Next Generation Sequencing of mouse melanoma tumors generated with a repressible BRAF construct
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE152722 | GEO | 2020/07/15
REPOSITORIES: GEO
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Project description:Purpose: The goals of this study are to compare NGS-derived transciptomes from human melanoma cell lines before and after vemurafenib in order to compare to a mouse model of melanoma using doxycycline that permits control of mutant BRAF expression. Treatment with doxycycline leads to loss of mutant BRAF expression and tumor regression, but tumors reoccur after a prolonged period of dormancy. Results: We found conserved expression changes in histone methyltransferase genes (HMT), including Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), Suppressor Of Variegation 3-9 Homolog 1(SUV39H1), Suppressor Of Variegation 3-9 Homolog 2 (SUV39H2), ASH2 Like, Histone Lysine Methyltransferase Complex Subunit (ASH2L), SET And MYND Domain Containing 2 (SYMD2), and Protein arginine methyltransferase 5 (PRMT5) and histone deacetylases (HDACs) HDACS 7, 9,10, and 11.
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Project description:Melanoma cells are highly plastic and have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanoma cell lines and patient specimens to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia and UV-irradiation leads to an increase in histone deacetylase 8 (HDAC8) expression/activity, and in turn, the adoption of a drug-resistant, invasive phenotype. Systems level analyses using mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling pathways. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in in vivo xenograft models. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase (RTK) activation leading to Ras/CRAF/MEK/ERK signaling. Although HDACs primarily function at the histone level, they also regulate signaling through the modulation of non-histone substrates. In line with this, HDAC8 introduction decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine residue 273 reduced the transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition through increased RTK expression and enhanced MAPK pathway activity. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both non-selective and HDAC8-specific inhibitors enhancing the durability of response to BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors could represent an excellent strategy to limit the adaptation of melanoma cells to multiple stresses and therapeutic interventions, including BRAF-MEK inhibitor combinations.
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Project description:Melanoma cells are highly plastic and have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanoma cell lines and patient specimens to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia and UV-irradiation leads to an increase in histone deacetylase 8 (HDAC8) expression/activity, and in turn, the adoption of a drug-resistant, invasive phenotype. Systems level analyses using mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling pathways. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in in vivo xenograft models. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase (RTK) activation leading to Ras/CRAF/MEK/ERK signaling. Although HDACs primarily function at the histone level, they also regulate signaling through the modulation of non-histone substrates. In line with this, HDAC8 introduction decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine residue 273 reduced the transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition through increased RTK expression and enhanced MAPK pathway activity. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both non-selective and HDAC8-specific inhibitors enhancing the durability of response to BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors could represent an excellent strategy to limit the adaptation of melanoma cells to multiple stresses and therapeutic interventions, including BRAF-MEK inhibitor combinations.
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Project description:SMYD3, a member of the SET and MYND domain-containing (SMYD) family, is a histone methyltransferase (HMT) and transcription factor that plays an important role in transcriptional regulation in human carcinogenesis.In an effort to better understanding the mechanistic role of SMYD3, affinity purification and mass spectrometry assays were used to identify proteins associated with SMYD3 in vivo. In these experiments, FLAG-tagged SMYD3 or an empty vector was stably expressed in LM3 cells. Cellular extracts were prepared and subjected to affinity purification using an anti-FLAG affinity gel. Immunocomplexed proteins were separated using SDS–PAGE and silver stained. Immunoprecipitated proteins in specific bands in comparison to the vector were gel extracted, trypsin digested and identified using liquid chromatography tandem mass spectrometry.
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Project description:Systems level analyses using mass spectrometry-based phosphoproteomics and RNA-Seq implicated HDAC8 in the regulation of MAPK and AP-1 signaling pathways. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in in vivo xenograft models. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase (RTK) activation leading to Ras/CRAF/MEK/ERK signaling. Although HDACs primarily function at the histone level, they also regulate signaling through the modulation of non-histone substrates. In line with this, HDAC8 introduction decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine residue 273 reduced the transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition through increased RTK expression and enhanced MAPK pathway activity.
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