Transient low doses of DNA-demethylating agents exert durable antitumor effects on hematological and epithelial tumor cells

Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses, without causing immediate cytotoxicity, produce an antitumor "memory" response, including inhibition of subpopulations of cancer stem-like cells. These effects are accompanied by sustained decreases in genomewide promoter DNA methylation, gene reexpression, and antitumor changes in key cellular regulatory pathways. Low-dose decitabine and azacitidine may have broad applicability for cancer management.     

GSTPl基因启动子甲基化与前列腺癌的相关性研究

背景与目的：谷胱甘S-转移酶P1(glutathione S-transferase P1,GSTP1)保护细胞避免DNA损伤和癌细胞形成,抑制GSTP1活性可以导致DNA损伤的敏感性增强和癌变发生的概率增加。该研究旨在研究前列腺癌组织中GSTPl基因甲基化与前列腺癌临床病理特征的关系。方法：收集2015年4月—2016年12月在海南省中医院和海口市人民医院住院的46例前列腺癌患者的前列腺癌组织及对应的包埋癌旁组织,应用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测GSTPl mRNA水平,通过甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MSP)检测GSTP1基因启动子区域CpG岛的甲基化表达水平,然后与性别、年龄及肿瘤TNM分期等临床数据进行关联分析。结果：与癌旁组织比较,前列腺癌组织中GSTP1 mRNA表达水平降低(P<0.01),并且GSTP1 mRNA降低与GSTPl基因甲基化呈负相关(P<0.05);前列腺癌和癌旁组织中甲基化阳性率分别为66.0%和23.5%,差异有统计学意义(P<0.05);GSTP1基因的启动子甲基化频率与肿瘤分期显著相关(r＝073,P<0.05),而与其他临床特征无明显相关性(P>0.05)。结论：GSTP1基因启动子甲基化可能造成GSTP1基因低表达,与前列腺癌发病明显相关,有望成为检测及诊断前列腺癌的新方法。     

Innovative approaches to the clinical development of DNA methylation inhibitors as epigenetic remodeling drugs

The most extensively studied inhibitors of DNA methylation are the cytidine analogs 5-azacytidine (5-aza-CR; azacitidine) and 5-aza-2'- deoxycytidine (5-aza-CdR; decitabine). Despite decades of nonclinical and clinical research, there remains considerable interest in finding innovative and better ways to use these DNA methyltransferase (DNMT) inhibitors. A mounting body of data supports the role of methylation in silencing genes involved in tumor growth and resistance. This information has fueled further nonclinical and clinical research on ways to use inhibitors of methylation to restore normal gene expression and function. As such, recent clinical strategies have shifted from simply evaluating cytotoxic effects to exploring and optimizing the ability of these agents to restore or reactivate gene expression and putative targets. This article considers innovative approaches to develop and evaluate inhibitors of DNA methylation as epigenetic remodeling agents for the treatment of cancer. These include optimization of dose and schedule, restoration or enhancement of sensitivity to other treatment modalities, and combinations with other agents including histone deacetylase inhibitors.     

Molecular detection of prostate cancer in urine by GSTP1 hypermethylation.

Novel approaches for the early detection and management of prostate cancer are urgently needed. Clonal genetic alterations have been used as targets for the detection of neoplastic cells in bodily fluids from many cancer types. A similar strategy for molecular diagnosis of prostate cancer requires a common and/or early genetic alteration as a specific target for neoplastic prostate cells. Hypermethylation of regulatory sequences at the glutathione S-transferase pi (GSTP1) gene locus is found in the majority (>90%) of primary prostate carcinomas, but not in normal prostatic tissue or other normal tissues. We hypothesized that urine from prostate cancer patients might contain shed neoplastic cells or debris amenable to DNA analysis. Matched specimens of primary tumor, peripheral blood lymphocytes (normal control), and simple voided urine were collected from 28 patients with prostate cancer of a clinical stage amenable to cure. Genomic DNA was isolated from the samples, and the methylation status of GSTP1 was examined in a blinded manner using methylation-specific PCR. Decoding of the results revealed that 22 of 28 (79%) prostate tumors were positive for GSTP1 methylation. In 6 of 22 (27%) cases, the corresponding urine-sediment DNA was positive for GSTP1 methylation, indicating the presence of neoplastic DNA in the urine. Furthermore, there was no case where urine-sediment DNA harbored methylation when the corresponding tumor was negative. Although we only detected GSTP1 methylation in under one-third of voided urine samples, we have demonstrated that molecular diagnosis of prostate neoplasia in urine is feasible. Larger studies focusing on carcinoma size, location in the prostate, and urine collection techniques, as well as more sensitive technology, may lead to the useful application of GSTP1 hypermethylation in prostate cancer diagnosis and management.     

