Epigenetics as a mechanism driving polygenic clinical drug resistance

Aberrant methylation of CpG islands located at or near gene promoters is associated with inactivation of gene expression during tumour development. It is increasingly recognised that such epimutations may occur at a much higher frequency than gene mutation and therefore have a greater impact on selection of subpopulations of cells during tumour progression or acquisition of resistance to anticancer drugs. Although laboratory-based models of acquired resistance to anticancer agents tend to focus on specific genes or biochemical pathways, such 'one gene : one outcome' models may be an oversimplification of acquired resistance to treatment of cancer patients. Instead, clinical drug resistance may be due to changes in expression of a large number of genes that have a cumulative impact on chemosensitivity. Aberrant CpG island methylation of multiple genes occurring in a nonrandom manner during tumour development and during the acquisition of drug resistance provides a mechanism whereby expression of multiple genes could be affected simultaneously resulting in polygenic clinical drug resistance. If simultaneous epigenetic regulation of multiple genes is indeed a major driving force behind acquired resistance of patients' tumour to anticancer agents, this has important implications for biomarker studies of clinical outcome following chemotherapy and for clinical approaches designed to circumvent or modulate drug resistance

Combined inhibition of DNA methylation and histone acetylation enhances gene re-expression and drug sensitivity in vivo

Histone deacetylation and DNA methylation have a central role in the control of gene expression in tumours, including transcriptional repression of tumour suppressor genes and genes involved in sensitivity to chemotherapy. Treatment of cisplatin-resistant cell lines with an inhibitor of DNA methyltransferases, 2-deoxy-5′azacytidine (decitabine), results in partial reversal of DNA methylation, re-expression of epigenetically silenced genes including hMLH1 and sensitisation to cisplatin both in vitro and in vivo. We have investigated whether the combination of decitabine and a clinically relevant inhibitor of histone deacetylase activity (belinostat, PXD101) can further increase the re-expression of genes epigenetically silenced by DNA methylation and enhance chemo-sensitisation in vivo at well-tolerated doses. The cisplatin-resistant human ovarian cell line A2780/cp70 has the hMLH1 gene methylated and is resistant to cisplatin both in vitro and when grown as a xenograft in mice. Treatment of A2780/cp70 with decitabine and belinostat results in a marked increase in expression of epigenetically silenced MLH1 and MAGE-A1 both in vitro and in vivo when compared with decitabine alone. The combination greatly enhanced the effects of decitabine alone on the cisplatin sensitivity of xenografts. As the dose of decitabine that can be given to patients and hence the maximum pharmacodynamic effect as a demethylating agent is limited by toxicity and eventual re-methylation of genes, we suggest that the combination of decitabine and belinostat could have a role in the efficacy of chemotherapy in tumours that have acquired drug resistance due to DNA methylation and gene silencing

Comprehensive Biostatistical Analysis of CpG Island Methylator Phenotype in Colorectal Cancer Using a Large Population-Based Sample

The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. METHOLODOLOGY/PRINCIPAL FINDINGS: DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and beta-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors

Epigenomic analysis of aberrantly methylated genes in colorectal cancer identifies genes commonly affected by epigenetic alterations.

Methylation profiling based on bead-chip arrays is an effective method for screening aberrantly methylated genes in CRC. In addition, we identified novel methylated genes that are candidate diagnostic or prognostic markers for CRC

Down-regulation of SFRP1 as a putative tumor suppressor gene can contribute to human hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. SFRP1 (the secreted frizzled-related protein 1), a putative tumor suppressor gene mapped onto chromosome 8p12-p11.1, the frequent loss of heterozygosity (LOH) region in human HCC, encodes a Wingless-type (Wnt) signaling antagonist and is frequently inactivated by promoter methylation in many human cancers. However, whether the down-regulation of SFRP1 can contribute to hepatocarcinogenesis still remains unclear.</p> <p>Methods</p> <p>We investigated the expression of SFRP1 through real time RT-PCR and immunohistochemistry staining. The cell growth and colony formation were observed as the overexpression and knockdown of SFRP1. The DNA methylation status within SFRP1 promoter was analyzed through methylation-specific PCR or bisulphate-treated DNA sequencing assays. Loss of heterozygosity was here detected with microsatellite markers.</p> <p>Results</p> <p>SFRP1 was significantly down-regulated in 76.1% (35/46) HCC specimens at mRNA level and in 30% (30/100) HCCs indicated by immunohistochemistry staining, as compared to adjacent non-cancerous livers. The overexpression of SFRP1 can significantly inhibit the cell growth and colony formation of YY-8103, SMMC7721, and Hep3B cells. The RNA interference against the constitutional SFRP1 in the offspring SMMC7721 cells, which were stably transfected by ectopic SFRP1, can markedly promote cell growth of these cells. LOH of both microsatellite markers D8S532 and D8SAC016868 flanking the gene locus was found in 13% (6 of 46 HCCs) and 6.5% (3 of 46 HCCs) of the informative cases, respectively, where 5 of 8 HCC specimens with LOH showed the down-regulation of SFRP1. DNA hypermethylation within SFRP1 promoter was identified in two of three HCC specimens without SFRP1 expression. Moreover, the DNA methylation of SFRP1 promoter was significantly reduced, along with the re-expression of the gene, in those HCC cell lines, Bel7404, QGY7701, and MHCC-H, as treated by DAC.</p> <p>Conclusion</p> <p>Our data suggested that the down-regulation of SFRP1 as a candidate tumor suppressor gene, triggered by the epigenetic and/or genetic events, could contribute to the oncogenesis of HCC.</p

