PO-119 Ferritin heavy subunit enhances apoptosis of non-small cell lung cancer cells through modulation of a miR-125B/P53 axis

IntroductionThe ferritin molecule is a nanocage composed by the variable assembly of 24 heavy and light subunits. As major intracellular iron storage protein, ferritin has been studied for many years in the context of iron metabolism. However, recent evidences highlight its role, and particularly that of the heavy subunit (FHC), in pathways related to cancer development and progression, such as cell proliferation, epithelial-mesenchymal transition, cell death and angiogenesis. This new role of FHC is largely due to its ability to regulate a repertoire of oncogenes and oncomiRNAs. Moreover, the existence of a feedback loop between FHC and the tumour suppressor p53 has been observed in different cell types.Material and methodsFHC was overexpressed in A549 and H460 non-small cell lung cancer (NSCLC) cells through transient transfection of a specific expression vector. FHC, p53 and miR-125b levels were measured by q-PCR and TaqMan analyses in cancer cell lines as well as in tumour tissue specimens. The analysis of the methylation status of miR-125b promoter region was achieved by Methylation Specific PCR (MSP). FHC-overexpressing and control A549 and H460 cells were monitored for changes in proliferation and apoptosis through PI and Annexin/7-AAD flow cytometry assays. Intrinsic and extrinsic apoptosis biomarkers were measured by Western Blot. Statistical analysis was performed by Student t-test or non-parametric Wilcoxon signed-rank test.Results and discussionsThe major finding of this study was that FHC is able to enhance p53 expression through the down-regulation of miR-125b in A549 and H460 NSCLC cell lines. Indeed, we found that FHC overexpression induces hypermethylation and thus the down-regulation of miR-125b which, in turn, is a direct repressor of the tumour suppressor p53. Absolute q-PCR highlighted a significant correlation among these three key molecules also in human tumour tissue specimens thus strongly suggesting the existence of a new regulatory axis In vitro, FHC overexpression also triggered p53-mediated cell apoptosis that is partially reverted by miR-125b reconstitution. Up-regulation of BAX, a pro-apoptotic member of Bcl-2 family, and the enhanced cleavage of caspase-9 demonstrated the activation of the intrinsic apoptotic pathway.ConclusionOverall, the identification of a FHC/miR-125b/p53 regulatory axis may provide a novel molecular strategy for the regulation of the apoptotic cell death in non small cell lung cancer.