From the ‡Cancer Program, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.
§Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Mol Cell Proteomics. 2018 Aug;17(8):1470-1486. doi: 10.1074/mcp.RA118.000633. Epub 2018 Apr 9.
Inhibition of the heat shock protein 90 (Hsp90) chaperone is a promising therapeutic strategy to target expression of the androgen receptor (AR) and other oncogenic drivers in prostate cancer cells. However, identification of clinically-relevant responses and predictive biomarkers is essential to maximize efficacy and treatment personalization. Here, we combined mass spectrometry (MS)-based proteomic analyses with a unique patient-derived explant (PDE) model that retains the complex microenvironment of primary prostate tumors. Independent discovery and validation cohorts of PDEs ( = 16 and 30, respectively) were cultured in the absence or presence of Hsp90 inhibitors AUY922 or 17-AAG. PDEs were analyzed by LC-MS/MS with a hyper-reaction monitoring data independent acquisition (HRM-DIA) workflow, and differentially expressed proteins identified using repeated measure analysis of variance (ANOVA; raw value <0.01). Using gene set enrichment, we found striking conservation of the most significantly AUY922-altered gene pathways between the discovery and validation cohorts, indicating that our experimental and analysis workflows were robust. Eight proteins were selectively altered across both cohorts by the most potent inhibitor, AUY922, including TIMP1, SERPINA3 and CYP51A (adjusted < 0.01). The AUY922-mediated decrease in secretory TIMP1 was validated by ELISA of the PDE culture medium. We next exploited the heterogeneous response of PDEs to 17-AAG in order to detect predictive biomarkers of response and identified PCBP3 as a marker with increased expression in PDEs that had no response or increased in proliferation. Also, 17-AAG treatment led to increased expression of DNAJA1 in PDEs that exhibited a cytostatic response, revealing potential drug resistance mechanisms. This selective regulation of DNAJA1 was validated by Western blot analysis. Our study establishes "proof-of-principle" that proteomic profiling of drug-treated PDEs represents an effective and clinically-relevant strategy for identification of biomarkers that associate with certain tumor-specific responses.
抑制热休克蛋白 90（Hsp90）伴侣是一种有前途的治疗策略，可靶向前列腺癌细胞中雄激素受体（AR）和其他致癌驱动因子的表达。然而，确定临床相关的反应和预测性生物标志物对于最大限度地提高疗效和治疗个性化至关重要。在这里，我们将基于质谱（MS）的蛋白质组分析与独特的患者衍生外植体（PDE）模型相结合，该模型保留了原发性前列腺肿瘤的复杂微环境。PDE 的独立发现和验证队列（分别为 16 和 30）在没有或存在 Hsp90 抑制剂 AUY922 或 17-AAG 的情况下进行培养。通过 LC-MS/MS 对 PDE 进行分析，并使用重复测量方差分析（ANOVA；原始 值 <0.01）进行差异表达蛋白鉴定。使用基因集富集，我们发现发现和验证队列之间 AUY922 改变最显著的基因途径非常相似，这表明我们的实验和分析工作流程是稳健的。两种抑制剂 AUY922 和 17-AAG 均能改变 8 种蛋白的表达，包括 TIMP1、SERPINA3 和 CYP51A（调整 值 <0.01）。通过对 PDE 培养物上清液进行 ELISA 验证，证实了 AUY922 介导的 TIMP1 分泌减少。接下来，我们利用 PDE 对 17-AAG 的异质性反应来检测反应的预测性生物标志物，并发现 PCBP3 是一种表达增加的标志物，在没有反应或增殖增加的 PDE 中表达增加。此外，17-AAG 处理导致在表现出细胞抑制反应的 PDE 中 DNAJA1 的表达增加，揭示了潜在的药物耐药机制。通过 Western blot 分析验证了 DNAJA1 的这种选择性调节。我们的研究确立了“原理证明”，即药物处理的 PDE 蛋白质组谱分析代表了一种有效且与临床相关的策略，可用于鉴定与特定肿瘤反应相关的生物标志物。
