Activating Transcription Factor 3 Activates p53 by Preventing E6-associated Protein from Binding to E6

Abstract

Genomic integration of human papillomavirus (HPV) DNA accounts for more than 90% of cervical cancers. High-risk genital HPVs encode E6 proteins that can interact with a cellular ubiquitin ligase E6-associated protein (E6AP) and target the tumor suppressor p53 for ubiquitin-mediated proteolysis. Currently, how this critical event is regulated is largely unknown. Here we report that activating transcription factor 3 (ATF3), a broad DNA damage sensor whose expression is frequently downregulated in cervical cancer, interacted with E6 and prevented p53 from ubiquitination and degradation mediated by the viral protein. Consistent with its role as a potent E6 antagonist, ATF3 expressed enforcedly in HPV-positive SiHa cells activated p53, leading to expression of p53-target genes (e.g. p21 and PUMA), cell cycle arrest and apoptotic cell death. The leucine zipper domain of ATF3 appears indispensable for these effects as an ATF3 mutant lacking this domain failed to interact with E6 and activate p53 in the cervical cancer cells. The prevention of p53 degradation was unlikely caused by binding of ATF3 to the tumor suppressor, but rather was a consequence of disruption of the E6-E6AP interaction by ATF3. These results indicate that ATF3 plays a key role in a mechanism defending against HPV-induced carcinogenesis, and could serve as a novel therapeutic target for HPV-positive cancers. Genomic integration of human papillomavirus (HPV) DNA accounts for more than 90% of cervical cancers. High-risk genital HPVs encode E6 proteins that can interact with a cellular ubiquitin ligase E6-associated protein (E6AP) and target the tumor suppressor p53 for ubiquitin-mediated proteolysis. Currently, how this critical event is regulated is largely unknown. Here we report that activating transcription factor 3 (ATF3), a broad DNA damage sensor whose expression is frequently downregulated in cervical cancer, interacted with E6 and prevented p53 from ubiquitination and degradation mediated by the viral protein. Consistent with its role as a potent E6 antagonist, ATF3 expressed enforcedly in HPV-positive SiHa cells activated p53, leading to expression of p53-target genes (e.g. p21 and PUMA), cell cycle arrest and apoptotic cell death. The leucine zipper domain of ATF3 appears indispensable for these effects as an ATF3 mutant lacking this domain failed to interact with E6 and activate p53 in the cervical cancer cells. The prevention of p53 degradation was unlikely caused by binding of ATF3 to the tumor suppressor, but rather was a consequence of disruption of the E6-E6AP interaction by ATF3. These results indicate that ATF3 plays a key role in a mechanism defending against HPV-induced carcinogenesis, and could serve as a novel therapeutic target for HPV-positive cancers. IntroductionHuman papillomavirus (HPV) 3The abbreviations used are: HPVhuman papillomavirusATFactivating transcription factorE6APE6-associated proteinGSTglutathione S-transferaseshRNAshort-hairpin RNATUNELterminal dUTP nick-end labelingDTTdithiothreitolGFPgreen fluorescent proteinBSAbovine serum albumin. infection is a major risk factor for cervical cancer (1zur Hausen H. J. Natl. Cancer Inst. 2000; 92: 690-698Crossref PubMed Google Scholar, 2Schiffman M. Castle P.E. Jeronimo J. Rodriguez A.C. Wacholder S. Lancet. 2007; 370: 890-907Abstract Full Text Full Text PDF PubMed Scopus (1960) Google Scholar). Genomic integration of HPV DNA, occurring in more than 90% of cervical cancers, results in expression of a viral protein E6, which in turn inactivates the tumor suppressor p53 by driving its proteolysis (3Scheffner M. Werness B. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3429) Google Scholar). The E6 protein can bind to p53 at both the C terminus and the central DNA-binding region (4Li X. Coffino P. J. Virol. 