Hypermethylation Of Specific Genes Research Articles

Recent advances in understanding DNA methylation of prostate cancer.

Epigenetic modifications, such as DNA methylation, is widely studied in cancer. DNA methylation patterns have been shown to distinguish between benign and malignant tumors in various cancers, including prostate cancer. It may also contribute to oncogenesis, as it is frequently associated with downregulation of tumor suppressor genes. Aberrant patterns of DNA methylation, in particular the CpG island hypermethylator phenotype (CIMP), have shown associative evidence with distinct clinical features and outcomes, such as aggressive subtypes, higher Gleason score, prostate-specific antigen (PSA), and overall tumor stage, overall worse prognosis, as well as reduced survival. In prostate cancer, hypermethylation of specific genes is significantly different between tumor and normal tissues. Methylation patterns could distinguish between aggressive subtypes of prostate cancer, including neuroendocrine prostate cancer (NEPC) and castration resistant prostate adenocarcinoma. Further, DNA methylation is detectable in cell-free DNA (cfDNA) and is reflective of clinical outcome, making it a potential biomarker for prostate cancer. This review summarizes recent advances in understanding DNA methylation alterations in cancers with the focus on prostate cancer. We discuss the advanced methodology used for evaluating DNA methylation changes and the molecular regulators behind these changes. We also explore the clinical potential of DNA methylation as prostate cancer biomarkers and its potential for developing targeted treatment of CIMP subtype of prostate cancer.

Gene-Specific DNA Methylation Linked to Postoperative Cognitive Dysfunction in Apolipoprotein E3 and E4 Mice.

Geriatric surgical patients are at higher risk of developing postoperative neurocognitive disorders (NCD) than younger patients. The specific mechanisms underlying postoperative NCD remain unknown, but they have been linked to genetic risk factors, such as the presence of APOE4, compared to APOE3, and epigenetic modifications caused by exposure to anesthesia and surgery. To test the hypothesis that compared to E3 mice, E4 mice exhibit a more pronounced postoperative cognitive impairment associated with differential DNA methylation in brain regions linked to learning and memory. 16-month-old humanized apolipoprotein-E targeted replacement mice bearing E3 or E4 were subjected to surgery (laparotomy) under general isoflurane anesthesia or sham. Postoperative behavioral testing and genome-wide DNA methylation were performed. Exposure to surgery and anesthesia impaired cognition in aged E3, but not E4 mice, likely due to the already lower cognitive performance of E4 prior to surgery. Cognitive impairment in E3 mice was associated with hypermethylation of specific genes, including genes in the Ephrin pathway implicated in synaptic plasticity and learning in adults and has been linked to Alzheimer's disease. Other genes, such as the Scratch Family Transcriptional Repressor 2, were altered after surgery and anesthesia in both the E3 and E4 mice. Our findings suggest that the neurocognitive and behavioral effects of surgery and anesthesia depend on baseline neurocognitive status and are associated with APOE isoform-dependent epigenetic modifications of specific genes and pathways involved in memory and learning.

Novel Epigenetic Eight-Gene Signature Predictive of Poor Prognosis and MSI-Like Phenotype in Human Metastatic Colorectal Carcinomas.

Simple SummaryThe global methylation profile of two human metastatic colorectal carcinoma subgroups with significantly different outcomes (primary-resistant versus drug-sensitive tumors) was analyzed and compared with the gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) metastatic colorectal carcinoma dataset with the aim to identify a prognostic signature of functionally methylated genes. A novel epigenetic eight-gene signature, with hypermethylation of the promoter regions, was identified and validated for its capacity to predict poor outcome, which had a CpG-island methylator phenotype (CIMP)-high status and microsatellite instability (MSI)-like phenotype.Epigenetics is involved in tumor progression and drug resistance in human colorectal carcinoma (CRC). This study addressed the hypothesis that the DNA methylation profiling may predict the clinical behavior of metastatic CRCs (mCRCs). The global methylation profile of two human mCRC subgroups with significantly different outcome was analyzed and compared with gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) and the NCBI GENE expression Omnibus repository (GEO) GSE48684 mCRCs datasets to identify a prognostic signature of functionally methylated genes. A novel epigenetic signature of eight hypermethylated genes was characterized that was able to identify mCRCs with poor prognosis, which had a CpG-island methylator phenotype (CIMP)-high and microsatellite instability (MSI)-like phenotype. Interestingly, methylation events were enriched in genes located on the q-arm of chromosomes 13 and 20, two chromosomal regions with gain/loss alterations associated with adenoma-to-carcinoma progression. Finally, the expression of the eight-genes signature and MSI-enriching genes was confirmed in oxaliplatin- and irinotecan-resistant CRC cell lines. These data reveal that the hypermethylation of specific genes may provide prognostic information that is able to identify a subgroup of mCRCs with poor prognosis.

