Department of Cell Stress Biology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, USA; Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA; Center of Excellence in Bioinformatics & Life Science, University at Buffalo, State University of New York, Buffalo, New York, USA.
Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA; Center of Excellence in Bioinformatics & Life Science, University at Buffalo, State University of New York, Buffalo, New York, USA.
Mol Cell Proteomics. 2022 Oct;21(10):100409. doi: 10.1016/j.mcpro.2022.100409. Epub 2022 Sep 7.
Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.
胰腺导管腺癌（PDAC）对治疗具有高度抗性。标准护理吉西他滨（Gem）仅提供适度的生存益处，而 Gem 耐药（GemR）的发展会影响其疗效。为了研究 GemR 的分子机制，开发了对 Gem 敏感的 MIAPaCa-2 细胞的高度 GemR 克隆，并实施了基于全面、可重复的离子电流 MS1 工作流程的全局定量差异蛋白质组学分析，以定量所有样本中约 6000 种蛋白质。在 GemR 克隆 MIA-GR8 中，改变了细胞代谢、增殖、迁移和“药物反应”机制等主要生物学过程，这与细胞周期和运动性的细胞表型改变一致。S100 钙结合蛋白 A4 是下调最明显的蛋白质，与糖酵解和氧化能量产生相关的蛋白质也是如此。这两种反应都会降低肿瘤增殖。间充质标志物的上调很明显，细胞侵袭性增加。Gem 代谢途径中的关键酶发生改变，从而减少 Gem 的细胞内利用。核糖核苷酸二磷酸还原酶大亚基是上调最明显的 Gem 代谢蛋白，支持其在 GemR 中的关键作用。较低的核糖核苷酸二磷酸还原酶大亚基表达与 PDAC 的更好临床结果相关，其下调与 MIAPaCa-2 增殖和迁移减少以及 Gem 敏感性增加平行。MIAPaCa-2 与 GemR 细胞系（固有 GemR PANC-1 和获得性 GemR MIA-GR8）的 Gem 时间蛋白反应表明，细胞增殖和药物转运和代谢的细胞反应系统的适应性变化，降低了细胞毒性 Gem 代谢物、DNA 修复和其他反应，是 GemR 在 PDAC 中复杂性的关键因素。这些发现还表明，针对 GemR PDAC 患者具有可靶向的治疗弱点。
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