NF-Y LOSS REACTIVATES p53 AND TRIGGERS p53-DEPENDENT APOPTOSIS IN HPV-POSITIVE HUMAN CERVICAL CARCINOMA CELLS

The two early HPV genes, E6 and E7, play an essential role in cellular transformation and abnormal proliferation. The oncoprotein E6 suppresses the activation of p53-dependent pathways through proteasomal degradation of p53 and inhibition of its transactivating activity. The CCAAT-binding factor NF-Y regulates the transcription of various genes related to the cell cycle. A biochemical and genetic link has been described between p53 and NF-Y: i) a large overlap of targets exists between NF-Y and p53, particularly many genes transactivated by NF-Y are repressed following p53 activation; ii) both wt and mutp53 proteins interact with NF-Y; iii) loss of NF-Y, as well as unrestricted activity, triggers cycle defects and p53-dependent cell death. Specifically, the inactivation of NF-YA, the DNA binding subunit of the NF-Y complex, leads to a delay in S phase progression and to the activation of a DNA-damage response. Here we show a novel mechanism through which NF-Y and p53 are connected in HPV-positive cells. The knock-down of NF-YA in Hela cervical carcinoma cells leads to p53 re-expression and re-activation of p53-dependent apoptosis. The analysis of microarray profiles of NF-YA inactivated versus control cells points at “p53 pathway” and “apoptosis” as the most represented KEGG pathways up-regulated following NF-Y loss. Western blot, RT-PCR, transient transfection and ChIP data clearly suggest that NF-Y positively regulates E6 expression, driven by HPV-18 upstream regulatory region, which contains two NF-Y binding sites. Consequently, NF-Y down-regulation by shRNA delivery results in E6 transcriptional repression and in the activation of p53 and its target genes. Our results demonstrate that NF-Y inactivation might represent an interesting anti-tumor strategy to induce apoptosis in high-risk HPV-infected cancer cells, through the re-establishment of p53 expression.