定量蛋白體學解析合併用藥對神經母細胞瘤細胞骨架組成與自噬誘導細胞死亡的協同效應

Abstract

神經母細胞瘤為常見且高度惡性的兒童腫瘤，目前仍缺乏有效治療策略。近年來，「藥物重新定位」被視為解決此臨床缺口的有望途徑。本研究使用 LINCS (Library of Integrated Network-Based Cellular Signatures)資料庫中的小分子誘導基因表現譜，系統性鑑定出兩種已獲 FDA 核准的藥物pyrvinium pamoate 及 sirolimus作為潛在的合併用藥組合。功能性試驗（集落形成以及三維病人來源類器官模型）顯示，雙藥併用相較於單獨施用任一藥物，具有顯著提升的抗腫瘤效力。由 LINCS 分析預測的多個靶基因在藥物處理後樣本中同步下調。為闡明協同效應的機制，我們採用 TMT (Tandem Mass Tag) 串聯質譜標籤進行定量蛋白體學的分析，於 20,623 條胜肽序列中鑑定出 3,416 種蛋白質。接著透過 GSEA 與 DAVID 生物資訊平台進行路徑分析，結果顯示合併用藥可大幅抑制細胞骨架形成，與觀察到的細胞遷移能力降低相符；同時，蛋白質表現譜亦指出合併用藥導致明顯的細胞週期停滯。進一步實驗證實，在雙藥併用條件下自噬活性顯著升高。 總結而言，本研究揭示了pyrvinium pamoate 及 sirolimus併用提升療效的分子機制，並支持其作為低毒性、可重新定位之藥物組合，用於改善神經母細胞瘤治療成果的潛力。

Neuroblastoma is a common and highly malignant pediatric tumor that currently lacks effective therapeutic strategies. Drug repurposing has recently emerged as a promising avenue to address this unmet clinical need. In this study, we systematically screened small-molecule-induced gene expression profiles from the Library of Integrated Network-Based Cellular Signatures (LINCS) and identified two FDA-approved compounds pyrvinium pamoate and sirolimus as promising candidates for combinatorial therapy against neuroblastoma. Functional assays, including colony formation and three-dimensional patient-derived organoid models, demonstrated that the combined application of these two agents produced significantly greater antitumor efficacy than either compound used individually. Expression levels of multiple target genes predicted through LINCS analysis were concurrently downregulated in treated samples. To elucidate the underlying mechanisms responsible for this enhanced efficacy, we employed tandem mass tag (TMT)-based quantitative proteomic profiling, identifying 3,416 proteins across 20,623 peptide sequences. Subsequent pathway analysis using Gene Set Enrichment Analysis (GSEA) and the DAVID bioinformatics resource revealed that the combination therapy led to a marked suppression of cytoskeletal assembly, which was consistent with the observed inhibition of cellular motility. In addition, protein expression profiles indicated that the dual-drug treatment resulted in significant cell cycle arrest. Further investigations confirmed that autophagic activity was notably elevated under combination treatment conditions. Collectively, these findings provide mechanistic insight into the enhanced therapeutic action of sirolimus and pyrvinium pamoate co-administration and support their potential use as a low-toxicity, repurposed drug combination for improving neuroblastoma treatment outcomes.