Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells

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.

Keywords: DNA repair; PARP1; Rad51; breast cancer; cysteine-mediated cross-linking; drug action; drug discovery; homologous recombination; nitro fatty acid; nitroalkene; signal transduction; triple negative breast cancer.

Figure 1.

OA-NO₂ inhibits TNBC cell growth, RAD51 foci formation, and sensitivity to ionizing radiation. A, MDA-MB-231 (MM231) cells (0.5 × 10⁶) were orthotopically injected into 6-week-old mice, and mice were gavaged with 15 mg/kg OA (black) or OA-NO₂ (red) for 4 weeks when tumors reached a volume of 100 mm³. Values indicate average, and error bars represent S.E.; n = 6–7 per group. B, tumoral γH2AX was increased in OA-NO₂–treated mice compared with OA control mice as assessed by immunoblotting (n = 6–7 per group). C, MDA-MB-231 (red), BT549 (blue), or Hs578T (green) cells were treated with increasing concentrations of OA-NO₂, and relative growth was measured by quantifying luminescent ATP levels (CellTiter-Glo). EC₅₀ values indicate average, and error bars represent S.E.; n = 3. D–F, MDA-MB-231 (red), BT549 (blue), or Hs578T (green) cells were treated with increasing concentrations of doxorubicin, cisplatin, or olaparib ± OA-NO₂ and measured as above. G, MDA-MB-231 (red) cells were treated with increasing concentrations of olaparib daily + vehicle, OA, or OA-NO₂ and measured as above. H and I, OA-NO₂ diminished RAD51 foci formation (green) and increased γH2AX (red) in MDA-MB-231 cells following irradiation with 5 Gy. Merged samples include 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei (blue). Cells on 16-well coverslips were dosed with 5 Gy and then treated with 5 μm OA-NO₂ or vehicle for 6 h prior to immunofluorescence processing. The average percentages of cells with five or more foci from confocal z-stacked images are indicated from 0- or 5-Gy samples, and error bars represent. * = p < 0.05.

Figure 2.

Inhibition of homologous recombination but not nonhomologous end joining by OA-NO₂. A and B, U2OS cells containing the HR reporter construct DR-GFP were transfected with an I-SceI plasmid and treated with vehicle (gray), 5 μm OA (green), or 5 μm OA-NO₂ (red). Negative control cells did not have I-SceI present. Values indicate average, and error bars represent S.E.; n ≥ 3. A, the number of GFP-positive cells was detected by flow cytometry at 48 h. B, emergence of GFP-positive cells over 68 h was quantified using live-cell fluorescence microscopy. GFP-positive cell counts were normalized to cell confluence and compared. C and D, U2OS cells containing the NHEJ reporter construct EJ5-GFP were transfected with an I-SceI plasmid and treated with vehicle (gray), 5 μm OA (green), or 5 μm OA-NO₂ (red). Negative control cells did not have I-SceI present. Values indicate average, and error bars represent S.E.; n = 3. C, the number of GFP-positive cells were detected by flow cytometry at 48 h. D, emergence of GFP-positive cells over 68 h was quantified using live-cell fluorescence microscopy. GFP-positive cell counts were normalized to cell confluence and compared.

Figure 3.

OA-NO₂ binds to RAD51 at Cys-319 and blocks ABL heterodimerization. A, U2OS cells containing the HR reporter construct DR-GFP were stably transfected with a control or RAD51 overexpression plasmid, and the cells were investigated as above with 5 μm OA-NO₂ in control (red) or RAD51-overexpressing cells (red striped). B, OA-NO₂ binds RAD51 in vitro. Purified RAD51 protein was incubated with control, biotinylated OA, OA-NO₂, or SA-NO₂ for 1 h; precipitated with streptavidin-coated agarose; and then detected by immunoblotting. C, OA-NO₂ binds to RAD51 Cys-319 in cells expressing RAD51. 293T cells expressing WT or cysteine mutant RAD51 were incubated with biotinylated OA-NO₂ for 1 h, precipitated, and detected. Three independent experiments were quantified and analyzed by one-way analysis of variance. *, p < 0.05. D, Alexa Fluor 488–conjugated DNA was incubated with purified RAD51, ATP, and 5 μm OA (black), 5 μm OA-NO₂ (gray), or 10 μm OA-NO₂ (white), and fluorescence polarization was quantified and normalized to a control lacking ATP. E, molecular modeling of RAD51 (blue) and OA-NO₂ (purple). Binding of OA-NO₂ with the Cys-319 residue (gold) of RAD51 is predicted to be further stabilized by hydrophobic interactions with Pro-318 and possible hydrogen bonding with Glu-322 of RAD51. F, OA-NO₂ disrupts ABL binding to RAD51 in vitro. Purified RAD51 and ABL core proteins were incubated with OA-NO₂ at 0, 100, or 500 nm for 1 h, and ABL was precipitated. The amount of bound RAD51 was detected by immunoblotting. G, OA-NO₂ disrupts RAD51 and ABL interactions by IP and reduces RAD51 Tyr-315 phosphorylation. 293T cells were transfected with FLAG-RAD51 and ABL core protein and then treated with OA-NO₂ for 1 h. RAD51 interactions with ABL and phosphorylated RAD51 Tyr-315 were probed by IP and immunoblotting. H, OA-NO₂ binds RAD51 in MDA-MB-231 or MDA-MB-468 cells. Cells were incubated with biotinylated OA-NO₂, and then lysates were precipitated with streptavidin-coated agarose and detected by immunoblotting. I, OA-NO₂ decreases HR and causes genomic instability and death in TNBC cells. All values indicate average, and error bars represent S.E.; n = 3.