Journal of Clinical Pharmacy and Therapeutics, 2014
doi: 10.1111/jcpt.12206
Review Article
DNA methylation and personalized medicine J. Tang*†a MD, Y. Xiong*†a MD, H.-H. Zhou*† MD PhD and X.-P. Chen*† MD PhD *Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, and †Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Hunan Key laboratory of Pharmacogenetics, Central South University, Changsha, China
Received 9 June 2013, Accepted 17 August 2014
Keywords: DNA methylation, drug response, single nucleotide polymorphisms
(7-mG). Clusters of cytosine–phosphate–guanine dinucleotides in a chromosomal region are referred to as CpG islands. In mammalian genomes, there are about 40 000 CpG islands, which are typically located in the promoters or in the first exon of the genes.1,2 CpG island methylation is a dynamic epigenetic process during mammalian development and is critical in regulating gene expression,3 maintaining the stability of the genome and its chromosomal structure,4 and inactivating genomic imprinting and X-chromosome dosage.5 Aberrant methylation of the CpG island may also result in human diseases such as cancer, amentia, autoimmunity and diabetes. Therefore, DNA methylation has become an important focus of recent epigenomics research. Understanding of the role of DNA methylation in pathogenesis has led to the development of several drugs targeting DNA methylation. Two DNMT inhibitors, 5-azacitidine6,7 and decitabine,8,9 are approved by the U.S. FDA for the treatment of myelodysplastic syndromes (MDS) and haematological malignancies. DNA methylation can also affect drug responses by altering the expression of genes encoding drug metabolism enzymes (DMEs),10 drug transporters11 and drug targets.12 Single nucleotide polymorphisms (SNPs) near DNA methylation sites can also function through affecting gene methylation and thus the expression of associated genes.13 Both these mechanisms can result in interindividual difference in drug response.
SUMMARY What is known and objective: Variation in the expression of drug-response-related genes contributes significantly to interindividual differences in drug response. DNA methylation is one of the most common epigenetic modifications that control gene expression. DNA methylation may occur in genes encoding drug metabolizing enzymes (DMEs), drug transporters and drug targets, and can thereby alter the pharmacokinetics and pharmacodynamics of drugs. In this review, we discuss recent advances in pharmacoepigenetics with a focus on DNA methylation. Methods: The literature search focusing on DNA methylation of drug-response-related genes and DNA methylation-related SNPs in pharmacogenomics was carried out using the PUBMED database and a combination of keywords including DNA methylation, drug response, DMEs, drug transporters, drug target and SNPs. Results and discussion: An extensive range of research has contributed to our understanding of how DNA methylation of drug-response-related genes alters their function. This is particularly well studied in cancer chemotherapy and drug resistance. The impact of polymorphisms of miRNAs in these processes requires further study. What is new and conclusion: DNA methylation-related genetic variation is an increasingly recognized mechanism for altered drug-response and disease susceptibility. These new discoveries require assimilation into the practice of personalized medicine.
METHODS A literature search was undertaken focusing on two main aspects – DNA methylation of drug-response-related genes and DNA methylation in relation to pharmacogenomics. The search interrogated the PUBMED database and the keywords included DNA methylation, drug response, DMEs, drug transporters, drug target and SNPs.
WHAT IS KNOWN AND OBJECTIVE Interest in the epigenetic modification of genomes has grown exponentially in recent years. Epigenetics plays an important role in regulating gene expression. DNA methylation, the biological methylation of specific nucleotides by DNA methyltransferases (DNMTs), is the epigenetic modification that has attracted most interest. DNA methylation can result in suppression of gene expression, a process that can be reactivated by demethylation. DNA methylation occurs mainly as 5-methylcytosine (5-mC), with a small fraction of N6-methylpurine (N6-mA) and 7-methylguanine
RESULTS AND DISCUSSION DNA methylation of drug-response-related genes and relevance to clinical pharmacology Variation in the expression of drug-response-related genes is an important contributor to interindividual difference in drug response. This variation is seen in the expression of DMEs, drug transporters, drug targets and downstream signalling molecules. DNA methylation has attracted increasing interest due to its universality and heritability in the regulation of gene expression (Table 1).
Correspondence: Professor X.-P. Chen, Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan, 410078, P. R. China. Tel.: 86-731-84805380; fax: 86731-82354476; e-mail: [email protected]. a These two authors contributed equally to this work.
© 2014 John Wiley & Sons Ltd
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DNA methylation and personalized medicine
Table 1. DNA methylation of drug-response-related gene
CYP450 family members
Phase II DMEs Drug transporters
Drug targets and signal pathway genes
DNA methylated regions
Affected drugs
Ref.
CYP1A1 promoter CYP1A2 promoter/enhancer
Polycyclic aromatic hydrocarbon Drug metabolism and synthesis of cholesterol, steroids and other lipids Oestrogen and 4-hydroxy estradiol Vitamin D Oxaliplatin and adriamycin Tamoxifen 5-azacitidine, Doxorubicin and irinotecan Dichloroacetate(DAC) Cisplatin Fluorouracil Camptothecin (CPT) Temozolomide
15,16 19,20
CYP1B1 promoter/enhancer CYP24A1 promoter GSTP1 promoter NAT1 MDR1/ABCC1 promoter SLC5A8 SLC22A1/OCT1 TFAP2E DEXI MGMT promoter
21,22 27 31 32 34,35 36 37 38 42 45
CYP, cytochrome P450; GSTP1, glutathione-S-transferase P1; NAT1, N-acetyl transferase 1; MDR1/ABCC1, multidrug resistance protein; ABC, ATP-binding cassette (ABC); SLC, soluble carrier (SLC); TFAP2E, transcription factor AP-2 epsilon; DEXI, dexamethasone-induced protein; MGM, O6-methylguanine-DNA methyltransferase.