Epigenetics in cancer: what's the future?

Epigenetics is a rapidly expanding field that focuses on stable changes in gene expression that are not accompanied by changes in DNA sequence and that are mediated primarily by DNA methylation and histone modifications. Disruption of the epigenome is a fundamental mechanism in cancer, and several epigenetic drugs that have proved to prolong survival and to be less toxic than conventional chemotherapy were recently approved by the FDA for cancer treatment. These include azacitidine (Vidaza), decitabine (Dacogen), vorinostat (Zolinza), and romidepsin (Istodax). Promising results of combination clinical trials with DNA methylation inhibitors and histone deacetylase inhibitors have recently been reported, and data are emerging that describe molecular determinants of clinical responses. Despite significant advances, challenges remain, including a lack of predictive markers, unclear mechanisms of response and resistance, and rare responses in solid tumors. Preclinical studies are ongoing with novel classes of agents that target various components of the epigenetic machinery. In this review, we focus on recent clinical and translational data in the epigenetics field that have potential in cancer therapy.     

Safety, efficacy and biological predictors of response to sequential azacitidine and lenalidomide for elderly patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is a disease of the elderly. Poor outcomes with standard therapies necessitate novel approaches. Outpatient regimens sufficiently potent and well tolerated to induce remissions and enable continuation therapy may be beneficial. In this phase-1 study, we determined the maximum tolerated dose (MTD) and the efficacy for sequential azacitidine and lenalidomide as remission induction and continuation therapy in elderly, previously untreated patients. We investigated the impact on global DNA methylation and bone marrow cytokines, and sought biological predictors of response. Eighteen patients were enrolled. The MTD was not reached. Median follow-up was 8.2 months (10.3 months for survivors). Common adverse events included fatigue, injection site reactions, constipation, nausea, pruritus and febrile neutropenia. Ten patients responded (56%), and the rate of complete remissions (CRs) or CRs with incomplete recovery of blood counts for evaluable patients was 44% (7/16). The median response duration was 6.2 months. DNA demethylation and changes in bone marrow cytokines were observed; responders had a unique cytokine profile and a trend towards lower methylation levels. Sequential azacitidine and lenalidomide was well tolerated with encouraging clinical and biological activity in previously untreated elderly AML patients. This trial is registered at ClinicalTrials.gov (NCT00890929).     

Methylation in benign prostate and risk of disease progression in men subsequently diagnosed with prostate cancer

In DNA from prostate tumors, methylation patterns in gene promoter regions can be a biomarker for disease progression. It remains unclear whether methylation patterns in benign prostate tissue—prior to malignant transformation—may provide similar prognostic information. To determine whether early methylation events predict prostate cancer outcomes, we evaluated histologically benign prostate specimens from 353 men who eventually developed prostate cancer and received “definitive” treatment [radical prostatectomy (58%) or radiation therapy (42%)]. Cases were drawn from a large hospital‐based cohort of men with benign prostate biopsy specimens collected between 1990 and 2002. Risk of disease progression associated with methylation was estimated using time‐to‐event analyses. Average follow‐up was over 5 years; biochemical recurrence (BCR) occurred in 91 cases (26%). In White men, methylation of the APC gene was associated with increased risk of BCR, even after adjusting for standard clinical risk factors for prostate cancer progression (adjusted hazard ratio (aHR) = 2.26; 95%CI 1.23–4.16). APC methylation was most strongly associated with a significant increased risk of BCR in White men with low prostate specific antigen at cohort entry (HR = 3.66; 95%CI 1.51–8.85). In additional stratified analyses, we found that methylation of the RARB gene significantly increased risk of BCR in African American cases who demonstrated methylation of at least one of the other four genes under study (HR = 3.80; 95%CI 1.07–13.53). These findings may have implications in the early identification of aggressive prostate cancer as well as reducing unnecessary medical procedures and emotional distress for men who present with markers of indolent disease.     

