Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells
Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells
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DOI：
10.1074/jbc.ac118.005899
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发表时间：
2019-01-11
影响因子：
4.8
通讯作者：
Neumann, Carola A.
中科院分区：
文献类型：
--
作者：
Asan, Alparslan;Skoko, John J.;Neumann, Carola A.
同源重组 (HR) 指导的 DNA 双链断裂 (DSB) 修复可实现模板指导的 DNA 修复，以维持基因组稳定性。 RAD51 重组酶 (RAD51) 是 HR 的关键组成部分，可促进 DSB 修复中的 DNA 链交换。我们在此报告，用脂肪酸硝基烯烃 10-硝基-十八-9-烯酸 (OA-NO2) 与抗肿瘤 DNA 损伤剂阿霉素、顺铂、奥拉帕尼和 IR 联合治疗三阴性乳腺癌 (TNBC) 细胞可增强这些药物的抗增殖作用。 OA-NO2 抑制 IR 诱导的 RAD51 灶形成并增强 TNBC 细胞中 H2A 组蛋白家族成员 X (H2AX) 的磷酸化。通过静态流式细胞术和动态活细胞研究对荧光 DSB 报告基因活性进行分析，实现重组的时间分辨率，结果表明 OA-NO2 抑制 HR，而不是非同源末端连接 (NHEJ)。 OA-NO2 烷基化 RAD51 中的 Cys-319，这种烷基化取决于 OA-NO2 的迈克尔受体性质，因为 OA-NO2 的非硝化和饱和非亲电类似物、十八烷酸和 10-硝基十八烷酸不与 Cys-319 反应。值得注意的是，RAD51 的 OA-NO2 烷基化抑制了其与 ssDNA 的结合。 RAD51 Cys-319 位于 ABL 原癌基因 1（非受体酪氨酸激酶 (ABL1)）的 SH3 结合位点内，因此我们研究了 OA-NO2 介导的 Cys-319 烷基化对 ABL1 结合的影响，发现 OA-NO2 在体外和基于细胞的免疫沉淀测定中均抑制 RAD51-ABL1 复合物形成。 RAD51-ABL1 复合物的抑制也抑制了下游 RAD51 Tyr-315 磷酸化。总之，RAD51 Cys-319 是 OA-NO2 等软亲电子试剂加合的功能上重要的位点，并建议进一步研究基于脂质亲电子试剂的 TNBC 联合疗法。
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 -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.