Growth Suppression Induced by Downregulation of E6-AP Expression in Human Papillomavirus-Positive Cancer Cell Lines Depends on p53

Abstract

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the antigrowth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.Modification of proteins by the covalent attachment of ubiquitin or ubiquitin-related proteins is involved in the regulation of many cellular and viral processes (3, 10, 23). Remarkably, not only do viral proteins represent substrates for such proteinprotein modification systems, but some are also intrinsically involved in the conjugation of ubiquitin (ubiquitination) to cellular proteins, thereby redirecting the ubiquitin conjugation system for viral purposes (3). A prominent example for viral proteins associated with the ubiquitin-conjugation system is provided by the E6 oncoprotein of high-risk human papillomaviruses (HPVs) that are etiologically associated with cervical cancer and other malignant lesions of the anogenital tract. The E6 oncoprotein binds to the cellular ubiquitin-protein ligase E6-AP and utilizes E6-AP to target the tumor suppressor protein p53 for ubiquitination and subsequent proteasomemediated degradation (13, 24). Furthermore, E6 has been reported to target additional proteins, including E6TP1, hScrib, hDlg, and Bak for ubiquitination and degradation in an E6-AP-dependent or E6-AP-independent manner (18, 25).Continuous expression of E6 and E7, the two major HPV oncoproteins, is required for the maintenance of the transformed phenotype of cervical cancer cell lines (28). On the functional level, E6 has p53-dependent as well as p53-independent antiapoptotic properties (22). Indeed, interference with E6 expression or E6 activity results in the induction of apoptosis in HPV-positive cells, which is accompanied by a significant increase in p53 levels (6, 7). Similarly, the downregulation of E6-AP expression by antisense approaches or overexpression of a catalytically inactive E6-AP mutant results in the accumulation of p53 in HPV-positive cells but not in HPVnegative cells (4, 26). Moreover, ribozyme-mediated reduction of E6-AP expression enhances the apoptotic response of HeLa cells, an HPV-18-positive cell line, to the DNA damage-inducing drug mitomycin C (16). However, since E6-AP has been implicated in E6-mediated degradation of proteins other than p53 (e.g., E6TP1 and hScrib) (9, 21), it remains unclear if this apoptosis-enhancing effect is directly linked to the ability of E6-AP to target p53 for degradation in the presence of E6.To determine if the pr...

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

“…Furthermore, four copies of BS2, upstream of a minimal promotor, were sufficient to confer transcriptional activation (BS2), whereas mutation of these sites (BS2Mut) resulted in loss of responsiveness ( Figure 1b). In further support for the p53-dependence of the PUMA induction, we did not observe an increase in PUMA transcript levels or promoter activities upon E6 inhibition in HeLa cells in which p53 expression is suppressed by RNAi (Hengstermann et al, 2005) (data not shown). These results show that the transcriptional activities of endogenous p53 are restored upon RNAi-mediated E6 inhibition in HeLa cells and induce the PUMA promoter via its p53 recognition sites.…”

“…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).…”

“…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). Since E6 can induce the degradation of p53 (Scheffner et al, 1990), apoptosis following siRNA-mediated E6 inhibition may be linked to the transcriptional induction of apoptosis-related p53 target genes.…”

“…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.…”

HPV16 E6 confers p53-dependent and p53-independent phenotypes in the epidermis of mice deficient for E6AP

“…E6's inactivation of human p53 has been argued to be E6AP dependent based upon the use of antisense oligomers (Traidej et al, 2000), a dominant-negative E6AP (Talis et al, 1998) and small interfering RNAs (siRNAs) (Hengstermann et al, 2001(Hengstermann et al, , 2005Kelley et al, 2005). In particular, siRNA-mediated knockdown of E6AP in HPV16-positive cervical cancer-derived cell lines resulted in the same upregulation of p53-responsive genes as seen with E6 siRNAs (Kelley et al, 2005;Nomine et al, 2006).…”