Fernando Setien

Distinct DNA methylomes of newborns and centenarians

Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine—phosphate—guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.

A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition

Disruption of histone acetylation patterns is a common feature of cancer cells, but very little is known about its genetic basis. We have identified truncating mutations in one of the primary human histone deacetylases, HDAC2, in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. The presence of the HDAC2 frameshift mutation causes a loss of HDAC2 protein expression and enzymatic activity and renders these cells more resistant to the usual antiproliferative and proapoptotic effects of histone deacetylase inhibitors. As such drugs may serve as therapeutic agents for cancer, our findings support the use of HDAC2 mutational status in future pharmacogenetic treatment of these individuals.

Small molecule enoxacin is a cancer-specific growth inhibitor that acts by enhancing TAR RNA-binding protein 2-mediated microRNA processing

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their processing machineries also contributes to the development of human tumors. A common scenario for miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.

Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma

Sotos syndrome is an autosomal dominant condition characterized by overgrowth resulting in tall stature and macrocephaly, together with an increased risk of tumorigenesis. The disease is caused by loss-of-function mutations and deletions of the nuclear receptor SET domain containing protein-1 (NSD1) gene, which encodes a histone methyltransferase involved in chromatin regulation. However, despite its causal role in Sotos syndrome and the typical accelerated growth of these patients, little is known about the putative contribution of NSD1 to human sporadic malignancies. Here, we report that NSD1 function is abrogated in human neuroblastoma and glioma cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also demonstrate that the epigenetic inactivation of NSD1 in transformed cells leads to the specifically diminished methylation of the histone lysine residues H4-K20 and H3-K36. The described phenotype is also observed in Sotos syndrome patients with NSD1 genetic disruption. Expression microarray data from NSD1-depleted cells, followed by ChIP analysis, revealed that the oncogene MEIS1 is one of the main NSD1 targets in neuroblastoma. Furthermore, we show that the restoration of NSD1 expression induces tumor suppressor-like features, such as reduced colony formation density and inhibition of cellular growth. Screening a large collection of different tumor types revealed that NSD1 CpG island hypermethylation was a common event in neuroblastomas and gliomas. Most importantly, NSD1 hypermethylation was a predictor of poor outcome in high-risk neuroblastoma. These findings highlight the importance of NSD1 epigenetic inactivation in neuroblastoma and glioma that leads to a disrupted histone methylation landscape and might have a translational value as a prognostic marker.

EMP3, a Myelin-Related Gene Located in the Critical 19q13.3 Region, Is Epigenetically Silenced and Exhibits Features of a Candidate Tumor Suppressor in Glioma and Neuroblastoma

The presence of common genomic deletions in the 19q13 chromosomal region in neuroblastomas and gliomas strongly suggests the presence of a putative tumor suppressor gene for these neoplasms in this region that, despite much effort, has not yet been identified. In an attempt to address this issue, we compared the expression profile of 89 neuroblastoma tumors with that of benign ganglioneuromas by microarray analysis. Probe sets (637 of 62,839) were significantly down-regulated in neuroblastoma tumors, including, most importantly, a gene located at 19q13.3: the epithelial membrane protein 3 (EMP3), a myelin-related gene involved in cell proliferation and cell-cell interactions. We found that EMP3 undergoes hypermethylation-mediated transcriptional silencing in neuroblastoma and glioma cancer cell lines, whereas the use of the demethylating agent 5-aza-2-deoxycytidine restores EMP3 gene expression. Furthermore, the reintroduction of EMP3 into neuroblastoma cell lines displaying methylation-dependent silencing of EMP3 induces tumor suppressor-like features, such as reduced colony formation density and tumor growth in nude mouse xenograft models. Screening a large collection of human primary neuroblastomas (n = 116) and gliomas (n = 41), we observed that EMP3 CpG island hypermethylation was present in 24% and 39% of these tumor types, respectively. Furthermore, the detection of EMP3 hypermethylation in neuroblastoma could be clinically relevant because it was associated with poor survival after the first 2 years of onset of the disease (Kaplan-Meier; P = 0.03) and death of disease (Kendall tau, P = 0.03; r = 0.19). Thus, EMP3 is a good candidate for being the long-sought tumor suppressor gene located at 19q13 in gliomas and neuroblastomas.