1996; 70: 4509-4516Crossref PubMed Google Scholar) and recruit a cellular protein E6AP to p53 (5Huibregtse J.M. Scheffner M. Howley P.M. EMBO J. 1991; 10: 4129-4135Crossref PubMed Scopus (689) Google Scholar, 6Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1952) Google Scholar). E6AP belongs to the HECT family of E3 ubiquitin ligases and can catalyze the addition of ubiquitin moieties to p53, leading to its degradation by the 26 S proteasomes (6Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1952) Google Scholar). Because the p53 gene is rarely mutated in cervical cancer, the E6-mediated degradation serves as the major mechanism inactivating p53 and promoting cervical carcinogenesis (7Hengstermann A. Linares L.K. Ciechanover A. Whitaker N.J. Scheffner M. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1218-1223Crossref PubMed Scopus (175) Google Scholar).In addition to p53 and E6AP, the E6 protein interacts with many other cellular proteins including Bak (8Thomas M. Banks L. Oncogene. 1998; 17: 2943-2954Crossref PubMed Scopus (243) Google Scholar), Bax (9Vogt M. Butz K. Dymalla S. Semzow J. Hoppe-Seyler F. Oncogene. 2006; 25: 4009-4015Crossref PubMed Scopus (52) Google Scholar), CBP/p300 (10Patel D. Huang S.M. Bagila L.A. McCane D.J. EMBO J. 1999; 18: 5061-5072Crossref PubMed Scopus (340) Google Scholar), and BRCA1 (11Zhang Y. Fan S. Meng Q. Ma Y. Katiyar P. Schlegel R. Rosen E.M. J. Biol. Chem. 2005; 280: 33165-33177Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), presumably through the four CXXC motifs sparsely distributed in the viral protein (12Huibregtse J.M. Beaudenon S.L. Semin. Cancer Biol. 1996; 7: 317-326Crossref PubMed Scopus (57) Google Scholar). These interactions account for the oncogenic activities of E6, which include not only promoting transformation but also enhancing cell proliferation and survival (13zur Hausen H. Nat. Rev. Cancer. 2002; 2: 342-350Crossref PubMed Scopus (3078) Google Scholar). Therefore, it is important to dissect the E6 interaction network for a better understanding of the molecular basis for cervical cancer and identification of therapeutic targets for the disease.ATF3 is a member of the ATF/CREB family of transcription factors and can be rapidly induced by DNA damage and other oncogenic stimuli (14Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expression. 1999; 7: 321-335PubMed Google Scholar, 15Hai T. Hartman M.G. Gene. 2001; 273: 1-11Crossref PubMed Scopus (645) Google Scholar). Whereas consequences of ATF3 induction are unclear, it is often assumed that ATF3 functions as a transcription factor to regulate gene expression thereby contributing to cellular responses to oncogenic stresses (14Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expression. 1999; 7: 321-335PubMed Google Scholar, 15Hai T. Hartman M.G. Gene. 2001; 273: 1-11Crossref PubMed Scopus (645) Google Scholar). However, emerging evidence indicates that this DNA-binding protein can also regulate cellular functions through mechanisms beyond transcriptional regulation (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar). ATF3 contains a central leucine zipper domain (Zip) that is well characterized as a mediator of protein-protein interaction (17Phizicky E.M. Fields S. Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar). ATF3 binds to p53 via this domain, and as a consequence, p53 ubiquitination catalyzed by MDM2, the major ubiquitin ligase in HPV-negative cells (18Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3656) Google Scholar, 19Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2812) Google Scholar), is blocked, leading to up-regulation of the p53 tumor suppressor activity independent of the ATF3 transcriptional activity (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar). These findings are consistent with recent reports that ATF3 expression is down-regulated in a wide range of human cancers (20Beer D.G. Kardia S.L. Huang C.C. Giordano T.J. Levin A.M. Misek D.E. Lin L. Chen G. Gharib T.G. Thomas D.G. Lizyness M.L. Kuick R. Hayasaka S. Taylor J.M. Iannettoni M.D. Orringer M.B. Hanash S. Nat. Med. 2002; 8: 816-824Crossref PubMed Scopus (1644) Google Scholar, 21Chen X. Cheung S.T. So S. Fan S.T. Barry C. Higgins J. Lai K.M. Ji J. Dudoit S. Ng I.O. Van De Rijn M. Botstein D. Brown P.O. Mol. Biol. Cell. 2002; 13: 1929-1939Crossref PubMed Scopus (730) Google Scholar, 22Lapointe J. Li C. Higgins J.P. Van De Rijn M. Bair E. Montgomery K. Ferrari M. Egevad L. Rayford W. Bergerheim U. Ekman P. DeMarzo A.M. Tibshirani R. Botstein D. Brown P.O. Brooks J.D. Pollack J.R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 811-816Crossref PubMed Scopus (1044) Google Scholar) including cervical cancer (23Yan C. Boyd D.D. Cell Cycle. 