Laboratory for Radiopharmaceutical Research, Department of Pharmacy and Pharmacology, KU Leuven, Leuven, Belgium.
Switch Laboratory, VIB-KU Leuven Center for Brain & Disease Research, KU Leuven, Leuven, Belgium.
Theranostics. 2019 Jan 1;9(2):554-572. doi: 10.7150/thno.27213. eCollection 2019.
Heat shock protein 90 is an ATP-dependent molecular chaperone important for folding, maturation and clearance of aberrantly expressed proteins and is abundantly expressed (1-2% of all proteins) in the cytosol of all normal cells. In some tumour cells, however, strong expression of HSP90 is also observed on the cell membrane and in the extracellular matrix and the affinity of tumoural HSP90 for ATP domain inhibitors was reported to increase over 100-fold compared to that of HSP90 in normal cells. Here, we explore [C]NMS-E973 as a PET tracer for visualisation of HSP90 and as a potential tool for quantification of occupancy of HSP90 inhibitors. HSP90 expression was biochemically characterized in a panel of established cell lines including the melanoma line B16.F10. B16.F10 melanoma xenograft tumour tissue was compared to non-malignant mouse tissue. NMS-E973 was tested for HSP90 inhibitory activity in several tumour cell lines. HSP90-specific binding of [C]NMS-E973 was evaluated in B16.F10 melanoma cells and B16.F10 melanoma, prostate cancer LNCaP and PC3, SKOV-3 xenograft tumour slices and in a B16.F10 melanoma mouse model. Strong intracellular upregulation and abundant membrane localisation of HSP90 was observed in the different tumour cell lines, in the B16.F10 tumour cell line and in B16.F10 xenograft tumours compared to non-malignant tissue. NMS-E973 showed HSP90-specific inhibition and reduced proliferation of cells. [C]NMS-E973 showed strong binding to B16.F10 melanoma cells, which was inhibited by 200 µM of PU-H71, a non-structurally related HSP90 inhibitor. HSP90-specific binding was observed by autoradiography of murine B16.F10 melanoma, LNCaP and PC3 prostate cancer and SKOV-3 ovary carcinoma tissue slices. Further, B16.F10 melanoma-inoculated mice were subjected to a µPET study, where the tracer showed fast and persistent tumour uptake. Pretreatment of B16.F10 melanoma mice with PU-H71 or Ganetespib (50 mg/kg) completely blocked tumour accumulation of [C]NMS-E973 and confirmed HSP90 binding specificity. HSP90-specific binding of [C]NMS-E973 was observed in blood, lungs and spleen of tumour-bearing animals but not in control animals. [C]NMS-E973 is a PET tracer for visualisation of tumour HSP90 expression and can potentially be used for quantification of HSP90 occupancy. Further translational evaluation of [C]NMS-E973 is warranted.
热休克蛋白 90 是一种依赖于 ATP 的分子伴侣，对于折叠、成熟和清除异常表达的蛋白质非常重要，并且在所有正常细胞的细胞质中大量表达（占所有蛋白质的 1-2%）。然而，在一些肿瘤细胞中，HSP90 也强烈表达在细胞膜和细胞外基质中，并且肿瘤 HSP90 与 ATP 结构域抑制剂的亲和力据报道比正常细胞中的 HSP90 高 100 多倍。在这里，我们探索 [C]NMS-E973 作为 HSP90 的 PET 示踪剂，以及作为 HSP90 抑制剂占有率定量的潜在工具。在包括黑色素瘤系 B16.F10 在内的一系列已建立的细胞系中，对 HSP90 的表达进行了生化特征分析。将 B16.F10 黑色素瘤异种移植肿瘤组织与非恶性小鼠组织进行了比较。在几种肿瘤细胞系中测试了 NMS-E973 的 HSP90 抑制活性。在 B16.F10 黑色素瘤细胞和 B16.F10 黑色素瘤、前列腺癌 LNCaP 和 PC3、SKOV-3 异种移植肿瘤切片以及 B16.F10 黑色素瘤小鼠模型中评估了 [C]NMS-E973 的 HSP90 特异性结合。与非恶性组织相比，在不同的肿瘤细胞系、B16.F10 肿瘤细胞系和 B16.F10 异种移植肿瘤中观察到 HSP90 的细胞内强烈上调和丰富的膜定位。NMS-E973 表现出 HSP90 特异性抑制作用，并降低了细胞的增殖能力。[C]NMS-E973 对 B16.F10 黑色素瘤细胞表现出强烈的结合，而这种结合被非结构相关 HSP90 抑制剂 PU-H71 抑制 200µM。通过对小鼠 B16.F10 黑色素瘤、LNCaP 和 PC3 前列腺癌和 SKOV-3 卵巢癌组织切片进行放射自显影观察到 HSP90 特异性结合。此外，B16.F10 黑色素瘤接种小鼠进行了 µPET 研究，示踪剂显示出快速和持续的肿瘤摄取。用 PU-H71 或 Ganetespib（50mg/kg）预处理 B16.F10 黑色素瘤小鼠可完全阻断 [C]NMS-E973 的肿瘤积聚，并证实 HSP90 结合特异性。在携带肿瘤的动物的血液、肺和脾脏中观察到 [C]NMS-E973 的 HSP90 特异性结合，但在对照动物中没有观察到。[C]NMS-E973 是一种用于可视化肿瘤 HSP90 表达的 PET 示踪剂，可潜在用于 HSP90 占有率的定量。需要进一步的转化评估 [C]NMS-E973。
