Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Cancer Lett. 2015 Aug 10;364(2):142-55. doi: 10.1016/j.canlet.2015.05.004. Epub 2015 May 7.
Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.
神经母细胞瘤是一种具有挑战性的儿童恶性肿瘤，很大比例的患者在获得耐药性后复发，因此需要确定耐药机制以及导致疾病侵袭性的新的生物学靶点。为了研究与神经母细胞瘤耐药相关的分子途径，我们开发并鉴定了顺铂耐药亚系 SK-N-ASCis24、KellyCis83 和 CHP-212Cis100，整合了细胞行为、细胞毒性、基因组改变和蛋白表达调节的数据。所有三种顺铂耐药细胞系均表现出对替莫唑胺、依托泊苷和伊立替康的交叉耐药性，这些药物均为再起始治疗药物。阵列 CGH 分析表明，耐药株获得了额外的基因组失衡。通过质谱鉴定差异表达蛋白，并通过生物信息学工具根据其分子和细胞功能及其参与的生物学途径进行分类。参与肌动蛋白细胞骨架信号转导（p=9.28E-10）、整合素连接激酶（ILK）信号转导（p=4.01E-8）、上皮黏附连接信号转导（p=5.49E-8）和上皮黏附连接重塑（p=5.87E-8）等途径的蛋白表达显著变化，表明顺铂耐药 SK-N-ASCis24 发展为间充质表型。Western blot 和 MYH9、ACTN4 和 ROCK1 的共聚焦显微镜与侵袭实验相结合，证明 MYH9 和 ACTN4 水平升高和 ROCK1 水平降低导致 SK-N-ASCis24 的 ROCK1 非依赖性迁移潜力增加。因此，我们的结果表明，上皮-间充质转化是神经母细胞瘤耐药发展的一个特征。
