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 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.
Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.|Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Alfeu
A
Zanotto-Filho
Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.|Molecular Oncology Research Institute, Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts, United States of America.
Ravi
R
Dashnamoorthy
Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Eva
E
Loranc
Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Luis H T
LH
de Souza
Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
José C F
JC
Moreira
Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.|Department of Epidemiology and Biostatistics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Uthra
U
Suresh
Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.|Department of Epidemiology and Biostatistics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Yidong
Y
Chen
Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.|Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Alexander J R
http://orcid.org/0000-0002-5742-4387
AJ
Bishop
D000478:Alkylation; D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D004260:DNA Repair / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D016678:Genome; D057166:High-Throughput Screening Assays; D006801:Humans; D055432:Metabolomics / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D060066:Protein Interaction Maps; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D056811:Unfolded Protein Response / Q000235:genetics
D011506:Proteins