目的:NF1是抑癌基因及其蛋白产物,神经纤维蛋白,是RAS途径的负调节因子。NF1是散发性乳腺癌的主要驱动突变之一,因此27%的乳腺癌表现出破坏性的NF1改变。NF1功能丧失是雌激素受体(ER)+乳腺癌转移和内分泌抵抗的基因组进化中的常见事件。患有1型神经纤维瘤病(NF)的个体-由种系NF1突变引起的疾病-死于乳腺癌的风险增加[1-4]。与散发性乳腺癌相比,NF相关乳腺癌与总体生存率降低有关。尽管有大量研究询问RAS突变在肿瘤代谢中的作用,目前还没有一项研究对缺乏NF1的乳腺癌代谢组进行全面分析,以确定能量和代谢重编程的模式.这项研究的目标是(1)定义NF1缺乏在雌激素受体阳性(ER)乳腺癌代谢重编程中的作用,以及(2)确定NF1缺陷型ER+乳腺癌的潜在靶向途径和代谢抑制剂组合疗法。
方法:我们采用了两种ER+NF1缺陷型乳腺癌模型:(1)NF1缺陷型MCF7乳腺癌细胞系对散发性乳腺癌进行建模,和(2)三个不同的,Nf1缺陷大鼠模型与NF相关乳腺癌模型[1]。IncuCyte增殖分析用于测量NF1缺乏对细胞增殖和药物反应的影响。通过蛋白质印迹分析评估蛋白质量。然后,我们使用RNAseq研究NF1缺乏对整体和代谢相关转录的转录作用。我们使用Agilent海马XF-96Glyco应激测试和Mito应激测试测定来测量细胞能量学。我们进行了稳定的同位素标记,并使用质谱法测量了[U-13C]-葡萄糖和[U-13C]-谷氨酰胺代谢物的掺入,并测量了总代谢物池。最后,我们使用Bliss协同作用模型来研究NF1驱动的靶向和代谢抑制剂协同作用的变化.
结果:我们的结果显示NF1缺乏增强了细胞增殖,神经纤维蛋白表达改变,和增加RAS和PI3K/AKT途径信号,同时限制氧化ATP的产生和限制能量的灵活性。神经纤维蛋白缺乏也增加了谷氨酰胺流入TCA中间体,并急剧增加了脂质库,尤其是甘油三酯(TG)。最后,NF1缺乏改变了代谢抑制剂和传统靶向抑制剂之间的协同作用。这包括增加与靶向糖酵解的抑制剂的协同作用,谷氨酰胺代谢,线粒体脂肪酸运输,和TG合成。
结论:NF1缺乏驱动ER+乳腺癌的代谢重编程。这种重编程的特征是氧化ATP约束,谷氨酰胺TCA流入,和脂质池扩张,这些代谢变化引入了新的代谢-靶向抑制剂协同作用。
OBJECTIVE: NF1 is a tumor suppressor gene and its protein product, neurofibromin, is a negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27 % of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) - a disorder caused by germline NF1 mutations - have an increased risk of dying from breast cancer [1-4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-deficient breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER + breast cancer.
METHODS: We employed two ER+ NF1-deficient breast cancer models: (1) an NF1-deficient MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF-related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U-13C]-glucose and [U-13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy.
RESULTS: Our results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis.
CONCLUSIONS: NF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies.