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MISSION esiRNA for RNAi Screening in Mammalian Cells
Published on: May 12, 2010
Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human
Alfeu Zanotto-Filho1,2, Ravi Dashnamoorthy1,3, Eva Loranc1
1Greehey Children´s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Abstract:
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.
Insights
Combining RNAi screening and gene expression data reveals novel pathways for alkylation damage survival. This includes glutathione (GSH)-mediated detoxification and Endoplasmic Reticulum stress/Unfolded Protein Responses (UPR), crucial for cancer chemotherapy efficacy.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Alkylating agents are vital in cancer chemotherapy, but cellular survival mechanisms beyond DNA repair and detoxification are not fully understood.
- Genomic screens suggest additional cellular components influence survival and chemoresistance.
- Identifying novel survival pathways can improve chemotherapy and address chemoresistance.
Purpose of the Study:
- To reevaluate Drosophila RNAi screening data alongside gene expression arrays to identify novel processes in alkylation damage survival.
- To explore conserved cross-species pathways involved in cellular response to alkylating agents.
Main Methods:
- Comparative analysis of Drosophila RNAi screening data and gene expression arrays.
- Pathway/process level analysis of combined gene lists from RNAi and microarray datasets.
- Validation of identified pathways using metabolomics, protein studies, and functional cell assays.
Main Results:
- A conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification, and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in alkylation survival.
- Previously identified pathways like p53 signaling, Nucleotide Excision Repair, DNA-mRNA-protein metabolism, and proteasome machinery were also confirmed.
- Combining RNAi and gene expression datasets prior to analysis yielded better results.
Conclusions:
- Fusion of RNAi and gene expression data is an effective strategy for rapidly identifying key cellular processes involved in damage survival.
- NRF2, GSH-mediated detoxification, and UPR are critical, conserved pathways for alkylation survival.
- Findings can inform strategies to enhance chemotherapeutic efficacy and overcome chemoresistance.
