ZFIN ID: ZDB-PUB-190422-7
Automated in vivo screen in zebrafish identifies clotrimazole as targeting a metabolic vulnerability in a melanoma model
Precazzini, F., Pancher, M., Gatto, P., Tushe, A., Adami, V., Anelli, V., Mione, M.C.
Date: 2019
Source: Developmental Biology   457(2): 215-225 (Journal)
Registered Authors: Anelli, Viviana, Precazzini, Francesca
Keywords: Anticancer drugs, Chemical screening, Clotrimazole, Melanocytes, Metabolism, Zebrafish
MeSH Terms:
  • Animals
  • Animals, Genetically Modified
  • Antineoplastic Agents/pharmacology*
  • Antineoplastic Combined Chemotherapy Protocols/pharmacology
  • Cell Line, Tumor
  • Cell Proliferation/drug effects
  • Cell Survival/drug effects
  • Clotrimazole/pharmacology*
  • Disease Models, Animal
  • Drug Screening Assays, Antitumor/methods
  • Farnesyltranstransferase/antagonists & inhibitors
  • Humans
  • Melanocytes/metabolism
  • Melanoma/drug therapy*
  • Melanoma/metabolism
  • Miconazole/pharmacology
  • Piperidines/pharmacology*
  • Pyridines/pharmacology*
  • Skin Neoplasms/drug therapy*
  • Skin Neoplasms/metabolism
  • Zebrafish
PubMed: 30998907 Full text @ Dev. Biol.
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ABSTRACT
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.
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