PO.MCB09.01 · 分子与细胞生物学

β-羟基丁酸改变葡萄糖限制下ER+乳腺癌细胞的一碳代谢和支链氨基酸代谢

Beta-hydroxybutyrate alters one-carbon and branched-chain amino acid metabolism in glucose-restricted ER+ breast cancer cells

编号 2015 展板 8 时间 4/20 09:00–12:00 区域 Section 24 主讲 Michael Weichhaus, PhD

分会场 Metabolic Regulation in Breast and Gynecologic Cancers

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作者与单位 Authors & Affiliations

Cynthia Cheung¹, Natalija Glibetic², Rylee Maldonado³, Tia Skaggs³, Scott Bowman⁴, Louise Torres³, Katelynn A. Perrault Uptmor⁵, Michael Weichhaus⁶

¹Laboratory of Forensic and Bioanalytical Chemistry, School of Natural Sciences and Mathematics, Chaminade University of Honolulu, Honolulu, HI,²Department of Cell and Molecular Biology, John A Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI,³Undergraduate Biology Program, School of Natural Sciences and Mathematics, Chaminade University of Honolulu, Honolulu, HI,⁴Undergraduate Biochemistry Program, School of Natural Sciences and Mathematics, Chaminade University of Honolulu, Honolulu, HI,⁵Nontargeted Separations Laboratory, Chemistry Department, William and Mary, Williamsburg, VA,⁶Laboratory of Molecular Cancer Research, School of Natural Sciences and Mathematics, Chaminade University of Honolulu, Honolulu, HI

摘要 Abstract

中文摘要

生酮饮食干预在乳腺癌治疗中显示出前景，但其精确的代谢适应机制仍未被完整阐明。我们研究了雌激素受体阳性（ER+）乳腺癌细胞在葡萄糖限制并补充β-羟基丁酸（BHB）以模拟生酮条件下的氨基酸代谢重编程。将MCF-7和T47D细胞在葡萄糖限制培养基（标准浓度的5%）中培养，并给予梯度BHB补充（2.5-15 mM），持续四天。使用全二维气相色谱-质谱联用（GC×GC-MS）结合Fisher比值分析进行代谢组学分析。使用比色法和荧光法对甘氨酸和支链氨基酸（BCAA）进行靶向定量。通过ELISA检测SHMT1蛋白表达。对TCGA乳腺浸润性癌数据（n=1,084）进行生物信息学分析，评估一碳代谢基因（SHMT1/2）、BCAA代谢基因（BCAT1）和酮体分解酶（OXCT1、ACAT1、BDH1）之间的共表达模式。对来自GSE58135的配对ER+肿瘤与邻近正常组织样本（n=20）进行差异表达分析。用于DEG分析的R代码部分由Anthropic Claude（Sonnet 4.5版本）生成。代谢组学分析显示一碳代谢和BCAA代谢通路发生显著扰动。直接定量证实甘氨酸呈剂量依赖性积累，在两种细胞系中均于5 mM BHB时达到峰值升高，MCF-7细胞为51.1±29.5 μM对比低葡萄糖时的27.7±3.9 μM（p<0.05），T47D细胞为40.7±9.8 μM对比29.6±9.5 μM（p<0.05）。尽管甘氨酸水平改变，但两种细胞系中SHMT1蛋白表达在各处理组间保持稳定。TCGA分析显示SHMT1与酮体分解基因BDH1（r=0.18，p=2.88×10⁻⁹）和ACAT1（r=0.05，p=0.133）之间呈弱正相关，而BCAT1与BDH1呈中度负相关（r=-0.36，p=3.69×10⁻³⁵）。差异表达分析显示，与正常组织相比，ER+肿瘤中OXCT1（log2FC=-1.41，p=5.75×10⁻⁷）、SHMT1（log2FC=-1.31，p=1.62×10⁻⁵）和ACAT1（log2FC=-1.07，p=1.07×10⁻⁴）显著下调，表明酮体分解和胞质一碳代谢受到选择性抑制。葡萄糖限制下补充BHB可诱导甘氨酸稳态发生特异性改变，而SHMT1无相应变化，提示存在翻译后调控或代谢通量改变。ER+肿瘤中酮体分解酶的下调可能代表一种可通过生酮干预加以利用的代谢脆弱性，值得进一步研究将一碳代谢作为ER+乳腺癌的治疗靶点。

查看英文原文 English abstract

Ketogenic diet interventions show promise in breast cancer treatment, yet precise metabolic adaptations remain incompletely characterized. We investigated amino acid metabolic reprogramming in estrogen receptor-positive (ER+) breast cancer cells subjected to glucose restriction with beta-hydroxybutyrate (BHB) supplementation to model ketogenic conditions. MCF-7 and T47D cells were cultured in glucose-restricted medium (5% standard concentration) with graded BHB supplementation (2.5-15 mM) for four days. Metabolomic profiling was performed using comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) with Fisher ratio analysis. Targeted quantification of glycine and branched-chain amino acids (BCAAs) was conducted using colorimetric and fluorometric assays. SHMT1 protein expression was measured by ELISA. Bioinformatic analysis of TCGA Breast Invasive Carcinoma data (n=1,084) assessed co-expression patterns between one-carbon metabolism genes (SHMT1/2), BCAA metabolism (BCAT1), and ketone body catabolic enzymes (OXCT1, ACAT1, BDH1). Differential expression analysis was performed on paired ER+ tumor and adjacent normal tissue samples (n=20) from GSE58135. R-code for DEG analysis was partially created using Anthropic Claude (version Sonnet 4.5). Metabolomics analysis revealed significant perturbations in one-carbon and BCAA metabolism pathways. Direct quantification confirmed dose-dependent glycine accumulation, with peak elevation at 5 mM BHB in both MCF-7 (51.1±29.5 μM vs. 27.7±3.9 μM low glucose, p<0.05) and T47D cells (40.7±9.8 μM vs. 29.6±9.5 μM, p<0.05). Despite altered glycine levels, SHMT1 protein expression remained stable across treatments in both cell lines. TCGA analysis showed weak positive correlations between SHMT1 and ketone catabolic genes BDH1 (r=0.18, p=2.88×10⁻⁹) and ACAT1 (r=0.05, p=0.133), while BCAT1 showed moderate negative correlation with BDH1 (r=-0.36, p=3.69×10⁻³⁵). Differential expression analysis revealed significant downregulation of OXCT1 (log2FC=-1.41, p=5.75×10⁻⁷), SHMT1 (log2FC=-1.31, p=1.62×10⁻⁵), and ACAT1 (log2FC=-1.07, p=1.07×10⁻⁴) in ER+ tumors versus normal tissue, indicating selective suppression of ketone catabolism and cytoplasmic one-carbon metabolism. BHB supplementation under glucose restriction induces specific alterations in glycine homeostasis without corresponding SHMT1 changes, suggesting post-translational regulation or altered metabolic flux. Downregulation of ketone catabolic enzymes in ER+ tumors may represent a metabolic vulnerability exploitable through ketogenic interventions, warranting investigation of one-carbon metabolism as a therapeutic target in ER+ breast cancer.

利益披露 Disclosure

C. Cheung, None.. N. Glibetic, None.. R. Maldonado, None.. T. Skaggs, None.. S. Bowman, None.. L. Torres, None.. K. A. Perrault Uptmor, None.. M. Weichhaus, None.

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