Targeting Breast Cancer with Small Molecule Inhibitors of Estrogen Receptor

用雌激素受体小分子抑制剂治疗乳腺癌

• 批准号: 8448699
• 负责人: DAVID J SHAPIRO
• 金额: $ 30.42万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448699
• 关键词: American; Amino Acids; Anchorage-Independent Growth; Apoptosis; Aromatase Inhibitors; Binding; Biological Assay; Breast; Breast Cancer Cell; Bypass; C-terminal; Calorimetry; Cancer cell line; Cells; Chemicals; Chimera organism; Collaborations; Collection; Complex; Consensus; Crystallography; DNA; DNA Binding Domain; Detection; Development; Dimerization; Dissociation; Dose; Drug Targeting; Drug resistance; Effectiveness; Estradiol; Estrogen Antagonists; Estrogen Receptors; Estrogen receptor negative; Estrogen receptor positive; Estrogens; Fluorescein; Fluorescence Anisotropy; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Growth; Health; Human; Immune; Immunoprecipitation; In Vitro; Label; Lead; Ligand Binding; Link; Longitudinal Studies; MAPK3 gene; MCF7 cell; Malignant Neoplasms; Mammary Gland Parenchyma; Mediating; Methods; Microarray Analysis; Modeling; Mus; Mutation; Natural Killer Cells; Nucleic Acid Regulatory Sequences; Nude Mice; Pathway interactions; Pattern; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Photoaffinity Labels; Play; Point Mutation; Pre-Clinical Model; Production; Proteins; Purinergic P1 Receptors; Reporter Genes; Reporting; Resistance; Resistance development; Response Elements; Role; SP1 gene; Selective Estrogen Receptor Modulators; Signal Transduction Pathway; Site; Specificity; Structure; Structure-Activity Relationship; Tamoxifen; Testing; Titrations; Toxic effect; Toxicity Tests; Transcription Factor AP-1; Transfection; VP 16; Western Blotting; Work; Xenograft Model; base; cell growth; chromatin immunoprecipitation; efficacy testing; follow-up; high throughput screening; hormone therapy; improved; inhibitor/antagonist; killings; malignant breast neoplasm; member; mouse model; mutant; neoplastic cell; receptor; receptor binding; research study; response; restoration; scaffold; screening; small molecule; stable cell line; stoichiometry; tumor; tumor growth

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.

描述(由申请人提供)：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作用的理解方面发挥了关键作用。抗雌激素化合物(Tamoxifen)被广泛用于治疗乳腺癌，但其长期使用受到限制，因为肿瘤最终会对他莫昔芬产生抗药性。我们开发了一种高通量筛选策略，以在ER与其DNA反应元件结合的水平上识别靶向ER作用的小分子，而不是传统的靶向雌激素结合拮抗作用的方法。我们的先导ER抑制剂TPSF有效且特异地抑制了敏感的MCF-7细胞和三种耐药的乳腺癌细胞株的雌激素-ER介导的基因表达和雌激素依赖的生长。但三七总皂甙对ER阴性细胞的生长无明显影响。这项建议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)利用构效关系来优化TPSF，以指导新化合物的合成，以筛选和评估以敏感和耐药的乳腺癌细胞为基础的细胞检测中的抑制剂有效性、特异性和细胞内作用。(目的2)确定ER1上与抑制剂相互作用的部位及其作用方式，(目的3)在敏感和耐药的小鼠异种移植瘤模型中，检测TPSF，以及优化后出现的最佳抑制剂。方法和方法：(目的1)我们将在使用ER、AR和GR的基因表达分析中测试每种抑制剂的有效性、效力和特异性，并检查对不需要ER与DNA直接结合的ER作用的抑制。使用敏感和耐药的乳腺癌细胞，我们将评估该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杀伤重新敏感的能力。我们将在长期研究中测试ERK通路和其他信号转导通路的核外效应以及毒性。为了分析先导抑制剂对基因表达模式的影响，我们将在ER阳性的乳腺癌细胞和非致瘤的乳腺细胞中进行微阵列分析。(目的2)为了评估抑制剂-ER在体外和细胞内的相互作用，我们将使用包含大ER结构域的ER突变体，然后用较小的突变进行研究。光亲和标记法、核磁共振法比较游离ER和ER抑制剂复合体的化学位移，以及等温滴定热法将被用来评估抑制剂与ER的直接相互作用。如果可行，将进行ER结构域-抑制剂复合体的结构研究。我们将评估潜在的抑制剂对内质网二聚化、降解、磷酸化以及与已知拮抗剂的协同作用的影响。(目的3)利用敏感和耐药的乳腺癌细胞，评价其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ER在乳腺癌中作用的强有力的新探针。