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

基本信息

批准号：
7655786
负责人：
DAVID J SHAPIRO
金额：
$ 35.77万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
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/7655786
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 Figs - dietary Fluorescein Fluoresceins Fluorescence Anisotropy Gene Expression Genes Genetic Transcription Growth Human Immune Immunoprecipitation In Vitro Label Lead Ligand Binding Link Longitudinal Studies MCF7 cell MDA MB 231 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 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 public health relevance receptor receptor binding research study response restoration scaffold small molecule stable cell line stoichiometry tumor tumor growth tumorigenic

项目摘要

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）：结合在雌激素受体（ER）配体结合袋中的雌激素和抗雌激素化合物在加深我们对 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 反应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 Tam 敏感的 MCF-7 细胞和三种 Tam 耐药乳腺癌细胞系的雌激素 ER 介导的基因表达和雌激素 ER 依赖性生长。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以我们最近确定的 TPSF 为基础。（目标 1）利用构效关系优化 TPSF，指导新化合物的合成，以在使用 Tam 敏感和 Tam 抗性乳腺癌细胞的细胞检测中筛选和评估抑制剂功效、特异性和细胞内作用。（目标 2）确定 ER1 上与抑制剂相互作用的位点及其作用模式，以及（目标 3）在 Tam 敏感和 Tam 抗性乳腺小鼠异种移植模型中测试 TPSF 以及优化后出现的最佳抑制剂癌症。测定和方法：（目标 1）我们将使用 ER、AR 和 GR 在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 抗性乳腺癌细胞，我们将评估该抑制剂改变内源基因表达、贴壁依赖性和贴壁非依赖性细胞生长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核外对 ERK 通路和其他信号转导通路的影响以及毒性。为了分析先导抑制剂对基因表达模式的影响，我们将对 ER 阳性乳腺癌细胞和非致瘤性乳腺癌细胞进行微阵列分析。（目标 2）为了在体外和细胞内评估抑制剂-ER 相互作用，我们将使用包含大 ER 结构域的 ER 突变体，然后使用较小的突变进行研究。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将用于评估抑制剂与 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。我们将评估抑制剂对 ER 二聚化、降解、磷酸化的潜在影响以及与已知拮抗剂的协同作用。（目标 3）使用 Tam 敏感和 Tam 抗性乳腺癌细胞，我们将评估抑制剂阻止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 ER 作用的强大新探针。公共健康相关性：依赖于与雌激素受体结合的雌激素生长的乳腺癌最终会对针对雌激素的药物产生耐药性。我们已经确定了新的抑制剂，可以绕过当前药物的靶向位点，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会产生临床上有用的药物，有效对抗对当前疗法耐药的肿瘤。