DNA异常甲基化在前列腺癌发生发展及诊疗中的研究进展

前列腺癌是全球男性常发的恶性肿瘤之一。近年来研究发现前列腺癌的发生发展很大程度上是由表观遗传修饰所驱动的,其中最重要的当属DNA异常甲基化。DNA启动子的异常甲基化会造成基因的异常表达,从而调控着前列腺癌的发生发展过程。此外,目前前列腺癌的诊断仍然依靠侵入性的前列腺穿刺活检,而基于DNA异常甲基化的无创性液体活检有望成为未来前列腺癌分子诊断的发展趋势,同时也为前列腺癌提供了新的治疗靶标。本文就DNA甲基化在前列腺癌发生发展、早期诊断、预后评估以及治疗中的研究进展进行综述。     

Epigenomic Alterations in Localized and Advanced Prostate Cancer

Although prostate cancer (PCa) is the second leading cause of cancer death among men worldwide, not all men diagnosed with PCa will die from the disease. A critical challenge, therefore, is to distinguish indolent PCa from more advanced forms to guide appropriate treatment decisions. We used Enhanced Reduced Representation Bisulfite Sequencing, a genome-wide high-coverage single-base resolution DNA methylation method to profile seven localized PCa samples, seven matched benign prostate tissues, and six aggressive castration-resistant prostate cancer (CRPC) samples. We integrated these data with RNA-seq and whole-genome DNA-seq data to comprehensively characterize the PCa methylome, detect changes associated with disease progression, and identify novel candidate prognostic biomarkers. Our analyses revealed the correlation of cytosine guanine dinucleotide island (CGI)-specific hypermethylation with disease severity and association of certain breakpoints (deletion, tandem duplications, and interchromosomal translocations) with DNA methylation. Furthermore, integrative analysis of methylation and single-nucleotide polymorphisms (SNPs) uncovered widespread allele-specific methylation (ASM) for the first time in PCa. We found that most DNA methylation changes occurred in the context of ASM, suggesting that variations in tumor epigenetic landscape of individuals are partly mediated by genetic differences, which may affect PCa disease progression. We further selected a panel of 13 CGIs demonstrating increased DNA methylation with disease progression and validated this panel in an independent cohort of 20 benign prostate tissues, 16 PCa, and 8 aggressive CRPCs. These results warrant clinical evaluation in larger cohorts to help distinguish indolent PCa from advanced disease.     

Hypomethylating agents for urologic cancers

Silencing of tumor suppressor genes by promoter-region methylation as an epigenetic mechanism of gene regulation is increasingly recognized as beneficial in cancer. Initially developed as cytotoxic high-dose therapies, azacitidine and decitabine are now being reinvestigated in lower-dose cancer treatment regimens with a different paradigm - hypomethylation. Recent evidence for benefit in myelodysplastic syndromes and acute myeloid leukemias has renewed interest in hypomethylation as a therapeutic option in epithelial cancers. In this article, we describe the mechanistic aspects of DNA methylation, which alters gene expression, and review the evidence for hypomethylation as a therapeutic option in urologic cancers. Potential correlative studies that may assist in developing tailored therapy with hypomethylating agents are reviewed. Given that the population with urologic cancers is typically elderly with multiple comorbidities, the excellent tolerability of lower-dose hypomethylating agents provides a high therapeutic index and rational development is warranted, bearing in mind that the cytostatic and delayed activity present challenges in the choice of appropriate trial end points.     

前列腺癌的p16基因甲基化程度定量分析

目的 建立和评价基因组DNA甲基化的定量分析方法,并比较正常前列腺组织和前列腺癌组织中的p16抑癌基因的DNA甲基化差异.方法 提取正常前列腺组织和前列腺癌组织中的基因组DNA,用亚硫酸氢钠将未甲基化的胞嘧啶转化为尿嘧啶,经过PCR扩增后,用BstUⅠ和HpaⅡ限制性内切酶消化,通过电泳分离消化的片段,定量分析基因组中...     

