Project description:Melanoma cell lines were assessed for differences in gene expression patterns between the lines sensitive and resistant to BRAF and MEK inhibitor drugs. 22 BRAF-mutant melanoma cell lines were assessed for response to BRAF and MEK inhibitors in a 3 day drug treatment dose response assay. Based on the IC50, 18 lines were found to be responsive to BRAF or MEK inhibition and 4 were resistant. Normalised gene expression data generated from experimental replicate affymetrix arrays was assessed to identify differential patterns of inherent gene expression between the cell lines grouped as drug-responsive or drug-resistant. This were used to idenify specific candidate genes and pathways associated with inherent BRAF/MEK inhibitor drug resistance in melanoma cells.
Project description:To investigate mechanisms of resistance to BRAF inhibitor therapy in melanoma, BRAF mutant cell lines have been chronically exposed to BRAFi to create phenotypes with acquired drug resistance. Expression proteomics is used to examine the differences between naive and drug-resistant cells.
Project description:In vitro cell cultures are frequently used to define the molecular background of drug resistance. In this study our major aim was to compare the gene expression signature of 2D and 3D cultured BRAFV600E mutant melanoma cell lines. We successfully developed BRAF-drug resistant cell lines from paired primary/metastatic melanoma cell lines in both 2D and 3D in vitro cultures. Using Affymetrix Human Gene 1.0 ST arrays, we determined the gene expression pattern of all cell lines. Our study highlights gene expression alterations that might help to understand the development of acquired resistance in melanoma cells in tumour tissue.
Project description:Melanoma cell lines were assessed for differences in gene expression patterns between the lines sensitive and resistant to BRAF and MEK inhibitor drugs.
Project description:Acquired BRAF/MEK inhibitor resistance in melanoma results in a new transcriptional state associated with increased risk of metastasis. Here, we identified non-canonical EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry-based proteomic and phenotypic assays to demonstrate that the expression of active non-canonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition (MAT) driven by Cdc42 activation. The induction of MAT promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous flow conditions, increased permeability of endothelial cell monolayers and stimulated melanoma transendothelial cell migration. In vivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention following tail-vain injection. Analysis of BRAF inhibitor-sensitive and -resistant melanoma cells demonstrated resistance to be associated with an MAT switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug resistant metastatic state was dependent upon histone deacetylase 8 (HDAC8) activity. Silencing of HDAC8 lead to inhibition of EphA2 and AKT phosphorylation, reduced invasion and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on non-canonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.
Project description:aCGH of human melanoma cell lines comparing parental (drug sensitve) vs isogenic drug resistant-derived subline Two condition experiment: two BRAF-V600E mutant cell lines (drug sensitive - parental baseline) vs two derived sublines after chronic exposure to the MEK inhibitor trametinib (drug resistant) are compared
Project description:Acquired BRAF/MEK inhibitor resistance in melanoma results in a new transcriptional state associated with increased risk of metastasis. Here, we identified non-canonical EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry-based proteomic and phenotypic assays to demonstrate that the expression of active non-canonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition (MAT) driven by Cdc42 activation. The induction of MAT promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous flow conditions, increased permeability of endothelial cell monolayers and stimulated melanoma transendothelial cell migration. In vivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention following tail-vain injection. Analysis of BRAF inhibitor-sensitive and -resistant melanoma cells demonstrated resistance to be associated with an MAT switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug resistant metastatic state was dependent upon histone deacetylase 8 (HDAC8) activity. Silencing of HDAC8 lead to inhibition of EphA2 and AKT phosphorylation, reduced invasion and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on non-canonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.
Project description:We generated four drug-resistant melanoma cell lines from paired primary/metastatic cell lines using PLX4720 and used for Affymetrix Human Gene 1.0 ST array