Activation of p53 in Cervical Cancer Cells by Human Papillomavirus E6 RNA Interference Is Transient, but Can Be Sustained by Inhibiting Endogenous Nuclear Export–Dependent p53 Antagonists

Abstract

Abstractp53 is degraded in cervical cancer cells by the human papillomavirus E6 and can be stabilized with short interfering RNA (siRNA) molecules targeting E6 mRNA. In this in vitro study, we show that E6 siRNA-induced p53 activation is transient in HeLa cervical cancer cells despite continuous suppression of E6 mRNA; activation can be sustained if the endogenous p53 antagonists COP1, MDM2, Pirh2, and c-Jun-NH 2 -kinase are also targeted by siRNAs or by inhibiting the nuclear export of p53 with leptomycin B. The direct targeting of any one of these four cellular p53 antagonists had no effect on p53 activity when E6 was intact, but inhibited the fading off of E6 siRNA-induced p53 activation in nonstress conditions. The effect was additive when multiple cellular antagonists were concomitantly inhibited, indicating that all these proteins degrade p53 when E6 is inactivated. The antiproliferative effect induced by E6 silencing was enhanced when the endogenous p53 antagonists were additionally targeted. In conclusion, if human papillomavirus E6 is inhibited under nonstress conditions, the subsequent p53 activation is quickly reversed by the endogenous p53 degenerative machinery. The present results indicate that several cellular p53 antagonists must be inhibited for sustained p53 activity if E6 siRNA therapy is attempted and if no combined genotoxic therapy is applied. (Cancer Res 2006; 66(24): 11817-24)

Rescue of p53 Function by Small-Molecule RITA in Cervical Carcinoma by Blocking E6-Mediated Degradation

“…Despite the fact that MDM2 plays a negligible role as a p53 inhibitor in HPV-infected cells, a recent report suggests that continuous silencing of E6 gives MDM2 a chance to take over and degrade p53; therefore, targeting E6 along with depletion of MDM2 prevents the fading of p53 (27). We detected MDM2 induction upon E6/E6AP depletion by siRNA, as well as upon RITA treatment.…”

p53, secreted by K-Ras–Snail pathway, is endocytosed by K-Ras-mutated cells; implication of target-specific drug delivery and early diagnostic marker

“…Next, we explored the relevance of a p53 nuclearexporting mechanism using p53 NES (p53 mutated in nuclear-exporting sequence; Nie et al, 2007) and Leptomycin B (LMB; nuclear-exporting blocker; Koivusalo et al, 2006). However, Snail reduced p53 NES expression ( Figure 2a) and LMB did not block the Snailmediated p53 reduction (Figures 2b and c).…”

NM23-H1 Tumor Suppressor and Its Interacting Partner STRAP Activate p53 Function

“…Luciferase Reporter Assay-HeLa (32,33), HCT116, and MCF7 cells were transiently transfected according to the WelFect-Ex TM Plus method with the p53-Luc reporter plasmid, along with the appropriate plasmids as indicated. Luciferase activity was monitored with a luciferase assay kit (Promega) following the manufacturer's instructions as described previously (25).…”