Neuroprotective effect of 3,3\u2019-Diindolylmethane against perinatal asphyxia involves inhibition of the AhR and NMDA signaling and hypermethylation of specific genes

Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3’-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.

Analysis of healthy and tumour DNA methylation distributions in kidney-renal-clear-cell-carcinoma using Kullback-Leibler and Jensen-Shannon distance measures.

DNA methylation is an epigenetic phenomenon in which methyl groups get bonded to the cytosines of the DNA molecule altering the expression of the associated genes. Cancer is linked with hypo or hyper-methylation of specific genes as well as global changes in DNA methylation. In this study, the authors study the probability density function distribution of DNA methylation in various significant genes and across the genome in healthy and tumour samples. They propose a unique 'average healthy methylation distribution' based on the methylation values of several healthy samples. They then obtain the Kullback-Leibler and Jensen-Shannon distances between methylation distributions of the healthy and tumour samples and the average healthy methylation distribution. The distance measures of the healthy and tumour samples from the average healthy methylation distribution are compared and the differences in the distances are analysed as possible parameters for cancer. A classifier trained on these values was found to provide high values of sensitivity and specificity. They consider this to be a computationally efficient approach to predict tumour samples based on DNA methylation data. This technique can also be improvised to consider other differentially methylated genes significant in cancer or other epigenetic diseases.

A Pharmacologically Reversible Epigenomic Signature of Relapse and Chemotherapy Resistance in Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) are orphan entities representing around 20% of all Peripheral T-cell lymphoma. ALCLs can be stratified into primary cutaneous ALCL and systemic ALCL. Patients with systemic ALCL are further distinguished based on anaplastic lymphoma kinase (ALK) translocations. Unlike ALK+ patients, ALK- ALCLs are more likely to be refractory or relapse upon treatment with CHOP (Cyclophosphamide, Hydroxydaunorubicin, Oncovin, Prednisone). A small number of genetic defects and genomic translocations have been identified and associated with clinical outcome in ALK- ALCL, but cannot fully explain the poor outcomes of ALK- patients. To further probe the molecular bases of ALCL, we performed systematic epigenomic profiling in systemic ALCL through Enhanced Reduced Representation Bisulfite Sequencing (ERRBS).To investigate the methylation profile of systemic ALCLs, we first interrogated 8 ALK+ ALCL and 16 ALK- ALCL tumors. Combining methylation levels from all interrogated CpG sites, we observed overall hypermethylation at CpG-islands (CGIs; p=0.02, t-test) in ALK- tumors compared ALK+ patients but hypomethylation outside of CGIs (>10kb away from known CGIs; p=0.03, t-test). There were no significant differences on the average methylation levels at CpG-island shores, which indicate varied methylation profiles among different genomic locations. To define methylation-based signatures, we then performed supervised analysis between ALK+ and ALK- patients. We have identified 254 ALK- patients linked consistently differentially methylated promoters (>20% DNA methylation changes and FDR<0.1, t-test). Out of 254 promoters, 252 genes were hypermethylated in ALK- patients confirming the association between systemic ALCL subtypes and hypermethylation.We then sought to investigate how methylation evolves in ALCL. We performed ERRBS of 2 primary ALCL tumors and their matched relapse samples (2 for each patient). We observed a significant hypermethylation at CGIs in relapsed samples (p=1.4e-5, paired t-test), suggesting that hypermethylation changes may be associated with tumor evolution.To test whether the hypermethylation of specific genes is linked to tumor aggressiveness, we analyzed serial passages of patient-derived tumor xenograft (PDTX) derived from primary and relapse ALCL samples. We observed consistently hypermethylation at CGIs along different implants and conserved hypermethylation of genes at the same time (p<0.05, 1959). To confirm the behavior of those genes, we estimate their methylation trend in ALCL primary and relapse samples with GSEA. Target genes showed a very strong hypermethylation in relapsed ALCL compared to primary samples (FDR<0.01). Next we confirmed analogous methylation changes in a cohort of primary and relapse PDTX (Primary, n=2, FDR<0.01; Relapse, n=3, FDR<0.01). Interestingly, when comparing this to our previous diffuse large b-cell lymphoma (DLBCL) study (Pan et.al., 2015), we found similar hypermethylated profiles, suggesting commonalities among different type of human lymphomas.Lastly, we investigated whether a treatment with the 5-azacytidine (5-Aza) could reverse the hypermethylation profile of ALCL PDTX. Tumors from in vivo 5-Aza treated and control mice were extracted and subjected to ERRBS. As expected, we only found global hypomethylation in 5-Aza-treated samples closely mimicked methylation profiles in naive primary ALCL and early passages of PDTX.In summary, this study provides the first comprehensive characterization of methylome in ALCL. Hypermethylation is firstly associated with the aggressiveness of ALK- patients in ALCL. Also, we identified a group of hypermethylated genes associated with relapsed ALCL, a phenotype mirrored by serial passages of PDTX in vivo. As 5-Aza treatments can revert the methylome of late passages of ALCL PDTX, we speculate that this treatment may represent a novel therapeutic approach in chemo-relapsed ALCL patients. DisclosuresMartin:Gilead: Consultancy, Other: travel, accommodations, expenses; Acerta: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Honoraria; Teva: Research Funding; Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses.

