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，对乳腺癌高风险女性的长期治疗只能将浸润性和非浸润性ER+乳腺癌的发病率降低约50%，同时也会适度增加子宫内膜癌的风险。乳腺肿瘤的生长也可能依赖于这种药物。因此，它莫西芬还有很大的改进空间。他莫昔芬的临床局限性体现在乳腺和子宫内膜异种肿瘤移植模型中。了解他莫昔芬的不同分子机制对改进其降低乳腺肿瘤发病率和复发率的作用是必要的。雌激素（E2）作用的一个值得注意的未充分研究的方面是基因抑制，这通常是由他莫昔芬（去抑制）预防的。我们已经建立了一种新的非经典机制，E2直接抑制基因，其中ER形成tafii30相关的协同抑制复合物；他莫昔芬通过简单地解离复合体来防止这种抑制。我们已经在多种模型启动子和基因中鉴定了模仿他莫西芬的合成内质网配体；然而，这些化合物与他莫昔芬的不同之处在于，就上述直接基因抑制机制而言，它们的行为与E2类似。值得注意的是，新化合物不仅阻断e2刺激的MCF-7乳腺肿瘤细胞增殖，而且对体内培养的耐他莫昔芬MCF-7细胞具有抗增殖作用；与他莫昔芬相比，它们还能阻断e2刺激的石川子宫内膜癌细胞的生长。这些化合物不影响er阴性细胞的生长。研究结果提出了他莫昔芬对不同基因去抑制机制的生理关联，特别是他莫昔芬对乳腺和子宫内膜肿瘤发生和生长的影响。我们假设，他莫昔芬的基因去抑制提供了独特的机制，可以为识别具有优越治疗效果的内质网拮抗剂的机制类别提供基础。换句话说，他莫昔芬的去抑制某些基因的特性，其本身或与其他作用相结合，可能有助于创造一个条件，允许不同的生存/增殖机制的开始或成功，从而限制药物在某些细胞环境中的有益作用；在某些内质网拮抗剂中取消这种机制可能会间接抑制细胞存活/增殖，从而增加肿瘤抑制的持续时间。我们已经确定的内质网拮抗剂（以及我们希望在未来确定的内质网拮抗剂）是解决这一假设的绝佳工具，因为它们具有与基因抑制/去抑制相关的共同机制差异。由于这些化合物与他莫昔芬在基因去抑制谱方面的预期差异相对较小，因此使用生物统计分析结合多种基因敲低方法，最终将他莫昔芬去抑制的E2靶点与药物的特定生理效应联系起来，甚至可能是可行的。无论结果如何，此时启动系统研究来阐明他莫昔芬基因组作用的新方面和未研究方面可能影响的性质和程度是必要和可行的。本文提出的基于机制的化学生物学方法是及时的，因为大量的部分表征的内质网配体的可用性，其分类是有限的，很大程度上是经验主义的。目的1：进一步研究他莫昔芬对基因去抑制的分子机制，并确定代表每种机制的其他模式基因。目标2：继续识别新的内质网拮抗剂机制类别；比较他莫昔芬和新化合物对体外ER+乳腺癌和子宫内膜癌细胞系的细胞效应；确定不同的基因抑制模式，并试图确定关键的基因目标。目的3：在异种移植模型中，使用ER+人类肿瘤细胞，对他莫昔芬和选定的新试剂在乳腺和子宫内膜肿瘤的抑制和潜伏期方面的作用进行比较评估，这些模型先前为他莫昔芬提供了最具预测性的临床信息，并检查了子宫效应。公共卫生相关性：他莫昔芬是抑制乳腺癌生长和降低发病率/复发率的主要药物。为了改进这种药物，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，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他莫昔芬的作用机制，并在此过程中找到性能优于他莫昔芬的先导化合物。

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.