promoter/enhancer is involved in the pathogenesis of prostate cancer.21 Habano et al. have also reported that CYP1B1 methylation in colorectal-cancer cell lines but not in corresponding normal tissue, and the methylation status can be decreased by treating with DNMT inhibitor 5-aza-20 -deoxycytidine.22 For the CYP2 family genes, CpG islands are observed in the promoters of CYP2A6, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP2R1, CYP2S1 and CYP2W1, indicating that these genes may also undergo regulation by DNA methylation.23 Demethylation of CYP2W1 CpG island appears to be associated with high CYP2W1 expression.24 CYP3A is the most important DME in adult human liver that contributes to metabolism of approximately 70% of clinically used drugs. DNMT inhibitors are shown to influence the expression of CYP3A4, CYP3A5 and CYP3A7 in HepG2 cells.23 Studies with adult and foetal human livers show highly variable CpG methylation sites in important CYP3A4 transcription factor-binding sites, and hypermethylation of CYP3A4 promoter may account for lack of CYP3A4 expression in foetal liver.25 Therefore, methylation status of CYP3A4 may contribute to interindividual differences in CYP3A4 activity in adult liver. As DNMT inhibitors are used in the clinic, and given the importance of CYP3A in drug metabolism, potential drug–drug interactions between DNMTs inhibitors and CYP3A substrates should be studied further. CYP24A1 is a key enzyme involved in regulating vitamin D metabolism. Methylation of CYP24A1 promoter is tissue specific. Novakovic et al. showed that CYP24A1 is methylated in human placenta, and this may be relevant to adverse effects of gestation.26 Significant variance in the methylation status of CYP24A1 promoter between tumour and normal endothelium cells is also observed, and this may lead to interindividual difference in cellular response to calcitriol, an active metabolite of vitamin D3.27 In addition, CYP24A1 methylation may play a role in the formation of tumour microenvironment in prostate cancer.28
Methylation of CYP450 family members The cytochrome P450 (CYP450) superfamily contributes to the phase I metabolism of about 75% clinically used drugs.14 Evidence has shown that DNA methylation plays important roles in regulating the expression of members of the CYP1, CYP2 and CYP3 subfamilies. CYP1A1, the major enzyme that metabolizes polycyclic aromatic hydrocarbons, can be induced by smoking. Therefore, the enzyme is potentially important in the aetiology of lung cancer.15 An epidemiological study has shown that in lung tissues of nonsmokers, light smokers and heavy smokers, the rate of complete or partial methylation for CYP1A1 promoter was 98%, 71% and 33%, respectively. This indicates hypomethylation of CYP1A1 promoter by cigarette smoking. CYP1A1 promoter methylation is increased within 1–7 days of quitting smoking.16 A recent study by Tekpli et al. indicated that the CYP1A1 enhancer was hypermethylated, whereas CYP1A1 mRNA level was reduced in lung cancer.17 Another study also shows that DNA methylation can modulate CYP1A1 expression in prostate tissue. In the prostate cell line LNCaP, CYP1A1 hypermethylation results in deficiency of CYP1A1 expression. For the non-cancerous cell lines RWPE-1, the environmental contaminant dioxin can upregulate CYP1A1 expression by decreasing its methylation.18 CYP1A2, another member of the CYP450 family, is involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. CYP1A2 activity is also regulated by smoking and some dietary components. Miyajima et al. observed that the CpG methylation status of CYP1A2 promoter was strongly associated with CYP1A2 mRNA expression in the liver.19 This report indicates that CpG methylation may account for the tissue-specific regulation of CYP1A2 gene expression. The extent of CpG island DNA methylation close to the CYP1A2 transcriptional start site correlates inversely with hepatic CYP1A2 mRNA levels.20 CYP1B1 is important in oestrogen metabolism and is involved in the pathogenesis of breast cancer and endometrial cancer by regulating the production of 4-hydroxy estradiol. A study by Tokizane et al. has shown that aberrant methylation in CYP1B1 promoter/enhancer can influence the binding of transcription factor AhR/ARNT and SP-1, and hypomethylation of CYP1B1
DNA methylation of phase II DMEs Glutathione-S-transferase P1 (GSTP1) is an important phase II DME that is involved in the metabolism of drugs such as
© 2014 John Wiley & Sons Ltd
Journal of Clinical Pharmacy and Therapeutics, 2014 2
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DNA methylation and personalized medicine
oxaliplatin and adriamycin. GSTP1 promoter is generally methylated in most patients with prostate cancer, and the level of methylation is a marker useful for distinguishing benign prostatic hyperplasia from prostate cancer or to predict the prognosis of prostate cancer.29,30 DNMTs inhibitors 5-azacitidine and decitabine demethylate GSTP1 and promote GSTP1 re-expression.31 Therefore, GSTP1 promoter methylation status may serve as a potential biomarker to predict 5-azacitidine response. N-acetyl transferase 1 (NAT1) is a phase II DME that is important in metabolizing drugs and several other xenobiotics. In breast cancer tissues, NAT1 was observed to be methylated in 622% of the tamoxifen-resistant tumours, a proportion significantly higher than the 338% seen in normal tissues.32 At the same time, NAT1 mRNA expression was found to be significantly lower in tamoxifen-resistant tumours.32 These findings indicate that NAT1 methylation may contribute to tamoxifen resistance, although further studies are needed.
