Epigenetic Alterations In Human Cancers Research Articles

Genetic and epigenetic alterations in human cancers

Genetic and epigenetic alterations in human cancers

Increased Local Disorder of DNA Methylation Forms the Basis of High Intra-Leukemic Epigenetic Heterogeneity and Enhances CLL Evolution

We recently demonstrated that genetic heterogeneity contributes to evolution and clinical outcome in chronic lymphocytic leukemia (CLL) (Landau et al., Cell 2013). As both genetic and epigenetic alterations contribute to the cellular phenotype and its fitness for selection, we hypothesized that heterogeneity in DNA methylation impacts leukemic evolution as well.We analyzed existing CLL methylation data (Kulis et al., Nat Gen 2012), and found higher heterogeneity in 450K arrays of 139 CLLs compared to 23 normal B cell samples, with a higher proportion of sites having intermediate levels of methylation (13.7% vs 10.7%, P<0.0001). Next, we re-analyzed whole genome bisulfite sequencing data from 2 CLL and 3 normal B cell samples to distinguish between 2 possible explanations for heterogeneity: 1) a mixture of cell subpopulations with ordered but differing methylation states, and 2) increased proportion of cells with ‘noisy' or disordered methylation states. We found that the bulk of heterogeneity resulted from the latter, as evidenced by a high proportion in CLL of individual sequencing reads with a discordant methylation state in neighboring CpGs.To validate this observation, we applied reduced representation bisulfite sequencing (RRBS) to generate genome-scale information of methylation at critical sites (e.g., CG islands) of 108 genetically-characterized CLLs. As predicted, we found higher proportions of discordant reads in CLL than either samples from 30 normal B cells or from 35 diverse healthy human tissues (average of 24.7% vs. 17.6% or 16.3%, respectively, P<0.00001). Similarly high proportions of discordant reads were also found in 31 cancer cell lines. Samples with somatic mutations in methylation modulators (e.g., DNMT3A) had increased proportion of discordant reads (P<0.001). Importantly, the presence of methylation ‘noise' was associated with reduced correlation between promoter methylation and gene silencing (P<0.0001), and with substantial allele specific expression, both measured by RNA-seq of 48 CLLs. These findings suggest that much of DNA methylation heterogeneity in CLL stems from locally disordered methylation, and shed light on how epigenetic alterations in human cancer arise in part from epigenetic drift.Increased epigenetic ‘noise' is expected to result in a more plastic evolutionary landscape that facilitates the emergence of fitness-enhancing genetic and epigenetic alterations. Indeed, the increase in discordant reads in CLL compared to normal samples was most prominent in genomic regions important to transcription (promoters, CG islands [P <1*e-14] vs. introns, CGI shelves [P <1*e-2]). In addition, significantly higher levels of discordant reads were found in promoters of genes silenced in CLL, in differentially methylated regions and in genes important to pluripotency potential (e.g., stem cell modules, Q<1*e-14). Finally, samples with increased disordered methylation had higher numbers of subclonal, but not clonal mutations (P<0.001 vs. P=0.26, respectively).To observe the relationship between genetic and epigenetic evolution, we used RRBS to longitudinally study 14 CLLs with characterized patterns of genetic evolution (median time between samples 3.5 yrs; 6 unevolved, 8 evolved). While unevolved CLLs revealed largely unchanged epigenetic landscapes, evolved CLLs demonstrated decreased methylation over time in promoters enriched for stem cell and MYC targets gene sets (N=347, Q<0.0001), and increased methylation in TP53 targets (N=336, Q<0.00001). Moreover, in evolved CLLs, specific promoters with disordered methylation across all samples (e.g., TERT, WT1), revealed changes over time in methylation proportions corresponding to increases in subclone size inferred from the genetic analysis.Increased heterogeneity was found to be associated with shorter retreatment-free survival in a cohort of 46 patients treated after sampling (P=0.03, HR of 3.13), in a regression model including established risk indicators (IGHV unmutated status, del(17p), del(11q) and treatment prior to sample).Our data suggest that heterogeneity in CLL DNA methylation results from stochastic drift that subsequently undergoes positive selection. Thus, we propose that locally disordered DNA methylation may have a similar role to genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories. Disclosures:No relevant conflicts of interest to declare.

