Journal article
Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells
The Journal of biological chemistry, Vol.294(2), pp.397-404
01/11/2019
DOI: 10.1074/jbc.AC118.005899
PMCID: PMC6333886
PMID: 30478172
Abstract
Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO2, octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO2–mediated Cys-319 alkylation on ABL1 binding and found that OA-NO2 inhibits RAD51–ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51–ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile–based combinational therapies for TNBC.
Details
- Title: Subtitle
- Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells
- Creators
- Alparslan Asan - University of Pittsburgh Medical CenterJohn J. Skoko - UPMC Hillman Cancer CenterChen-Shan Chen Woodcock - University of PittsburghBentley M. Wingert - University of PittsburghSteven R. Woodcock - University of PittsburghDaniel Normolle - University of PittsburghYi Huang - UPMC Hillman Cancer CenterJeremy M. Stark - Cancer Genetics (United States)Carlos J. Camacho - University of PittsburghBruce A. Freeman - University of PittsburghCarola A. Neumann - UPMC Hillman Cancer Center
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.294(2), pp.397-404
- DOI
- 10.1074/jbc.AC118.005899
- PMID
- 30478172
- PMCID
- PMC6333886
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Publisher
- Elsevier Inc
- Grant note
- T32-EB009403 / UPMC Hillman Cancer Center
- Language
- English
- Date published
- 01/11/2019
- Academic Unit
- Internal Medicine
- Record Identifier
- 9984383308102771
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