is a major cause of decreased TFAP2E expression in primary and metastatic colorectal-cancer and corresponding cell lines.38 Overexpression of the gene coding dickkopf homolog 4 protein (DKK4), a target of TFAP2E, has been implicated in fluorouracil resistance. More recently, it was shown that TFAP2E hypomethylation can lead to fluorouracil resistance by increasing DKK4 expression.38 Therefore, for colorectal-cancer patients with TFAP2E hypermethylation and resistant to fluorouracil therapy, DKK4-targeting drugs may potentially overcome this chemoresistance. Transcriptional silencing by CpG islands hypermethylation of tumour suppressor genes is one of the most important mechanisms in the tumorigenesis of colorectal cancer. Methylation status of tumour suppressor genes may also affect drug response. It is reported that with fluoropyrimidine-based adjuvant chemotherapy, the recurrence-free survival of patients with high CpG island methylator phenotype (CIMP) was significantly higher than that of those who were low.39 In colorectal-cancer patients treated with irinotecan and cetuximab, those with hypermethylated epidermal growth factor receptor (EGFR) promoter exhibit higher percentage of resistance, quicker disease progression and shorter median overall survival.40 Glucocorticoid-induced protein-coding gene (DEXI) is involved in camptothecin (CPT) resistance via inhibition of apoptosis.41 Silencing of DEXI by siRNA reduces CPT-induced apoptosis, whereas restoring of DEXI expression by 5-aza-20 -deoxycytidine enhances CPT sensitivity.41 DEXI is frequently methylated in colorectal (49% patients) and gastric cancer (31% patients), and colorectal-cancer patients harbouring DEXI methylation showed significantly poor response and worse outcome after 5-Fluorouracil +CPT-11 therapy.41 It is supposed that DEXI methylation is a potential biomarker for determining whether a patient will benefit from CPT-11-based chemotherapy. The breast cancer 1 and 2 (BRCA1 and BRCA2) genes are tumour suppressor genes encoding proteins responsible for DNA repair. Cells with inactivating mutations of the BRCA1 or BRCA2 show increased sensitivity to Poly-ADP ribose polymerase-inhibitors, while hypermethylation of BRCA1 promoter accounts for loss of BRCA1 expression, disruption of BRCA-associated DNA repair and increased sensitivity to BRCA-directed chemotherapies.42 The mitotic checkpoint with forkhead-associated and ring finger (CHFR) gene controls mitotic process and cell-cycle progression. Aberrant methylation of CHFR may serve as a diagnostic marker for micrometastasis in gastric cancer.43 Similar findings were also observed in endometrial cancer. Hypermethylation of CHFR is associated with reduced CHFR expression and increased taxanes sensitivity in SNG-II and HEC108 cells, whereas CHFR demethylation leads to taxanes resistant in these cells.44 The findings suggest that methylation status of CHFR may be a new molecular marker for personalized therapy in endometrial cancer. O6-methylguanine methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from the O6 position of guanine and thus compromises the cytotoxic effects of alkylating agents. Silencing of MGMT gene by promoter methylation is associated with longer survival in glioblastoma patients receiving alkylating agents.45 Hegi et al. has also demonstrated that MGMT methylation status is an independent predictor of drug response to temozolomide, a alkylating agent, in glioblastoma patients,46 only patients with methylated MGMT promoter benefited from temozolomide therapy. This was confirmed in a phase III clinical trial in patients ≥60 years of age.47
DNA methylation of genes encoding drug transporters Drug transporters are membrane proteins that are important in transmembrane transporting of drugs. Two broad groups of drug transporters have been identified in humans: the energy-dependent ATP-binding cassette (ABC) transporters and the solute carrier (SLC) transporters. Members of the ABC transporter family mainly act as efflux transporters, whereas the SLC family members act as uptake transporters. A large number of researches have focused on the relationship between ABC transporters and multidrug resistance (MDR) in cancer. MDR is a common reason for chemotherapeutic failure, cancer reoccurrence and cancerrelated death.33 Increase in the expression and/or activity of one or more of the ABC transporters can lead to MDR by decreasing the intracellular chemotherapeutic drug concentration in cancer cells. The expression of many drug transporters is regulated by DNA methylation. Treatment of human breast cancer cell MCF-7 with 5-azacitidine increased the expression of multidrug resistance protein (MDR1/ABCC1) significantly.34 Doxorubicin and irinotecan can also affect the expression of MDR1 in a MDR1 promoter methylation dependent manner: both drugs promote the transcription of MDR1 only when the MDR1 promoter is hypomethylated.35 SLC5A8 is a plasma membrane transporter that also acts as a tumour suppressor. Silencing of SLC5A8 in tumour cells is also related to methylation modification, which can be reversed by DNMT inhibitors. Therefore, it is postulated that DNMT inhibitors may alleviate, at least partially, the side-effects of the antitumor reagent dichloroacetate (DAC).36 Recently, Schaeffeler et al. investigated the role of DNA methylation in regulating the expression of SLC22A1/OCT1 in hepatocellular carcinoma.37 Downregulation of SLC22A1 expression due to SLC22A1 promoter hypermethylation was observed in hepatocellular carcinoma, and this may explain the low response rates and interindividual differences in response to cisplatin-based chemotherapy. DNA methylation of drug targets and signal pathway genes Drug sensitivity can be altered by DNA methylation-regulated expression of drug targets and the downstream signalling molecules. Transcription factor AP-2 epsilon (TFAP2E) is a key member of the AP-2 transcription factor family. Hypermethylation of TFAP2E
© 2014 John Wiley & Sons Ltd
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DNA methylation and personalized medicine