Mesures peropératoires naviguées de la laxité du genou sain comparé au genou lésé controlatéral, avant et après reconstruction du LCA

Epigenetic information is characterized by its plasticity during development and differentiation as well as its stable transmission during mitotic cell divisions in somatic tissues. This duality contrasts to genetic information, which is essentially static and identical in every cell in an organism with only a few exceptions such as immunoglobulin genes in lymphocytes. Epigenetics is traditionally perceived as a means to regulate gene expression without a change in DNA sequence. This, however, does not exclude a potential role for genetic variations in providing differential backgrounds on which epigenetic modulations and their regulatory consequences are achieved. An effective approach to investigating the interplay between genetic variations and epigenetic variations is through allele-specific analysis of epigenetics and gene expression. Such studies have generated many new insights into functions of genetic variations, mechanisms of gene expression regulation, and the role of mutations and epigenetic alterations in human cancer. This article is part of a Special Issue entitled: Chromatin in time and space.

Covalent histone modifications — miswritten, misinterpreted and mis-erased in human cancers

Post-translational modification of histones provides an important regulatory platform for processes such as gene transcription and DNA damage repair. It has become increasingly apparent that the misregulation of histone modification, which is caused by the deregulation of factors that mediate the modification installation, removal and/or interpretation, actively contributes to human cancer. In this Review, we summarize recent advances in understanding the interpretation of certain histone methylations by plant homeodomain finger-containing proteins, and how misreading, miswriting and mis-erasing of histone methylation marks can be associated with oncogenesis and progression. These observations provide us with a greater mechanistic understanding of epigenetic alterations in human cancers and might also help direct new therapeutic interventions in the future.

Cause and consequences of genetic and epigenetic alterations in human cancer.

Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells.

MicroRNA epigenetic alterations in human cancer: One step forward in diagnosis and treatment

MicroRNAs (miRNAs) are approximately 22 nt non-coding RNAs, which regulate gene expression in a sequence-specific manner via translational inhibition or messenger RNA (mRNA) degradation. Since the discovery of their fundamental mechanisms of action, the field of miRNAs has opened a new era in the understanding of small noncoding RNAs. By molecular cloning and bioinformatic approaches, miRNAs have been identified in viruses, plants and animals. miRNAs are predicted to negatively target up to one-third of human mRNAs. Cancer is a complex genetic disease caused by abnormalities in gene structure and expression. Previous studies have heavily focused on protein-coding genes; however, accumulating evidence is revealing an important role of miRNAs in cancer. Epigenetics is defined as mitotically and/or meiotically heritable changes in gene expression that are not accompanied by changes in DNA sequence. Given the critical roles of miRNAs and epigenetics in cancer, characterizing the epigenetic regulation of miRNAs will provide novel opportunities for the development of cancer biomarkers and/or the identification of new therapeutic targets in the foreseeable future.

Adiposity and Age are Statistically Related to EnhancedRASSF1ATumor Suppressor Gene Promoter Methylation in Normal Autopsy Kidney Tissue

Age, adiposity, and smoking are risk factors for the development of renal cell carcinoma. Hypermethylation of the RAS association domain family 1A gene (RASSF1A) promoter belongs to the most frequently detected epigenetic alterations in human cancers including renal cell carcinoma. RASSF1A is functionally involved in cell cycle control in normal cells and depletion promotes a number of cellular changes increasing the risk for neoplastic growth. We investigated the hypothesis that age, modulated by the factors adiposity and anthracosis as a surrogate for smoking, is a predictor of RASSF1A promoter methylation in normal kidney tissue. Using a cross-sectional study design, we quantitatively analyzed RASSF1A methylation in 78 normal autopsy kidney tissues by quantitative combined bisulfite and restriction analysis and bisulfite sequencing, and statistically evaluated the degree of relative methylation for a relationship with the predictor age and study factors adiposity and state of anthracosis. Statistical analysis showed that age (regression analysis; P < 0.001), adiposity (univariate analysis; P = 0.016), and state of anthracosis (t test; P = 0.005) are each significantly associated with an increase of RASSF1A promoter methylation in normal kidney tissue. However, only age (P = 0.008) and adiposity (P = 0.008) were identified as independent predictors of RASSF1A promoter methylation using covariance analysis. This study provides statistical evidence that the common cancer risk factors age and adiposity enhance RASSF1A promoter methylation in nonmalignant kidney tissue.

Inflammation-Mediated Cytosine Damage: A Mechanistic Link between Inflammation and the Epigenetic Alterations in Human Cancers

Aberrant methylation patterns have long been known to exist in the promoter regions of key regulatory genes in the DNA of tumor cells. However, the mechanisms by which these methylation patterns become altered during the transformation of normal cells to tumor cells have remained elusive. We have recently shown in in vitro studies that inflammation-mediated halogenated cytosine damage products can mimic 5-methylcytosine in directing enzymatic DNA methylation and in enhancing the binding of methyl-binding proteins whereas certain oxidative damage products inhibit both. We have therefore proposed that cytosine damage products could potentially interfere with normal epigenetic control by altering DNA-protein interactions critical for gene regulation and the heritable transmission of methylation patterns. These inflammation-mediated cytosine damage products may provide, in some cases, a mechanistic link between inflammation and cancer.

Molecular Mechanism of Adaphostin-mediated G1 Arrest in Prostate Cancer (PC-3) Cells

Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.

The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis.

The RASSF1A isoform of RASSF1 is frequently inactivated by epigenetic alterations in human cancers, but it remains unclear if and how it acts as a tumor suppressor. RASSF1A overexpression reduces in vitro colony formation and the tumorigenicity of cancer cell lines in vivo. Conversely, RASSF1A knockdown causes multiple mitotic defects that may promote genomic instability. Here, we have used a genetic approach to address the function of RASSF1A as a tumor suppressor in vivo by targeted deletion of Rassf1A in the mouse. Rassf1A null mice were viable and fertile and displayed no pathological abnormalities. Rassf1A null embryonic fibroblasts displayed an increased sensitivity to microtubule depolymerizing agents. No overtly altered cell cycle parameters or aberrations in centrosome number were detected in Rassf1A null fibroblasts. Rassf1A null fibroblasts did not show increased sensitivity to microtubule poisons or DNA-damaging agents and showed no evidence of gross genomic instability, suggesting that cellular responses to genotoxins were unaffected. Rassf1A null mice showed an increased incidence of spontaneous tumorigenesis and decreased survival rate compared with wild-type mice. Irradiated Rassf1A null mice also showed increased tumor susceptibility, particularly to tumors associated with the gastrointestinal tract, compared with wild-type mice. Thus, our results demonstrate that Rassf1A acts as a tumor suppressor gene.

Identification of maspin and S100P as novel hypomethylation targets in pancreatic cancer using global gene expression profiling.

DNA hypomethylation is one of the major epigenetic alterations in human cancers. We have previously shown that genes identified as hypomethylated in pancreatic cancer are expressed in pancreatic cancer cell lines, but not in normal pancreatic ductal epithelium and can be reexpressed in nonexpressing cells using 'epigenetic modifying agents' such as DNA methyltransferase inhibitors. To identify additional targets for aberrant hypomethylation in pancreatic cancer, we used oligonucleotide microarrays to screen for genes that displayed expression patterns associated with hypomethylation. This analysis identified a substantial number of candidates including previously reported hypomethylated genes. A subset of eight genes were selected for further methylation analysis, and two cancer-related genes, maspin and S100P, were found to be aberrantly hypomethylated in a large fraction of pancreatic cancer cell lines and primary pancreatic carcinomas. Combined treatment with 5-aza-2'-deoxycytidie and trichostatin A resulted in synergistic induction of maspin and S100P mRNA in MiaPaCa2 cells where both genes were methylated. Furthermore, there was an inverse correlation between methylation and mRNA expression level for maspin and S100P in a large panel of pancreatic cancer cell lines. We also found a significant difference in the methylation patterns of maspin and two previously identified hypomethylated genes (trefoil factor 2 and lipocalin 2) between pancreatic and breast cancer cell lines, suggesting cancer-type specificity for some hypomethylation patterns. Thus, our present results confirm that DNA hypomethylation is a frequent epigenetic event in pancreatic cancer, and suggest that gene expression profiling may help to identify potential targets affected by this epigenetic alteration.

Restriction landmark genome scanning for aberrant methylation in primary refractory and relapsed acute myeloid leukemia; involvement of the WIT-1 gene.

There is substantial evidence to suggest that aberrant DNA methylation in the regulatory regions of expressed genes may play a role in hematologic malignancy. In the current report, the Restriction Landmark Genomic Scanning (RLGS) method was used to detect aberrant DNA methylation (M) in acute myeloid leukemia (AML). RLGS-M profiles were initially performed using DNA from diagnostic, remission, and relapse samples from a patient with AML. Rp18, one of the eight spots found that was absent in the relapse sample, was cloned. Sequence analysis showed that the spot represented a portion of the WIT-1 gene on human chromosome 11p13. Rp18 was missing in the relapse sample due to a distinct DNA methylation pattern of the WIT-1 gene. Twenty-seven AML patients that entered CR after therapy (i.e., chemosensitive) were studied and only 10 (37%) of the diagnostic bone marrow (BM) samples showed methylation of WIT-1. However, seven of eight (87.5%) diagnostic BM samples from primary refractory AML (chemosensitive) showed methylation of WIT-1. The incidence of WIT-1 methylation in primary refractory AML was significantly higher than that noted in chemosensitive AML (P=0.018). Together, these results indicate that RLGS-M can be used to find novel epigenetic alterations in human cancer that are undetectable by standard methods. In addition, these results underline the potential importance of WIT-1 methylation in chemoresistant AML.