2006; 5: 926-929Crossref PubMed Scopus (59) Google Scholar), arguing for a link between ATF3 expression and tumor suppression (23Yan C. Boyd D.D. Cell Cycle. 2006; 5: 926-929Crossref PubMed Scopus (59) Google Scholar). In line with this notion, we report here the identification of ATF3 as a novel E6 repressor that can compete with E6AP for binding to the viral protein thereby activating p53 in HPV-positive cervical cancer cells. These results indicate that ATF3 could contribute to suppression of HPV-induced carcinogenesis.DISCUSSIONExpression of HPV proteins such as E6 is a driving force for cervical carcinogenesis (1zur Hausen H. J. Natl. Cancer Inst. 2000; 92: 690-698Crossref PubMed Google Scholar, 2Schiffman M. Castle P.E. Jeronimo J. Rodriguez A.C. Wacholder S. Lancet. 2007; 370: 890-907Abstract Full Text Full Text PDF PubMed Scopus (1960) Google Scholar). The HPV proteins promote malignant transformation by intervening in functions of various cellular proteins through protein-protein interactions (38Boulet G. Horvath C. Vanden Broeck D. Sahebali S. Bogers J. Int. J. Biochem. Cell Biol. 2007; 39: 2006-2011Crossref PubMed Scopus (99) Google Scholar). While cellular proteins regulated by the viral proteins have been well documented (38Boulet G. Horvath C. Vanden Broeck D. Sahebali S. Bogers J. Int. J. Biochem. Cell Biol. 2007; 39: 2006-2011Crossref PubMed Scopus (99) Google Scholar), little is known as to how the oncogenic activities of the HPV proteins are regulated. In this study, we identified ATF3 as a novel repressor of the HPV E6 protein. ATF3 bound to E6 and prevented p53 degradation mediated by the viral protein. Because it is widely induced by oncogenic stimuli (e.g. DNA damage) (14Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expression. 1999; 7: 321-335PubMed Google Scholar, 15Hai T. Hartman M.G. Gene. 2001; 273: 1-11Crossref PubMed Scopus (645) Google Scholar), ATF3 could serve as a key activator of p53 in HPV-infected cervix epithelium thereby defending against oncogenic transformation, a notion which is supported by our recent observation that ATF3 expression is frequently down-regulated in cervical cancers (23Yan C. Boyd D.D. Cell Cycle. 2006; 5: 926-929Crossref PubMed Scopus (59) Google Scholar). Whereas the mechanism(s) for ATF3 down-regulation in cervical cancer remains elusive, decreased ATF3 expression allows E6 to drive p53 degradation thereby contributing to cervical carcinogenesis. Moreover, the demonstration of ATF3 as a direct binding partner of E6 suggests that ATF3 could regulate other oncogenic activities of E6 such as activation of telomerase and the Src family kinase Blk (39Veldman T. Horikawa I. Barrett J.C. Schlegel R. J. Virol. 2001; 75: 4467-4472Crossref PubMed Scopus (232) Google Scholar, 40Oda H. Kumar S. Howley P.M. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9557-9562Crossref PubMed Scopus (137) Google Scholar). Activation of the latter proteins promotes cell proliferation, which acts in concert with p53 inactivation to promote the genesis and progression of cervical cancer (13zur Hausen H. Nat. Rev. Cancer. 