DNA methylation can also influence IGFBP3 expression by altering the binding of transcription factor. At baseline, IGFBP-3 levels were higher in SGA children with the -202 AA genotype than in those carrying the -202 C allele. Compared to children carrying the -202A/-185C haplotype, children with the -202C/ -185C haplotype showed lower IGFBP-3 levels. The methylation status of CpGs affecting transcription factor binding is higher in short young SGA adults than in controls. With growth hormone treatment, children with the -202A/-185C haplotype showed greater increase in both IGFBP3 level and growth in height than children with the -202C/-185T haplotype. These findings indicate that -202A/C and -185C/T polymorphisms can affect growth hormone response via altering the methylation of IGBP3 promoter.57 Moreover, the -667G/A and -185 C/T polymorphisms in IGFBP3 promoter can affect the transcriptional activity of the gene.58 5-Lipoxygenase (5-LOX) is an enzyme essential for arachidonic acid metabolism and leukotriene biosynthesis. It is also a target for anti-asthmatic drugs. The 5-LOX promoter contains 5 tandem Sp1/Egr-1 consensus binding sites. So far, 5-LOX is the only gene that has been found to contain 5 Sp1/Egr1 tandems in the human genome. The binding of Sp1/Egr1 to this Sp1/Egr1 recognition site can promote the transcription and expression of 5-LOX. Interestingly, repeat number variants in the Sp1/Egr1 recognition site with 3, 4 or 6 Sp1/Egr1 binding elements are found,59 and the frequency of the mutant alleles is about 35%. Clinical studies have shown that the 5-LOX genotype is associated with response to 5LOX inhibitors. Wild-type homozygotes and heterozygotes are responders, whereas mutant homozygotes are non-responders.60 The promoter activity was reduced by 25–30% due to decreased Sp1 or Egr1 binding to the 5-LOX mutant alleles.61 However, a recent study reported that the shorter repeat alleles can lead to higher 5-LOX expression by altering CpG island methylation.62 The C3435T polymorphism in MDR1 exon 26 is functional by altering MDR1 mRNA stability.63 The polymorphism can affect the absorption and distribution of drugs such as digoxin,64 phenytoin,65 tacrolimus,66 carbamazepine,67 doxorubicin and vincristine.68 Ulcerative colitis patients with the 3435C allele (especially those with T SNP (rs16906252) in colorectal cancer and normal colonic mucosa. Mod Pathol, 2009;22:1588–1599. Leng S, Bernauer AM, Hong C et al. The A/ G allele of rs16906252 predicts for MGMT methylation and is selectively silenced in premalignant lesions from smokers and in lung adenocarcinomas. Clin Cancer Res, 2011;17:2014–2023. McDonald KL, Rapkins RW, Olivier J et al. The T genotype of the MGMT C>T (rs16906252) enhancer single-nucleotide polymorphism (SNP) is associated with promoter methylation and longer survival in glioblastoma patients. Eur J Cancer, 2013;49:360–368. Candiloro IL, Dobrovic A. Detection of MGMT promoter methylation in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter single nucleotide polymorphism. Cancer Prev Res (Phila), 2009;2:862–867. Yuferov V, Ji F, Nielsen DA et al. A functional haplotype implicated in vulnerability to develop cocaine dependence is associated with reduced PDYN expression in human brain. Neuropsychopharmacology, 2009;34: 1185–1197. Taqi MM, Bazov I, Watanabe H et al. Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation
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58.
59.
60.
61. 62.
63.
64.
65.
66.
67.
68.
in the brain of human alcoholics. Addict Biol, 2011;16:499–509. van der Kaay DC, Hendriks AE, Ester WA et al. Genetic and epigenetic variability in the gene for IGFBP-3 (IGFBP3): correlation with serum IGFBP-3 levels and growth in short children born small for gestational age. Growth Horm IGF Res, 2009;19:198– 205. Faienza MF, Marzano F, Ventura AM et al. Regulation of IGFBP3 gene expression in short children born small for gestational age. Growth Horm IGF Res, 2011;21:349– 355. Alvarez V, Gonzalez P, Corao AI et al. The Sp1/Egr1-tandem repeat polymorphism in the 5-lipoxygenase gene promoter is not associated with late onset Alzheimer disease. Alzheimer Dis Assoc Disord, 2008;22: 177–180. Qu T, Manev R, Manev H. 5-Lipoxygenase (5-LOX) promoter polymorphism in patients with early-onset and late-onset Alzheimer’s disease. J Neuropsychiatry Clin Neurosci, Spring 2001;13:304–305. Fenech A, Hall IP. Pharmacogenetics of asthma. Br J Clin Pharmacol, 2002;53:3–15. Vikman S, Brena RM, Armstrong P, Hartiala J, Stephensen CB, Allayee H. Functional analysis of 5-lipoxygenase promoter repeat variants. Hum Mol Genet, 2009; 18:4521–4529. Wang D, Johnson AD, Papp AC, Kroetz DL, Sadee W. Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability. Pharmacogenet Genomics, 2005;15:693–704. Chowbay B, Li H, David M, Cheung YB, Lee EJ. Meta-analysis of the influence of MDR1 C3435T polymorphism on digoxin pharmacokinetics and MDR1 gene expression. Br J Clin Pharmacol, 2005;60:159–171. Ponnala S, Chaudhari JR, Jaleel MA et al. Role of MDR1 C3435T and GABRG2 C588T gene polymorphisms in seizure occurrence and MDR1 effect on anti-epileptic drug (phenytoin) absorption. Genet Test Mol Biomarkers, 2012;16:550–557. Li Y, Hu X, Cai B et al. Meta-analysis of the effect of MDR1 C3435 polymorphism on tacrolimus pharmacokinetics in renal transplant recipients. Transpl Immunol, 2012;27:12–18. Sterjev Z, Trencevska GK, Cvetkovska E et al. The association of C3435T singlenucleotide polymorphism, Pgp-glycoprotein gene expression levels and carbamazepine maintenance dose in patients with epilepsy. Neuropsychiatric Dis Treat, 2012;8:191–196. Efferth T, Sauerbrey A, Steinbach D et al. Analysis of single nucleotide polymorphism
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C3435T of the multidrug resistance gene MDR1 in acute lymphoblastic leukemia. Int J Oncol, 2003;23:509–517. 69. Tahara T, Shibata T, Nakamura M et al. Effect of MDR1 gene promoter methylation in patients with ulcerative colitis. Int J Mol Med, 2009;23:521–527.
70. McDonald KL, Aw G, Kleihues P. Role of Biomarkers in the Clinical Management of Glioblastomas: what are the Barriers and How Can We Overcome Them? Front Neurol, 2012;3:188. 71. MDxHealth. MDxHealth Signs Worldwide Development & Commercialization Agree-
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ment With Merck KGaA for MGMT Diagnostic Test. 5 2012 2012. 72. Toyota M, Suzuki H, Yamashita T et al. Cancer epigenomics: implications of DNA methylation in personalized cancer therapy. Cancer Sci, 2009;100:787–791.
