Cancer Program, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, 3800, Australia.
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia.
Cell Commun Signal. 2022 Dec 22;20(1):197. doi: 10.1186/s12964-022-01006-y.
Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression. Video Abstract.
Nima 相关激酶 (NEK) 家族的特定成员与癌症的发生和发展有关，最近有人提出，NEK5（研究最少的成员之一）在乳腺癌中起作用。然而，虽然已知 NEK5 调节中心体分离和有丝分裂纺锤体组装，但 NEK5 在这种恶性肿瘤中的信号机制和功能需要进一步表征。为此，我们建立了一个模型系统，在永生化乳腺上皮细胞系 MCF-10A 中过表达 NEK5。过表达 NEK5 的 MCF-10A 细胞在单层条件下克隆形成能力增加，在 3D Matrigel 培养中腺泡大小增加和形态异常。有趣的是，它们还表现出Src 激活和下游信号的明显减少。为了以无偏倚的方式研究 NEK5 的信号和功能，我们应用了各种基于 MS 的蛋白质组学方法。通过 Bio-ID 确定的 NEK5 相互作用组鉴定了各种蛋白质类别，包括驱动蛋白 KIF2C 和 KIF22、线粒体蛋白 TFAM、TFB2M 和 MFN2、RhoH 效应物和 Src 的负调节剂 CSK。通过基于全局 MS 的（磷酸化）蛋白质组学谱分析过表达 NEK5 时调节的蛋白质和磷酸化位点的特征，揭示了对细胞周期、DNA 合成和修复、Rho GTPase 信号、微管细胞骨架和半桥粒组装的影响。总体而言，该研究表明 NEK5 影响乳腺上皮细胞中的多种途径和过程，并且可能在乳腺癌的发生和发展中发挥多方面的作用。视频摘要。