Cutaneous Squamous Cell Carcinoma: A Review of High-Risk and Metastatic Disease.

Non-melanoma skin cancer represents one-third of all malignancies and its incidence is expected to rise until the year 2040. Cutaneous squamous cell carcinoma (cSCC) represents 20% of all non-melanoma skin cancer and is a deadly threat owing to its ability to metastasize to any organ in the body. Therefore, a better understanding of cSCC is essential to strengthen preventative measures and curable treatment options. Currently, research demonstrates that cSCC is diagnosed at a rate of 15-35 per 100,000 people and is expected to increase 2-4% per year. With respect to metastatic cSCC, this disease is more common in men; people over the age of 75years; and inhabitants of the south and mid-west USA. In 2010, the American Joint Committee on Cancer updated the Cancer Staging Manual's primary tumor designation to now include high-risk factors; however, factors such as immunosuppression and tumor recurrence were not included. Other staging systems such as Brigham and Women's Hospital have allowed for increased stratification of cSCC. High-risk cSCC is defined as a cSCC that is staged as N0, extends beyond basement membrane, and has high-risk features associated with sub-clinical metastasis. High-risk features are depth of invasion (>2mm), poor histological differentiation, high-risk anatomic location (face, ear, pre/post auricular, genitalia, hands, and feet), perineural involvement, recurrence, multiple cSCC tumors, and immunosuppression. Epidermal growth factor receptor and nuclear activeIκB kinase (IKK) expression are also predictive of metastatic capabilities. Clinically, the initial lesions of a cSCC tumor can present as a painless plaque-like or verrucous tumor that can ultimately progress to being large, necrotic, and infected. Tumors can also present with paresthesias or lymphadenopathy depending on the location involved. With respect to prognosis, metastatic cSCC is lethal, with several large studies demonstrating a mortality rate of >70%. Therefore, treatment of metastatic cSCC is difficult and depends on the location involved and extent of metastasis. Treatment options include surgery, radiation therapy, chemotherapy, and any combination of the above. Surgery alone can be used for metastatic cSCC treatment, but is not as effective as surgery in conjunction with radiation therapy. Radiation therapy has some success as a monotherapy in low-risk or cosmetically sensitive areas such as the external ear, eyelid or nose. According to the 2013 National Comprehensive Cancer Network Guidelines, cisplatin as a single agent or combined with 5-fluorouracil hold the strongest support for the treatment of metastatic cSCC; however, the supporting evidence is inconsistent and a curative chemotherapeutic approach is still lacking. Epidermal growth factor receptor inhibitors are a newer class of agents being used in metastatic cSCC and hold some promise as a therapy for this disease. Other areas of interest in finding curative treatments for metastatic cSCC include p53, hypermethylation of specific genes, chromatin remodeling genes, and the RAS/RTK/PI3K pathway. This review addresses the epidemiology, staging, risk factors, clinical presentation, management, and new trends in the treatment of high-risk and metastatic cSCC.

