Laboratory of Experimental Cancer Biology, CIBIO Department, University of Trento, Via Sommarive 9, 38123 Povo TN, Italy.
High Throughput Screening (HTS) Facility, CIBIO Department, University of Trento, Via Sommarive 9, 38123 Povo TN, Italy.
Dev Biol. 2020 Jan 15;457(2):215-225. doi: 10.1016/j.ydbio.2019.04.005. Epub 2019 Apr 15.
Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies. The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.
治疗皮肤黑色素瘤的方法层出不穷，但尽管有令人鼓舞的结果，临床上越来越多的报道表明，对不断增加的治疗药物组合存在原发性或继发性耐药。联合治疗方法可能会克服耐药性，因为它们可以针对黑色素瘤细胞的特定弱点，这些弱点本身并不足以解决问题，但与其他疗法联合使用则可能有效。转基因斑马鱼线 kita:ras 高频发生黑色素瘤。在 3 dpf 时，转基因 kita:ras 幼虫表现出超色素沉着表型，这是异常黑素细胞生长的最早证据。我们使用该模型，针对库中 1280 种 FDA 或 EMA 批准的药物中的任何一种，基于自动检测黑素细胞数量减少的情况，进行了化学筛选。分析表明，有 55 种药物能够使每个胚胎中的黑素细胞数量减少 60%或更多。我们进一步测试了 5 个类别中的每一个的两种化合物，以及一种法尼基转移酶抑制剂（Lonafarnib），该抑制剂抑制 HRAS 的必要翻译后修饰，并抑制超色素沉着表型。酮康唑和 Lonafarnib 的组合在斑马鱼胚胎中显示出最有前途的结果，允许两种药物的剂量减少。我们在转移性人黑色素瘤细胞系 A375M 中以及在正常人类上皮黑素细胞（NHEM）中验证了这些观察结果，以研究酮康唑在阻断转化的黑素细胞增殖中的作用机制。酮康唑处理细胞的活力测定和能量代谢分析表明，该药物特异性地影响体外黑色素瘤细胞和体内转化的黑素细胞，对 NHEM 或野生型幼虫没有影响。同一类别的另一种药物咪康唑也观察到类似的效果。此外，我们表明酮康唑的作用是通过抑制己糖激酶活性介导的，己糖激酶活性在体外和体内对黑色素瘤细胞异常代谢谱具有致死性。因此，我们的研究表明，斑马鱼可以提供一种富含表型的测定方法，用于完全自动化的筛选方法，以鉴定黑色素瘤的合成致死性治疗药物，并建议进一步测试酮康唑在组合治疗中的应用。
