Metabolic mechanisms of antiestrogen resistance in breast cancer

乳腺癌抗雌激素抵抗的代谢机制

• 批准号: 9128565
• 负责人: Ubaldo Martinez Outschoorn
• 金额: $ 16.54万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-12 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9128565
• 关键词: Acetylcysteine; Aftercare; Antiestrogen Therapy; Antioxidants; Apoptosis; Biological Markers; Breast Cancer Cell; Cancer Center; Catabolism; Cell Culture Techniques; Cell Line; Cell model; Cells; Clinical Research; Clinical Trials; Clinical Trials Design; Coculture Techniques; Core Facility; Coupled; Coupling; Development Plans; Drug Targeting; Drug resistance; Educational workshop; Emergency Department patient; Enrollment; Ensure; Epigallocatechin Gallate; Epithelial; Epithelial Cells; Estrogen Antagonists; Estrogen receptor positive; Fibroblasts; Foundations; Fulvestrant; Gene Expression; Gene Expression Profiling; Generations; Genes; Glycolysis; Goals; Health; Human; Immunohistochemistry; In Vitro; Knowledge; Laboratories; Laboratory Research; Lead; Leadership; Learning; Link; MCF7 cell; Malignant Epithelial Cell; Malignant Neoplasms; Measurement; Measures; Mentors; Mentorship; Metabolic; Metabolism; Mitochondria; Modeling; Molecular Biology Techniques; NCI-Designated Cancer Center; Nuclear; Oxidative Phosphorylation; Oxidative Stress; Pathway interactions; Patients; Pharmaceutical Preparations; Physicians; Progression-Free Survivals; Proteins; Public Health; Reactive Oxygen Species; Recurrence; Recurrent disease; Refractory; Refractory Disease; Relapse; Research; Research Personnel; Resistance; Respiration; Risk Factors; Role; Scientist; Selective Estrogen Receptor Modulators; Signal Pathway; Signal Transduction; Staining method; Stains; Stromal Cells; Subgroup; T47D; Tamoxifen; Testing; Tissues; Up-Regulation; Work; Xenograft Model; base; cancer cell; career; career development; caveolin 1; cell growth; cohort; design; drug development; drug testing; follow-up; genetically modified cells; glucose uptake; improved; malignant breast neoplasm; meetings; mitochondrial metabolism; mortality; overexpression; predictive marker; programs; protein expression; skills; treatment strategy; tumor; tumor metabolism; tumor microenvironment; uncoupling protein 1; uptake

DESCRIPTION (provided by applicant): My long term career goal is to become an independent physician scientist who focuses on drug resistance in breast cancer and combines laboratory based mechanistic research and clinical trial design and implementation. My current efforts are devoted to studying how to overcome antiestrogen resistance by modulating epithelial stromal metabolic interactions in breast cancer utilizing molecular biology techniques. My mentor Dr. Richard Pestell has expertise in breast cancer metabolism and the tumor microenvironment. My co-mentor Dr. Scott Waldman has expertise in clinical trial design and implementation. The Kimmel Cancer Center where I work is an NCI designated Cancer Center with a well-established and vibrant research program and multiple core facilities that will allow me to carry out this proposal. A career development plan based on experimental work in Dr. Pestell's lab, twice weekly interactions with Dr. Pestell, and weekly with Dr. Waldman as well as mentorship committee meetings every two months and participation in workshops and seminars is being implemented to learn technical and leadership skills. Relapsed or refractory cancer after antiestrogen therapy defines antiestrogen resistance clinically and this is a major public health issue. Antiestrogen resistance occurs in 40% of ER+ patients and it is most often fatal. We lack good biomarkers and treatments for antiestrogen resistance. It has recently been discovered that metabolic coupling with high mitochondrial metabolism in epithelial cells with low metabolism in the stroma is associated