2002; 2: 342-350Crossref PubMed Scopus (3078) Google Scholar). Therefore, this study has identified a novel mechanism by which the oncogenic activities of HPV proteins are antagonized. Because enforced expression of ATF3 can restore p53 activity and induce apoptosis of HPV-positive cancer cells, targeting ATF3 expression through chemical interventions or gene therapy could be a promising strategy to treat cervical cancer. Interestingly, recent studies have revealed a causal role of HPV infection in the genesis of head and neck cancer (41Tran N. Rose B.R. O'Brien C.J. Head Neck. 2007; 29: 64-70Crossref PubMed Scopus (80) Google Scholar, 42Chung C.H. Gillison M.L. Clin. Cancer Res. 2009; 15: 6758-6762Crossref PubMed Scopus (308) Google Scholar). Therefore, the significance of the current findings may extend beyond the understanding of the etiology of cervical cancer. Indeed, we recently reported that ATF3 promotes anti-cancer effects of curcumin (43Yan C. Jamaluddin M.S. Aggarwal B. Myers J. Boyd D.D. Mol. Cancer Ther. 2005; 4: 233-241Crossref PubMed Scopus (1) Google Scholar), an agent currently in clinical trials, in a p53-wild-type head and neck cancer cell line (44Xu L. Davidson B.J. Murty V.V. Li R.G. Sacks P.G. Garinchesa P. Schantz S.P. Chaganti R.S. Int. J. Cancer. 1994; 59: 383-387Crossref PubMed Scopus (46) Google Scholar). It is very likely that the HPV-antagonizing activity of ATF3 described here contributes to such an effect.ATF3 is well recognized as a stress responsive protein (14Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expression. 1999; 7: 321-335PubMed Google Scholar). Although ATF3 induction occurs frequently in the early stage of the DNA damage response, the role of ATF3 in cancer development remains undecided. Whereas earlier studies indicate that ATF3 may promote growth and dissemination of malignant cells (45Ishiguro T. Nakajima M. Naito M. Muto T. Tsuruo T. Cancer Res. 1996; 56: 875-879PubMed Google Scholar, 46Perez S. Vial E. van Dam H. Castellazzi M. Oncogene. 2001; 20: PubMed Scopus Google Scholar), results from many other studies for a role that ATF3 plays in cancer development Y. B. M. Mol. Cancer Ther. 2005; 4: PubMed Scopus Google Scholar, Y. Chen J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, ATF3 to cellular transformation induced by the (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar, D. Wolfgang C.D. Hai T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). current findings that ATF3 the oncogenic activity of a HPV viral protein for the notion that ATF3 induction can as an to against oncogenic transformation (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar, C. Boyd D.D. Cell Cycle. 2006; 5: 926-929Crossref PubMed Scopus (59) Google Scholar).In addition to suppression of E6-mediated p53 we that ATF3 and p53 in HPV-negative cells (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar) the p53 is by D. Oren M. Semin. Cancer Biol. 13: PubMed Scopus Google Scholar). Interestingly, it appears that ATF3 p53 degradation mediated by and E6 through While ATF3 ubiquitination by binding to p53 and its (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar), we have here that the interaction was for suppression of the E6-mediated Indeed, ATF3 binding may the at the p53 C terminus (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar), p53 ubiquitination mediated by E6 not in the C terminus S. Higgins M. S. PubMed Scopus Google Scholar, S. T. Oncogene. 2002; PubMed Scopus Google Scholar). Moreover, it can also bind to p53 at the DNA-binding domain (4Li X. Coffino P. J. Virol. 1996; 70: 4509-4516Crossref PubMed Google Scholar), E6 not compete with ATF3 for binding to the C terminus of the tumor suppressor, a supported by the results that ATF3 failed to the interaction results that ATF3 rather E6-mediated p53 degradation through direct binding to the viral protein. an interaction E6AP from binding to E6 and the of the ubiquitin ligase to p53, leading to ubiquitination and proteolysis of the latter protein Therefore, these results the of ATF3 in activating p53 in to oncogenic the binding of E6 to the p53 C terminus not E6AP, the of the E3 ubiquitin ligase is likely indispensable for E6 binding to the p53 DNA binding domain (4Li X. Coffino P. J. Virol. 