Journal of Clinical Pharmacy and Therapeutics, 2014 7
doi: 10.1111/jcpt.12206
Review Article
DNA methylation and personalized medicine J. Tang*†a MD, Y. Xiong*†a MD, H.-H. Zhou*† MD PhD and X.-P. Chen*† MD PhD *Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, and †Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Hunan Key laboratory of Pharmacogenetics, Central South University, Changsha, China
Received 9 June 2013, Accepted 17 August 2014
Keywords: DNA methylation, drug response, single nucleotide polymorphisms
(7-mG). Clusters of cytosine–phosphate–guanine dinucleotides in a chromosomal region are referred to as CpG islands. In mammalian genomes, there are about 40 000 CpG islands, which are typically located in the promoters or in the first exon of the genes.1,2 CpG island methylation is a dynamic epigenetic process during mammalian development and is critical in regulating gene expression,3 maintaining the stability of the genome and its chromosomal structure,4 and inactivating genomic imprinting and X-chromosome dosage.5 Aberrant methylation of the CpG island may also result in human diseases such as cancer, amentia, autoimmunity and diabetes. Therefore, DNA methylation has become an important focus of recent epigenomics research. Understanding of the role of DNA methylation in pathogenesis has led to the development of several drugs targeting DNA methylation. Two DNMT inhibitors, 5-azacitidine6,7 and decitabine,8,9 are approved by the U.S. FDA for the treatment of myelodysplastic syndromes (MDS) and haematological malignancies. DNA methylation can also affect drug responses by altering the expression of genes encoding drug metabolism enzymes (DMEs),10 drug transporters11 and drug targets.12 Single nucleotide polymorphisms (SNPs) near DNA methylation sites can also function through affecting gene methylation and thus the expression of associated genes.13 Both these mechanisms can result in interindividual difference in drug response.
SUMMARY What is known and objective: Variation in the expression of drug-response-related genes contributes significantly to interindividual differences in drug response. DNA methylation is one of the most common epigenetic modifications that control gene expression. DNA methylation may occur in genes encoding drug metabolizing enzymes (DMEs), drug transporters and drug targets, and can thereby alter the pharmacokinetics and pharmacodynamics of drugs. In this review, we discuss recent advances in pharmacoepigenetics with a focus on DNA methylation. Methods: The literature search focusing on DNA methylation of drug-response-related genes and DNA methylation-related SNPs in pharmacogenomics was carried out using the PUBMED database and a combination of keywords including DNA methylation, drug response, DMEs, drug transporters, drug target and SNPs. Results and discussion: An extensive range of research has contributed to our understanding of how DNA methylation of drug-response-related genes alters their function. This is particularly well studied in cancer chemotherapy and drug resistance. The impact of polymorphisms of miRNAs in these processes requires further study. What is new and conclusion: DNA methylation-related genetic variation is an increasingly recognized mechanism for altered drug-response and disease susceptibility. These new discoveries require assimilation into the practice of personalized medicine.
METHODS A literature search was undertaken focusing on two main aspects – DNA methylation of drug-response-related genes and DNA methylation in relation to pharmacogenomics. The search interrogated the PUBMED database and the keywords included DNA methylation, drug response, DMEs, drug transporters, drug target and SNPs.
WHAT IS KNOWN AND OBJECTIVE Interest in the epigenetic modification of genomes has grown exponentially in recent years. Epigenetics plays an important role in regulating gene expression. DNA methylation, the biological methylation of specific nucleotides by DNA methyltransferases (DNMTs), is the epigenetic modification that has attracted most interest. DNA methylation can result in suppression of gene expression, a process that can be reactivated by demethylation. DNA methylation occurs mainly as 5-methylcytosine (5-mC), with a small fraction of N6-methylpurine (N6-mA) and 7-methylguanine
RESULTS AND DISCUSSION DNA methylation of drug-response-related genes and relevance to clinical pharmacology Variation in the expression of drug-response-related genes is an important contributor to interindividual difference in drug response. This variation is seen in the expression of DMEs, drug transporters, drug targets and downstream signalling molecules. DNA methylation has attracted increasing interest due to its universality and heritability in the regulation of gene expression (Table 1).
Correspondence: Professor X.-P. Chen, Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan, 410078, P. R. China. Tel.: 86-731-84805380; fax: 86731-82354476; e-mail: [email protected]. a These two authors contributed equally to this work.
© 2014 John Wiley & Sons Ltd
1
J. Tang et al.
DNA methylation and personalized medicine
Table 1. DNA methylation of drug-response-related gene
CYP450 family members
Phase II DMEs Drug transporters
Drug targets and signal pathway genes
DNA methylated regions
Affected drugs
Ref.
CYP1A1 promoter CYP1A2 promoter/enhancer
Polycyclic aromatic hydrocarbon Drug metabolism and synthesis of cholesterol, steroids and other lipids Oestrogen and 4-hydroxy estradiol Vitamin D Oxaliplatin and adriamycin Tamoxifen 5-azacitidine, Doxorubicin and irinotecan Dichloroacetate(DAC) Cisplatin Fluorouracil Camptothecin (CPT) Temozolomide
15,16 19,20
CYP1B1 promoter/enhancer CYP24A1 promoter GSTP1 promoter NAT1 MDR1/ABCC1 promoter SLC5A8 SLC22A1/OCT1 TFAP2E DEXI MGMT promoter
21,22 27 31 32 34,35 36 37 38 42 45
CYP, cytochrome P450; GSTP1, glutathione-S-transferase P1; NAT1, N-acetyl transferase 1; MDR1/ABCC1, multidrug resistance protein; ABC, ATP-binding cassette (ABC); SLC, soluble carrier (SLC); TFAP2E, transcription factor AP-2 epsilon; DEXI, dexamethasone-induced protein; MGM, O6-methylguanine-DNA methyltransferase.