Epigenetic probes for lung cancer monitoring: Line-1 methylation pattern in blood-circulating DNA

The main components of DNA aberrant methylation in malignant cells are promoter hypermethylation of specific genes and hypomethylation of the main part of DNA, in particular, the repeated sequences of retrotransposons. The hypomethylation of retrotransposons from the LINE-1 family was revealed in the lung cancer (LC) tissue. The composition of circulating DNA (cirDNA) from the plasma and blood cell surface bound circulating DNA (csb-cirDNA) was shown earlier to be altered in the blood of cancer patients due to the accumulation of tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. The present study compares LINE-1 retrotransposon methylation patterns in the cirDNA and csb-cirDNA plasma from 21 untreated LC patients and 23 healthy donors. Concentrations of methylated LINE-1 region 1 copies (LINE-1met) were assessed by quantitative real-time methylation-specific PCR. In order to normalize the LINE-1 methylation level, the LINE-1 region 2 concentration was evaluated, which was independent of the methylation status (LINE-1Ind). The LINE1met concentration in csb-cirDNA tended to decrease (by a factor of 1.4) in blood from LC patients compared to healthy donors (Mann–Whitney test, P = 0.16). The LINE-1Ind (methylation-independent) concentration in csb-cirDNA was found to be lower by a factor of three in LC patients and by a factor of four in patients with adenocarcinoma than in healthy donors. Thus, along with the expected decrease in the LINE1met concentration in csb-cirDNA from LC patients’ blood, we recorded an unexpected statistically significant increase of the LINE-1 methylation index determined as (LINE-1met/LINE-1Ind) due to the profound decrease of the total LINE-1Ind number. There were no differences between the plasma cirDNA LINE-1 methylation indexes in the LC patients and healthy donors (Mann–Whitney test, P = 0.40). The data obtained agreed with our earlier results, which showed that csb-cirDNA was highly informative material for lung cancer diagnostics.

The significance of epigenetic alterations in lung carcinogenesis

Lung cancer is recognized as a leading cause of cancer-related death worldwide and its frequency is still increasing. The prognosis in lung cancer is poor and limited by the difficulties of diagnosis at early stage of disease, when it is amenable to surgery treatment. Therefore, the advance in identification of lung cancer genetic and epigenetic markers with diagnostic and/or prognostic values becomes an important tool for future molecular oncology and personalized therapy. As in case of other tumors, aberrant epigenetic landscape has been documented also in lung cancer, both at early and late stage of carcinogenesis. Hypermethylation of specific genes, mainly tumor suppressor genes, as well as hypomethylation of oncogenes and retrotransposons, associated with histopathological subtypes of lung cancer, has been found. Epigenetic aberrations of histone proteins and, especially, the lower global levels of histone modifications have been associated with poorer clinical outcome in lung cancer. The recently discovered role of epigenetic modifications of microRNA expression in tumors has been also proven in lung carcinogenesis. The identified epigenetic events in lung cancer contribute to its specific epigenotype and correlated phenotypic features. So far, some of them have been suggested to be cancer biomarkers for early detection, disease monitoring, prognosis, and risk assessment. As epigenetic aberrations are reversible, their correction has emerged as a promising therapeutic target.

Prenatal Exposure to Polycyclic Aromatic Hydrocarbons, Benzo[ a ]pyrene–DNA Adducts, and Genomic DNA Methylation in Cord Blood

Background: Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic environmental pollutants generated during incomplete combustion. After exposure and during metabolism, PAHs can form reactive epoxides that can covalently bind to DNA. These PAH–DNA adducts are established markers of cancer risk. PAH exposure has been associated with epigenetic alterations, including genomic cytosine methylation. Both global hypomethylation and hypermethylation of specific genes have been associated with cancer and other diseases in humans. Experimental evidence suggests that PAH–DNA adduct formation may preferentially target methylated genomic regions. Early embryonic development may be a particularly susceptible period for PAH exposure, resulting in both increased PAH–DNA adducts and altered DNA methylation.Objective: We explored whether prenatal exposure to PAHs is associated with genomic DNA methylation in cord blood and whether methylation levels are associated with the presence of detectable PAH–DNA adducts.Methods: In a longitudinal cohort study of nonsmoking women in New York City, we measured PAH exposure during pregnancy using personal air monitors, assessed PAH internal dose using prenatal urinary metabolites (in a subset), and quantified benzo[a]pyrene–DNA adducts and genomic DNA methylation in cord blood DNA among 164 participants.Results: Prenatal PAH exposure was associated with lower global methylation in umbilical cord white blood cells (p = 0.05), but global methylation levels were positively associated with the presence of detectable adducts in cord blood (p = 0.01).Conclusions: These observations suggest that PAH exposure was adequate to alter global methylation in our study population. Additional epidemiologic studies that can measure site-specific cytosine methylation and adduct formation will improve our ability to understand this complex molecular pathway in vivo.