Antiproliferative Effects of DNA Methyltransferase 3B Depletion Are Not Associated with DNA Demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs

CpG-island methylation study of liver fluke-related cholangiocarcinoma

Background: Genetic changes have been widely reported in association with cholangiocarcinoma (CCA), while epigenetic changes are poorly characterised. We aimed to further evaluate CpG-island hypermethylation in CCA at candidate loci, which may have potential as diagnostic or prognostic biomarkers. Methods: We analysed methylation of 26 CpG-islands in 102 liver fluke related-CCA and 29 adjacent normal samples using methylation-specific PCR (MSP). Methylation of interest loci was confirmed using pyrosequencing and/or combined bisulfite restriction analysis, and protein expression by immunohistochemistry. Results: A number of CpG-islands (OPCML, SFRP1, HIC1, PTEN and DcR1) showed frequency of hypermethylation in >28% of CCA, but not adjacent normal tissues. The results showed that 91% of CCA were methylated in at least one CpG-island. The OPCML was the most frequently methylated locus (72.5%) and was more frequently methylated in less differentiated CCA. Patients with methylated DcR1 had significantly longer overall survival (Median; 41.7 vs 21.7 weeks, P=0.027). Low-protein expression was found in >70% of CCA with methylation of OPCML or DcR1. Conclusion: Aberrant hypermethylation of certain loci is a common event in liver fluke-related CCA and may potentially contribute to cholangiocarcinogenesis. The OPCML and DcR1 might serve as methylation biomarkers in CCA that can be readily examined by MSP

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours’ biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription–quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes

Expression of methylation-related genes is associated with overall survival in patients with non-small cell lung cancer

The abnormality of DNA methylation is involved in tumour progression, and thus has a modulating effect on clinical outcome of cancer patients. In this study, we measured the mRNA expression levels of three methylation-regulating genes (DNMT1, DNMT3b, and MBD2) in 148 tumour samples from patients with non-small cell lung cancer (NSCLC) using quantitative real-time polymerase chain reaction and then determined their prognostic values. Our data showed that the high level of DNMT1 expression was significantly associated with an increased risk of death in all NSCLC patients (hazard ratio (HR), 1.74; 95% confidence interval (95% CI), 1.04–2.90). However, the high level of DNMT3b expression was significantly associated with poor prognosis only in young patients (<65 years). The high level of MBD2 expression had a significantly reduced risk for death only in male patients and in squamous cell lung carcinoma (SQLC) patients. All three combination groups with DNMT1 and DNMT3b, DNMT1 and MBD2 or DNMT3b and MBD2 revealed significant combined effects in male patients and SQLC patients. Our results suggest that DNMT1, DNMT3b, and MBD2 may play important roles in modulating NSCLC patient survival and thus be useful for identifying NSCLC patients who would benefit most from aggressive therapy

Clinicopathological Significance and Prognostic Value of DNA Methyltransferase 1, 3a, and 3b Expressions in Sporadic Epithelial Ovarian Cancer

Altered DNA methylation of tumor suppressor gene promoters plays a role in human carcinogenesis and DNA methyltransferases (DNMTs) are responsible for it. This study aimed to determine aberrant expression of DNMT1, DNMT3a, and DNMT3b in benign and malignant ovarian tumor tissues for their association with clinicopathological significance and prognostic value. A total of 142 ovarian cancers and 44 benign ovarian tumors were recruited for immunohistochemical analysis of their expression. The data showed that expression of DNMT1, DNMT3a, and DNMT3b was observed in 76 (53.5%), 92 (64.8%) and 79 (55.6%) of 142 cases of ovarian cancer tissues, respectively. Of the serious tumors, DNMT3a protein expression was significantly higher than that in benign tumor samples (P = 0.001); DNMT3b was marginally significant down regulated in ovarian cancers compared to that of the benign tumors (P = 0.054); DNMT1 expression has no statistical difference between ovarian cancers and benign tumor tissues (P = 0.837). Of the mucious tumors, the expression of DNMT3a, DNMT3b, and DNMT1 was not different between malignant and benign tumors. Moreover, DNMT1 expression was associated with DNMT3b expression (P = 0.020, r = 0.195). DNMT1 expression was associated with age of the patients, menopause status, and tumor localization, while DNMT3a expression was associated with histological types and serum CA125 levels and DNMT3b expression was associated with lymph node metastasis. In addition, patients with DNMT1 or DNMT3b expression had a trend of better survival than those with negative expression. Co-expression of DNMT1 and DNMT3b was significantly associated with better overall survival (P = 0.014). The data from this study provided the first evidence for differential expression of DNMTs proteins in ovarian cancer tissues and their associations with clinicopathological and survival data in sporadic ovarian cancer patients