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Originally published as JCO Early Release 10.1200/JCO.2005.11.923 on March 7 2005 © 2005 American Society of Clinical Oncology.
Promoter Hypermethylation: A New Therapeutic Target Emerges in Urothelial CancerDepartments of Pathology and Urology; Biochemistry, Molecular Biology, and Surgery; and Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA Significant changes in the global levels and regional patterns of DNA methylation are among the earliest and most frequent events known to occur in human cancers.1 Alterations in DNA methylation have a direct impact on both the mutational and epigenetic components of neoplastic transformation. Important effects of DNA methylation on the genome include mutational burden of 5-methylcytosine, epigenetic effects of promoter methylation on gene transcription and potential gene activation, and induction of chromosomal instability by DNA hypomethylation.2,3 Hypermethylation of cytosine-guanine dinucleotide (CpG) islands is associated with transcriptional repression, whereas hypomethylation may lead to increased potential for gene activity or chromosomal instability. CpG islands located in tumor suppressor gene promoters are normally unmethylated. Abnormal methylation of these regions may lead to progressive reduction in gene expression, thus transcriptionally silencing suppressor genes by a variety of mechanisms, including remodeling of local chromatin structure or inhibition of transcription factor binding, ultimately altering normal cellular growth properties. Application of novel molecular biologic techniques has increased our appreciation of the widespread changes in methylation patterns that occur during urinary bladder carcinogenesis. Epigenetic events, such as methylation, occur throughout all stages of tumorigenesis, including the early phases, and are increasingly recognized as major mechanisms involved in silencing tumor suppressor genes. A large number of genes have been reported to be hypermethylated in bladder cancer. The frequency of such events suggests that widespread alterations in the patterns of DNA methylation are common in bladder carcinogenesis. Some examples include the p16 gene (at CDKN2AINK4a locus on chromosome 9p21), E-cadherin gene (CDH1, encoding a transmembrane glycoprotein that modulates calcium-dependent intercellular adhesion), and RASSF1A gene (ras association domain family gene 1, isoform A).4-6 Diminished CDH1 expression may lead to increase in beta-catenin activity and proliferation in urothelial carcinomas.7 Aside from deletion and mutation, the p16 gene has been shown to be inactivated by promoter methylation.8-11 Mutation or deletion of one p16 allele and hypermethylation of the remaining allele may be sufficient for loss of functional activity. Progressive inhibition of p16 expression by DNA methylation may result in loss of adequate growth regulatory function. Methylation of p16 has been observed in 27% to 60% of primary urothelial carcinomas.12,13 Both p16INKa and p14ARF hypermethylation may be involved in bladder carcinogenesis.14 p14ARF promoter hypermethylation in plasma DNA may also be an indicator of disease recurrence in bladder cancer patients.15 A variety of techniques are available for assessment of DNA methylation. Methylation-specific polymerase chain reaction16 and methylation-sensitive single nucleotide primer extension17 are among the most widely used techniques for identification and characterization of novel methylation changes associated with bladder carcinogenesis. This method has been used to detect methylation differences between the genomes of normal and tumor bladder cancer tissues and cell lines.18 The development of MethyLight technology has further improved the ability to assess the methylation patterns in a high-throughput manner.19 Gene expression microarray analysis has also been used to characterize methylation changes in bladder cancer cells after treatment with 5-aza-2'-deoxycytidine.20 Given that DNA-based tumor markers can be characterized by unique specificity, they make an attractive target for molecular diagnosis of cancer in body fluids such as blood serum, plasma, and urine. Methylation changes in DNA isolated from body