Regulation of p14^ARF expression by miR-24: a potential mechanism compromising the p53 response during retinoblastoma development

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.