Department of Radiology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
University of Wisconsin Carbone Cancer Center, Madison, Wisconsin; and.
J Nucl Med. 2019 Sep;60(9):1247-1252. doi: 10.2967/jnumed.118.224667. Epub 2019 Mar 8.
The purpose of this study was to determine the effect of estrogen receptor-α gene () mutations at the tyrosine (Y) 537 amino acid residue within the ligand binding domain on F-fluoroestradiol (F-FES) binding and tumor uptake compared with wild-type (WT)-estrogen receptor α (ER). ER-negative MDA-MB-231 breast cancer cells were used to generate stable cell lines that express WT-ER, Y537S, or Y537C mutant ER. Receptor expression and localization were confirmed by Western blot and immunofluorescence, respectively. ER transcriptional function was measured using an estrogen response element-luciferase reporter gene assay and quantitative polymerase chain reaction analysis of ER-regulated endogenous target genes. Saturation binding and competition assays were performed to determine equilibrium dissociation constant (K) and half maximal inhibitory concentration (IC50) values. F-FES uptake was measured in tumor xenografts grown in female athymic nude mice by small-animal PET/CT imaging and tissue biodistribution using 5.55 MBq (150 μCi) of F-FES. A 10-fold-lower injected dose of 0.555 MBq (15 μCi) of F-FES was also used for tissue biodistribution. Statistical significance was determined using ANOVA. Y537S and Y537C mutations resulted in increased ER transcriptional activity in the absence of estrogen compared with WT-ER (11.48 ± 2.42 fold; = 0.0002, and 5.89 ± 0.94 fold; = 0.04, respectively). Constitutive ER activation of two target genes ( and ) in the absence of estrogen was also observed in Y537S- and Y537C-ER cells compared with WT-ER. K values for F-FES were 0.98 ± 0.54 nM for Y537S-ER ( = 0.27) and 0.24 ± 0.03 nM for Y537C-ER ( = 0.95) compared with 0.07 ± 0.03 nM for WT-ER. IC50 values were 0.22 ± 0.09 nM for Y537S-ER ( = 0.97), 0.18 ± 0.09 nM for Y537C-ER ( = 0.99), and 0.19 ± 0.11 nM for WT-ER. Tumor xenografts expressing Y537S-ER (mean percentage injected dose per gram, 1.45 ± 0.06; = 0.77) and Y537C-ER (2.09 ± 0.20; = 0.21) had similar F-FES uptake compared with WT-ER (1.68 ± 0.12). Comparable F-FES uptake between Y537S-, Y537C-, and WT-ER xenografts was also observed using a 10-fold-lower injected dose with the tissue biodistribution assay. Since tumoral uptake of F-FES is not significantly impacted by Y537S-ER or Y537C-ER mutations, the potential diagnostic utility of F-FES PET imaging is expected to be equally valid for patients with or without these activating mutations.
这项研究的目的是确定配体结合域中酪氨酸（Y）537 位氨基酸残基处的雌激素受体-α（）基因突变对 F-氟雌二醇（F-FES）结合和肿瘤摄取的影响，与野生型（WT）-雌激素受体（ER）相比。使用稳定表达 WT-ER、Y537S 或 Y537C 突变型 ER 的 MDA-MB-231 乳腺癌细胞系来生成。受体表达和定位分别通过 Western blot 和免疫荧光来确认。使用雌激素反应元件-荧光素酶报告基因测定和定量聚合酶链反应分析 ER 调节的内源性靶基因来测量 ER 转录功能。进行了饱和结合和竞争测定，以确定平衡解离常数（K）和半最大抑制浓度（IC50）值。通过小动物 PET/CT 成像和使用 5.55 MBq（150 μCi）的 F-FES 进行组织生物分布，在雌性无胸腺裸鼠中生长的肿瘤异种移植物中测量 F-FES 的摄取。还使用组织生物分布的 0.555 MBq（15 μCi）的 10 倍低注射剂量。使用方差分析确定统计学意义。与 WT-ER 相比，Y537S 和 Y537C 突变导致 ER 在没有雌激素的情况下转录活性增加（11.48 ± 2.42 倍； = 0.0002，和 5.89 ± 0.94 倍； = 0.04）。在没有雌激素的情况下，两个靶基因（和）的 ER 组成型激活也在 Y537S-和 Y537C-ER 细胞中观察到，与 WT-ER 相比。Y537S-ER 的 F-FES K 值为 0.98 ± 0.54 nM（ = 0.27），Y537C-ER 的 F-FES K 值为 0.24 ± 0.03 nM（ = 0.95），而 WT-ER 的 F-FES K 值为 0.07 ± 0.03 nM。Y537S-ER 的 IC50 值为 0.22 ± 0.09 nM（ = 0.97），Y537C-ER 的 IC50 值为 0.18 ± 0.09 nM（ = 0.99），而 WT-ER 的 IC50 值为 0.19 ± 0.11 nM。表达 Y537S-ER（平均每克注射剂量的百分比，1.45 ± 0.06； = 0.77）和 Y537C-ER（2.09 ± 0.20； = 0.21）的肿瘤异种移植物与 WT-ER（1.68 ± 0.12）相比，F-FES 的摄取相似。在用组织生物分布测定法进行 10 倍低注射剂量时，也观察到 Y537S-、Y537C-和 WT-ER 异种移植物之间的 F-FES 摄取相似。由于 Y537S-ER 或 Y537C-ER 突变对 F-FES 的肿瘤摄取没有显著影响，因此 F-FES PET 成像的潜在诊断效用预计对具有或不具有这些激活突变的患者同样有效。