promoter/enhancer is involved in the pathogenesis of prostate cancer.21 Habano et al. have also reported that CYP1B1 methylation in colorectal-cancer cell lines but not in corresponding normal tissue, and the methylation status can be decreased by treating with DNMT inhibitor 5-aza-20 -deoxycytidine.22 For the CYP2 family genes, CpG islands are observed in the promoters of CYP2A6, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP2R1, CYP2S1 and CYP2W1, indicating that these genes may also undergo regulation by DNA methylation.23 Demethylation of CYP2W1 CpG island appears to be associated with high CYP2W1 expression.24 CYP3A is the most important DME in adult human liver that contributes to metabolism of approximately 70% of clinically used drugs. DNMT inhibitors are shown to influence the expression of CYP3A4, CYP3A5 and CYP3A7 in HepG2 cells.23 Studies with adult and foetal human livers show highly variable CpG methylation sites in important CYP3A4 transcription factor-binding sites, and hypermethylation of CYP3A4 promoter may account for lack of CYP3A4 expression in foetal liver.25 Therefore, methylation status of CYP3A4 may contribute to interindividual differences in CYP3A4 activity in adult liver. As DNMT inhibitors are used in the clinic, and given the importance of CYP3A in drug metabolism, potential drug–drug interactions between DNMTs inhibitors and CYP3A substrates should be studied further. CYP24A1 is a key enzyme involved in regulating vitamin D metabolism. Methylation of CYP24A1 promoter is tissue specific. Novakovic et al. showed that CYP24A1 is methylated in human placenta, and this may be relevant to adverse effects of gestation.26 Significant variance in the methylation status of CYP24A1 promoter between tumour and normal endothelium cells is also observed, and this may lead to interindividual difference in cellular response to calcitriol, an active metabolite of vitamin D3.27 In addition, CYP24A1 methylation may play a role in the formation of tumour microenvironment in prostate cancer.28
Methylation of CYP450 family members The cytochrome P450 (CYP450) superfamily contributes to the phase I metabolism of about 75% clinically used drugs.14 Evidence has shown that DNA methylation plays important roles in regulating the expression of members of the CYP1, CYP2 and CYP3 subfamilies. CYP1A1, the major enzyme that metabolizes polycyclic aromatic hydrocarbons, can be induced by smoking. Therefore, the enzyme is potentially important in the aetiology of lung cancer.15 An epidemiological study has shown that in lung tissues of nonsmokers, light smokers and heavy smokers, the rate of complete or partial methylation for CYP1A1 promoter was 98%, 71% and 33%, respectively. This indicates hypomethylation of CYP1A1 promoter by cigarette smoking. CYP1A1 promoter methylation is increased within 1–7 days of quitting smoking.16 A recent study by Tekpli et al. indicated that the CYP1A1 enhancer was hypermethylated, whereas CYP1A1 mRNA level was reduced in lung cancer.17 Another study also shows that DNA methylation can modulate CYP1A1 expression in prostate tissue. In the prostate cell line LNCaP, CYP1A1 hypermethylation results in deficiency of CYP1A1 expression. For the non-cancerous cell lines RWPE-1, the environmental contaminant dioxin can upregulate CYP1A1 expression by decreasing its methylation.18 CYP1A2, another member of the CYP450 family, is involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. CYP1A2 activity is also regulated by smoking and some dietary components. Miyajima et al. observed that the CpG methylation status of CYP1A2 promoter was strongly associated with CYP1A2 mRNA expression in the liver.19 This report indicates that CpG methylation may account for the tissue-specific regulation of CYP1A2 gene expression. The extent of CpG island DNA methylation close to the CYP1A2 transcriptional start site correlates inversely with hepatic CYP1A2 mRNA levels.20 CYP1B1 is important in oestrogen metabolism and is involved in the pathogenesis of breast cancer and endometrial cancer by regulating the production of 4-hydroxy estradiol. A study by Tokizane et al. has shown that aberrant methylation in CYP1B1 promoter/enhancer can influence the binding of transcription factor AhR/ARNT and SP-1, and hypomethylation of CYP1B1
DNA methylation of phase II DMEs Glutathione-S-transferase P1 (GSTP1) is an important phase II DME that is involved in the metabolism of drugs such as
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oxaliplatin and adriamycin. GSTP1 promoter is generally methylated in most patients with prostate cancer, and the level of methylation is a marker useful for distinguishing benign prostatic hyperplasia from prostate cancer or to predict the prognosis of prostate cancer.29,30 DNMTs inhibitors 5-azacitidine and decitabine demethylate GSTP1 and promote GSTP1 re-expression.31 Therefore, GSTP1 promoter methylation status may serve as a potential biomarker to predict 5-azacitidine response. N-acetyl transferase 1 (NAT1) is a phase II DME that is important in metabolizing drugs and several other xenobiotics. In breast cancer tissues, NAT1 was observed to be methylated in 622% of the tamoxifen-resistant tumours, a proportion significantly higher than the 338% seen in normal tissues.32 At the same time, NAT1 mRNA expression was found to be significantly lower in tamoxifen-resistant tumours.32 These findings indicate that NAT1 methylation may contribute to tamoxifen resistance, although further studies are needed.
