Epigenetic Mechanisms of Carcinogenesis: Commentary

Jay I. Goodman, Ph.D.

Department of Pharmacology and Toxicology, Michigan State University, B-440 Life Sciences Building

East Lansing, MI 48824 U.S.A.

Tel: (517)-353-9346

Fax: (517)-353-8915

E-mail: goodman3@pilot.msu.edu

There is, particularly among many toxicologists, an excessive focus upon mutagenesis as the (read the one and only) mechanism underlying carcinogenesis. Therefore, I am delighted to see this provocative, timely review of epigenetic mechanisms of carcinogenesis by Klaunig et al. I would like to take this opportunity to respond, in a succinct fashion, by amplifying and extending some of the points made. Specifically, I will 1) expand upon the definition of the term "epigenetic" and its role in carcinogenesis, 2) discuss the role that increased gene expression, without mutation, may play in transformation, 3) provide an example of a dose-response relationship between a tumor promoter and its effect on DNA methylation, 4) indicate that the initiation stage of carcinogenesis may have an epigenetic rather than a mutagenic basis, and 5) discuss the possible interrelationships between a genotoxic event and altered DNA methylation, an epigenetic event.

Epigenetics and Its Role in Carcinogenesis

Epigenetic regulation of gene expression is based upon a modulation of gene expression by heritable mechanisms superimposed on that conferred by the primary DNA sequence, e.g. DNA methylation, 5-methylcytosine content of DNA (Holiday, 1994). In contrast to mutation, this involves a heritable alteration of gene expression that is not based upon a change in DNA base sequence. Altered DNA methylation leading to aberrant gene expression due, in part, to affecting the ability of methylated DNA-binding proteins to interact with their cognate cis elements, may underlie some of the crucial changes in gene expression involved in carcinogenesis (Samiec and Goodman, 1999). The key role that altered DNA methylation may play in carcinogenesis, as an epigenetic, nongenotoxic mechanism, has been the subject of several reviews (Holiday, 1987; Counts and Goodman, 1995a; Counts and Goodman, 1995b; Counts and Goodman, 1995c; Baylin, 1997; Trosko et al., 1998; and Jones and Laird, 1999).

Increased Gene Expression, Without Mutation, May Play a Role in Carcinogenesis

The role that mutation plays in carcinogenesis by activating proto-oncogenes to oncogenes and silencing tumor suppresser genes is appreciated widely. It is axiomatic that, in order to affect the phenotype of a cell, a mutated oncogene must be expressed. However, aberrant increased expression of nonmutated genes has been shown to play a key roll too (Shastry, 1995). Proto-oncogene overexpression may be a mechanism of activation of the ras pathway, alternative to point mutation (Mangues et a., 1994; Clark et al., 1996). Overexpression of myc as well as K-ras can contribute to transformation (Schwab, 1983; Lee, 1991). Furthermore, overexpression of HER2/c-erbB2 receptor tyrosine kinase induces the transformed phenotype of NIH3T3 cells and is required for tumor formation and progression in nude mice (Baasner et al., 1996). In this context, it is important to note that the U.S. Environmental Protection Agency's proposed Cancer Risk Assessment Guidelines include a section (section entitled "Nonmutagenic and Other Effects" which refers explicitly to a role for altered DNA methylation as a basis for the altered gene expression involved in carcinogenesis (U.S. Environmental Protection agency, 1996).

Tumor Promoter-Induced Alteration in DNA Methylation: A Dose-Response Relationship

Administration of a tumor-promoting dose of phenobarbital (PB) in the drinking water (500 ppm) for 14 days resulted in hypomethylation of raf in the liver of the tumor-prone B6C3F1mouse, while the methylation status of the gene was not affected in the relatively resistant C57BL/6 mouse (Ray et al., 1994). Importantly, when B6C3F1 mice were administered drinking water containing 20 ppm PB the methylation of raf was not affected.

Initiation of Carcinogenesis May Have an Epigenetic Basis

The view of carcinogenesis as consisting of three biological stages, initiation, promotion and progression provides a very useful conceptual framework for understanding the cancer problem and approaching it experimentally (Dragan et al., 1993). The first stage, initiation, involves a heritable alteration to the genome that facilitates the clonal expansion of initiated cells in response to a promotion stimulus. It is usually assumed that mutagenesis provides the basis for initiation. However, there could be an epigenetic basis too. Despite the fact that many initiators of carcinogenesis have been shown to be capable of acting as mutagens under certain experimental circumstances, one does not have to assume that all of their biological effects stem from mutagenesis. Hypermethylation-induced silencing of a tumor suppresser gene(s) (Counts and Goodman, 1995; Baylin, 1997; Jones and Laird, 1999) and/or hypomethylation-facilitated aberrant increase in expression of an oncogene(s) (Counts and Goodman, 1995) are plausible mechanisms that could underlie initiation. The involvement of epigenetics in initiation is not mutually incompatible with a role for mutation. Indeed, one or the other, or perhaps both, may play key roles depending upon the particular circumstances, e.g., causative agent, dose, target organ and species.

