Dissecting the Mechanisms of Tamoxifen Action

描述(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，对乳腺癌高危女性进行长期治疗，侵袭性和非侵袭性ER+乳腺癌的发病率仅降低约50%，并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，与他莫昔芬相比，还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。了解他莫昔芬的不同分子机制对于改进该药在降低乳腺癌发病率和复发率方面是必要的。雌激素(E2)作用的一个明显未被研究的方面是基因抑制，这通常是由他莫昔芬(去抑制)防止的。我们已经建立了一种新的非经典的E2直接基因抑制机制，其中ER形成一个与TAFII30相关的共抑制物复合体；他莫昔芬通过简单地解离该复合体来防止这种抑制。我们已经在各种模式启动子和基因中发现了模拟他莫昔芬的人工合成ER配体；然而，这些化合物与他莫昔芬的不同之处在于，它们在上述直接基因抑制机制方面的行为类似于E2。值得注意的是，新化合物不仅抑制了E2刺激的MCF-7乳腺癌细胞的增殖，而且在体内培养的耐他莫昔芬的MCF-7细胞中也具有抗增殖作用；与他莫昔芬相比，它们还阻止了E2刺激的石川子宫癌细胞的生长。这些化合物不影响ER阴性细胞的生长。这些发现提出了一个问题，即他莫昔芬下调基因抑制的不同机制可能存在什么生理关联，特别是与他莫昔芬对乳腺和子宫内膜肿瘤的发病率和生长的影响有关。我们推测，他莫昔芬对基因的抑制提供了独特的机制，可以为识别乳腺癌治疗效果更好的ER拮抗剂的机制类别提供基础。换句话说，他莫昔芬解除抑制某些基因的特性本身或通过与其他效应相结合，可能有助于创造一种条件，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ER拮抗剂中取消这种机制(S)可能具有间接抑制细胞生存/增殖的净效应，从而延长肿瘤抑制的持续时间。我们已经确定的ER拮抗剂(以及我们希望在未来发现的那些)是解决这一假说的优秀工具，因为它们在基因抑制/去抑制方面有共同的机制差异。由于化合物和他莫昔芬在基因抑制谱上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除方法最终将一组被他莫昔芬解除抑制的E2靶标与药物的特定生理效应联系起来甚至是可行的。无论结果如何，此时启动系统研究是必要和可行的，以阐明他莫昔芬基因组作用的新的和未被研究的方面可能产生的影响的性质和程度。这里提出的基于机理的化学生物学方法是及时的，因为可以获得大量部分表征的ER配体，其分类一直是有限的，并且在很大程度上是经验性的。目的1：进一步研究他莫昔芬下调基因表达的分子机制，并寻找代表每种机制的其他模型基因。目的2：继续鉴定ER拮抗剂的新机制类别；比较他莫昔芬和新化合物在体外对ER+乳腺癌细胞和子宫内膜癌细胞的细胞作用；确定差异基因抑制模式，并尝试确定关键基因靶点。目的：利用ER+的人肿瘤细胞建立的异种移植模型，比较评价他莫昔芬和选定的新试剂对乳腺和子宫内膜肿瘤的抑制作用和潜伏期，并检查亲子宫作用。

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.