fluids such as urine or blood may serve as useful adjuncts to current techniques for disease detection or surveillance. The potential clinical value of DNA-based analysis of body fluids for the initial diagnosis and the follow-up of urologic cancer patients was reviewed by Goessl et al.21 In this issue of the Journal of Clinical Oncology, Catto et al22 have analyzed hypermethylation at 11 CpG islands in a large cohort of upper urinary tract transitional cell carcinomas (UTTs) and lower tract (bladder) urothelial carcinomas (UCs), and have provided some interesting insights into differential epigenetic features of the two types of malignancies. Despite morphologic similarities between the two, more extensive promoter hypermethylation was found in UTTs (96%) than UCs (76%). Compared with tumors without methylation, the presence of methylation was significantly associated with advanced stage, higher tumor progression rates, and higher and mortality rates. The authors have shown that methylation occurs more extensively in UTTs compared with bladder UCs. Furthermore, there is an association between methylation and advanced tumor stage. The authors have also shown an association between methylation at the RASSF1A and DAPK loci and cumulative survival, suggesting their utility as potential predictive and prognostic markers and as likely therapeutic targets. In combining the data regarding the genes studied here with those from numerous publications reporting the role of methylation in many other genes relevant in bladder cancer, it is becoming obvious that not only these marker genes, but also that the process of aberrant methylation itself, may provide a therapeutic target in bladder cancer. Because of its global role in cancer progression, there has been intense interest in the process of DNA methylation as a potential therapeutic target. Inhibitors of DNA methylation act through reactivation of the expression of genes that have undergone epigenetic silencing. Initially developed as cytotoxic agents, the methylation inhibitors such as 5-azacytidine and 5-aza-2'-deoxycytidine have been shown to induce gene expression and differentiation in vitro.23 When incorporated in place of cytosine into replicating DNA, such nucleoside analogs serve as powerful mechanism-based inhibitors of DNA methylation active only in S-phase cells, heritably demethylating DNA. Recent additions of other analogs include 5-fluorocytosine, pseudoisocytosine or zebularine (5-fluoro-2'-deoxycytidine), and azacytosine. Of these, zebularine might be orally active.24 Another approach to target the process of hypermethylation is to block the activity of DNA methyltransferases; clinical trials with antisense oligonucleotides that target these enzymes are also underway.25 Decitabine (2'-deoxy-5-azacytidine) has been shown to inhibit DNA methyltransferases and reverse epigenetic silencing of aberrantly methylated genes.26 Epigenetic changes are also promising targets for cancer chemopreventive drug development and can be reversed by small molecules.27 Manipulation of DNA methylation can also be used advantageously in enhancing immune surveillance of tumors.28 Deregulated methylation of CpG dinucleotides may impair the immunogenic potential of cancer cells, via contribution to the absent or downregulated expression of different components of the tumor recognition complex (ie, HLA class I antigens, cancer testis antigens, and accessory or costimulatory molecules) in solid and hematopoietic human malignancies. Hence, pharmacologic agents that induce DNA hypomethylation can reverse these effects, restoring the defective expression of selected components of the tumor recognition complex in cancer cells. Improved immune recognition of cancer cells via epigenetic drugs thus provides an attractive approach to design new combined chemotherapeutic and immunotherapeutic strategies for the treatment of cancer. Potential pitfalls of epigenetic therapy have been summarized by Egger et al.29 Concerns regarding the clinical applications of these agents center mainly on the potential for nonspecific activation of genes in normal cells, aside from their potential mutagenicity and carcinogenicity. Early studies, which have indicated that DNA methylation