1996; 70: 4509-4516Crossref PubMed Google Scholar). the ATF3 binding the E6-E6AP interaction it the as to how this and how the interaction occurs in the of E6 is a protein four CXXC motifs that Y. J.D. 13: PubMed Scopus Google Scholar). It be that ATF3 with E6AP for binding to the of the viral protein the p53 by ATF3 interaction with its C terminus (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar) E6 bind to p53 via the central DNA binding domain the p53 C terminus be bound by E6 the of ATF3. Interestingly, the domain of ATF3 not the leucine that in E6-associated proteins J. K. B. 2000; PubMed Scopus Google Scholar), it leucine and can a which is of the of proteins Y. E. J.D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). it be of to other leucine proteins also interact with E6 and regulate the oncogenic activities of the viral of its activity to drive p53 E6 also p53 activity by of p53 and (10Patel D. Huang S.M. Bagila L.A. McCane D.J. EMBO J. 1999; 18: 5061-5072Crossref PubMed Scopus (340) Google Scholar, Mol. Cell. 2005; 17: Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The results from our indicate that ATF3 not only the p53 activity decreased by E6, but also the p53 activity by These results for a that ATF3 the p53 transcriptional activity independent of its The ATF3 binding p53 to a its binding to DNA, or its interaction with transcription (e.g. S.M. M. Proc. Natl. Acad. Sci. U.S.A. 2007; PubMed Scopus Google Scholar, H. A. B. R. S. D. C.J. Mol. Cell Biol. 1994; PubMed Scopus Google Scholar, A.J. S. J.M. J.M. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). ATF3 recruit these transcription to p53-target It also be that ATF3 with transcription for binding to p53 and its target M. F. Mol. Cell. 29: Full Text Full Text PDF PubMed Scopus Google Scholar). of the the of ATF3 to promote p53 the cell with an of activating p53 in to a of oncogenic we have identified ATF3 as a novel repressor of the HPV E6 protein. ATF3 p53 by E6AP from the viral protein thereby p53 Because E6 is the major protein that inactivates p53 in HPV-positive cells, these findings for a in which ATF3 induction by DNA damage the oncogenic activity of the E6 protein and thereby serves as an to carcinogenesis HPV IntroductionHuman papillomavirus (HPV) 3The abbreviations used are: HPVhuman papillomavirusATFactivating transcription factorE6APE6-associated proteinGSTglutathione S-transferaseshRNAshort-hairpin RNATUNELterminal dUTP nick-end labelingDTTdithiothreitolGFPgreen fluorescent proteinBSAbovine serum albumin. infection is a major risk factor for cervical cancer (1zur Hausen H. J. Natl. Cancer Inst. 2000; 92: 690-698Crossref PubMed Google Scholar, 2Schiffman M. Castle P.E. Jeronimo J. Rodriguez A.C. Wacholder S. Lancet. 2007; 370: 890-907Abstract Full Text Full Text PDF PubMed Scopus (1960) Google Scholar). Genomic integration of HPV DNA, occurring in more than 90% of cervical cancers, results in expression of a viral protein E6, which in turn inactivates the tumor suppressor p53 by driving its proteolysis (3Scheffner M. Werness B. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3429) Google Scholar). The E6 protein can bind to p53 at both the C terminus and the central DNA-binding region (4Li X. Coffino P. J. Virol. 1996; 70: 4509-4516Crossref PubMed Google Scholar) and recruit a cellular protein E6AP to p53 (5Huibregtse J.M. Scheffner M. Howley P.M. EMBO J. 1991; 10: 4129-4135Crossref PubMed Scopus (689) Google Scholar, 6Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1952) Google Scholar). E6AP belongs to the HECT family of E3 ubiquitin ligases and can catalyze the addition of ubiquitin moieties to p53, leading to its degradation by the 26 S proteasomes (6Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1952) Google Scholar). Because the p53 gene is rarely mutated in cervical cancer, the E6-mediated degradation serves as the major mechanism inactivating p53 and promoting cervical carcinogenesis (7Hengstermann A. Linares L.K. Ciechanover A. Whitaker N.J. Scheffner M. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1218-1223Crossref PubMed Scopus (175) Google Scholar).In addition to p53 and E6AP, the E6 protein interacts with many other cellular proteins including Bak (8Thomas M. Banks L. Oncogene. 1998; 17: 2943-2954Crossref PubMed Scopus (243) Google Scholar), Bax (9Vogt M. Butz K. Dymalla S. Semzow J. Hoppe-Seyler F. Oncogene. 