权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells

使用循环肿瘤细胞模拟转移和获得性耐药性

基本信息

批准号：
10655155
负责人：
Daniel A. Haber
金额：
$ 42万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10655155
关键词：
Address Binding Biopsy Blood Circulation Blood specimen Breast CRISPR screen CRISPR-mediated transcriptional activation Cell Culture Techniques Cell Line Cell Survival Cell model Cells Chemicals Chemistry Chromatin Clinical Treatment Clustered Regularly Interspaced Short Palindromic Repeats Cultured Tumor Cells Cysteine Data Dependence Drug Targeting Drug resistance ESR1 gene Endocrine Epigenetic Process Estrogen Receptor alpha Estrogen receptor positive Funding Genes Genomics Goals Heterogeneity In Vitro Intervention Invaded Knock-out Laboratories Libraries Ligands Malignant Neoplasms Mass Spectrum Analysis Microfluidic Microchips Microfluidics Modeling Molecular Mutation Neoplasm Circulating Cells Neoplasm Metastasis Oncogenic Outcome PIK3CA gene Pathway interactions Patients Pharmaceutical Preparations Phenotype Predisposition Proliferating Property Proteins Proteome Proteomics Refractory Resistance Resources Sampling Signal Transduction Source Technology Testing Tumor Cell Line Woman acquired drug resistance advanced breast cancer cancer cell cohort crosslink drug resistance development epigenetic regulation functional genomics genome-wide hormone receptor-positive hormone therapy in vivo individual patient inhibitor malignant breast neoplasm neoplastic cell new therapeutic target preservation prevent protein expression restoration screening tool tumor tumorigenic

项目摘要

PROJECT SUMMARY Advanced hormone receptor positive (HR+) breast cancers, the most common subtype, are initially responsive to multiple endocrine interventions, but they ultimately develop drug resistance. Circulating tumor cells (CTCs) underlie the blood-borne metastatic spread of cancer, and they also provide a noninvasive source to sample tumor cells during the course of therapy and acquired resistance. Using a microfluidic enrichment technology that preserves CTC viability, we successfully established a cohort of 15 CTC-derived longterm cultures from women with refractory HR+ breast cancer. A subset of these shows the expected acquired ESR1 and PIK3CA mutations, confirmed in matched metastatic tumor biopsies. However, the most common, and least well understood, correlate of endocrine resistance is loss of estrogen receptor-α (ER) expression by cancer cells. In these cases, cultured CTCs recapitulate ER silencing, and some are biphenotypic, with coexisting ER+ and ER- subpopulations derived from the same patient. Single cell-derived colonies show that ER+ CTCs can produce ER- progeny, pointing to likely epigenetic mechanisms and underlying cell plasticity. We propose to investigate the epigenetic regulation of ER expression loss and its potential restoration, using genomic analyses combined with CRISPR functional screens. In Aim 1, we will compare chromatin landscapes of isogenic ER+ and ER- CTC subpopulations, define their distinct functional properties and identify factors that modulate their interconversion in vitro. In Aim 2, we will use undertake CRISPR screens to identify genes capable of restoring ER expression to CTC lines from HR+ breast cancers that have lost endogenous ER expression following endocrine therapy. Preliminary data indicate the feasibility of this approach, and we will explore mechanisms underlying restoration of ER expression, and whether this is accompanied by a return to ER-dependent proliferation. Together, these Aims address the plasticity of ER expression in patient-derived cultured CTCs that recapitulate clinical treatment exposures and tumor adaptation mechanisms. Loss of ER expression in HR + breast cancer may also lead to dependence on alternative oncogenic drivers, that may be constitute drug targets in refractory HR+ breast cancer. In Aim 3, we apply a chemical proteomic strategy in CTC lines, combining cysteine cross-linking with mass spectrometry to identify all ligandable cysteine residues within cellular proteins. Preliminary data show multiple cysteine-targetable proteins in CTCs that are not present in untreated breast cancer lines, and we will use CRISPR screens to identify those required for proliferation, followed by identification of tool compounds from a specialized library of cysteine-reactive covalent inhibitors for functional analyses. Together, these complementary approaches address the loss of ER expression in advanced HR+ breast cancers, with the goal of overcoming acquired resistance to endocrine therapy.

项目总结晚期激素受体阳性(HR+)乳腺癌是最常见的亚型，包括最初对多种内分泌干预有反应，但最终产生抗药性。循环中的肿瘤细胞(CTCs)是癌症通过血液传播转移的基础，它们还提供了一种非侵入性来源，在治疗过程中和获得性抵抗。使用保持CTC活性的微流控浓缩技术，我们成功地从患有难治性HR+乳房的妇女中建立了15个来自CTC的长期培养的队列癌症。其中一个子集显示了预期获得的ESR1和PIK3CA突变，已在与转移性肿瘤活检结果相匹配。然而，最常见、也最不为人所知的是内分泌耐药是指癌细胞失去雌激素受体α(ER)的表达。在这些情况下，培养的CTCs概括为ER沉默，有些是双表型，ER+和ER-共存来自同一患者的亚群。单细胞来源的克隆表明ER+CTCs可以产生内质网后代，指向可能的表观遗传机制和潜在的细胞可塑性。我们建议研究ER表达缺失的表观遗传调控及其潜在的恢复，使用基因组分析和CRISPR功能筛查相结合。在目标1中，我们将比较等位基因ER+和ER-CTC亚群的染色质景观，定义了它们不同的功能性质并确定在体外调节它们相互转化的因素。在目标2中，我们将使用Engage CRISPR筛选能够恢复HR+乳腺CTC细胞ER表达的基因内分泌治疗后失去内源性ER表达的癌症。初步数据指出这种方法的可行性，我们将探索潜在的修复机制 ER的表达，以及这是否伴随着ER依赖的增殖的回归。一起，这些目的是解决患者来源的CTCs中ER表达的可塑性概述临床治疗暴露和肿瘤适应机制。高血压大鼠雌激素受体表达缺失 +乳腺癌还可能导致对替代致癌驱动因素的依赖，这可能构成难治性HR+乳腺癌的药物靶点。在目标3中，我们在CTC中应用了化学蛋白质组学策略品系，结合半胱氨酸交联和质谱学来鉴定所有可连接的半胱氨酸残基在细胞蛋白质中。初步数据显示CTCs中存在多个半胱氨酸靶向蛋白存在于未经治疗的乳腺癌株系中，我们将使用CRISPR筛查来确定哪些是增殖，然后从半胱氨酸反应的专门文库中鉴定工具化合物用于功能分析的共价抑制剂。这些互补的方法共同解决了这一损失 ER在晚期HR+乳腺癌中的表达，目的是克服获得性耐药内分泌治疗。