InterrelationshipsBetween Genotoxicity and Altered DNA Methylation

Carcinogen adducts in DNA involving chemical carcinogens (Wilson and Jones, 1983; Wilson et al., 1987; Hepburn et al., 1991) or free radical adducts (Weitzman et al., 1994) have been shown to result in decreased DNA methylation. Additionally, depending upon the location of a particular alkylated (O6-methyl) guanine in relation to a potentially methylatable cytosine residue, DNA methylation may be either increased or decreased (Tan and Li, 1990). Thus, a genotoxic compound may produce an epigenetic change that can persist, even though the DNA damage may be repaired.

Summary Statement

It is now quite clear that carcinogenesis involves more than mutagenesis and an increased focus on epigenetic events underlying the transformation of a normal cell into a frank malignancy is appropriate.


Baasner, S, von Melchner, H, Klenner, T, Hilgard, P, Beckers, T. (1996). Reversible tumorigenesis in mice by conditional expression of the HER2/c-erbB2 receptor tyrosine kinase. Oncogene 13: 901-911.

Baylin, SB. (1997). Tying it all together: Epigenetics, Genetics, cell cycle, and cancer. Science 277: 1948-1949.

Clark, GJ, Kinch, MS, Gilmer, TM, Burridge, K , Der, CJ. (1996). Overexpression of the ras-related TC21/R-ras2 protein may contribute to the development of human breast cancers. Oncogene 12: 169-176.

Counts, JL, Goodman, JI. (1995a). Alterations in DNA methylation may play a variety of roles in carcinogenesis. Cell 83: 13-15.

Counts, JL, Goodman, JI. (1995b). Hypomethylation of DNA: A possible epigenetic mechanism involved in tumor promotion. In, Growth Factors and Tumor Promotion: Implications for Risk Assessment, McLain, RM, Slaga, TJ, LeBoeuf, RA, Pitot, HC, Eds., Wiley-Liss, Inc., NY, pp. 81-101.

Counts, JL, Goodman, JI. (1995c). Hypomethylation of DNA: An epigenetic mechanism that can facilitate the aberrant oncogene expression involved in liver carcinogenesis. In, Liver Regeneration and Carcinogenesis, Jirtle, R, Ed., Academic Press, Inc., NY, p.227-255.

Dragan, YP, Sargent, L, Xu, YD, Xu, YH, Pitot, HC. (1993). The initiation, promotion, progression model of rat hepatocarcinogenesis. Proc. Soc. Exp. Biol. Med. 202: 16-24.

Hepburn, PA, Margison, GP, Tisdale, MJ. (1991). Enzymatic methylation of cytosine inDNA is prevented by adjacent O6-methylguanine residues. J. Biol. Chem. 266: 7985-7987.

Holiday, R. (1987). DNA methylation and epigenetic defects in carcinogenesis. Mutation Res. 181: 215-217.

Holiday, R. (1994). Epigenetics: An overview. Develop. Genetics 15: 453-457.

Jones, PA, Laird, PW. (1999). Cancer epigenetics comes of age. Nature Genetics 21: 163-167.

Lee, LW, Raymond, VW, Tsao, M-S, Lee, DC, Earp, HS, Grisham, JW. (1991). Clonal cosegregation of tumorigenicity with over expression of c-myc and transforming growth factor a genes in chemically transformed rat liver epithelial cells. Cancer Res. 51: 5238-5244.

Mangues, R, Kahn, JM, Seidman, I, Pellicer, A. (1994). An overexpressed N-ras proto-oncogene cooperates with N-methylnitrosourea in mouse mammary carcinogenesis. Cancer Res. 54: 6395-6401.

Ray, JS, Harbison, ML, McClain, RM, Goodman, JI. (1994). Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors. Mol. Carcinogen. 9: 155-166.

Samiec, PS, Goodman, JI. (1999). Evaluation of methylated DNA binding protein-1 in mouse liver. Toxicol. Sci. 49: 255-262.

Schwab, M, Alitalo, K, Varmus, HE, Bishop, JM. (1983). Nature 303: 497-501.

Shastry, BS (1995). Overexpression of genes in health and sickness. Comp. Biochem. Physiol. 112B: 1-13.

Singh, P, Wong, SH, Hong, W. (1994). Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation. EMBOJ. 13: 3329-3338.

Tan, N-W, Li, BF. (1990). Interaction of oligonucleotides containing 6-O-methylguanine with human DNA (cytosine-5-)-methyltransferase. Biochem. 29: 9234-9240.

Trosko, JE, Chang, CC, Upham, B, Wilson, M. (1998). Epigenetic toxicology as toxicant-induced changes in intracellular signaling leading to altered gap junctional intercellular communication. Toxicol. Letters 102-103: 71-78.

U.S. Environmental Protection Agency. (1996). Proposed guidelines for carcinogen risk assessment. Federal Register 61: 17960-18011.

Weitzman, SA, Turk, PW, Milkowski, DH, Kozlowski, K. (1994). Free radical adducts induce alterations in DNA cytosine methylation. Proc. Natl. Acad. Sci. USA 91: 1261-1264.

Wilson, VL, Jones, PA. (1983). Inhibition of DNA methylation by chemical carcinogens in vitro. Cell 32: 239-246.

Wilson, VL, Smith, RA, Longoria, J, Liotta, MA, Harper, CM, Harris, CC. (1987). Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine content of normal bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 84: 3298-3301.