2006; 25: 4009-4015Crossref PubMed Scopus (52) Google Scholar), CBP/p300 (10Patel D. Huang S.M. Bagila L.A. McCane D.J. EMBO J. 1999; 18: 5061-5072Crossref PubMed Scopus (340) Google Scholar), and BRCA1 (11Zhang Y. Fan S. Meng Q. Ma Y. Katiyar P. Schlegel R. Rosen E.M. J. Biol. Chem. 2005; 280: 33165-33177Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), presumably through the four CXXC motifs sparsely distributed in the viral protein (12Huibregtse J.M. Beaudenon S.L. Semin. Cancer Biol. 1996; 7: 317-326Crossref PubMed Scopus (57) Google Scholar). These interactions account for the oncogenic activities of E6, which include not only promoting transformation but also enhancing cell proliferation and survival (13zur Hausen H. Nat. Rev. Cancer. 2002; 2: 342-350Crossref PubMed Scopus (3078) Google Scholar). Therefore, it is important to dissect the E6 interaction network for a better understanding of the molecular basis for cervical cancer and identification of therapeutic targets for the disease.ATF3 is a member of the ATF/CREB family of transcription factors and can be rapidly induced by DNA damage and other oncogenic stimuli (14Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expression. 1999; 7: 321-335PubMed Google Scholar, 15Hai T. Hartman M.G. Gene. 2001; 273: 1-11Crossref PubMed Scopus (645) Google Scholar). Whereas consequences of ATF3 induction are unclear, it is often assumed that ATF3 functions as a transcription factor to regulate gene expression thereby contributing to cellular responses to oncogenic stresses (14Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expression. 1999; 7: 321-335PubMed Google Scholar, 15Hai T. Hartman M.G. Gene. 2001; 273: 1-11Crossref PubMed Scopus (645) Google Scholar). However, emerging evidence indicates that this DNA-binding protein can also regulate cellular functions through mechanisms beyond transcriptional regulation (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar). ATF3 contains a central leucine zipper domain (Zip) that is well characterized as a mediator of protein-protein interaction (17Phizicky E.M. Fields S. Microbiol. Rev. 1995; 59: 94-123Crossref PubMed Google Scholar). ATF3 binds to p53 via this domain, and as a consequence, p53 ubiquitination catalyzed by MDM2, the major ubiquitin ligase in HPV-negative cells (18Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3656) Google Scholar, 19Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2812) Google Scholar), is blocked, leading to up-regulation of the p53 tumor suppressor activity independent of the ATF3 transcriptional activity (16Yan C. Lu D. Hai T. Boyd D.D. EMBO J. 2005; 24: 2425-2435Crossref PubMed Scopus (161) Google Scholar). These findings are consistent with recent reports that ATF3 expression is down-regulated in a wide range of human cancers (20Beer D.G. Kardia S.L. Huang C.C. Giordano T.J. Levin A.M. Misek D.E. Lin L. Chen G. Gharib T.G. Thomas D.G. Lizyness M.L. Kuick R. Hayasaka S. Taylor J.M. Iannettoni M.D. Orringer M.B. Hanash S. Nat. Med. 2002; 8: 816-824Crossref PubMed Scopus (1644) Google Scholar, 21Chen X. Cheung S.T. So S. Fan S.T. Barry C. Higgins J. Lai K.M. Ji J. Dudoit S. Ng I.O. Van De Rijn M. Botstein D. Brown P.O. Mol. Biol. Cell. 2002; 13: 1929-1939Crossref PubMed Scopus (730) Google Scholar, 22Lapointe J. Li C. Higgins J.P. Van De Rijn M. Bair E. Montgomery K. Ferrari M. Egevad L. Rayford W. Bergerheim U. Ekman P. DeMarzo A.M. Tibshirani R. Botstein D. Brown P.O. Brooks J.D. Pollack J.R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 811-816Crossref PubMed Scopus (1044) Google Scholar) including cervical cancer (23Yan C. Boyd D.D. Cell Cycle. 2006; 5: 926-929Crossref PubMed Scopus (59) Google Scholar), arguing for a link between ATF3 expression and tumor suppression (23Yan C. Boyd D.D. Cell Cycle. 2006; 5: 926-929Crossref PubMed Scopus (59) Google Scholar). In line with this notion, we report here the identification of ATF3 as a novel E6 repressor that can compete with E6AP for binding to the viral protein thereby activating p53 in HPV-positive cervical cancer cells. These results indicate that ATF3 could contribute to suppression of HPV-induced carcinogenesis.