in breast cancer. In other words, tamoxifen's property of de-repressing certain genes may, in itself or by association with other effects, help in creating a condition that would be permissive to the onset or success of different survival/proliferation mechanisms, thereby constraining the drug's beneficial effects in certain cell contexts; the abrogation of such a mechanism(s) in certain ER antagonists may have a net effect of indirectly suppressing cell survival/proliferation, thereby increasing the duration of tumor suppression. The ER antagonists that we have identified (as well as those we expect to identify in the future) are excellent tools to address the hypothesis since they share a common mechanistic difference related to gene repression/de- repression. Since the expected differences in gene de-repression profiles of the compounds vs. tamoxifen are relatively small, it may even be feasible to use bio-statistical analysis coupled with multiple gene knockdown approaches to ultimately relate a group of E2 targets de-repressed by tamoxifen to specific physiological effects of the drug. Regardless of the outcome, it is necessary and feasible at this time to initiate systematic studies to shed light on the nature and extent of the possible impacts of novel and uninvestigated aspects of the genomic action of tamoxifen. The mechanism-based chemical biology approach proposed here is timely because of the availability of a large collection of partially characterized ER ligands, whose classification has been limited and largely empiric. Aim 1: Further investigate molecular mechanisms of gene de-repression by tamoxifen and identify additional model genes to represent each mechanism. Aim 2: Continue to identify new mechanistic classes of ER antagonists; compare tamoxifen and the new compounds with respect to cellular effects in ER+ breast and endometrial cancer cell lines in vitro; determine differential gene repression patterns and attempt to identify critical gene targets. Aim 3: Undertake a comparative evaluation of the effects of tamoxifen and selected new reagents in relation to inhibition and latency of breast and endometrial tumors using ER+ human tumor cells in xenograft models that have previously provided the most predictive clinical information for tamoxifen and also examine uterotropic effects. PUBLIC HEALTH RELEVANCE: The drug, tamoxifen is a mainstay in suppressing the growth and decreasing the incidence/recurrence of breast cancer. To improve upon this drug, it is necessary to understand all aspects of its mechanism of action. We have discovered a new aspect of tamoxifen's molecular actions which, under the appropriate conditions, could limit the drug's effectiveness by counteracting its own ability to inhibit tumor growth. We propose to further investigate this and other mechanisms of tamoxifen action and in the process, to identify lead compounds whose properties are superior to that of tamoxifen.