Department of Hematology and Oncology, Children's Hospital of Chongqing Medical University, 136 Zhongshanerlu, Yuzhong district, Chongqing, 400014, China.
National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China.
Cell Commun Signal. 2022 Aug 16;20(1):122. doi: 10.1186/s12964-022-00935-y.
B-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown.
The expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway.
Netrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b.
Taken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b. Video Abstract.
B 细胞急性淋巴细胞白血病（B-ALL）占所有急性淋巴细胞白血病（ALL）病例的 85%以上，是最常见的儿童恶性肿瘤。尽管 B-ALL 患者的 5 年总生存率超过 90%，但复发或难治性 B-ALL 患者可能预后不良并出现不良事件。轴突导向因子 netrin-1 已被报道参与多种类型癌症的肿瘤发生。然而，netrin-1 对 B-ALL 的影响尚不清楚。
通过酶联免疫吸附测定（ELISA）试剂盒测量儿童 B-ALL 患者和无肿瘤儿童外周血样本中的 netrin-1 表达水平。然后，使用浓度为 0、25、50 和 100ng/ml 的外源性重组 netrin-1 处理 B-ALL 细胞（Reh 和 Sup B15），通过 CCK-8 细胞增殖测定和流式细胞术分析检测 B-ALL 细胞的活力和凋亡。此外，通过共免疫沉淀（co-IP）检测 netrin-1 的受体。用重组慢病毒诱导 B-ALL 细胞中 UNC5B 的表达干扰，然后通过 CCK-8 测定、流式细胞术测定和 Western blot 测定验证 netrin-1 可能通过受体 Unc5b 作用于 B-ALL 细胞。最后，应用 Western blot 和激酶抑制剂处理检测下游信号通路。
netrin-1 在 B-ALL 中表达增加，高危和中危分层组患者的 netrin-1 表达上调。然后，我们发现 netrin-1 在 B-ALL 细胞中诱导抗凋亡作用，表明 netrin-1 在 B-ALL 中发挥致癌作用。co-IP 结果表明，netrin-1 在 B-ALL 细胞中与受体 Unc5b 相互作用。在 B-ALL 细胞中干扰 UNC5B 并消除了 netrin-1 的抗凋亡作用。进一步的 Western blot 用于检测用重组 netrin-1 处理的 B-ALL 细胞中常见信号转导通路中关键分子的磷酸化水平，发现 FAK-MAPK 信号通路被激活。UNC5B 干扰消除了 netrin-1 的抗凋亡作用和 FAK-MAPK 磷酸化。应用 FAK 抑制剂处理和 ERK 抑制剂处理并验证了 FAK-MAPK 途径可能是 Unc5b 的下游。
总之，我们的研究结果表明，netrin-1 通过与受体 Unc5b 结合激活 FAK-MAPK 信号通路，诱导 B-ALL 细胞的抗凋亡作用。