is a major cause of decreased TFAP2E expression in primary and metastatic colorectal-cancer and corresponding cell lines.38 Overexpression of the gene coding dickkopf homolog 4 protein (DKK4), a target of TFAP2E, has been implicated in fluorouracil resistance. More recently, it was shown that TFAP2E hypomethylation can lead to fluorouracil resistance by increasing DKK4 expression.38 Therefore, for colorectal-cancer patients with TFAP2E hypermethylation and resistant to fluorouracil therapy, DKK4-targeting drugs may potentially overcome this chemoresistance. Transcriptional silencing by CpG islands hypermethylation of tumour suppressor genes is one of the most important mechanisms in the tumorigenesis of colorectal cancer. Methylation status of tumour suppressor genes may also affect drug response. It is reported that with fluoropyrimidine-based adjuvant chemotherapy, the recurrence-free survival of patients with high CpG island methylator phenotype (CIMP) was significantly higher than that of those who were low.39 In colorectal-cancer patients treated with irinotecan and cetuximab, those with hypermethylated epidermal growth factor receptor (EGFR) promoter exhibit higher percentage of resistance, quicker disease progression and shorter median overall survival.40 Glucocorticoid-induced protein-coding gene (DEXI) is involved in camptothecin (CPT) resistance via inhibition of apoptosis.41 Silencing of DEXI by siRNA reduces CPT-induced apoptosis, whereas restoring of DEXI expression by 5-aza-20 -deoxycytidine enhances CPT sensitivity.41 DEXI is frequently methylated in colorectal (49% patients) and gastric cancer (31% patients), and colorectal-cancer patients harbouring DEXI methylation showed significantly poor response and worse outcome after 5-Fluorouracil +CPT-11 therapy.41 It is supposed that DEXI methylation is a potential biomarker for determining whether a patient will benefit from CPT-11-based chemotherapy. The breast cancer 1 and 2 (BRCA1 and BRCA2) genes are tumour suppressor genes encoding proteins responsible for DNA repair. Cells with inactivating mutations of the BRCA1 or BRCA2 show increased sensitivity to Poly-ADP ribose polymerase-inhibitors, while hypermethylation of BRCA1 promoter accounts for loss of BRCA1 expression, disruption of BRCA-associated DNA repair and increased sensitivity to BRCA-directed chemotherapies.42 The mitotic checkpoint with forkhead-associated and ring finger (CHFR) gene controls mitotic process and cell-cycle progression. Aberrant methylation of CHFR may serve as a diagnostic marker for micrometastasis in gastric cancer.43 Similar findings were also observed in endometrial cancer. Hypermethylation of CHFR is associated with reduced CHFR expression and increased taxanes sensitivity in SNG-II and HEC108 cells, whereas CHFR demethylation leads to taxanes resistant in these cells.44 The findings suggest that methylation status of CHFR may be a new molecular marker for personalized therapy in endometrial cancer. O6-methylguanine methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from the O6 position of guanine and thus compromises the cytotoxic effects of alkylating agents. Silencing of MGMT gene by promoter methylation is associated with longer survival in glioblastoma patients receiving alkylating agents.45 Hegi et al. has also demonstrated that MGMT methylation status is an independent predictor of drug response to temozolomide, a alkylating agent, in glioblastoma patients,46 only patients with methylated MGMT promoter benefited from temozolomide therapy. This was confirmed in a phase III clinical trial in patients ≥60 years of age.47
DNA methylation of genes encoding drug transporters Drug transporters are membrane proteins that are important in transmembrane transporting of drugs. Two broad groups of drug transporters have been identified in humans: the energy-dependent ATP-binding cassette (ABC) transporters and the solute carrier (SLC) transporters. Members of the ABC transporter family mainly act as efflux transporters, whereas the SLC family members act as uptake transporters. A large number of researches have focused on the relationship between ABC transporters and multidrug resistance (MDR) in cancer. MDR is a common reason for chemotherapeutic failure, cancer reoccurrence and cancerrelated death.33 Increase in the expression and/or activity of one or more of the ABC transporters can lead to MDR by decreasing the intracellular chemotherapeutic drug concentration in cancer cells. The expression of many drug transporters is regulated by DNA methylation. Treatment of human breast cancer cell MCF-7 with 5-azacitidine increased the expression of multidrug resistance protein (MDR1/ABCC1) significantly.34 Doxorubicin and irinotecan can also affect the expression of MDR1 in a MDR1 promoter methylation dependent manner: both drugs promote the transcription of MDR1 only when the MDR1 promoter is hypomethylated.35 SLC5A8 is a plasma membrane transporter that also acts as a tumour suppressor. Silencing of SLC5A8 in tumour cells is also related to methylation modification, which can be reversed by DNMT inhibitors. Therefore, it is postulated that DNMT inhibitors may alleviate, at least partially, the side-effects of the antitumor reagent dichloroacetate (DAC).36 Recently, Schaeffeler et al. investigated the role of DNA methylation in regulating the expression of SLC22A1/OCT1 in hepatocellular carcinoma.37 Downregulation of SLC22A1 expression due to SLC22A1 promoter hypermethylation was observed in hepatocellular carcinoma, and this may explain the low response rates and interindividual differences in response to cisplatin-based chemotherapy. DNA methylation of drug targets and signal pathway genes Drug sensitivity can be altered by DNA methylation-regulated expression of drug targets and the downstream signalling molecules. Transcription factor AP-2 epsilon (TFAP2E) is a key member of the AP-2 transcription factor family. Hypermethylation of TFAP2E
