CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Nicotinamide N-methyltransferase (NNMT) regulates the glucocorticoid signaling pathway during the early phase of adipogenesis.

Obesity is associated with adipose tissue dysfunction through the differentiation and expansion of pre-adipocytes to adipocytes (hyperplasia) and/or increases in size of pre-existing adipocytes (hypertrophy). A cascade of transcriptional events coordinates the differentiation of pre-adipocytes into fully differentiated adipocytes; the process of adipogenesis. Although nicotinamide N-methyltransferase (NNMT) has been associated with obesity, how NNMT is regulated during adipogenesis, and the underlying regulatory mechanisms, remain undefined. In present study we used genetic and pharmacological approaches to elucidate the molecular signals driving NNMT activation and its role during adipogenesis. Firstly, we demonstrated that during the early phase of adipocyte differentiation NNMT is transactivated by CCAAT/Enhancer Binding Protein beta (CEBPB) in response to glucocorticoid (GC) induction. We found that Nnmt knockout, using CRISPR/Cas9 approach, impaired terminal adipogenesis by influencing the timing of cellular commitment and cell cycle exit during mitotic clonal expansion, as demonstrated by cell cycle analysis and RNA sequencing experiments. Biochemical and computational methods showed that a novel small molecule, called CC-410, stably binds to and highly specifically inhibits NNMT. CC-410 was, therefore, used to modulate protein activity during pre-adipocyte differentiation stages, demonstrating that, in line with the genetic approach, chemical inhibition of NNMT at the early stages of adipogenesis impairs terminal differentiation by deregulating the GC network. These congruent results conclusively demonstrate that NNMT is a key component of the GC-CEBP axis during the early stages of adipogenesis and could be a potential therapeutic target for both early-onset obesity and glucocorticoid-induced obesity.

Epigenetic Profiling and Response to CD19 Chimeric Antigen Receptor T-Cell Therapy in B-Cell Malignancies.

BACKGROUND: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. METHODS: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. RESULTS: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). CONCLUSIONS: We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy.

Epigenetic Deregulation of the Histone Methyltransferase KMT5B Contributes to Malignant Transformation in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor in adulthood. Epigenetic mechanisms are known to play a key role in GBM although the involvement of histone methyltransferase KMT5B and its mark H4K20me2 has remained largely unexplored. The present study shows that DNA hypermethylation and loss of DNA hydroxymethylation is associated with KMT5B downregulation and genome-wide reduction of H4K20me2 levels in a set of human GBM samples and cell lines as compared with non-tumoral specimens. Ectopic overexpression of KMT5B induced tumor suppressor-like features in vitro and in a mouse tumor xenograft model, as well as changes in the expression of several glioblastoma-related genes. H4K20me2 enrichment was found immediately upstream of the promoter regions of a subset of deregulated genes, thus suggesting a possible role for KMT5B in GBM through the epigenetic modulation of key target cancer genes.

Conservation of Aging and Cancer Epigenetic Signatures across Human and Mouse.

Aging and cancer are two interrelated processes, with aging being a major risk factor for the development of cancer. Parallel epigenetic alterations have been described for both, although differences, especially within the DNA hypomethylation scenario, have also been recently reported. Although many of these observations arise from the use of mouse models, there is a lack of systematic comparisons of human and mouse epigenetic patterns in the context of disease. However, such comparisons are significant as they allow to establish the extent to which some of the observed similarities or differences arise from pre-existing species-specific epigenetic traits. Here, we have used reduced representation bisulfite sequencing to profile the brain methylomes of young and old, tumoral and nontumoral brain samples from human and mouse. We first characterized the baseline epigenomic patterns of the species and subsequently focused on the DNA methylation alterations associated with cancer and aging. Next, we described the functional genomic and epigenomic context associated with the alterations, and finally, we integrated our data to study interspecies DNA methylation levels at orthologous CpG sites. Globally, we found considerable differences between the characteristics of DNA methylation alterations in cancer and aging in both species. Moreover, we describe robust evidence for the conservation of the specific cancer and aging epigenomic signatures in human and mouse. Our observations point toward the preservation of the functional consequences of these alterations at multiple levels of genomic regulation. Finally, our analyses reveal a role for the genomic context in explaining disease- and species-specific epigenetic traits.

Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19.

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

Epigenetic downregulation of TET3 reduces genome-wide 5hmC levels and promotes glioblastoma tumorigenesis.

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

Deregulation of the imprinted DLK1-DIO3 locus ncRNAs is associated with replicative senescence of human adipose-derived stem cells.

BACKGROUND: Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential. AIM AND SCOPE: A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures. METHODS: We compared hADSC cultures at short (PS) and prolonged (PL) passages, both in standard and low [O2] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster. RESULTS: Comparison of hADSCs cultured at PL or PS surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in PL cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O2], was also significantly higher in PL than in PS passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions. CONCLUSION: A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice.

