DataSheet_1_A Novel Mechanism of 17-AAG Therapeutic Efficacy on HSP90 Inhibition in MYCN-Amplified Neuroblastoma Cells.docx

BackgroundNeuroblastoma is the most common pediatric extra-cranial nervous system tumor, originating from neural crest elements and giving rise to tumors in the adrenal medulla and sympathetic chain ganglia. Amplification of MYCN confers increased malignancy and poorer prognosis in high-risk neuroblastoma. Our SILAC proteomics analysis revealed over-expression of HSP90 in MYCN-amplified IMR-32 compared to the non-MYCN amplified SK-N-SH human neuroblastoma cells, rendering them highly resistant to therapeutic intervention.
MethodsWe used cellular bio-functional (proliferation, migration/invasion, apoptosis, viability and stem-cell self-renewal) assays and Western blot analysis to elucidate the therapeutic efficacy of HSP90 inhibition with 17-AAG.
Results17-AAG treatment significantly inhibited cellular proliferation, viability and migration/invasion and increased apoptosis in both cell lines. Moreover, drug treatment significantly abrogated stem-cell self-renewal potential in the MYCN-amplified IMR-32 cells. Differential tumorigenic protein expression revealed a novel mechanism of therapeutic efficacy after 17-AAG treatment with a significant downregulation of HMGA1, FABP5, Oct4, MYCN, prohibitin and p-L1CAM in SK-N-SH cells. However, we observed a significant up-regulation of p-L1CAM, MYCN and prohibitin, and significant down-regulation of Oct4, FABP5, HMGA1, p-ERK, cleaved/total caspase-3 and PARP1 in IMR-32 cells.
ConclusionsHSP90 inhibition revealed a novel therapeutic mechanism of antitumor activity in MYCN-amplified neuroblastoma cells that may enhance therapeutic sensitivity.

背景：神经母细胞瘤（Neuroblastoma）是儿童最常见的颅外神经系统肿瘤，起源于神经嵴细胞组分，可发生于肾上腺髓质与交感神经节链部位。MYCN基因扩增可增加高危神经母细胞瘤的恶性程度并缩短患者预后。本研究通过细胞培养氨基酸稳定同位素标记（Stable Isotope Labeling by Amino acids in Cell culture, SILAC）蛋白质组学分析发现，相较于非MYCN扩增的人神经母细胞瘤SK-N-SH细胞，MYCN扩增的IMR-32细胞中热休克蛋白90（Heat Shock Protein 90, HSP90）表达上调，使其对治疗干预产生高度耐药性。
方法：本研究采用细胞生物学功能实验（包括增殖、迁移/侵袭、凋亡、细胞活力及干细胞自我更新实验）与蛋白质免疫印迹（Western blot）分析，以阐明17-AAG介导的HSP90抑制疗法的治疗效果。
结果：17-AAG处理可显著抑制两种细胞系的细胞增殖、活力及迁移/侵袭能力，并促进细胞凋亡。此外，药物处理可显著抵消MYCN扩增的IMR-32细胞的干细胞自我更新潜能。通过对致瘤相关差异蛋白表达的分析发现，17-AAG处理后SK-N-SH细胞中高迁移率族蛋白A1（High Mobility Group AT-hook 1, HMGA1）、脂肪酸结合蛋白5（Fatty Acid Binding Protein 5, FABP5）、八聚体结合转录因子4（Octamer-binding Transcription Factor 4, Oct4）、MYCN、prohibitin及磷酸化L1细胞黏附分子（phosphorylated L1 Cell Adhesion Molecule, p-L1CAM）的表达显著下调，这揭示了一种全新的治疗效应机制。而在IMR-32细胞中，我们观察到p-L1CAM、MYCN及prohibitin的表达显著上调，Oct4、FABP5、HMGA1、磷酸化细胞外调节蛋白激酶（phosphorylated Extracellular Regulated Protein Kinase, p-ERK）、切割型/总半胱氨酸天冬氨酸蛋白酶3（cleaved/total Cysteine Aspartate Protease 3, caspase-3）及多聚ADP核糖聚合酶1（Poly(ADP-ribose) Polymerase 1, PARP1）的表达显著下调。
结论：HSP90抑制疗法在MYCN扩增的神经母细胞瘤细胞中展现出全新的抗肿瘤活性治疗机制，或可提升该类肿瘤的治疗敏感性。