with antiestrogen resistance. We have recently demonstrated that a tumor stroma with increased reactive oxygen species (ROS), low oxidative phosphorylation metabolism (OXPHOS) and high glycolysis is found in aggressive breast cancers. This type of stromal metabolism leads to metabolic coupling and transfer of high energy catabolites to the epithelial cancer cells and is associated with antiestrogen resistance. My overall hypothesis is that metabolic coupling drives antiestrogen resistance and reversal of epithelial-stromal metabolic coupling will overcome antiestrogen resistance in breast cancer. The project aims are: i) To test the hypothesis that OXPHOS metabolic coupling is sufficient to induce antiestrogen resistance in breast cancer. I will use an in vitro stromal-epithelial cell model of estrogen receptor positive (ER+) breast cancer. I will genetically modify cells in order to generate tight epithelial-stromal metabolic coupling with epithelial cancer cells with high OXPHOS metabolism via upregulation of monocarboxylate transporter 1 (MCT1), nuclear respiration factor 1 (NRF1) and mitoNEET and stromal cells with low OXPHOS metabolism and high catabolism via upregulation of monocarboxylate transporter 4 (MCT4) and uncoupling protein 1 (UCP1) to determine if changes in the metabolism of the epithelial or stromal compartment are sufficient to increase antiestrogen resistance. These cell lines that I generate will be cultured with either fibroblasts or ER+ carcinoma cells. Antiestrogen resistance will be measured by quantifying apoptosis and proliferation of the breast cancer cells after treatment with tamoxifen and fulvestrant. We will also determine if these cell lines induce antiestrogen resistance using xenograft models. ii) To test the hypothesis that expression of genes linked to OXPHOS metabolic coupling are associated with antiestrogen resistance in a cohort of patients. I will stain a human tumor microarray (TMA) of patients with ER+ breast cancer treated with tamoxifen for the proteins listed in aim 1. I will correlate the expression of these proteins by immunohistochemistry (IHC) in the stromal and epithelial compartments with progression free survival (PFS). We will also perform gene expression profiling (GEP) of the carcinoma cells that I generate to determine if we can generate a signature that predicts antiestrogen resistance. iii) To test the hypothesis that drugs that modulate OXPHOS, glycolysis or reactive oxygen species will overcome antiestrogen resistance. I will use our epithelial-stromal coculture models of antiestrogen resistant breast cancer, including the genetically modified cells generated for aim 1 to determine if drugs that metabolically uncouple epithelial and stromal cells can overcome antiestrogen resistance. Specifically, I will test drugs that increase or decrease OXPHOS, inhibit glycolysis or inhibit oxidative stress to determine their effects on carcinoma cell growth. I will also study the functional effects of these drugs in vitro by studying glucose uptake, mitochondrial activity and ROS measurement to ensure expected effects. I will also study the effects of the antioxidant n-acetylcysteine (NAC) on OXPHOS metabolic coupling in humans. Subjects with breast cancer are being enrolled in a pilot clinical trial with NAC where cancer tissue is obtained pre-NAC and post-NAC treatment. The effects of NAC on stromal Caveolin-1 (Cav-1) and MCT4 expression will be studied by IHC. This study and career development plan will allow me to gain the skills to become an independent investigator and the research will discover mechanisms of antiestrogen drug resistance, develop biomarkers and lay the foundations for drug development. I hope to become a physician scientist who links the laboratory and clinical research aspects to improve the lives of patients with breast cancer.