DNA methylation pathway alterations in an autochthonous murine model of prostate cancer

We examined the DNA methylation pathway in an autochthonous murine prostate cancer model, transgenic adenocarcinoma of mouse prostate (TRAMP). We observed that, compared with strain-matched normal prostates, primary and metastatic TRAMP tumors display increased cytosine DNA methyltransferase (Dnmt) activity, Dnmt1 and Dnmt3b protein expression, and Dnmt1, Dnmt3a, and Dnmt3b mRNA expression. Increased expression of Dnmt genes correlates with increased expression of cyclin A and E2F target genes, implicating increased cell proliferation and Rb inactivation in Dnmt overexpression. We analyzed DNA methylation in TRAMP and found that global levels of 5-methyl-2'-deoxycytidine are unaltered, whereas specific tumors display centromeric repeat hypomethylation. To interrogate locus-specific methylation, we did restriction landmark genomic scanning (RLGS) on normal prostates and primary tumors. In primary tumors, 2.3% of approximately 1,200 analyzed loci display aberrant DNA hypermethylation, whereas a considerably smaller number of events show hypomethylation. The pattern of RLGS changes was nonrandom, indicating a coordinated methylation defect. Two specific genes identified by RLGS were studied in detail. Surprisingly, methylation of a downstream exon of p16(INK4a) (p16) was the highest frequency hypermethylation event identified in TRAMP, where it is associated with increased p16 mRNA and protein expression. In contrast, hypermethylation of the 5' CpG island region of the homeobox gene Irx3 in TRAMP is associated with reduced gene expression. In summary, our data reveal a systemic DNA methylation pathway defect in TRAMP reminiscent of human prostate cancer, supporting the use of this model to investigate the functional role of DNA methylation pathway alterations in prostate cancer development.     

Polymorphisms in the DNA methyltransferase 3b gene and prostate cancer risk

Inactivation of tumor suppressor genes by promoter methylation is an important mechanism of tumorigenesis. Increased expression of DNA methyltransferases has been commonly observed in cancer. A C/T polymorphism in the DNA methyltransferase 3b (DNMT3b) promoter region results in increased activity and has recently been identified as a risk factor for lung cancer. In this study, we examined the C/T polymorphism of the DNMT3b gene in specimens from 81 patients with prostate cancer and 42 controls selected from patients with benign prostatic hypertrophy (BPH). Genomic DNA was isolated from archived formaldehyde-fixed and paraffin-embedded tissue blocks. DNMT3b genotypes were determined by restriction-fragment-length-polymorphism polymerase chain reaction. The DNMT3b polymorphism frequencies in the prostate cancer and BPH specimens were, respectively, 20 and 26% for CC, 42 and 52% for CT, and 38 and 21% for TT. Although such differences fall within the realm of chance variation (P>0.05), the data suggest that the TT genotype may be associated with an increased risk of prostate cancer: the age-adjusted odds ratio (aOR) was 2.6 [95% confidence interval: 0.8-8.0]; the increase in odds ratio was seen in both blacks and whites (aOR=4.3 in blacks, and 2.0 in whites). The samples used in this study have previously been examined for methylation index (MI) based on the number of genes methylated, the range being 0 to 5. A trend toward an increase in MI was detected for the DNMT3b polymorphisms in prostate cancer patients but not for BPH subjects (mean MI 2.6, 2.9, 3.1 for CC, CT, and TT genotype in prostate cancer; 0.8, 0.8, 0.7 for CC, CT, and TT genotype in BPH subjects). These findings suggest that the DNMT3b polymorphisms may be associated with an increase in promoter methylation of tumor-suppressor genes related to the development of prostate cancer, and may thereby increase the risk of this disease.     

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.     

Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer

Epigenetic alterations are strongly associated with the development of cancer. We conducted a phase I/II trial of combined epigenetic therapy with azacitidine and entinostat, inhibitors of DNA methylation and histone deacetylation, respectively, in extensively pretreated patients with recurrent metastatic non-small cell lung cancer. This therapy is well tolerated, and objective responses were observed, including a complete response and a partial response in a patient who remains alive and without disease progression approximately 2 years after completing protocol therapy. Median survival in the entire cohort was 6.4 months (95% CI 3.8-9.2), comparing favorably with existing therapeutic options. Demethylation of a set of 4 epigenetically silenced genes known to be associated with lung cancer was detectable in serial blood samples in these patients and was associated with improved progression-free (P = 0.034) and overall survival (P = 0.035). Four of 19 patients had major objective responses to subsequent anticancer therapies given immediately after epigenetic therapy. Significance: This study demonstrates that combined epigenetic therapy with low-dose azacitidine and entinostat results in objective, durable responses in patients with solid tumors and defines a blood-based biomarker that correlates with clinical benefit.