Regulation of p14ARF expression by miR-24: a potential mechanism compromising the p53 response during retinoblastoma development

Abstract

Most human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14^ARF protein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression. In human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14 ARF mRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14^ARF. To study p14^ARF biogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24. In human retinoblastoma cell lines, p14 ARF mRNA was disproportionally high relative to the level of p14^ARF protein expression, suggesting a perturbation of p14^ARF regulation. When p14^ARF was over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14^ARF-p53 axis. To investigate the discrepancy between p14 ARF mRNA and protein in retinoblastoma, we examined p14^ARF biogenesis. The proteasome inhibitor, MG132, did not cause p14^ARF accumulation, although p14^ARF normally is degraded by proteasomes. miR-24, a microRNA that represses p14^ARF expression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14 ARF mRNA. Transient over-expression of siRNA against miR-24 led to elevated p14^ARF protein in retinoblastoma cells. In retinoblastoma cells where high levels of p14 ARF mRNA are not accompanied by high p14^ARF protein, we found a correlation between miR-24 expression and low p14^ARF protein. p14^ARF protein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss.