背景:NUAK家族激酶2(NUAK2)已被确定为多种恶性肿瘤中肿瘤进展的重要介质。然而,其在肺腺癌(LUAD)中的作用尚不清楚。
方法:进行生物信息学分析以评估LUAD患者NUAK2的表达和预后。在多个LUAD细胞系中测量NUAK2表达,并进行了功能丧失实验。使用CCK-8和集落形成测定评估细胞增殖能力。球样形成,碱性磷酸酶(AP)染色,进行管形成和SA-β-gal染色测定以检查干性,血管生成和衰老。通过TBARS产生和脂质ROS评估脂质过氧化物酶。Westernblot用于检测关键蛋白。此外,A549细胞用铁凋亡抑制剂铁抑制素-1(Fer-1)处理用于拯救测定。最后,将A549细胞皮下注射入小鼠右侧腹,建立LUAD荷瘤小鼠模型,并检测肿瘤的重量和大小。
结果:NUAK2在LUAD和LUAD细胞系患者中上调。NUAK2耗竭抑制细胞活力,殖民地,肿瘤球和降低Oct4和Nanog表达,证实NUAK2耗竭抑制A549细胞的增殖和干性。同时,NUAK2消耗通过减少形成的管和VEGFR1/2表达来阻断血管生成,并通过升高SA-β-gal阳性细胞和p16,p21和p53的表达来促进A549细胞的衰老。此外,NUAK2耗竭脂质ROS升高,TBARS生产和Fe2+水平,证明NUAK2耗竭可引发A549细胞的铁凋亡。此外,拯救实验表明,额外的Fer-1治疗部分削弱了NUAK2耗竭对A549细胞恶性行为的影响。最后,体内实验表明,NUAK2敲低可以极大地抑制LUAD小鼠的肿瘤生长。
结论:总之,NUAK2耗竭部分通过触发铁凋亡阻碍了A549细胞的致癌表型,表明NUAK2是治疗LUAD的新靶点。
BACKGROUND: NUAK family kinase 2 (
NUAK2) has been identified as an important mediator for tumor progression in multiple malignancies. Nevertheless, its role in lung adenocarcinoma (LUAD) remains unclear.
METHODS: Bioinformatic analysis was performed to assess the expression and prognosis of NUAK2 in patients with LUAD. The NUAK2 expression was measured in multiple LUAD cell lines, and the loss-of-function experiment was conducted. Cell proliferation ability was assessed using CCK-8 and colony formation assays. Spheroid formation, alkaline phosphatase (AP) staining, tube formation and SA-β-gal staining assays were performed to examine stemness, angiogenesis and senescence. Lipid peroxidase was assessed by TBARS production and lipid ROS. Western blot was used to detect critical proteins. In addition, A549 cells were treated with ferroptosis inhibitor ferrostatin-1 (Fer-1) for a rescue assay. Finally, A549 cells were subcutaneously injected into the right flank of mice to establish LUAD-bearing mouse model, and the tumor weight and size were detected.
RESULTS: NUAK2 was upregulated in patients with LUAD and LUAD cell lines. NUAK2 depletion inhibited cell viability, colonies, tumor spheres and decreased Oct4 and Nanog expression, confirming
NUAK2 depletion inhibited proliferation and stemness of A549 cells. Meanwhile, NUAK2 depletion blocked angiogenesis via reducing formed tubes and VEGFR1/2 expression, and promoted senescence of A549 cells by elevating SA-β-gal-positive cells and p16, p21 and p53 expression. Moreover,
NUAK2 depletion elevated lipid ROS, TBARS production and Fe2+ level, demonstrating that
NUAK2 depletion could trigger ferroptosis in A549 cells. Furthermore, the rescue experiments revealed that the impacts of NUAK2 depletion on malignant behaviors in A549 cells were partly weakened by additional Fer-1 treatment. Finally, in vivo experiments demonstrated that NUAK2 knockdown greatly inhibited tumor growth in LUAD-bearing mice.
CONCLUSIONS: In summary, NUAK2 depletion impeded oncogenic phenotypes of A549 cells partly via triggering ferroptosis, suggesting
NUAK2 as a novel target for treating LUAD.