EDITORIALS

Zn2+ Fingers and Cervical Cancer

Scott A. Foster, William C. Phelps

Affiliation of authors: Department of Virology, GlaxoWellcome Co., Research Triangle Park, NC.

Correspondence to: William C. Phelps, Ph.D., Department of Virology, GlaxoWellcome Co., Bldg. RC2, Rm. 3521, Research Triangle Park, NC 27709 (e-mail: wcp41432{at}glaxowellcome.com).

Cervical cancer will kill several hundred thousand women around the world during the next year. It ranks second only to breast cancer as the leading cause of cancer death in women worldwide (1).

The widespread use of the Papanicolaou (Pap) smear since its introduction in the 1940s as a cancer-screening tool has dramatically reduced the incidence of cervical cancer and has undoubtedly prevented the deaths of thousands of women in developed nations. Management of cervical cancer has focused on prevention through early detection and ablation of dysplastic tissues. Carcinoma in situ and invasive cancers, when diagnosed, are treated more aggressively with surgery, radiation therapy, and chemotherapy as the standard therapeutic options. Unfortunately, the standard therapies are not highly specific. In the article by Beerheide et al. (2) in this issue of the Journal, an alternative therapeutic strategy is proposed that focuses on inhibition of a critical oncoprotein of the human papillomavirus (HPV).

ANTIVIRAL CHEMOTHERAPY FOR CANCER

Among the myriad human cancers, those etiologically related to viral infections offer unique targets for potentially novel therapies. As an alternative to traditional antitumor therapy that seeks to exploit sometimes subtle differences in tumor cells, an antiviral approach to cancer therapy would specifically target the viral gene products that contribute to the transformed phenotype. Theoretically, since the target is viral rather than cellular, it should be possible to develop much less toxic therapies than many of those that are currently available. The relationship between cervical cancer and HPV infection provides a clinically important opportunity to test this hypothesis.

The initial link between HPV infection and cervical cancer was forged when it was demonstrated that a high percentage of cervical cancers contained HPV DNA (3). Since that time, a large number of epidemiologic studies (4,5) have confirmed the relationship between HPV infection and cervical cancer. The E6 and E7 genes from "high-risk" HPVs, those most commonly associated with cervical cancer, have been identified as the major viral oncogenes by their activity in transformation assays and by their ability to immortalize primary human epithelial cells (6). Further studies (7) revealed that E6 and E7 inactivate two cellular tumor suppressors, p53 and Rb, respectively. The E6 and E7 from "low-risk" HPV types, not commonly associated with cancer, are either inactive or reduced in their ability to transform cells or inactivate p53 and Rb. However, infection with high-risk HPV does not itself produce cancer. Most infections are cleared naturally, and the minority that do progress require many years to become malignant. Clearly, additional cellular events are required for progression to cancer. The observation that HPV integration results in selective retention and expression of E6 and E7, however, is suggestive of a continued requirement for these oncogenes in the maintenance of the malignant phenotype. The frequent inactivation of the p53 and Rb pathways by mutation in non-HPV-associated cancers further underscores the importance of these tumor suppressors in the control of cell growth.

E6 BIOCHEMISTRY

Beerheide et al. (2) examine one possibility for selectively inactivating the HPV16 E6 protein. E6 binds to p53 in conjunction with the E6-associated protein (E6AP), a ubiquitin ligase and, in so doing, inactivates p53 by targeting it for degradation via the ubiquitin proteolysis pathway (8). p53 is a multifunctional tumor suppressor involved in the cellular response to stress, including, among others, DNA damage, hypoxia, low ribonucleotide levels, or oncogene expression. In response, p53 can induce cell cycle arrest, modulate DNA repair, or induce apoptosis (9). Although E6-mediated p53 degradation is its most carefully studied activity, E6 clearly does other things as well. For example, E6 binds paxillin, thereby disrupting the actin cytoskeleton. E6 interacts with the human homologue of the Drosophila discs large tumor suppressor (hDLG), disrupting the ability of hDLG to interact with another tumor suppressor, APC, the product of the adenomatous polyposis coli gene. E6 can associate with a calcium-binding protein known as E6-binding protein (E6BP; also known as ERC-55), although the functional significance of this interaction is not clear (10). Finally, E6 activates telomerase activity in epithelial cells—activation of telomerase activity is a common feature of cancer cells (11). Therefore, if E6 could be selectively inactivated, a cascade of events leading to repression of the transformed phenotype would be expected to occur in tumor cells.

ZN2+ FINGERS AS TARGETS

The approach used by Beerheide et al. (2) is to target E6 for inactivation by displacing the bound Zn2+. Two pairs of highly conserved Cys-X-X-Cys motifs chelate two atoms of Zn2+, and coordination of the metal is thought to play an important structural role in the E6 protein. Is the Zn2+ finger a good therapeutic target? A cursory examination of the 15 most clinically successful drugs of 1998 shows that the majority of the pharmacologic targets are either enzymes (five of 15) or receptor/ion channel antagonists (eight of 15). For antiviral inhibitors, the most notable have come from targeting kinases, proteases, and polymerases. Therefore, targeting a nonenzymatic viral protein such as HPV E6 through binding to a Zn2+ finger leading to allosteric inactivation does not follow a traditionally successful avenue for drug discovery. In spite of the history, there is no reason a priori that a Zn2+ finger should not represent a viable therapeutic target if it satisfies several important criteria.

