Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human

Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components...

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Bibliographic Details
Published in:PloS one 2016-04, Vol.11 (4), p.e0153970-e0153970
Main Authors: Zanotto-Filho, Alfeu, Dashnamoorthy, Ravi, Loranc, Eva, de Souza, Luis H T, Moreira, José C F, Suresh, Uthra, Chen, Yidong, Bishop, Alexander J R
Format: Article
Language:English
Subjects:
Alkylating agents
Alkylation
Analysis
Animals
Biology and Life Sciences
Blotting, Western
Cancer
Cancer therapies
Cell Survival
Cells, Cultured
Chemoresistance
Chemotherapy
Conservation
Damage detection
Deoxyribonucleic acid
Detoxification
DNA
DNA damage
DNA microarrays
DNA repair
DNA Repair - genetics
Drosophila
Drosophila melanogaster
Drosophila melanogaster - genetics
Drosophila melanogaster - growth & development
Drosophila melanogaster - metabolism
Embryo, Mammalian - cytology
Embryo, Mammalian - metabolism
Embryos
Endoplasmic reticulum
Endoplasmic Reticulum Stress - genetics
Epidemiology
Fibroblasts
Fibroblasts - cytology
Fibroblasts - metabolism
Gene expression
Gene Expression Profiling
Gene Expression Regulation
Gene Regulatory Networks
Genetic aspects
Genome
Genomes
Glutathione
High-Throughput Screening Assays
Humans
Insects
Mammals
Medical research
Metabolism
Metabolomics
Metabolomics - methods
Mice
Mice, Inbred C57BL
Nucleotide excision repair
p53 Protein
Penicillin
Physical Sciences
Physiological aspects
Proteasomes
Protein folding
Protein Interaction Maps
Protein metabolism
Protein turnover
Proteins - genetics
Proteins - metabolism
Repair
Research and Analysis Methods
RNA Interference
RNA-mediated interference
Science
Screening
Signal Transduction
Signaling
Survival
Transcription
Unfolded Protein Response - genetics
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Summary:Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0153970