Epigenetic dysregulation of TET2 in human glioblastoma.

Ten-eleven translocation (TET) enzymes are frequently deregulated in cancer, but the underlying molecular mechanisms are still poorly understood. Here we report that TET2 shows frequent epigenetic alterations in human glioblastoma including DNA hypermethylation and hypo-hydroxymethylation, as well as loss of histone acetylation. Ectopic overexpression of TET2 regulated neural differentiation in glioblastoma cell lines and impaired tumor growth. Our results suggest that epigenetic dysregulation of TET2 plays a role in human glioblastoma.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Epigenome-wide analysis reveals specific DNA hypermethylation of T cells during human hematopoietic differentiation.

AIM: Epigenetic regulation plays an important role in cellular development and differentiation. A detailed map of the DNA methylation dynamics that occur during cell differentiation would contribute to decipher the molecular networks governing cell fate commitment. METHODS: Illumina MethylationEPIC BeadChip platform was used to describe the genome-wide DNA methylation changes observed throughout hematopoietic maturation by analyzing multiple myeloid and lymphoid hematopoietic cell types. RESULTS: We identified a plethora of DNA methylation changes that occur during human hematopoietic differentiation. We observed that T lymphocytes display substantial enhancement of de novo CpG hypermethylation as compared with other hematopoietic cell populations. T-cell-specific hypermethylated regions were strongly associated with open chromatin marks and enhancer elements, as well as binding sites of specific key transcription factors involved in hematopoietic differentiation, such as PU.1 and TAL1. CONCLUSION: These results provide novel insights into the role of DNA methylation at enhancer elements in T-cell development.

DNA methylation changes in human lung epithelia cells exposed to multi-walled carbon nanotubes.

Humans are increasingly exposed to nanoparticles and, although many of their physiological effects have been described, the molecular mechanisms underlying them are still largely unknown. The present study aimed to determine the possible role of certain epigenetic mechanisms in the cellular response of human lung epithelial cells that are triggered by long-term exposure to titanium dioxide nanoparticles (TiO2NPs) and multi-walled carbon nanotubes (MWCNTs). The results showed that exposure to TiO2NPs had only minor effects on genome-wide DNA methylation. However, we identified 755 CpG sites showing consistent DNA hypomethylation in cells exposed to MWCNTs. These sites were mainly located at low density CpG regions and enhancers, and very frequently on the X chromosome. Our results thus suggest that long-term MWCNT exposure may have important effects on the epigenome.

Age-associated hydroxymethylation in human bone-marrow mesenchymal stem cells.

BACKGROUND: Age-associated changes in genomic DNA methylation have been primarily attributed to 5-methylcytosine (5mC). However, the recent discovery of 5-hydroxymethylcytosine (5hmC) suggests that this epigenetic mark might also play a role in the process. METHODS: Here, we analyzed the genome-wide profile of 5hmc in mesenchymal stem cells (MSCs) obtained from bone-marrow donors, aged 2-89 years. RESULTS: We identified 10,685 frequently hydroxymethylated CpG sites in MSCs that were, as in other cell types, significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypo-hydroxymethylated CpG sites in the MSCs obtained from older individuals. CONCLUSIONS: DNA hyper-hydroxymethylation in the advanced-age group was associated with loss of 5mC, which suggests that, at specific CpG sites, this epigenetic modification might play a role in DNA methylation changes during lifetime. Since bone-marrow MSCs have many clinical applications, and the fact that the epigenomic alterations in this cell type associated with aging identified in this study could have associated functional effects, the age of donors should be taken into account in clinical settings.

HERG1A potassium channel is the predominant isoform in head and neck squamous cell carcinomas: evidence for regulation by epigenetic mechanisms.

Evidences indicate that HERG1 voltage-gated potassium channel is frequently aberrantly expressed in various cancers including head and neck squamous cell carcinomas (HNSCC), representing a clinically and biologically relevant feature during disease progression and a potential therapeutic target. The present study further and significantly extends these data investigating for the first time the expression and individual contribution of HERG1 isoforms, their clinical significance during disease progression and also the underlying regulatory mechanisms. Analysis of HERG1A and HERG1B expression using real-time RT-PCR consistently showed that HERG1A is the predominant isoform in ten HNSCC-derived cell lines tested. HERG2 and HERG3 were also detected. Immunohistochemical analysis of HERG1A expression on 133 HNSCC specimens demonstrated that HERG1A expression increased during tumour progression and correlated significantly with reduced disease-specific survival. Furthermore, our study provides original evidence supporting the involvement of histone acetylation (i.e. H3Ac and H4K16Ac activating marks) in the regulation of HERG1 expression in HNSCC. Interestingly, this mechanism was also found to regulate the expression of another oncogenic channel (Kv3.4) as well as HERG2 and HERG3. These data demonstrate that HERG1A is the predominant and disease-relevant isoform in HNSCC progression, while histone acetylation emerges as an important regulatory mechanism underlying Kv gene expression.