is only one of the mechanisms enforcing silencing in normal cells, suggest they are less sensitive to drug-induced gene activation. However, these studies support the hope that global demethylation may not be as harmful to normal cells as feared.30 On the positive side, because multiple genes become methylated in individual cancers,31 it may be possible to target multiple candidate genes with one drug. An opposite scenario, however, may present itself when one considers genes such as MDR1, encoding P-gp protein responsible for multidrug resistance in many cancers. Hypomethylation of the MDR1 promoter has been observed to be a clinically important event in patients with recurrent bladder cancers that are resistant to therapy.32 When expression of the MDR1 gene in bladder cancer was examined, the tumors that recur after treatment with chemotherapy had 3.5- to 5.7-fold higher mRNA levels of the MDR1 gene compared with untreated tumors. Furthermore, the MDR1 gene was overexpressed in the majority of tumors (89%) that later recurred, whereas overexpression was observed in 25% of the tumors that did not recur (and thus also responded to chemotherapy). Overexpression of the MDR1 gene in cancers is shown to be inversely correlated with DNA methylation at CpG sites in the promoter. Thus, hypomethylation of the MDR1 promoter may result in development of a multidrug-resistant phenotype in patients with bladder cancer. Recent use of small interfering RNAs targeted to CpG islands within the promoter of a specific gene, which induce transcriptional gene silencing by means of DNA-methyltransferase–dependent methylation of DNA, may help resolve such situations.33 Finally, inactivation of tumor suppressor genes by aberrant DNA methylation of the promoter region is complemented by another epigenetic event that alters the structure of chromatin—the hypoacetylation of lysines in histones, brought about by histone deacetylase. Histone deacetylase inhibitors, therefore, also have potential in cancer therapy. Because a cross-talk can occur between DNA methylation and histone deacetylation, a combination of these two epigenetic modifications represents an interesting target for therapeutic intervention. Inhibitors of these two pathways in combination have been shown to produce a synergistic reactivation of tumor suppressor genes and an enhanced antineoplastic effect against tumor cells.34 Bladder cancer represents a unique model for targeted therapy because of the availability of a number of avenues for drug delivery, which include intravesical, systemic, systemic with concentration in bladder, and continuous administration. Rational targeting in bladder cancer, as in most other malignancies, will involve development of new therapeutic strategies aimed at specific defects that characterize the malignancy. As elucidation of the molecular biology of bladder cancer progression proceeds, effective prediction of risk of progression and chemotherapeutic response is likely to become a reality.35 Development of epigenetic therapeutic drugs can be expected to add to the armamentarium of rational drugs, and efficacy of such rationally designed drugs can be assayed for the targeted effect in appropriately designed clinical trials. Authors' Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. REFERENCES 1. Jones PA, Baylin, SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet, 3:415-428, 2002[Medline] 2. Gonzalgo ML, Jones PA: Mutagenic and epigenetic effects of DNA methylation. Mutat Res 386:107-118, 1997[CrossRef][Medline]
3. Jones PA, Gonzalgo ML: Altered DNA methylation and genome instability: A new pathway to cancer? Proc Natl Acad Sci U S A 94:2103-2105, 1997 4. Chang LL, Yeh WT, Yang SY, et al: Genetic alterations of p16INK4a and p14ARF genes in human bladder cancer. J Urol 170:595-600, 2003[CrossRef][Medline] 5. Chan MW, Chan LW, Tang NL, et al: Frequent hypermethylation of promoter region of RASSF1A in tumor tissues and voided urine of urinary bladder cancer patients. Int J Cancer 104:611-616, 2003[CrossRef][Medline] 6. Horikawa Y, Sugano K, Shigyo M, et al: Hypermethylation of an E-cadherin (CDH1) promoter region in high grade transitional cell carcinoma of the bladder comprising carcinoma in situ. J Urol 169:1541-1545, 2003[CrossRef][Medline] 7. Thievessen I, Seifert HH, Swiatkowski S, et al: E-cadherin involved in inactivation of WNT/beta-catenin signalling in urothelial carcinoma and normal urothelial cells. Br J Cancer 88:1932-1938, 2003[CrossRef][Medline] 8. Spruck CH, Gonzalez-Zulueta M, Shibata A, et al: p16 gene in uncultured tumors. Nature 370:183-184, 1994[CrossRef][Medline]
