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Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling of drug-tolerant cancer cell subpopulation

ABSTRACT: Cancer relapse occurs even after curative treatment, suggesting the existence of undetectable cancer cell subpopulations and their drug-tolerant potential. We found that the number of drug-tolerant colonies (DTCs) was significantly suppressed by an RNA polymerase II (RNAPII) inhibitor, alpha-amanitin (alpha-AMA). To identify potential molecular target of alpha-AMA, we performed transcriptional profiling by Agilent-028004 SurePrint G3 Human GE 8x60K Microarray using untreated colonies and DTCs derived from MCF7. Among the top 2.5% of specifically induced genes in DTCs, we focused on TAF15 gene because its gene product binds RNAPII. A subsequent colony formation assay revealed that TAF15 knockdown suppressed the emergence of both DTCs and untreated colonies, suggesting that TAF15 is a crucial target of alpha-AMA in the context of DTC suppression. Total RNA was extracted from two biological replicates of bulk untreated MCF7 colonies and MCF7 DTCs emerged in the presence of 0.8 ÂµM CIS. For identification of the TAF15 gene, gene ontology analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software tools (http://david.abcc.ncifcrf.gov/).

ORGANISM(S): Homo sapiens

SUBMITTER: Kohei Kume

PROVIDER: E-GEOD-65419 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptional profiling of drug-tolerant cancer cell subpopulations

Project description:Cancer relapse occurs even after curative treatment, suggesting the existence of undetectable cancer cell subpopulations and their drug-tolerant potential. We found that the number of drug-tolerant colonies (DTCs) was significantly suppressed by an RNA polymerase II (RNAPII) inhibitor, alpha-amanitin (alpha-AMA). To identify potential molecular target of alpha-AMA, we performed transcriptional profiling by Agilent-028004 SurePrint G3 Human GE 8x60K Microarray using untreated colonies and DTCs derived from MCF7. Among the top 2.5% of specifically induced genes in DTCs, we focused on TAF15 gene because its gene product binds RNAPII. A subsequent colony formation assay revealed that TAF15 knockdown suppressed the emergence of both DTCs and untreated colonies, suggesting that TAF15 is a crucial target of alpha-AMA in the context of DTC suppression.

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Genome-wide analysis of DNA methylation in drug-tolerant cancer cell subpopulations

Project description:Cancer relapse after curative treatment is thought to originate from drug-tolerant and invisible cancer cell subpopulations. Using cancer cell colonies emerging in the presence of drugs (drug-tolerant colonies, DTCs), we found that the drug-tolerant properties of DTCs are lost through a reversible mechanism. To examine whether epigenetic regulation is responsible for the phenotypic changes in DTCs, we performed a genome-wide analysis for relative CpG methylation between the DTCs and untreated colonies derived from MKN45 by NimbleGen Human Meth 385K Prom Plus CpG Arrays. Global changes in the methylation levels were evident in a chromosomal location-dependent manner. The methylation status of the upstream regions of the transcription start sites of the pluripotency-inducing genes showed good agreement with the qRT-PCR data. These results suggest that reversible drug-tolerant properties in DTCs are epigenetically regulated and associated with transcriptional regulation, including pluripotency-inducing factors. Comparison of untreated colonies v.s. DTCs derived from MKN45 cells.

2015-05-26 | E-GEOD-69203 | biostudies-arrayexpress

Genome-wide analysis of DNA methylation in drug-tolerant cancer cell subpopulations

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Condensates induced by transcription inhibition mislocalizes active chromatin to nucleoli

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2023-03-11 | PXD035817 | Pride

Characterization of RAS/RAF/ERK signal cascade as a novel regulating factor in alpha-amanitin-induced liver injury

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Estrogen receptor α-mediated signaling inhibits type I interferon response to promote breast cancer

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Characterization of complement C3 as a marker of alpha-amanitin toxicity by comparative secretome profiling

Project description:In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they represent the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed via secretome analysis, which can be used to discover toxic mechanisms or exposure markers. Alpha-amanitin (-AMA) is the most widely studied amatoxin and inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, secretory proteins released during hepatic failure caused by -AMA have not been fully characterized. In this study, we analyzed secretome of -AMA-treated Huh-7 cells and mice using a comparative proteomics technique. Overall, 1,505 and 1,440 proteins were identified and quantified, respectively. Among them, 4 and 127 proteins were significantly upregulated and downregulated, respectively. Based on the bioinformatics results for the significantly downregulated proteins, we identified complement component 3 (C3) as a marker for -AMA-induced hepatotoxicity. Through western blot and C3 ELISA assays, we validated -AMA-induced downregulation of C3. In conclusion, using comparative proteomics and molecular biology techniques, we found that -AMA-induced hepatotoxicity reduced C3 levels in the secretome. We expect that this study will aid in identifying new toxic mechanisms, therapeutic targets, and exposure markers of -AMA-induced hepatotoxicity.

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