DNA methylation signatures identify biologically distinct thyroid cancer subtypes.

OBJECTIVE: The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer. RESEARCH DESIGN AND METHODS: We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors. RESULTS: We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer. CONCLUSIONS: Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors.

A DNA methylation signature associated with the epigenetic repression of glycine N-methyltransferase in human hepatocellular carcinoma.

The basic mechanisms underlying promoter DNA hypermethylation in cancer are still largely unknown. It has been proposed that the levels of the methyl donor group in DNA methylation reactions, S-adenosylmethionine (SAMe), might be involved. SAMe levels depend on the glycine-N-methyltransferase (GNMT), a one-carbon group methyltransferase, which catalyzes the conversion of SAMe to S-adenosylhomocysteine in hepatic cells. GNMT has been proposed to display tumor suppressor activity and to be frequently repressed in hepatocellular carcinoma (HCC). In this study, we show that GNMT shows aberrant DNA hypermethylation in some HCC cell lines and primary tumors (20 %). GNMT hypermethylation could contribute to gene repression and its restoration in cell lines displaying hypermethylation-reduced tumor growth in vitro. In agreement, human primary tumors expressing GNMT were of smaller size than tumors showing GNMT hypermethylation. Genome-wide analyses of gene promoter methylation identified 277 genes whose aberrant methylation in HCC was associated with GNMT methylation/expression. The findings in this manuscript indicate that DNA hypermethylation plays an important role in the repression of GNMT in HCC and that loss of GNMT in human HCC could promote the establishment of aberrant DNA methylation patterns at specific gene promoters.

Immune-dependent and independent antitumor activity of GM-CSF aberrantly expressed by mouse and human colorectal tumors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2) is a cytokine produced in the hematologic compartment that may enhance antitumor immune responses, mainly by activation of dendritic cells. Here, we show that more than one-third of human colorectal tumors exhibit aberrant DNA demethylation of the GM-CSF promoter and overexpress the cytokine. Mouse engraftment experiments with autologous and homologous colon tumors engineered to repress the ectopic secretion of GM-CSF revealed the tumor-secreted GM-CSF to have an immune-associated antitumor effect. Unexpectedly, an immune-independent antitumor effect was observed that depended on the ectopic expression of GM-CSF receptor subunits by tumors. Cancer cells expressing GM-CSF and its receptor did not develop into tumors when autografted into immunocompetent mice. Similarly, 100% of the patients with human colon tumors that overexpressed GM-CSF and its receptor subunits survived at least 5 years after diagnosis. These data suggest that expression of GM-CSF and its receptor subunits by colon tumors may be a useful marker for prognosis as well as for patient stratification in cancer immunotherapy.

Frequent aberrant expression of the human ether à go-go (hEAG1) potassium channel in head and neck cancer: pathobiological mechanisms and clinical implications.

Compelling evidence indicates that the human ether-à-go-go voltage-gated potassium channels (hEAG1) may represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression of hEAG1 potassium channel subunit in both primary tumors and HNSCC-derived cell lines to ascertain its clinical and biological role in tumor progression. Our findings demonstrate that hEAG1 is frequently aberrantly expressed in a high percentage of primary tumors (83 %, 45/54 cases) and HNSCC-derived cell lines (83 %, 10/12 cell lines). hEAG1 expression increased during HNSCC progression and was more frequent in advanced tumors. Strikingly, hEAG1 expression was also detected in a notable proportion (39 %, 17/44 cases) of patient-matched normal adjacent mucosa, whereas no expression was detected in normal epithelia from non-oncologic patients without exposure to tobacco carcinogens. In an attempt to identify the underlying mechanisms of aberrant hEAG1 expression in HNSCC, we found that hEAG1 gene copy gain occurred at a low frequency (15 %, 13/88 cases) in primary tumors but was not observed in early stages of HNSCC tumorigenesis. Furthermore, this study provides original evidence supporting the involvement of histone acetylation (i.e., H3Ac and H4K16Ac activating marks) in the regulation of hEAG1 expression in HNSCC. In addition, functional studies in HNSCC cells further revealed that hEAG1 expression is a biologically relevant feature that promotes cell proliferation and invasion, although independently of its ion-conducting function. Our findings strongly support the notion that hEAG1 may represent a promising candidate as tumor marker and membrane therapeutic target for HNSCC treatment.

A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer.

Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.