PB1096 - Prolonged exposure of cells to exogenous tissue factor alters the regulations of G1/S checkpoint protein p16INKa

Background: Tissue factor (TF) expression is upregulated as a result of inflammation and has been associated with more aggressive cancers. Reduction in the expression of the cell cycle regulating protein p16INKa is a known step in the initiation of some cancers and also the onset of malignancy. Therefore, the regulation of the expression of p16INKa may be a mechanism by which inflammatory TF influences cancer.

Aims: To assess the influence of prolonged treatment of cells with recombinant TF on the expression of p16INKa and identify the underlying mechanism.

Methods: hTERT-HPNE cells were supplemented with recombinant TF (0.5 U/mL) every 2-3 days for up to 5 weeks, and the outcome on p16INKa mRNA and protein levels examined by RT-qPCR and western blotting, respectively. To investigate the underlying mechanism, genomic DNA was extracted from the TF-treated and untreated cells, converted with bisulphite, and the methylation status of p16INKa promoter analysed by MS-PCR. Additionally, since p16INKa protein levels may also be regulated by p14ARF, the expression of p14ARF mRNA and protein was examined.

Results: Treatment of HPNE cells with recombinant TF (0.5 U/ml) significantly reduced p16INKa protein within 4 weeks of treatment, while p16INKa mRNA was only significantly reduced after 5 weeks. In agreement with these data, the ratio of methylated:unmethylated DNA within the p16INKa promotor region increased by week 4 of supplementation with TF. Moreover, increased levels of p14ARF mRNA and protein were detected in the HPNE cells after 4 weeks of TF treatment.

Conclusion(s): Prolonged treatment of HPNE cells with recombinant TF resulted in decreased p16INKa expression. This occurred as a combination of both the reduction in expression of p16INKa mRNA due to promotor methylation and also increased degradation of p16INKa protein induced by interaction with p14ARF. The regulation of p16INKa by TF is likely to have implications in the initiation and progression of cancers.