Metabolic mechanisms of antiestrogen resistance in breast cancer

7.项目总结/摘要：我的长期职业目标是成为一名独立的内科科学家 专注于乳腺癌的耐药性，并将基于实验室的机制研究与临床相结合 试行设计和实施。我目前致力于研究如何克服抗雌激素。 利用分子生物学调控乳腺癌上皮间质代谢相互作用的耐药性 技巧。我的导师理查德·佩斯特尔博士在乳腺癌新陈代谢和肿瘤方面有专长。 微环境。我的合作导师Scott Waldman博士在临床试验设计和实施方面拥有专业知识。 我工作的坎摩尔癌症中心是一家NCI指定的癌症中心，拥有完善的 充满活力的研究计划和多个核心设施将使我能够实施这一提议。一份事业 开发计划基于佩斯特尔博士实验室的实验工作，每周与佩斯特尔博士进行两次互动， 每周与Waldman博士以及指导委员会每两个月举行一次会议，并参与 正在举办讲习班和研讨会，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 这是一个重大的公共卫生问题。40%的ER+患者出现抗雌激素耐药，最常见的是 致命的。我们缺乏良好的生物标记物和抗雌激素耐药性的治疗方法。最近发现， 间质低代谢上皮细胞的代谢偶联与高线粒体代谢 与抗雌激素抵抗有关。我们最近证明了一种肿瘤间质增加 活性氧(ROS)、低氧化磷酸化代谢(OXPHOS)和高糖酵解 在侵袭性乳腺癌中发现。这种基质代谢导致代谢偶联和转移 对上皮性癌细胞的高能分解代谢，并与抗雌激素抵抗有关。我的 总体假设是代谢偶联驱动上皮-间质的抗雌激素抵抗和逆转 代谢偶联将克服乳腺癌患者的抗雌激素耐药性。该项目的目标是： I)检验OXPHOS代谢偶联足以产生抗雌激素的假设 乳腺癌中的耐药性。我将使用雌激素受体阳性的体外基质上皮细胞模型 (ER+)乳腺癌。我将对细胞进行基因改造，以产生紧密的上皮-间质代谢 通过上调单羧酸与高氧磷代谢的上皮癌细胞偶联 转运蛋白1(MCT1)、核呼吸因子1(NRF1)与低氧磷的丝裂原和基质细胞 单羧酸转运蛋白4(MCT4)上调和解偶联引起的代谢和高分解代谢 蛋白1(UCP1)，以确定上皮或间质间隔的代谢变化是否 足以增加抗雌激素抵抗力。我产生的这些细胞系将与 成纤维细胞或ER+癌细胞。抗雌激素耐药性将通过量化细胞凋亡和 三苯氧胺和氟维斯特对乳腺癌细胞增殖的影响。我们还将确定是否 这些细胞系通过异种移植模型诱导抗雌激素抵抗。 Ii)检验与OXPHOS代谢偶联相关的基因表达是 在一组患者中与抗雌激素耐药性有关。我要对人类肿瘤基因芯片进行染色 三苯氧胺治疗ER阳性乳腺癌患者(TMA)对AIM 1中列出的蛋白质的影响。我将相关 免疫组织化学(IHC)法检测这些蛋白在间质和上皮室的表达 无进展生存(PFS)。我们还将进行癌症的基因表达谱(GEP) 我产生的细胞，以确定我们是否可以产生一个预测抗雌激素耐药性的信号。 三)检验如下假设：调节氧磷酶、糖酵解或活性氧的药物 物种将克服抗雌激素耐药性。我将使用我们的上皮-间质共培养模型 抗雌激素耐药性乳腺癌，包括为AIM 1产生的转基因细胞，以确定是否 新陈代谢解偶联上皮细胞和基质细胞的药物可以克服抗雌激素耐药性。 具体地说，我将测试增加或降低OXPHOS、抑制糖酵解或抑制氧化应激的药物 确定它们对癌细胞生长的影响。我还将在体外研究这些药物的功能效应。 通过研究葡萄糖摄取、线粒体活性和ROS测量来确保预期效果。这就做 同时研究抗氧化剂N-乙酰半胱氨酸(NAC)对人体OXPHOS代谢偶联的影响。 患有乳腺癌的受试者正在接受NAC的一项试点临床试验，在那里获得癌症组织。 NAC治疗前和治疗后。NAC对间质小窝蛋白-1和MCT4表达的影响 将由IHC进行研究。 这个学习和职业发展计划将使我获得成为一个独立的人的技能 研究人员和研究人员将发现抗雌激素耐药性的机制，开发生物标记物 并为药物开发奠定基础。我希望成为一名连接实验室的内科科学家 以及临床研究方面，以改善乳腺癌患者的生活。 1

7. Project Summary/Abstract: 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. 1