描述（由申请人提供）：我的长期职业目标是成为一名独立的医生科学家，专注于乳腺癌的耐药性，并结合基于实验室的机制研究和临床试验设计和实施。我目前的工作是致力于研究如何克服抗雌激素抵抗，通过调节上皮基质代谢的相互作用，在乳腺癌利用分子生物学技术。我的导师Richard Pestell博士在乳腺癌代谢和肿瘤微环境方面具有专业知识。我的共同导师Scott Waldman博士在临床试验设计和实施方面具有专业知识。我工作的Kimmel癌症中心是NCI指定的癌症中心，拥有完善和充满活力的研究计划和多个核心设施，使我能够实施这项建议。正在实施一项职业发展计划，其基础是在Pestell博士实验室的实验工作、每周两次与Pestell博士的互动、每周一次与Waldman博士的互动以及每两个月一次的导师委员会会议和参加讲习班和研讨会，以学习技术和领导技能。 抗雌激素治疗后复发或难治性癌症在临床上定义为抗雌激素抵抗，这是一个主要的公共卫生问题。抗雌激素抵抗发生在40%的ER+患者中，并且通常是致命的。我们缺乏抗雌激素抵抗的良好生物标志物和治疗方法。最近发现，上皮细胞中高线粒体代谢与间质中低代谢的代谢偶联与抗雌激素抗性相关。我们最近已经证明，在侵袭性乳腺癌中发现了具有增加的活性氧（ROS）、低氧化磷酸化代谢（OXPHOS）和高糖酵解的肿瘤间质。这种类型的基质代谢导致代谢偶联和高能量催化剂转移到上皮癌细胞，并与抗雌激素抗性有关。我的总体假设是，代谢偶联驱动抗雌激素抵抗，逆转上皮-间质代谢偶联将克服乳腺癌中的抗雌激素抵抗。该项目的目标是：i）测试OXPHOS代谢偶联足以诱导乳腺癌抗雌激素抵抗的假设。我将使用雌激素受体阳性（ER+）乳腺癌的体外基质上皮细胞模型。我将对细胞进行遗传修饰，以便通过上调单羧酸转运蛋白1（MCT 1）与具有高OXPHOS代谢的上皮癌细胞产生紧密的上皮-基质代谢偶联，核呼吸因子1（NRF 1）和mitoNEET以及基质细胞，通过上调单羧酸转运蛋白4（MCT 4）和解偶联蛋白1（UCP 1），具有低OXPHOS代谢和高catalysts以确定上皮或间质区室的代谢变化是否足以增加抗雌激素抗性。我产生的这些细胞系将与成纤维细胞或ER+癌细胞一起培养。抗雌激素抗性将通过量化用他莫昔芬和氟维司群治疗后乳腺癌细胞的凋亡和增殖来测量。我们还将使用异种移植模型确定这些细胞系是否诱导抗雌激素抗性。 ii）在一组患者中检验与OXPHOS代谢偶联相关的基因的表达与抗雌激素抗性相关的假设。我将对接受他莫昔芬治疗的ER+乳腺癌患者的人类肿瘤微阵列（TMA）进行染色，以检测目标1中列出的蛋白质。我将通过免疫组织化学（IHC）将这些蛋白在间质和上皮区室中的表达与无进展生存期（PFS）相关联。我们还将对我产生的癌细胞进行基因表达谱分析（GEP），以确定我们是否可以产生预测抗雌激素抵抗的特征。 （三） 为了验证调节OXPHOS、糖酵解或活性氧的药物将克服抗雌激素抵抗的假设。我将使用我们的抗雌激素抵抗乳腺癌的上皮-间质共培养模型，包括为aim 1生成的基因修饰细胞，以确定代谢性解偶联上皮和间质细胞的药物是否可以克服抗雌激素抵抗。具体来说，我将测试增加或减少OXPHOS，抑制糖酵解或抑制氧化应激的药物，以确定它们对癌细胞生长的影响。我还将通过研究葡萄糖摄取、线粒体活性和ROS测量来研究这些药物在体外的功能作用，以确保预期的效果。我还将研究抗氧化剂N-乙酰半胱氨酸（NAC）对人类OXPHOS代谢偶联的影响。患有乳腺癌的受试者正在被招募到NAC的试点临床试验中，其中在NAC治疗前和NAC治疗后获得癌症组织。将通过IHC研究NAC对基质小窝蛋白-1（Cav-1）和MCT 4表达的影响。 这项研究和职业发展计划将使我获得成为独立研究者的技能，研究将发现抗雌激素药物耐药性的机制，开发生物标志物并为药物开发奠定基础。我希望成为一名医生科学家，将实验室和临床研究方面联系起来，以改善乳腺癌患者的生活。