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DNA methylation can also influence IGFBP3 expression by altering the binding of transcription factor. At baseline, IGFBP-3 levels were higher in SGA children with the -202 AA genotype than in those carrying the -202 C allele. Compared to children carrying the -202A/-185C haplotype, children with the -202C/ -185C haplotype showed lower IGFBP-3 levels. The methylation status of CpGs affecting transcription factor binding is higher in short young SGA adults than in controls. With growth hormone treatment, children with the -202A/-185C haplotype showed greater increase in both IGFBP3 level and growth in height than children with the -202C/-185T haplotype. These findings indicate that -202A/C and -185C/T polymorphisms can affect growth hormone response via altering the methylation of IGBP3 promoter.57 Moreover, the -667G/A and -185 C/T polymorphisms in IGFBP3 promoter can affect the transcriptional activity of the gene.58 5-Lipoxygenase (5-LOX) is an enzyme essential for arachidonic acid metabolism and leukotriene biosynthesis. It is also a target for anti-asthmatic drugs. The 5-LOX promoter contains 5 tandem Sp1/Egr-1 consensus binding sites. So far, 5-LOX is the only gene that has been found to contain 5 Sp1/Egr1 tandems in the human genome. The binding of Sp1/Egr1 to this Sp1/Egr1 recognition site can promote the transcription and expression of 5-LOX. Interestingly, repeat number variants in the Sp1/Egr1 recognition site with 3, 4 or 6 Sp1/Egr1 binding elements are found,59 and the frequency of the mutant alleles is about 35%. Clinical studies have shown that the 5-LOX genotype is associated with response to 5LOX inhibitors. Wild-type homozygotes and heterozygotes are responders, whereas mutant homozygotes are non-responders.60 The promoter activity was reduced by 25–30% due to decreased Sp1 or Egr1 binding to the 5-LOX mutant alleles.61 However, a recent study reported that the shorter repeat alleles can lead to higher 5-LOX expression by altering CpG island methylation.62 The C3435T polymorphism in MDR1 exon 26 is functional by altering MDR1 mRNA stability.63 The polymorphism can affect the absorption and distribution of drugs such as digoxin,64 phenytoin,65 tacrolimus,66 carbamazepine,67 doxorubicin and vincristine.68 Ulcerative colitis patients with the 3435C allele (especially those with T SNP (rs16906252) in colorectal cancer and normal colonic mucosa. Mod Pathol, 2009;22:1588–1599. Leng S, Bernauer AM, Hong C et al. The A/ G allele of rs16906252 predicts for MGMT methylation and is selectively silenced in premalignant lesions from smokers and in lung adenocarcinomas. Clin Cancer Res, 2011;17:2014–2023. McDonald KL, Rapkins RW, Olivier J et al. The T genotype of the MGMT C>T (rs16906252) enhancer single-nucleotide polymorphism (SNP) is associated with promoter methylation and longer survival in glioblastoma patients. Eur J Cancer, 2013;49:360–368. Candiloro IL, Dobrovic A. Detection of MGMT promoter methylation in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter single nucleotide polymorphism. Cancer Prev Res (Phila), 2009;2:862–867. Yuferov V, Ji F, Nielsen DA et al. A functional haplotype implicated in vulnerability to develop cocaine dependence is associated with reduced PDYN expression in human brain. Neuropsychopharmacology, 2009;34: 1185–1197. Taqi MM, Bazov I, Watanabe H et al. Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation
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in the brain of human alcoholics. Addict Biol, 2011;16:499–509. van der Kaay DC, Hendriks AE, Ester WA et al. Genetic and epigenetic variability in the gene for IGFBP-3 (IGFBP3): correlation with serum IGFBP-3 levels and growth in short children born small for gestational age. Growth Horm IGF Res, 2009;19:198– 205. Faienza MF, Marzano F, Ventura AM et al. Regulation of IGFBP3 gene expression in short children born small for gestational age. Growth Horm IGF Res, 2011;21:349– 355. Alvarez V, Gonzalez P, Corao AI et al. The Sp1/Egr1-tandem repeat polymorphism in the 5-lipoxygenase gene promoter is not associated with late onset Alzheimer disease. Alzheimer Dis Assoc Disord, 2008;22: 177–180. Qu T, Manev R, Manev H. 5-Lipoxygenase (5-LOX) promoter polymorphism in patients with early-onset and late-onset Alzheimer’s disease. J Neuropsychiatry Clin Neurosci, Spring 2001;13:304–305. Fenech A, Hall IP. Pharmacogenetics of asthma. Br J Clin Pharmacol, 2002;53:3–15. Vikman S, Brena RM, Armstrong P, Hartiala J, Stephensen CB, Allayee H. Functional analysis of 5-lipoxygenase promoter repeat variants. Hum Mol Genet, 2009; 18:4521–4529. Wang D, Johnson AD, Papp AC, Kroetz DL, Sadee W. Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability. Pharmacogenet Genomics, 2005;15:693–704. Chowbay B, Li H, David M, Cheung YB, Lee EJ. Meta-analysis of the influence of MDR1 C3435T polymorphism on digoxin pharmacokinetics and MDR1 gene expression. Br J Clin Pharmacol, 2005;60:159–171. Ponnala S, Chaudhari JR, Jaleel MA et al. Role of MDR1 C3435T and GABRG2 C588T gene polymorphisms in seizure occurrence and MDR1 effect on anti-epileptic drug (phenytoin) absorption. Genet Test Mol Biomarkers, 2012;16:550–557. Li Y, Hu X, Cai B et al. Meta-analysis of the effect of MDR1 C3435 polymorphism on tacrolimus pharmacokinetics in renal transplant recipients. Transpl Immunol, 2012;27:12–18. Sterjev Z, Trencevska GK, Cvetkovska E et al. The association of C3435T singlenucleotide polymorphism, Pgp-glycoprotein gene expression levels and carbamazepine maintenance dose in patients with epilepsy. Neuropsychiatric Dis Treat, 2012;8:191–196. Efferth T, Sauerbrey A, Steinbach D et al. Analysis of single nucleotide polymorphism
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C3435T of the multidrug resistance gene MDR1 in acute lymphoblastic leukemia. Int J Oncol, 2003;23:509–517. 69. Tahara T, Shibata T, Nakamura M et al. Effect of MDR1 gene promoter methylation in patients with ulcerative colitis. Int J Mol Med, 2009;23:521–527.
70. McDonald KL, Aw G, Kleihues P. Role of Biomarkers in the Clinical Management of Glioblastomas: what are the Barriers and How Can We Overcome Them? Front Neurol, 2012;3:188. 71. MDxHealth. MDxHealth Signs Worldwide Development & Commercialization Agree-
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