Metabolic alterations contributing to enzalutamide resistance in prostate cancer

项目摘要/摘要 对雄激素受体(AR)拮抗剂苯扎鲁胺的耐药性仍然是导致 前列腺癌(PCA)患者。因此，至关重要的是，我们要确定哪些患者会产生抗药性， 定义抗药性形成的机制。因为新陈代谢的改变对决定 癌细胞是否会抵抗压力和增殖，这项提议旨在定义 导致对苯扎鲁胺的耐药性。6对苯扎鲁胺敏感药(EnzS)的代谢比较研究 而对苯扎鲁胺耐药(EnzR)的PCA细胞株表明EnzR细胞具有增强的谷氨酰胺 新陈代谢，更依赖谷氨酰胺。这一点得到了之前数据的支持，这些数据表明AR促进了 谷氨酰胺摄取和谷氨酰胺分解。进一步的鉴定表明耐药细胞增加了 氧化应激和EnzR细胞的生长由抗氧化剂促进，如谷胱甘肽(GSH)。这个 数据表明，苯扎鲁胺抑制抗氧化剂和诱导 活性氧(ROS)。鉴于谷氨酰胺的一个重要作用是产生抗氧化剂，我们 认为谷氨酰胺代谢增强通过以下途径促进前列腺癌对苯扎鲁胺的耐药性 上调抗氧化剂计划以耐受增加的氧化应激，这种抵抗机制是 有针对性。这项提议将通过使用无偏的稳态和代谢流量来检验这一假设。 体外EnzS和EnzR细胞、体内小鼠异种移植和患者肿瘤的技术。两者之间的联系 代谢依赖性和ROS将通过荧光显微镜和酶学进一步表征 活动接近。RNAi、CRISPR-Cas9和慢病毒介导的谷氨酰胺酶(GLS)过表达 谷氨酰胺分解的关键酶，以及谷胱甘肽合成的限速酶谷氨酸-半胱氨酸连接酶 用来确定这些改装对于驾驶阻力的必要性和充分性。已知的抑制剂 这些酶将用于体外、体内和体外培养的患者来源的肿瘤中，以确定 这些治疗策略在EnzR PCa的治疗中是否具有潜在的疗效。这 拟议的研究将提供对苯扎鲁胺进化的机制的理解 耐药性，并允许开发治疗方法，导致对苯扎鲁胺更持久的反应 诊所。

Project Summary/Abstract Resistance to the androgen receptor (AR) antagonist enzalutamide remains a major cause of mortality among prostate cancer (PCa) patients. Thus, it is crucial that we identify which patients will become resistant and define the mechanism by which resistance develops. Because metabolic alterations are critical for determining whether cancer cells will resist stress and proliferate, this proposal aims to define the metabolic program that drives enzalutamide resistance. Comparative metabolic studies in six paired enzalutamide-sensitive (EnzS) and enzalutamide-resistant (EnzR) PCa cell lines indicate that EnzR cells have enhanced glutamine metabolism and are more glutamine dependent. This is supported by previous data showing that AR promotes glutamine uptake and glutaminolysis. Further characterization indicates that resistant cells have increased oxidative stress, and the growth of EnzR cells is promoted by antioxidants, such as glutathione (GSH). The role of oxidative stress is corroborated by data suggesting that enzalutamide inhibits antioxidants and induces reactive oxygen species (ROS). Given that an important role of glutamine is to generate antioxidants, we propose that enhanced glutamine metabolism drives enzalutamide resistance in prostate cancer by upregulating antioxidant programs to tolerate increased oxidative stress, and this resistance mechanism is targetable. This proposal will test this hypothesis through the use of unbiased steady-state and metabolic flux techniques in EnzS and EnzR cells in vitro, mouse xenografts in vivo, and patient tumors. The link between metabolic dependencies and ROS will be further characterized by fluorescence microscopy and enzymatic activity approaches. RNAi, CRISPR-Cas9, and lentiviral-mediated overexpression of glutaminase (GLS), a critical enzyme in glutaminolysis, and glutamate-cysteine ligase, a rate-limiting enzyme for GSH synthesis, will be used to establish the necessity and sufficiency of these alterations for driving resistance. Known inhibitors of these enzymes will be used in vitro, in vivo, and in patient-derived tumors cultured ex vivo to determine whether these therapeutic strategies will be potentially efficacious in the treatment of EnzR PCa. This proposed research will provide the understanding of mechanisms that enable the evolution of enzalutamide resistance and allow for the development of therapies leading to more durable responses to enzalutamide in the clinic.