DNA methylation alterations in urothelial carcinoma

In urothelial cancer, hypermethylation of specific genes and genome-wide hypomethylation, reflected in decreased methylation of LINE-1 retrotransposons, have both been reported, but were never investigated in the same specimens. We analyzed hypermethylation of 6 genes by methylation-specific PCR and LINE-1 hypomethylation by Southern blotting in 96 carcinoma tissues. Hypermethylation frequencies were: SFRP1 (55%), APC (45%), RASSF1A (35%), DAPK1 (29%), RARB2 (19%), and CDKN2A (2%). Three groups of cancers could be discerned, with escalating hypermethylation. Hypermethylation increased with tumor stage, particularly at the transition to invasive cancers, and RARB2 hypermethylation was indicative of lymph node involvement. A comparison to a previous study on prostate cancer using the same techniques suggests that hypermethylation in urothelial carcinoma occurs in a random rather than coordinated manner. LINE-1 hypomethylation was present in 90% of specimens, largely independent of hypermethylation. Lack of hypomethylation indicated a significantly better clinical prognosis. Bisulfite sequencing of SFRP1 demonstrated dense or patchy hypermethylation in tumor tissues that likely accounts for discrepant reported frequencies. In urothelial carcinoma cell lines, the same genes as in tissues were frequently hypermethylated. SFRP1 hypermethylation was concordant with lack of expression. 5-Aza-deoxycytidine induced its reexpression in some lines, whereas additional treatment with a histone deacetylase inhibitor was required in others. Thus, epigenetic SFRP1 inactivation occurs in a graduated manner. In conclusion, markers of genome-wide hypomethylation seem optimally suited for urothelial carcinoma detection, whereas combinations of hypermethylation and hypomethylation assays hold promise for classification.

Differential DNA hypermethylation and hypomethylation signatures in colorectal cancer

Cancer cells are characterized by a generalized disruption of the DNA methylation pattern involving an overall decrease in the level of 5-methylcytosine together with regional hypermethylation of particular CpG islands. The extent of both DNA hypomethylation and hypermethylation in the tumor cell is likely to reflect distinctive biological and clinical features, although no studies have addressed its concurrent analysis until now. DNA methylation profiles in sporadic colorectal carcinomas, synchronous adenoma-carcinoma pairs and their matching normal mucosa were analyzed by using the amplification of inter-methylated sites (AIMS) method. A total of 208 AIMS generated sequences were tagged and evaluated for differential methylation. Global indices of hypermethylation and hypomethylation were calculated. All tumors displayed altered patterns of DNA methylation in reference to normal tissue. On average, 24% of the tagged sequences were differentially methylated in the tumor in regard to the normal pair with an overall prevalence of hypomethylations to hypermethylations. Carcinomas exhibited higher levels of hypermethylation than did adenomas but similar levels of hypomethylation. Indices of hypomethylation and hypermethylation showed independent correlations with patient's sex, tumor staging and specific gene hypermethylation. Hierarchical cluster analysis revealed two main patterns of DNA methylation that were associated to particular mutational spectra in the K-ras and the p53 genes and alternative correlates of hypomethylation and hypermethylation with survival. We conclude that DNA hypermethylation and hypomethylation are independent processes and appear to play different roles in colorectal tumor progression. Subgroups of colorectal tumors show specific genetic and epigenetic signatures and display distinctive correlates with overall survival.

Methylation profile and amplification of proto-oncogenes in rat pancreas induced with phytoestrogens.

Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 micrograms of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras proto-oncogene. No hypermethylation or hypomethylation was observed in the proto-oncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders.