以基因表現策略探討驅蟲藥Niclosamide於高危險性神經母細胞瘤之治療效果

Abstract

神經母細胞瘤是源自於交感神經系統的小兒腫瘤，其具有不同的臨床表徵。MYCN基因是神經母細胞瘤發病機制中確立的致癌驅動因子，對於MYCN基因擴增的高危險性神經母細胞瘤病患而言，即使結合多種治療方式，治療效果仍然不佳，而且其遺傳異質性已大大阻礙了分子靶標藥物的治療效果。在這裡，我們基於基因表現的方法，將與高危險性神經母細胞瘤相關的基因表現與從多達一百萬個藥物擾動基因資訊建構出藥物擾動與高風險神經母細胞瘤基因表現的關係，藉由評估給定化合物或其組合可能“反轉“高危險性神經母細胞瘤基因表現的效果，我們找到了FDA批准的驅蟲藥氯硝柳胺作為潛在的抗神經母細胞瘤藥物。進一步，我們進行了即時聚合酶鏈式反應（qPCR）來驗證參與DNA複製的前五名候選基因：包括細胞週期蛋白A2（CCNA2）、微染色體維持10複製起始因子（MCM10）、紡錘體組裝檢查點解旋酶ERCC切除修復6（ERCC6L）、驅動蛋白家族成員20A（KIF20A）和RuvB AAA腺苷酶1（RUVBL1）。實驗結果表明，這些基因在氯硝柳胺處理的細胞中表現量下降，表示氯硝柳胺抑制DNA複製並進一步抑制細胞增殖。進一步，我們利用細胞增殖、細胞群落生成實驗以及流式細胞儀測定了氯硝柳胺在MYCN基因擴增的SK-N-DZ和MYCN基因未擴增的SK-N-AS細胞中的細胞毒性作用。實驗結果顯示氯硝柳胺不僅能夠有效減少細胞增殖和細胞群落形成，還能夠誘導細胞週期停滯和細胞凋亡。此外，我們在裸鼠模型中進行人類腫瘤異種移植以評估氯硝柳胺的治療效果，發現氯硝柳胺能夠顯著抑制腫瘤生長和延長裸鼠存活率，但是不會造成器官損傷和改變體重。為了探討氯硝柳胺的分子機制，我們於MYCN基因擴增的SK-N-DZ和MYCN基因未擴增的SK-N-AS細胞與動物模型進行穩定同位素二甲基標記的蛋白體學進行分析。我們在高危險群神經母細胞瘤細胞中除了確認先前研究報導關於氯硝柳胺會調控線粒體電子傳遞鏈系中的功能外，也發現了其他新的調控功能。這些結果表明，我們開發基於基因表現的策略對於老藥新用是有成效的，也提供了驅蟲藥物氯硝柳胺老藥新用於治療高危險性神經母細胞瘤療法的可能性。

Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with diverse clinical behaviors. Even with multimodal therapies, high-risk neuroblastoma has an unfavorable outcome irrespective of MYCN amplification, a well-established oncogenic driver in neuroblastoma pathogenesis, and its genetic heterogeneity has largely impeded efforts to correlate molecular targets with biological consequences for more effective treatment strategies. Here, using a gene expression-based approach, we identified the FDA-approved anthelmintic niclosamide as a potential anti-neuroblastoma drug. By combining the gene expression signature associated with high-risk neuroblastoma and the recurrent drug−transcript relationships inferred from up to one million perturbational gene expression profiles, our algorithm predicted effective therapeutic candidates by evaluating the extent to which a given compound or their combinations could ‘reverse’ the high-risk signature. Furthermore, we performed quantitative polymerase chain reaction (qPCR) to validate top five candidate reverse genes which are involved in DNA replication, including cyclin A2 (CCNA2), minichromosome maintenance 10 replication initiation factor (MCM10), ERCC excision repair 6 like, spindle assembly checkpoint helicase (ERCC6L), kinesin family member 20A (KIF20A), and RuvB like AAA ATPase 1 (RUVBL1). Indeed, those five genes were downregulated in niclosamide-treated cells, indicating niclosamide suppressed DNA replication and then inhibited cell proliferation. Using cell proliferation and clonogenic assays as well as flow cytometry, we determined the cytotoxic effects of niclosamide in MYCN-amplified SK-N-DZ and non-amplified SK-N-AS cells. The results showed that niclosamide could effectively reduce not only cell proliferation and colony formation but also trigger cell cycle arrest and apoptosis. Moreover, we conducted human tumor xenografts in a nude mice model to evaluate the in vivo efficacy of niclosamide and found that it significantly suppressed tumor growth and prolonged survival rate, but doesn’t cause organ damage and change body weight. To explore the molecular mechanism of niclosamide, stable-isotope dimethyl labeling strategy for quantitative proteomics was performed on both cell-based or xenograft-based MYCN-amplified SK-N-DZ and MYCN-nonamplified SK-N-AS models. We confirmed niclosamide not only mediated the function of mitochondrial electron transport chain but also the other functions in high risk neuroblastoma cell lines. The results suggest that our developed expression-based strategy is useful for drug discovery and provides the possibility of repurposing the anthelminthic drug niclosamide for treating high-risk neuroblastoma therapy.