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ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells.
Mol Cancer. 2017 06 08; 16(1):102.MC

Abstract

BACKGROUND

Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism.

METHODS

Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested.

RESULTS

We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells.

CONCLUSIONS

In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut).

Authors+Show Affiliations

Life Sciences Research Unit, University of Luxembourg, 6, Ave. du Swing, L-4367, Belvaux, Luxembourg.Life Sciences Research Unit, University of Luxembourg, 6, Ave. du Swing, L-4367, Belvaux, Luxembourg.Life Sciences Research Unit, University of Luxembourg, 6, Ave. du Swing, L-4367, Belvaux, Luxembourg.Life Sciences Research Unit, University of Luxembourg, 6, Ave. du Swing, L-4367, Belvaux, Luxembourg. stephanie.kreis@uni.lu.Life Sciences Research Unit, University of Luxembourg, 6, Ave. du Swing, L-4367, Belvaux, Luxembourg.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28595656

Citation

Cesi, Giulia, et al. "ROS Production Induced By BRAF Inhibitor Treatment Rewires Metabolic Processes Affecting Cell Growth of Melanoma Cells." Molecular Cancer, vol. 16, no. 1, 2017, p. 102.
Cesi G, Walbrecq G, Zimmer A, et al. ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells. Mol Cancer. 2017;16(1):102.
Cesi, G., Walbrecq, G., Zimmer, A., Kreis, S., & Haan, C. (2017). ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells. Molecular Cancer, 16(1), 102. https://doi.org/10.1186/s12943-017-0667-y
Cesi G, et al. ROS Production Induced By BRAF Inhibitor Treatment Rewires Metabolic Processes Affecting Cell Growth of Melanoma Cells. Mol Cancer. 2017 06 8;16(1):102. PubMed PMID: 28595656.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells. AU - Cesi,Giulia, AU - Walbrecq,Geoffroy, AU - Zimmer,Andreas, AU - Kreis,Stephanie, AU - Haan,Claude, Y1 - 2017/06/08/ PY - 2016/09/21/received PY - 2017/05/24/accepted PY - 2017/6/10/entrez PY - 2017/6/10/pubmed PY - 2018/3/27/medline KW - BRAF inhibitors KW - MEK inhibitors KW - Melanoma KW - Metabolism KW - NRAS KW - PDK inhibitors KW - Pyruvate dehydrogenase KW - Pyruvate dehydrogenase kinases KW - RAS/RAF/MEK/ERK pathway KW - Reactive oxygen species SP - 102 EP - 102 JF - Molecular cancer JO - Mol. Cancer VL - 16 IS - 1 N2 - BACKGROUND: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism. METHODS: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. RESULTS: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. CONCLUSIONS: In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut). SN - 1476-4598 UR - https://www.unboundmedicine.com/medline/citation/28595656/ROS_production_induced_by_BRAF_inhibitor_treatment_rewires_metabolic_processes_affecting_cell_growth_of_melanoma_cells_ L2 - https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-017-0667-y DB - PRIME DP - Unbound Medicine ER -