Targeting Breast Cancer with Small Molecule Inhibitors of Estrogen Receptor

描述（由申请人提供）：结合在雌激素受体（ER）配体结合口袋中的雌激素和抗雌激素化合物在发展我们对ER作用的理解中发挥了关键作用。抗雌激素化合物他莫昔芬（Tam）广泛用于治疗乳腺癌，但其长期使用受到限制，因为肿瘤最终会产生他莫昔芬耐药性。我们开发了一种高通量筛选策略，以识别在ER与其DNA应答元件结合水平上靶向ER作用的小分子，而不是传统的靶向雌激素结合拮抗的方法。我们的主要ER抑制剂TPSF有效且特异性地抑制雌激素ER介导的基因表达和雌激素ER依赖的tam敏感MCF-7细胞和三种tam耐药乳腺癌细胞系的生长。然而，TPSF对ER阴性细胞的生长没有影响。该提案的三个具体目标建立在我们最近确定的TPSF的基础上。（目的1）利用结构-活性关系来优化TPSF，以指导新化合物的合成，以筛选和评估抑制剂的功效、特异性和细胞内作用，在基于细胞的检测中使用tam敏感和tam耐药的乳腺癌细胞。（目标2）确定ER1上与抑制剂相互作用的位点及其作用模式；（目标3）在tam敏感和tam耐药的小鼠异种移植乳腺癌模型中测试TPSF，以及优化后出现的最佳抑制剂。实验和方法：（目标1）我们将使用ER、AR和GR测试每种抑制剂在基因表达分析中的功效、效力和特异性，并检查不需要将ER与DNA直接结合的ER作用的抑制作用。使用tam敏感和tam耐药的乳腺癌细胞，我们将评估抑制剂改变内源性基因表达、锚定依赖性和锚定非依赖性细胞生长的能力，以及使乳腺癌细胞对免疫细胞的杀伤再敏感的能力。我们将在长期研究中测试ERK途径和其他信号转导途径的核外效应以及毒性。为了分析铅抑制剂对基因表达模式的影响，我们将在ER阳性乳腺癌细胞和非致瘤性乳腺细胞中进行微阵列分析。（目的2）为了在体外和细胞中评估抑制剂-内质网相互作用，我们将使用含有较大内质网结构域的内质网突变体，然后用较小的突变体进行研究。光亲和标记、游离内质网和内质网抑制剂配合物化学位移的核磁共振比较以及等温滴定量热法将用于评估抑制剂与内质网的直接相互作用。如果可行，将进行ER结构域抑制剂配合物的结构研究。我们将评估潜在的抑制剂对内质网二聚化、降解、磷酸化和与已知拮抗剂的协同作用的影响。（目的3）使用tam敏感和tam耐药的乳腺癌细胞，我们将评估抑制剂阻断肿瘤生长或诱导肿瘤消退的能力。这些小分子抑制剂是研究雌激素受体在乳腺癌中的作用的强有力的新探针。

DESCRIPTION (provided by applicant): Estrogenic and antiestrogenic compounds that bind in the estrogen receptor (ER) ligand binding pocket have played key roles in developing our understanding of ER action. The antiestrogenic compound, tamoxifen (Tam), is used extensively to treat breast cancer, but its long-term use is limited because tumors eventually develop tamoxifen resistance. We developed a high throughput screening strategy to identify small molecules that target ER action at the level of ER binding to its DNA response element, rather than the traditional approach of targeting antagonism of estrogen binding. Our lead ER inhibitor, TPSF, potently and specifically inhibits estrogen-ER-mediated gene expression and estrogen-ER-dependent growth of Tam-sensitive MCF-7 cells and three Tam-resistant breast cancer cell lines. However, TPSF has no effect on growth of ER negative cells. The three Specific Aims of this proposal build on our recent identification of TPSF. (Aim 1) Optimize TPSF by using structure-activity relationships to guide synthesis of new compounds for screening and assess inhibitor efficacy, specificity and intracellular actions in cell-based assays using Tam-sensitive and Tam-resistant breast cancer cells. (Aim 2) Identify the site on ER1 that interacts with the inhibitors and their mode of action, and (Aim 3) Test TPSF, and the best inhibitor to emerge from optimization, in mouse xenograft models of Tam-sensitive and Tam-resistant breast cancer. Assays and methods: (Aim 1) We will test the efficacy, potency and specificity of each inhibitor in gene expression assays using ER, AR and GR and examine inhibition of ER actions that do not require direct binding of ER to DNA. Using Tam-sensitive and Tam-resistant breast cancer cells, we will evaluate the inhibitor's ability to alter endogenous gene expression, anchorage-dependent and anchorage-independent cell growth and to re-sensitize breast cancer cells to killing by immune cells. We will test for extranuclear effects on the ERK pathway and other signal transduction pathways and for toxicity in long-term studies. To analyze the lead inhibitor's effects on gene expression patterns, we will perform microarray analysis in ER positive breast cancer cells and in non-tumorigenic breast cells. (Aim 2) To evaluate inhibitor-ER interaction in vitro and in cells, we will use ER mutants containing large ER domains and then do studies with smaller mutations. Photoaffinity labeling, NMR comparison of chemical shifts of free ER and ER-inhibitor complexes, and isothermal titration calorimetry will be used to assess direct interaction of the inhibitors with ER. If feasible, structural studies of ER domain-inhibitor complexes will be performed. We will evaluate potential inhibitor effects on ER dimerization, degradation, phosphorylation and synergy with known antagonists. (Aim 3) Using Tam-sensitive and Tam-resistant breast cancer cells, we will assess the ability of the inhibitors to block tumor growth or induce tumor regression. These small molecule inhibitors are powerful new probes for ER action in breast cancer.