Aversa I, Zolea F, Ieranò C, Bulotta S, Trotta A M, Faniello M C, De Marco C, Malanga D, Biamonte F, Viglietto G, Cuda G, Scala S, Costanzo F
Research Center of Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Græcia University of Catanzaro, Salvatore Venuta Campus, Viale Europa, 88100, Catanzaro, Italy.
Genomica Funzionale, INT Pascale, Napoli, Italy.
J Exp Clin Cancer Res. 2017 Aug 3;36(1):104. doi: 10.1186/s13046-017-0571-8.
Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms.
Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry.
Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7, H460) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis.
Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.
铁蛋白在细胞内铁代谢中起核心作用；该分子是由重链和轻链两种类型的24个亚基组成的纳米笼。重链亚基（FHC）具有亚铁氧化酶活性，因此能将铁转化为无毒形式，发挥关键作用。最近研究表明，FHC还参与了其他一些与铁无关的关键途径，包括p53调控、肿瘤微小RNA表达的调节以及趋化因子信号传导。上皮-间质转化（EMT）是一种细胞机制，通过该机制细胞获得成纤维细胞样表型，同时细胞黏附性降低，运动性增强。在本研究中，我们聚焦于FHC在人细胞系MCF-7和H460中诱导EMT的作用，以阐明其潜在的分子机制。
采用慢病毒驱动的短发夹RNA（shRNA）策略对FHC进行靶向沉默。通过使用Lipofectamine 2000转染全长FHC cDNA来实现FHC基因产物的重建。采用MTT法和细胞计数法评估细胞活力和增殖情况；通过伤口愈合试验和Transwell法检测细胞迁移能力。采用流式细胞术对CXCR4表面表达进行定量分析。
实验数据表明，FHC沉默的MCF-7和H460细胞（MCF-7 sh、H460 sh）获得了间质表型，同时其迁移和增殖能力显著增强。这种转变伴随着活性氧（ROS）产生的增加以及CXCR4/CXCL12信号通路的激活。我们提供的实验数据表明，胞质内ROS水平的升高是FHC沉默细胞增殖增强的原因，而较高的迁移率则归因于CXCR4/CXCL12轴的失调。
我们的研究结果表明，在MCF-7和H460细胞中，EMT的诱导、迁移增加和存活依赖于FHC对铁/ROS代谢和CXCR4/CXCL12轴这两条途径控制的解除。除了进一步证实FHC的多功能性质外，这些数据还表明，对FHC含量/功能的分析可能是预测肿瘤侵袭性的一个重要辅助工具。
