IGFBP-3 interacts with NONO and SFPQ in PARP-dependent DNA damage repair in triple-negative breast cancer
IGFBP-3 interacts with NONO and SFPQ in PARP-dependent DNA damage repair in triple-negative breast cancer
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DOI：
10.1007/s00018-019-03033-4
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发表时间：
2019-05-01
影响因子：
8
通讯作者：
Baxter, Robert C.
中科院分区：
文献类型：
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作者：
de Silva, Hasanthi C.;Lin, Mike Z.;Baxter, Robert C.
患有三阴性乳腺癌(TNBC)的女性通常接受化疗，但DNA双链断裂(DSB)修复可能会削弱她们的反应性。我们先前报道IGFBP-3与EGFR和DNA依赖的蛋白激酶(DNA-PKcs)形成核复合体，通过非同源末端连接(NHEJ)调节TNBC细胞中的DSB修复。为了发现IGFBP-3结合伙伴通过刺激DSB修复参与化疗耐药，我们用LC-MS/MS分析了IGFBP-3相互作用组，并通过免疫共沉淀和邻近连接实验证实了相互作用。体外DNA末端连接和H_2AX焦点测定证实了其功能效应。在基底样型TNBC细胞系HCC1806和MDA-MB-468中，DNA/RNA结合蛋白、非POU结构域的八聚体结合蛋白(NONO)及其二聚体剪接因子(SFPQ)与IGFBP-3形成复合体。在无细胞生化实验中也显示了NONO与IGFBP-3的结合。IGFBP-3与NONO和SFPQ的复合体可被Gefitinib抑制EGFR或用NU7026抑制DNA-PKcs，并被PARP抑制剂veliparib和olaparib阻断，这也降低了DNA末端连接活性并延迟了H_2AX信号的分解(即抑制DNA DSB修复)。SiRNA下调NHEJ途径1(LINP1)中的长非编码RNA也阻断了IGFBP-3与NONO-SFPQ的相互作用。这些发现表明，NONO和SFPQ在IGFBP-3依赖的DSB修复中具有PARP依赖的作用，LINP1参与了复合体的形成。我们认为，靶向IGFBP-3的DNA修复功能可能会提高基底细胞样TNBC的化疗敏感性，从而改善患者的预后。
Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of H2AX foci. In response to 20 mu M etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the H2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.