siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells

Abstract

The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.

Inhibition of Bax activity is crucial for the antiapoptotic function of the human papillomavirus E6 oncoprotein

“…These differences may be due to the different experimental setups. Whereas, we specifically target endogenous E6 by RNAi (Butz et al, 2003), the cited studies involved expression of the bovine papillomavirus (BPV) E2 protein (in order to block endogenous HPV18 E6 and E7 expression in HeLa cells) in combination with retrovirally re-expressed HPV16 E7. Both the ectopic expression of two heterologous viral proteins as well as the fact that the multifunctional BPV E2 can modulate apoptosis in HeLa cells via p53-independent mechanisms (Desaintes et al, 1999) may complicate this system.…”

“…However, possible proapoptotic effects of IP10 that are HPV-independent were not investigated. In contrast, by the RNAi approach chosen in the present work, we target the E6 gene with high specificity (Butz et al, 2003) and the apoptotic effects are selectively observed in HPV-positive cells only.…”

“…The specific targeting of E6-encoding transcripts by RNAi results in an increase in p53 levels and the induction of apoptosis in HPV16 or HPV18-positive cancer cells (Butz et al, 2003;Hengstermann et al, 2005;Kelley et al, 2005). Apoptosis, as induced by inhibition of E6 expression (in the following termed 'E6 inhibition'), appears to be p53-dependent, because HeLa cells, in which p53 expression was stably suppressed by RNAi, become resistant to siRNAs against E6 (Hengstermann et al, 2005).…”

“…The viral E6 and E7 oncogenes are both regularly expressed in HPV-positive tumor cells and are necessary to maintain their transformed phenotype (zur Hausen, 2002). E6 exhibits antiapoptotic potential in various experimental settings (Pan and Griep, 1995;Sto¨ppler et al, 1998;Thomas and Banks, 1998;AguilarLemarroy et al, 2002;Filippova et al, 2004;Yuan et al, 2005), and the targeted inhibition of endogenous E6 activity induced apoptosis in HPV-positive cancer cells (Butz et al, 2000(Butz et al, , 2003Hengstermann et al, 2005;Kelley et al, 2005). Thus, the continuous presence of E6 is important for the viability of HPV-positive cancer cells, defining E6 as a promising target for therapeutic intervention.…”

“…These observations indicate that it is difficult to deduce the critical activities of E6 in HPV-positive cancer cells from the various effects observed after ectopic (over)expression of E6, in heterologous cell systems. In contrast to overexpression systems, the RNA interference (RNAi) technology should be useful in determining the critical functions of E6, since it allows the specific and direct inhibition of endogenous E6 expression in HPV-positive cancer cells (Butz et al, 2003). By employing this strategy, we identified the Bax-dependent proapoptotic pathway as a critical target for the E6 oncogene and provide evidence that E6-mediated inactivation of this pathway is crucial for the protection of HPV-positive cancer cells from apoptotic cell death.…”