First, E6 must represent a validated target in that inhibition of its activities should result in abrogation of the growth of tumor cells, and those critical biochemical activities must be dependent on the integrity of the Zn2+ finger that is being targeted. Antisense studies suggest that the continued expression of E6 is essential to maintain the transformed state (12) and, furthermore, a previous study (13) has shown that the Cys-X-X-Cys motifs are essential for E6 to retain full activity. Second, to be an attractive molecular target, E6 should possess the ability to avidly bind a low-molecular-weight ligand in an active site or within a regulatory pocket. Such binding pockets can be either detected through examination of the tertiary structure or empirically defined through screening of small molecule libraries. To our knowledge, to date, no tertiary structure or screening results have been published that indicate whether high-affinity ligands can be developed. Finally, can target specificity be achieved?

SPECIFICITY

Beerheide et al. (2) tested a series of 36 sulfhydryl residue-specific, redox-reactive compounds in hopes of finding one that specifically reacted with E6. First, the compounds were tested for their ability to "eject" Zn2+ from E6. The nine compounds that scored positive in the assay were further assayed for their ability to interfere with the interaction between E6 and E6AP or E6BP, and two seemed to inhibit these interactions. When examined for inhibition of cell growth, one compound (4,4'-dithiodimorpholine) seemed to selectively inhibit the proliferation of HPV-positive cervical cancer cells. It is interesting that p53 levels in the cells increased and the cells underwent apoptosis as evidenced by cleavage of poly-adenosine diphosphate ribose polymerase. These results are consistent with the idea that the compound interferes with the binding of Zn2+ by E6, thus allowing p53 to trigger apoptosis.

Where do we go from here? One lingering question is that of absolute specificity. To demonstrate that these or other compounds preferentially affect the Zn2+ fingers of E6, it will be important to examine their activities against a relevant battery of other proteins, including a series of Zn2+-containing metalloproteins. If clear biochemical specificity is observed, such data would support challenging HPV-infected cells. As more crystal structures become available, comparison of Zn2+ finger structures from different classes of Zn2+-binding proteins may provide valuable information for designing more specific "Zn2+-ejecting" compounds. Indeed the assumption that the Zn2+ fingers of E6 are distinct from those in cellular proteins could be best validated by a direct structural comparison because primary amino acid sequence can be misleading. Are these truly unique molecular motifs?

Finally, the chemistry itself may not represent ideal scaffolds for the initiation of a drug-discovery effort. In general, the reported compounds are likely to be associated with poor stability and high nonspecific reactivity to a variety of molecules in a cell. Such chemical reactivity might be associated with pleiotropic effects in cells, confounding mechanistic studies, and might ultimately be associated with toxicity in vivo. With that cautionary note, it should be pointed out that an azodicarbonamide that targets a Zn2+ finger in the p7 nucleocapsid (14) is currently in phase II trials for use against human immunodeficiency virus.

REFERENCES

1 Cervical cancer: summary of the National Institutes of Health (NIH) consensus. Aust NZ J Obstet Gynaecol 1997;37:339-41.[Medline]

2 Beerheide W, Bernard HU, Tan YJ, Ganesan A, Rice WG, Ting AE. Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein. J Natl Cancer Inst 1999;91:1211-1220.[Abstract/Free Full Text]

3 zur Hausen H. Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus types. In: zur Hausen H, editor. Human pathogenic papillomaviruses. Heidelberg (Germany): Springer-Verlag; 1994. p. 131-56.

4 Schiffman MH. Recent progress in defining the epidemiology of human papillomavirus infection and cervical neoplasia. J Natl Cancer Institute 1992;84:394-8.[Medline]

5 Schneider A, Koutsky LA. Natural history and epidemiological features of genital HPV infection. IARC Sci Publ 1992;119:25-52.[Medline]

6 McDougall JK. Immortalization and transformation of human cells by human papillomavirus. Curr Top Microbiol Immunol 1994;186:101-19.[Medline]

7 Munger K, Scheffner M, Huibregtse JM, Howley PM. Interactions of HPV E6 and E7 oncoproteins with tumour suppressor gene products. Cancer Surv 1992;12:197-217.[Medline]

8 Huibregtse JM, Beaudenon SL. Mechanism of HPV E6 proteins in cellular transformation. Semin Cancer Biol 1996;7:317-26.[Medline]

9 Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323-31.[Medline]

10 Kubbutat MH, Vousden KH. New HPV E6 binding proteins: dangerous liaisons? Trends Microbiol 1998;6:173-5.[Medline]

11 Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 1996;380: 79-82.[Medline]

12 He Y, Huang L. Growth inhibition of human papillomavirus 16 DNA-positive mouse tumor by antisense RNA transcribed from U6 promoter. Cancer Res 1997;57:3993-9.[Abstract]

13 Kanda T, Watanabe S, Zanma S, Sato H, Furuno A, Yoshiike K. Human papillomavirus type 16 E6 proteins with glycine substitutions for cysteine in the metal-binding motif. Virology 1991;185:536-43.[Medline]

14 Rice WG, Turpin JA, Huang M, Clanton D, Buckheit RW Jr, Covell DG, et al. Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein. Nat Med 1997;3:341-5.[Medline]



             
Copyright © 1999 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement