DNA Methylation and Colorectal Cancer

Colorectal cancer (CRC) is one of the major cancers in the world and the second death-causing cancer in the USA. CRC development involves genetic and epigenetic alterations. Changes in DNA methylation status are believed to be involved at different stages of CRC. Promoter silencing via DNA methylation and hypomethylation of oncogenes alters gene expression and can be used as a tool for the early detection of colonic lesions. DNA methylation use as a diagnostic and prognostic marker has been described for many cancers including CRC. CpG island methylator phenotype (CIMP) is one of the underlying CRC mechanisms. This review aims to define methylation signatures in CRC. The analysis of DNA methylation profile in combination with the pathological diagnosis would be useful in predicting CRC tumor evolution and their prognostic behavior.     

表观遗传学在结直肠癌中的应用进展

结直肠癌（CRC）的发生、发展伴随着许多基因的表达变化,但其具体调控机制至今仍未完全阐明。近年来对CRC表观遗传学尤其是微小RNA（miRNA）、异常DNA甲基化及组蛋白修饰状态等方面的研究受到广泛关注。研究证实,CRC进展过程中均存在异常的甲基化基因和miRNA的表达变化。与癌症基因组的基因突变一样,这类遗传学的分子改变在CRC中扮演着重要角色。表观遗传学的特异性改变可作为CRC诊断、治疗和预后的临床生物学标记物,对表观遗传学进行深入研究对CRC的防治具有重要指导意义。     

DNA methylation markers in colorectal cancer

Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation. Epigenetic modifications, particularly DNA methylation in selected gene promoters, are recognized as common molecular alterations in human tumors. Substantial efforts have been made to determine the cause and role of aberrant DNA methylation (“epigenomic instability”) in colon carcinogenesis. In the colon, aberrant DNA methylation arises in tumor-adjacent, normal-appearing mucosa. Aberrant methylation also contributes to later stages of colon carcinogenesis through simultaneous methylation in key specific genes that alter specific oncogenic pathways. Hypermethylation of several gene clusters has been termed CpG island methylator phenotype and appears to define a subgroup of colon cancer distinctly characterized by pathological, clinical, and molecular features. DNA methylation of multiple promoters may serve as a biomarker for early detection in stool and blood DNA and as a tool for monitoring patients with CRC. DNA methylation patterns may also be predictors of metastatic or aggressive CRC. Therefore, the aim of this review is to understand DNA methylation as a driving force in colorectal neoplasia and its emerging value as a molecular marker in the clinic.     

Genome-wide DNA methylation profiles in precancerous conditions and cancers

Alterations of DNA methylation, which result in chromosomal instability and silencing of tumor-related genes, are among the most consistent epigenetic changes observed in human cancers. Analysis of tissue specimens has revealed that DNA methylation alterations participate in multistage carcinogenesis, even from the early and precancerous stages, especially in association with chronic inflammation and/or persistent viral infection, such as chronic hepatitis or liver cirrhosis resulting from infection with hepatitis B or C virus. DNA methylation alterations can account for the histological heterogeneity and clinicopathological diversity of human cancers. Overexpression of DNA methyltransferase 1 is not a secondary result of increased cell proliferative activity, but is significantly correlated with accumulation of DNA hypermethylation in CpG islands of tumor-related genes. Alteration of DNA methyltransferase 3b splicing may result in chromosomal instability through DNA hypomethylation in pericentromeric satellite regions. Genome-wide analysis of DNA methylation status has revealed that the DNA methylation profile at the precancerous stage is basically inherited by the corresponding cancers developing in individual patients. DNA methylation status is not simply altered at the precancerous stage; rather, DNA methylation alterations at the precancerous stage may confer vulnerability to further genetic and epigenetic alterations, generate more malignant cancers, and thus determine patient outcome. Therefore, genome-wide DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication, and thus provide an avenue for cancer prevention and therapy on an individual basis. ( Cancer Sci 2009)     

Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer

Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.     

DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory

DNA methylation is commonly thought of as a "molecular lock" that leads to permanent gene silencing. To investigate this notion, we tested 24 different histone deacetylase inhibitors (HDACi) on colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated cytomegalovirus (CMV) promoter. We found that HDACi efficiently reactivated expression of GFP and many other endogenous genes silenced by DNA hypermethylation. After treatment, all promoters were marked with active chromatin, yet DNA hypermethylation did not change. Thus, DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state. In evaluating the relative contribution of DNA methylation and histone modifications to stable gene silencing, we followed expression levels of GFP and other genes silenced by DNA hypermethylation over time after treatment with HDACi or DNA-demethylating drugs. Reactivation of methylated loci by HDACi was detectable for only 2 weeks, whereas DNA-demethylating drugs induced permanent epigenetic reprogramming. Therefore, DNA methylation cannot be considered as a lock for gene expression but rather as a memory signal for long-term maintenance of gene silencing. These findings define chromatin as an important druggable target for cancer epigenetic therapy and suggest that removal of DNA methylation signals is required to achieve long-term gene reactivation.     

SPARC and DNA methylation: Possible diagnostic and therapeutic implications in gastrointestinal cancers

DNA methylation is a major contributor to epigenetic alterations and as such is a potential biomarker and therapeutic target in gastrointestinal malignancies. DNA methylation is commonly observed in several Gastrointestinal (GI) malignancies including pancreatic and colorectal cancer. Methylation results in decreased expression of tumor suppressor genes. Secreted protein acidic and rich in cysteine (SPARC) is a tumor suppressor gene that can be functionally inactivated through methylation. SPARC is commonly dysregulated in GI malignancies. Inhibition of DNA methylation can reverse the silencing of SPARC. In the present review, we will discuss recent advances in our understanding of the features of DNA methylation that pertain to SPARC, focusing on their functional and clinical relevance in GI carcinogenesis.     

Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma

Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.     

Epigenetic biomarkers of colorectal cancer: Focus on DNA methylation

The original theory of the multi-step process of colorectal cancer (CRC), suggesting that the disease resulted from the accumulation of mutations in oncogenes and tumor suppressor genes in colonic mucosa cells, has been largely revised following the observation that epigenetic modifications of several genes occur in the average CRC genome. Therefore, the current opinion is that CRCs are the consequence of the accumulation of both mutations and epigenetic modifications of several genes. This mini-review article focuses on DNA methylation biomarkers in CRC. Recent large-scale DNA methylation studies suggest that CRCs can be divided into at least three-four subtypes according to the frequency of DNA methylation and those of mutations in key CRC genes. Despite hundreds of genes might be epigenetically modified in CRC cells, there is interest in the identification of DNA methylation biomarkers to be used for CRC diagnosis, progression, tendency to tissue invasion and metastasis, prognosis, and response to chemotherapeutic agents. Moreover, DNA methylation largely depends on one-carbon metabolism, the metabolic pathway required for the production of S-adenosylmethionine, the major intracellular methylating agent. Complex interactions are emerging among dietary one-carbon nutrients (folates, vitamin B6, vitamin B12, methionine, and others), their metabolic genes, CRC risk, and DNA methylation profiles in CRC. Moreover, active research is also focused on the possible contribution of folic acid dietary fortification during pregnancy and the possible methylation of CRC-related genes in the offspring.     

Epigenetic changes in colorectal cancer

Epigenetic silencing is now recognized as a third pathway in Knudson's model of tumor-suppressor gene inactivation in cancer and can affect gene function without genetic changes. DNA methylation within gene promoters and alterations in histone modifications appear to be primary mediators of epigenetic inheritance in cancer cells. For selected genes, epigenetic changes are tightly related to neoplastic transformation in colorectal cancers (CRCs). In the colon, aberrant DNA methylation arises very early, initially in normal appearing mucosa, and may be part of the age-related field defect observed in sporadic CRCs. Aberrant methylation also contributes to later stages of colon cancer formation and progression through a hypermethylator phenotype termed CpG Island Methylator Phenotype (CIMP), which appears to be a defining event in about half of all sporadic tumors. CIMP+ CRCs are distinctly characterized by pathology, clinical and molecular genetic features. Histone modifications, recently recognized as a histone code that affects chromatin structure and gene expression also play an important role in the establishment of gene silencing during tumorigenesis. DNA methylation and histone H3 lysine 9 hypoacetylation and methylation appear to form a mutually reinforcing silencing loop that contributes to tumor-suppressor gene inactivation in CRCs. Understanding epigenetic alterations as a driving force in neoplasia opens new fields of research in epidemiology, risk assessment, and treatment in CRCs.     

Quantitative analysis of promoter hypermethylation in multiple genes in osteosarcoma

BACKGROUND: Osteosarcoma is the most common solid malignant diseases of childhood, occurring in approximately 6 children per million annually; however, to the authors' knowledge to date, the cause of osteosarcoma has remained mostly unknown. Genetic alterations of genes that are specific for osteosarcoma have not been identified. Genetic alternations in the status of DNA methylation, known as epigenetic alterations, are the most common molecular alterations in human neoplasia. Aberrant methylation in the promoter region of tumor-related genes is associated closely with epigenetically mediated gene silencing, which is a common feature in human tumors. METHODS: The authors analyzed CpG islands of 5 different gene loci for aberrant methylation profiles in 30 pairs of osteosarcoma and corresponding normal tissues by using the quantitative methylation-specific polymerase chain reaction method. The objectives of this study were to characterize the methylation changes in osteosarcoma more extensively and to identify epigenetic biomarkers that may be useful in the diagnosis and prevention of osteosarcoma. RESULTS: For the Ras effector homologue (RASSF1A), tissue inhibitor of metalloproteinase 3 (TIMP3), O-6-methylguanine DNA methyltransferase (MGMT), and death-associated protein kinase 1 (DAPK1) genes, significant differences were observed in the degree of hypermethylation between tumors and normal tissues (P < 0.01 and P < 0.001, respectively). Measurement of the cumulative multiple promoter hypermethylation revealed striking differences between tumor specimens and normal tissues (t = 7.31; P < .001). There also was a significant difference in the levels of DNA methylation between the metastatic and nonmetastatic high-grade osteosarcomas (t = 4.57; P < .01). In addition, the methylation levels were associated closely with gender (t = 6.44; P < .001). CONCLUSIONS: The results indicated that tumor tissues from patients with osteosarcoma had a significantly higher incidence of hypermethylation for several genes compared with corresponding normal tissues. The epigenetic changes observed in this study may have prognostic importance for patients with osteosarcoma.     

Should we invest in biological age predictors to treat colorectal cancer in older adults?

Colorectal cancer (CRC) is a chronic disease of the old population with slow development progressing into clinical signs and symptoms. Biological aging is characterized by e.g. mitochondrial dysfunction and epigenetic alterations (e.g. methylation) - mechanisms that are also important in cancer development. For CRC, specific types of tumors are distinguishable by their methylation patterns and several detection methods using different epigenetic marks have been developed as signatures for the disease. Biological age assessed by DNA methylation patterns from blood, i.e. the epigenetic clock, is higher in CRC patients compared to controls, and may be a tool for identifying individuals at increased risk for CRC. Other types of biomarkers of aging are useful to calculate biological age, such as metabolites, protein levels, inflammatory markers and clinical biomarkers, where composite scores of biomarkers have been used to assess the risk of CRC and colorectal adenomas.Clinical assessments of biological aging includes frailty, which is a geriatric syndrome characterized by increased vulnerability to adverse outcomes. More than half of the CRC patients are estimated to be frail or pre-frail, and these individuals are at increased risk of postoperative complications, poorer prognosis, treatment intolerance and death. Hence, considering frailty as part of biological age in CRC patients may help identifying those at need of close monitoring.In summary, future screening programs for CRC may make use of biological age assessments, e.g. by epigenetic clock or composite scores. Monitoring disease relapse and treatment response should be enhanced in frail individuals for better prognosis.     

Understanding Epigenetics: an Alternative Mechanism of Colorectal Carcinogenesis

Colorectal cancer (CRC) is a multifactorial disease that arises due to the accumulation of genetics as well as epigenetic alterations in a number of onco-, tumor suppressor-, mismatch repair-, and cell cycle-genes in colon mucosa cells. Epigenetic silencing of the key tumor suppressor genes has been identified as one of the new and distinct mechanisms driving the tumor initiation and progression. In this review we have explored the mechanism of hypermethylation of the CpG islands and its consequent role in colorectal carcinogenesis. We have also discussed the epigenetic view of molecular and pathological basis of colorectal neoplasia and also the role of 1-carbon metabolism in driving the methylation of key genes in CRC.     

Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma

The soluble frizzled receptor protein (SFRP) family encodes antagonists of the WNT pathway, and silencing of these genes, through promoter hypermethylation, leads to constitutive WNT signaling. In bladder cancers, hypermethylation of the SFRP genes occurs more often in current and former smokers and is a strong predictor of poor patient survival. Hence, we examined methylation of these genes in another tobacco-related epithelial cancer, head and neck squamous cell carcinoma (HNSCC), to determine if the pattern of tobacco exposure again predicts the epigenetic alteration of these genes. Using methylation-specific PCR, the prevalence of methylation of SFRP1, SFRP2, SFRP4 and SFRP5 was 35, 32, 35 and 29%, respectively among 350 HNSCC cases. Promoter methylation of SFRP1 occurred more often in both heavy (OR 3.5, 95% CI 0.9-13.7) and light drinkers (OR 3.7, 95% CI 1.0-14.3) compared to nondrinkers. SFRP4 promoter methylation, on the other hand, occurred at a higher prevalence in never smokers and former smokers than in current smokers, and also was independently associated with HPV16 viral DNA. A joint effects model of SFRP4 promoter methylation demonstrated that smoking status and HPV virus significantly interacted (p < 0.04) such that never smokers with HPV16 had an OR of SFRP4 methylation of 9.0 (95% CI 2.1-38.6). These results suggest that epigenetic alterations of the SFRP genes are highly prevalent in HNSCC, and that the clonal selection for these alterations is complex and may be related to the carcinogenic exposures that are known risk factors for this disease.     

Epigenetics and cancer, 2nd IARC meeting,Lyon, France, 6 and 7 December 2007

It is becoming widely accepted that epigenetic alterations are universally present in human malignancies and that cancer is as much a disease of abnormal epigenetics as it is a genetic disease. The potential utility of epigenetics and epigenomics in cancer research and cancer control is highlighted by the fact that many funding agencies put cancer epigenetics on the priority list. The goal of this meeting, held at the offices of the International Agency for Research on Cancer in Lyon, France, 6 and 7 December 2007, was to discuss recent conceptual and technological advances in cancer epigenetics and epigenomics, the future research needs in the field, and their implications for early detection, risk assessment and prevention of cancer.While epigenetics has been historically linked to phenomena that do not follow normal genetic principles of heritability, recent mechanistic advances have begun to change our understanding of complex diseases including cancer, traditionally viewed as genetic in origin. It is now known that epigenetic mechanisms play critical roles in regulation of many cellular functions and their deregulation may disrupt the control of fundamental processes leading to tumour formation. A flurry of screening and functional studies revealed that most key processes found in cancer cells, such as silencing of tumour suppressor genes, activation of oncogenes, aberrant cell cycle processes, defects in DNA repair, and deregulation of cell death, can be triggered by epigenetic deregulation. Two important features that distinguish epigenetic changes from genetic alterations are the gradual appearance and reversibility of epigenetic events. These features make epigenetic alterations an attractive target for therapeutic intervention and the development of preventive strategies. For example, aberrant patterns of DNA methylation and histone acetylation and methylation can be targeted by drugs aiming to re‐activate epigenetically silenced genes. Until now, most studies on epigenetic changes in cancer were generally focused on specific genes. However, this meeting also stressed the need to take advantage of recent progress in epigenomics and emergence of powerful technologies for detection of epigenetic changes in high throughput and genome‐wide settings. This may further advance our capacity to evaluate the contribution of epigenetic changes induced by environmental epimutagens to human cancer. This information may prove critical for the design of efficient strategies for early diagnosis, therapy, and prevention of cancer.     

Focus on genetic and epigenetic events of colorectal cancer pathogenesis: implications for molecular diagnosis

Originally, colorectal cancer (CRC) tumorigenesis was understood as a multistep process that involved accumulation of tumor suppressor genes and oncogenes mutations, such as APC, TP53 and KRAS. However, this assumption proposed a relatively limited repertoire of genetic alterations. In the last decade, there have been major advances in knowledge of multiple molecular pathways involved in CRC pathogenesis, particularly regarding cytogenetic and epigenetic events. Microsatellite instability, chromosomal instability and CpG island methylator phenotype are the most analyzed cytogenetic changes, while DNA methylation, modifications in histone proteins and microRNAs (miRNAs) were analyzed in the field of epigenetic alterations. Therefore, CRC development results from interactions at many levels between genetic and epigenetic amendments. Furthermore, hereditary cancer syndrome and individual or environmental risk factors should not be ignored. The difficulties in this setting are addressed to understand the molecular basis of individual susceptibility to CRC and to determine the roles of genetic and epigenetic alterations, in order to yield more effective prevention strategies in CRC patients and directing their treatment. This review summarizes the most investigated biomolecular pathways involved in CRC pathogenesis, their role as biomarkers for early CRC diagnosis and their possible use to stratify susceptible patients into appropriate screening or surveillance programs.     

Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high‐methylation CRC. The methylation levels of six Group‐1 and 14 Group‐2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group‐1 and Group‐2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high‐methylation CRC. But TSA showed low‐methylation levels of Group‐1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high‐methylation CRC. High‐methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1‐methylated SSA/P showed lower methylation level of MLH1 compared with high‐methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1‐methylated and MLH1‐unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF‐β and BMP signaling (but not in TP53 signaling) were significantly involved in high‐methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.