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: 8247814
• 负责人: DAVID J SHAPIRO
• 金额: $ 31.59万
• 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 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8247814
• 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 Delivery Systems; 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; Public Health; Purinergic P1 Receptors; Reporter Genes; Reporting; Resistance; Resistance development; Response Elements; Role; SP1 gene; Screening procedure; 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; 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. PUBLIC HEALTH RELEVANCE: Breast cancers that depend on estrogens bound to estrogen receptor for their growth eventually become resistant to drugs that target estrogen. We have identified new inhibitors that bypasses the site targeted by current drugs and instead target an essential action of the estrogen receptor. Further development of these inhibitors may lead to clinically useful drugs effective against tumors that are resistant to current therapies.

描述（由申请人提供）：结合雌激素受体（ER）配体结合口袋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。抗雌激素化合物他莫昔芬（Tamoxifen，Tam）被广泛用于治疗乳腺癌，但其长期使用受到限制，因为肿瘤最终会产生他莫昔芬耐药性。我们开发了一种高通量筛选策略来鉴定在ER结合其DNA反应元件的水平上靶向ER作用的小分子，而不是靶向雌激素结合的拮抗作用的传统方法。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依赖性的Tam敏感MCF-7细胞和三种Tam耐药乳腺癌细胞系的生长。但TPSF对ER阴性细胞的生长无影响。本提案的三个具体目标建立在我们最近对TPSF的识别基础上。(Aim 1）通过使用结构-活性关系来优化TPSF，以指导新化合物的合成，用于在使用TAM敏感性和TAM抗性乳腺癌细胞的基于细胞的测定中筛选和评估抑制剂功效、特异性和细胞内作用。(Aim 2）鉴定ER 1上与抑制剂相互作用的位点及其作用模式，以及（目的3）在TAM敏感性和TAM耐药性乳腺癌的小鼠异种移植模型中测试TPSF和从优化中出现的最佳抑制剂。测定和方法：（目的1）我们将在使用ER、AR和GR的基因表达测定中测试每种抑制剂的功效、效力和特异性，并检查不需要ER与DNA直接结合的ER作用的抑制。使用TAM敏感和TAM耐药乳腺癌细胞，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和重新敏感乳腺癌细胞杀死免疫细胞的能力。我们将在长期研究中测试对ERK通路和其他信号转导通路的核毒性作用。为了分析先导抑制剂对基因表达模式的影响，我们将在ER阳性乳腺癌细胞和非致瘤性乳腺细胞中进行微阵列分析。(Aim 2）为了在体外和细胞中评估EER与EER的相互作用，我们将使用含有大ER结构域的ER突变体，然后用较小的突变进行研究。将使用光亲和标记、游离ER和ER-抑制剂复合物的化学位移的NMR比较以及等温滴定量热法来评估抑制剂与ER的直接相互作用。如果可行，将进行ER结构域-抑制剂复合物的结构研究。我们将评估潜在的抑制剂对ER二聚化、降解、磷酸化的影响以及与已知拮抗剂的协同作用。(Aim 3）使用TAM敏感性和TAM抗性乳腺癌细胞，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乳腺癌中ER作用的强有力的新探针。公共卫生相关性：乳腺癌的生长依赖于与雌激素受体结合的雌激素，最终会对靶向雌激素的药物产生耐药性。我们已经确定了新的抑制剂，绕过目前药物靶向的位点，而是靶向雌激素受体的基本作用。这些抑制剂的进一步开发可能导致临床上有用的药物有效地对抗对当前疗法有抗性的肿瘤。