9. Herman JG, Merlo A, Mao L, et al: Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 55:4525-4530, 1995
10. Gonzalez-Zulueta M, Bender CM, Yang AS, et al: Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res 55:4531-4535, 1995 11. Serrano M, Hannon GJ, Beach D: A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366:704-707, 1993[CrossRef][Medline]
12. Chan MW, Chan LW, Tang NL, et al: Hypermethylation of multiple genes in tumor tissues and voided urine in urinary bladder cancer patients. Clin Cancer Res 8:464-470, 2002 13. Chang LL, Yeh WT, Yang SY, et al: Genetic alterations of p16INK4a and p14ARF genes in human bladder cancer. J Urol 170:595-600, 2003 14. Dominguez G, Silva J, Garcia JM, et al: Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors. Mutat Res 530:9-17, 2003[Medline]
15. Dominguez G, Carballido J, Silva J, et al: P14ARF promoter hypermethylation in plasma DNA as an indicator of disease recurrence in bladder cancer patients. Clin Cancer Res 8:980-985, 2002
16. Herman JG, Graff JR, Myohanen S, et al: Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821-9826, 1996
17. Gonzalgo ML, Jones PA: Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms- SNuPE). Nucleic Acids Res 25:2529-2531, 1997 18. Liang G, Salem CE, Yu MC, et al: DNA methylation differences associated with tumor tissues identified by genome scanning analysis. Genomics 53:260-268, 1998[CrossRef][Medline] 19. Eads CA, Danenberg KD, Kawakami K, et al: MethyLight: A high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:E32, 2000
20. Liang G, Gonzales FA, Jones PA, et al: Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine. Cancer Res 62:961-966, 2002 21. Goessl C, Muller M, Straub B, et al: DNA alterations in body fluids as molecular tumor markers for urological malignancies. Eur Urology 41:668-676, 2002
22. Catto JWF, Azzouzi A-R, Rehman I, et al: Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma. J Clin Oncol 23:2903-2910, 2005 23. Jones PA, Taylor SM: Cellular differentiation, cytidine analogs and DNA methylation. Cell 20:85-93, 1980
24. Cheng JC, Matsen CB, Gonzales FA, et al: Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 95:399-409, 2003 25. Yan L, Nass SJ, Smith D, et al: Specific inhibition of DNMT1 by antisense oligonucleotides induces re-expression of estrogen receptor-alpha (ER) in ER-negative human breast cancer cell lines. Cancer Biol Ther 2:552-556, 2003[Medline] 26. Brown R, Plumb JA: Demethylation of DNA by decitabine in cancer chemotherapy. Expert Rev Anticancer Ther 4:501-510, 2004[CrossRef][Medline]
27. Kopelovich L, Crowell JA, Fay JR: The epigenome as a target for cancer chemoprevention. J Natl Cancer Inst 95:1747-1757, 2003 28. Maio M, Coral S, Fratta E, et al: Epigenetic targets for immune intervention in human malignancies. Oncogene 22:6484-6488, 2003[CrossRef][Medline] 29. Egger G, Liang G, Aparicio A, et al: Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:457-463, 2004[CrossRef][Medline] 30. Wolf S, Migeon BR: Studies of X chromosome DNA methylation in normal human cells. Nature 295:667-671, 1982[CrossRef][Medline]
31. Esteller M, Corn PG, Baylin SB, et al: A gene hypermethylation profile of human cancer. Cancer Res 61:3225-3229, 2001
32. Tada Y, Wada M, Kuroiwa K, et al: MDR1 gene overexpression and altered degree of methylation at the promoter region in bladder cancer during chemotherapeutic treatment. Clin Cancer Res 6:4618-4627, 2000 33. Kawasaki H, Taira K: Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature 431:211-217, 2004[CrossRef][Medline] 34. Momparler RL: Cancer epigenetics. Oncogene 22:6479-6483, 2003[CrossRef][Medline] 35. Cote RJ, Datar RH: Therapeutic approaches to bladder cancer: Identifying targets and mechanisms. Crit Rev Oncol Hematol 46:S67-S83, 2003
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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