Inactivation of the Lamin A/C Gene by CpG Island Promoter Hypermethylation in Hematologic Malignancies, and Its Association With Poor Survival in Nodal Diffuse Large B-Cell Lymphoma

PURPOSE Lamins support the nuclear envelope and provide anchorage sites for chromatin, but they are also involved in DNA synthesis, transcription, and apoptosis. Although the lack of expression of A-type lamins in lymphoma and leukemia has been reported, the mechanism was unknown. We investigated the possible role of CpG island hypermethylation in lamin A/C silencing and its prognostic relevance. PATIENTS AND METHODS The promoter CpG island methylation status of the lamin A/C gene, encoding the A-type lamins, was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction in human cancer cell lines (n = 74; from 17 tumor types), and primary leukemias (n = 60) and lymphomas (n = 80). Lamin A/C expression was determined by reverse-transcription polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence. RESULTS seven (50%) of 14 leukemia- and five (42%) of 13 lymphoma cell lines. The presence of hypermethylation was associated with the loss of gene expression while a demethylating agent restored expression. In primary malignancies, lamin A/C hypermethylation was present in 18% (nine of 50) of acute lymphoblastic leukemias and 34% (14 of 41) of nodal diffuse large B-cell lymphomas. The presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphomas correlated strongly with a decrease in failure-free survival (Kaplan-Meier, P = .0001) and overall survival (Kaplan-Meier, P = .0005). CONCLUSION Epigenetic silencing of the lamin A/C gene by CpG island promoter hypermethylation is responsible for the loss of expression of A-type lamins in leukemias and lymphomas. The finding that lamin A/C hypermethylation is associated with poor outcome in diffuse large B-cell lymphomas suggests important clinical implications.

Epigenetic inactivation of the circadian clock gene BMAL1 in hematologic malignancies.

Disruption of circadian rhythms, daily oscillations in biological processes that are regulated by an endogenous clock, has been linked to tumorigenesis. Normal and malignant tissues often show asynchronies in cell proliferation and metabolic rhythms. Cancer chronotherapy takes biological time into account to improve the therapy. However, alterations of the circadian clock machinery genes have rarely been reported in human cancer. Herein, we show that the BMAL1 gene, a core component of the circadian clock, is transcriptionally silenced by promoter CpG island hypermethylation in hematologic malignancies, such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas BMAL1 depletion by RNA interference in unmethylated cells enhances tumor growth. We also show that BMAL1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in association with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylation-associated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets, further enhancing the perturbed circadian rhythm of the malignant cells. These findings suggest that BMAL1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting the cellular circadian clock.

Whole-Exome Sequencing Identifies MDH2 as a New Familial Paraganglioma Gene

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Students t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.

The impact of MECP2 mutations in the expression patterns of Rett syndrome patients

Rett syndrome (RTT), the second most common cause of mental retardation in females, has been associated with mutations in MeCP2, the archetypical member of the methyl-CpG binding domain (MBD) family of proteins. MeCP2 additionally possesses a transcriptional repression domain (TRD). We have compared the gene expression profiles of RTT- and normal female-derived lymphoblastoid cells by using cDNA microarrays. Clustering analysis allowed the classification of RTT patients according to the localization of the MeCP2 mutation (MBD or TRD) and those with clinically diagnosed RTT but without detectable MeCP2 mutations. Numerous genes were observed to be overexpressed in RTT patients compared with control samples, including excellent candidate genes for neurodevelopmental disease. Chromatin immunoprecipitation analysis confirmed that binding of MeCP2 to corresponding promoter CpG islands was lost in RTT-derived cells harboring a mutation in the region of the MECP2 gene encoding the MBD. Bisulfite genomic sequencing demonstrated that the majority of MeCP2 binding occurred in DNA sequences with methylation-associated silencing. Most importantly, the finding that these genes are also methylated and bound by MeCP2 in neuron-related cells suggests a role in this neurodevelopmental disease. Our results provide new data of the underlying mechanisms of RTT and unveil novel targets of MeCP2-mediated gene repression.

Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of methyl-CpG-binding domain proteins

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5′-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein γ-parvin and the fibroblast growth factor 19, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.