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 Riku Koivusalo, 1,2 Antoine Mialon, 3,4 Hanna Pitka ¨nen, 1,2 Jukka Westermarck, 3,4,6 and Sakari Hietanen 1,2,5 1 The Joint Clinical Biochemistry Laboratory of University of Turku, Turku University Central Hospital and Wallac Oy; 2 Medicity Research Laboratory and 3 Department of Medical Biochemistry and Molecular Biology, University of Turku; 4 Centre for Biotechnology, University of Turku and A ˚ bo Akademi University; 5 Department of Obstetrics and Gynecology, Turku University Central Hospital, Turku, Finland; and 6 Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland Abstract p53 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 antiproli- ferative 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) Introduction High-risk human papillomaviruses (HPV) play a pivotal role in the pathogenesis of cervical cancer. After infecting the genital mucosa and becoming integrated into the host genome, they start to overexpress E6 and E7 oncoproteins, which then immortalize the host cells by disrupting p53 and pRb function, respectively (1). E6 binds to p53 and targets it for ubiquitin-mediated degradation; E7 inactivates pRb in a similar manner. p53 coordinates cellular responses to different forms of stress, e.g., DNA damage. Activation of p53 may trigger apoptosis, cell cycle arrest, or attempts to repair the damaged DNA, depending on how extensive the damage is (2). As current treatment modalities are rather ineffective against advanced cervical cancer, new therapeutic approaches are needed. Because the p53 pathway is not irreversibly damaged in cervical cancer, means of reactivating p53 in this malignancy have been actively studied. Previously, gamma-irradiation, certain cytotoxic drugs, small molecule compounds, and some direct anti-E6 approaches, such as ones based on antisense and ribozyme techniques, have been studied and reported to activate p53 in cervical cancer cells (3–7). The discovery of RNA interference has enabled selective, highly efficient, and sustained degradation of the desired target mRNA. In RNA interference, small double-stranded RNA molecules, called short interfering RNA (siRNA), induce the degradation of homologous mRNA as part of the RNA-induced silencing complex (8, 9). The viral E6 expression in cervical cancer cells can be successfully silenced using siRNA, leading to the reactivation of p53 (10–14). However, these studies present controversial data on the biological consequences of E6 silencing: in one study, massive apoptosis was seen (14), whereas the main outcome of E6 siRNA treatment in other studies was either inhibition of cell growth or induction of cell senescence (10–13). In our previous study, siRNA-mediated E6 silencing produced only transient p53 activation in HeLa cervical cancer cells despite continuous suppression of E6 mRNA, together with a modest antiproliferative effect (13). These findings suggested that cellular p53-antagonizing mechanisms were activated in response to E6 depletion. There have been efforts to develop means of targeting HPV E6, with the hope that anti-E6 therapies would be clinically effective against cervical cancer. However, the quickly decreasing p53 activation may significantly limit the clinical usefulness of E6 siRNA monotherapy. Cellular negative regulation of p53 is principally mediated by certain ubiquitin ligases, such as MDM2. MDM2 forms a tight negative feedback loop with p53: active p53 stimulates MDM2 gene expression, and the resulting MDM2 protein binds to p53, exports it out of the nucleus and targets it for ubiquitin-mediated degradation (15, 16). Expression of E6 renders the MDM2 pathway silent in cervical cancer cells, but MDM2 becomes active in these cells in response to p53 activation induced by the small molecule compounds, actinomycin D and leptomycin B (LMB; refs. 5, 17). Also, the RING finger proteins, COP1 and Pirh2, can act as negative p53 regulators by working in a similar manner as MDM2, but independently of it (18, 19). c-Jun -NH 2 -kinase ( JNK), a p53 activator in stress conditions, targets p53 for ubiquitination and degradation in nonstressed cells (20). Before being degraded in the proteasome, p53 is transported from the nucleus to the cytoplasm. CRM1 is a central mediator of this nuclear export and works by binding to the nuclear export signal of the proteins to be exported, and guiding them to nuclear pores containing the actual export machinery (21). Requests for reprints: Sakari Hietanen, Department of Obstetrics and Gynecology, Turku University Central Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland. Phone: 358-2313-0200; Fax: 358-2313-2340; E-mail: sakari.hietanen@utu.fi. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-2185 www.aacrjournals.org 11817 Cancer Res 2006; 66: (24). December 15, 2006 Research Article Research. on October 27, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from