Expression of BRAF inhibitor resistant colon cancer lines
Ontology highlight
ABSTRACT: Colon cancer cell lines with partial sensitivity to the BRAF inhibitor PLX4720 were grown in increasing concentration of the drug to develop acquired resistance. Gene expression was performed for comparison of the resistant clones to the parental lines. Colon cancer cell lines with partial sensitivity to the BRAF inhibitor PLX4720 were grown in increasing concentration of the drug to develop acquired resistance. Gene expression was performed for comparison of the resistant clones to the parental lines.
Project description:Colon cancer cell lines with partial sensitivity to the BRAF inhibitor PLX4720 were grown in increasing concentration of the drug to develop acquired resistance. Gene expression was performed for comparison of the resistant clones to the parental lines.
Project description:Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to MAPK pathway inhibition we identified the combination PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ERBB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and RTK mutational status. Layered over base-line resistance was substantial upregulation of many ERBB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ERBB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes. 12 BRAF mutant melanomas and 4 melanomas with WT BRAF were exposed plx4720 treatment to evaluate their responses after 8 hours of treatment. 5 of the 12 BRAF mutant melanomas responses were also evaluated in response to the treatment of lapatinib alone, masitinib alone, the combination of lapatinib with plx4720, or the combination of masitinib with plx4720. All samples were run in at least triplicate.
Project description:A375P melanoma cells were treated with 1uM of the MEK inhibitor PD184352 or 0.4uM of the V600EBRAF inhibitor PLX4720 for 2hr, 6hr and 24hrs. DMSO treatment for 2hr, 6hr and 24hrs serves as the negative control Triplicate experiments were performed for DMSO, PD184352 and PLX4720 treatment at 3 timepoints - 2, 6, 24hrs.
Project description:Mutations in the BRAF proto-oncogene, which encodes the B-Raf kinase, are associated with more aggressive, less-differentiated and therapy-resistant colorectal cancers (CRC). However, the molecular mechanisms responsible for these correlations remain unknown. Here, we report the characterization of human isogenic CRC cell line models (Caco-2, HT29, Colo-205) in which we modulate the expression of the B-RafV600E oncoprotein either by conditional cDNA or shRNA expression. Using these models in conventional and three dimensional tissue culture systems, we demonstrate that genetic depletion of endogenous B-RafV600E decreases cellular motility and invasion, while it induces hallmarks of differentiated epithelia such as the formation of functional adherens and tight junctions. Importantly, these effects are recapitulated by exposing these lines to B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Furthermore, loss of endogenous B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces epithelial structures with marked expression of Cdx-2, a prognostic marker and master regulator of intestinal morphogenesis. By performing the first transcriptome profiles of B-Raf inhibitor treated 3D cultures of a primary adenocarcinoma and a metastasis derived CRC cell line, we establish functional links between B-RafV600E and proteins of known and potentially new prognostic relevance. We propose that B-Raf/MEK/ERK pathway inhibitors could be used to induce CRC differentiation and thereby to limit metastatic disease. To measure the time resolved gene responses, RNA was isolated from PLX4720 or DMSO treated Colo-205 and HT29 3D culture cell lysates at days 1,3 and 10 and days 1,3, and 8, respectively. The time points for HT29 cells were taken in biological duplicates.
Project description:microRNA expression profiles show altered patterns in cell lines with acquired resistance to the BRAF inhibitor compared to the parental cell lines, and common miRNAs regulated in the resistant variants
Project description:In an effort to understand the mechanisms of acquired resistance to BRAF inhibitors, we isolated clones that acquired resistance to the BRAF inhibitor GSK2118436 derived from the A375 BRAF V600E mutant melanoma cell line. This resistance clones acquired mutations in NRAS and MEK1. One clones, 16R6-4, acquired two mutations in NRAS – Q61K and A146T. Proliferation and western blot analyses demonstrated that these clones were insensitive to single agent GSK2118436 or GSK1120212 (an allosteric MEK inhibitor) but were sensitive to the combination of GSK2118436 and GSK1120212. To further characterize this combination, global transcriptomic analysis was performed in A375 and 16R6-4 after 24 hour treatment with GSK2118436, GSK1120212 or the combination of GSK2118436 and GSK1120212. This data set was published in Molecular Cancer Therapeutics with the title “Combined inhibition of BRAF and MEK, BRAF and PI3K/mTOR, or MEK and PI3K/mTOR overcomes acquired resistance to the BRAF inhibitor GSK2118436, mediated by NRAS or MEK mutations” by Greger, J.G., et.al.
Project description:In an effort to understand the mechanisms of acquired resistance to BRAF inhibitors, we isolated clones that acquired resistance to the BRAF inhibitor GSK2118436 derived from the A375 BRAF V600E mutant melanoma cell line. This resistance clones acquired mutations in NRAS and MEK1. One clones, 16R6-4, acquired two mutations in NRAS – Q61K and A146T. Proliferation and western blot analyses demonstrated that these clones were insensitive to single agent GSK2118436 or GSK1120212 (an allosteric MEK inhibitor) but were sensitive to the combination of GSK2118436 and GSK1120212. To further characterize this combination, global transcriptomic analysis was performed in A375 and 16R6-4 after 24 hour treatment with GSK2118436, GSK1120212 or the combination of GSK2118436 and GSK1120212. This data set was published in Molecular Cancer Therapeutics with the title “Combined inhibition of BRAF and MEK, BRAF and PI3K/mTOR, or MEK and PI3K/mTOR overcomes acquired resistance to the BRAF inhibitor GSK2118436, mediated by NRAS or MEK mutations” by Greger, J.G., et.al. A375 and 16R6-4 (an A375 derived GSK2118436 resistance clone) were treated for 24 hours with 0.1 micromolar GSK2118436, 1 micromolar GSK2118436, 0.01 micromolar GSK1120212, 0.1 micromolar GSK2118436 + 0.01 micromolar GSK1120212, or 1 micromolar GSK2118436 + 0.01 micromolar GSK1120212.
Project description:Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to MAPK pathway inhibition we identified the combination PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ERBB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and RTK mutational status. Layered over base-line resistance was substantial upregulation of many ERBB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ERBB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.
Project description:Two human colorectal adenocarcinoma cell lines (Colo-205 and RKO) were treated with PLX4720 and newly synthesized 2-Acetamido, 6-Carboxamide Substituted Benzothiazole Derivatives, as potential BRAF V600E inhibitors; IDK142, IDK145, IDK133 and DMSO as control.