Promoter hypermethylation as a mechanism for Lamin A/C silencing in a subset of neuroblastoma cells.

Nuclear lamins support the nuclear envelope and provide anchorage sites for chromatin. They are involved in DNA synthesis, transcription, and replication. It has previously been reported that the lack of Lamin A/C expression in lymphoma and leukaemia is due to CpG island promoter hypermethylation. Here, we provide evidence that Lamin A/C is silenced via this mechanism in a subset of neuroblastoma cells. Moreover, Lamin A/C expression can be restored with a demethylating agent. Importantly, Lamin A/C reintroduction reduced cell growth kinetics and impaired migration, invasion, and anchorage-independent cell growth. Cytoskeletal restructuring was also induced. In addition, the introduction of lamin Δ50, known as Progerin, caused senescence in these neuroblastoma cells. These cells were stiffer and developed a cytoskeletal structure that differed from that observed upon Lamin A/C introduction. Of relevance, short hairpin RNA Lamin A/C depletion in unmethylated neuroblastoma cells enhanced the aforementioned tumour properties. A cytoskeletal structure similar to that observed in methylated cells was induced. Furthermore, atomic force microscopy revealed that Lamin A/C knockdown decreased cellular stiffness in the lamellar region. Finally, the bioinformatic analysis of a set of methylation arrays of neuroblastoma primary tumours showed that a group of patients (around 3%) gives a methylation signal in some of the CpG sites located within the Lamin A/C promoter region analysed by bisulphite sequencing PCR. These findings highlight the importance of Lamin A/C epigenetic inactivation for a subset of neuroblastomas, leading to enhanced tumour properties and cytoskeletal changes. Additionally, these findings may have treatment implications because tumour cells lacking Lamin A/C exhibit more aggressive behaviour.

A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies.

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality.

X inactivation and progenitor cancer cells.

In mammals, silencing of one of the two X chromosomes is necessary to achieve dosage compensation. The 17 kb non-coding RNA called Xist triggers X inactivation. Gene silencing by Xist can only be achieved in certain contexts such as in cells of the early embryo and in certain hematopoietic progenitors where silencing factors are present. Moreover, these epigenetic contexts are maintained in cancer progenitors in which SATB1 has been identified as a factor related to Xist-mediated chromosome silencing.

ConteXt of change--X inactivation and disease.

Epigenetic regulation is important for stable maintenance of cell identity. For continued function of organs and tissues, illegitimate changes in cell identity must be avoided. Failure to do so can trigger tumour development and disease. How epigenetic patterns are established during cell differentiation has been explored by studying model systems such as X inactivation. Mammals balance the X-linked gene dosage between the sexes by silencing of one of the two X chromosomes in females. This is initiated by expression of the non-coding X-inactive specific transcript (Xist) RNA and depends on specific cellular contexts, in which essential silencing factors are expressed. Normally X inactivation is initiated in early embryogenesis, but recent reports identified instances where Xist is expressed and can initiate gene repression. Here we describe the features that characterize the cellular permissivity to initiation of X inactivation and note that these can also occur in cancer cells and in specific haematopoietic progenitors. We propose that embryonic pathways for epigenetic regulation are re-established in adult progenitor cells and tumour cells. Understanding their reactivation will deepen our understanding of tumourigenesis and may be exploited for cancer therapy.

X inactivation and disease.

X inactivation is the mechanism by which mammals adjust the X-linked gene dosage between the sexes. The dosage difference between XX females and XY males is functionally equalized by silencing one of the two X chromosomes in female cells. This dosage-compensation mechanism is based on the long functional Xist RNA. Here, we review our understanding of dosage compensation and Xist function in the context of disease.

Cancer progenitors and epigenetic contexts: an Xisting connection.

In mammals, silencing of one of the two X chromosomes is necessary to achieve dosage compensation. The non coding RNA Xist triggers X inactivation. Gene silencing by Xist is only possible in certain developmental contexts that only exist in cells of the early embryo and specific hematopoietic progenitors. Critical silencing factors may only be present in these contexts giving an explanation of why Xist is not operative outside these contexts. It has been demonstrated that Xist is functional in tumor cells, where SATB1 was identified as the first silencing factor for Xist mediated chromosome silencing.

SATB1 defines the developmental context for gene silencing by Xist in lymphoma and embryonic cells.

The noncoding Xist RNA triggers silencing of one of the two female X chromosomes during X inactivation in mammals. Gene silencing by Xist is restricted to a special developmental context in early embryos and specific hematopoietic precursors. Here, we show that Xist can initiate silencing in a lymphoma model. We identify the special AT-rich binding protein SATB1 as an essential silencing factor. Loss of SATB1 in tumor cells abrogates the silencing function of Xist. In lymphocytes Xist localizes along SATB1-organized chromatin and SATB1 and Xist influence each other's pattern of localization. SATB1 and its homolog SATB2 are expressed during the initiation window for X inactivation in ES cells. Importantly, viral expression of SATB1 or SATB2 enables gene silencing by Xist in embryonic fibroblasts, which normally do not provide an initiation context. Thus, our data establish SATB1 as a crucial silencing factor contributing to the initiation of X inactivation.

Cross-talk between aging and cancer: the epigenetic language.

The risk of having cancer increases with age probably because progenitor cells from mature organisms accumulate enough molecular lesions to evade the homeostatic control of their tissular contexts. Molecular lesions can be genetic (mutations, deletions, or translocations) and/or epigenetic. Epigenetic signaling, including DNA methylation and histone modification, is essential for normal development and becomes altered during Aging and by cancer. Several epigenetic alterations, such as global hypomethylation and CpG island hypermethylation, are progressively accumulated during Aging and directly contribute to cell transformation. Intriguingly, others, such as those involved in the control of telomere length and several epigenetic enzymes belonging to the family of nicotinamide adenine dinucleotide (NAD)(+) dependent deacetylases known as sirtuins, exhibit a well-defined progression during Aging that is dramatically reverted in transformed cells. We discuss the biological significance of both groups of epigenetic modifications in terms of their relative contribution to ontogenic development, senescence, and cell proliferation.

A new molecular model of cellular aging based on Werner syndrome.

In the Hayflick model, a decrease in the number of cells capable of undergoing proliferation constitutes the main criterion of cellular aging and is closely linked to organismal aging. Evidence suggests that a reduction of DNA replication capacity or a failure in the regulation systems of DNA replication occurs in aging cells, which leads to cellular replicative senescence. DNA replication depends on two parameters: the number of active replicons and the rate of chain elongation. Epigenetic parameters, in particular methylation, would be able to, either directly or indirectly, regulate replication origin activity of normal mammalian cells, as well as subsequent DNA replication. Werner syndrome (WS) is an autosomal recessive disorder that results in premature aging and is considered to be a model system for the study of cellular senescence and aging. WRN could involve DNA replication initiation, replication foci establishment, and the resolution of stalled replication forks during replication. In this paper, a molecular model of in vitro cellular aging is presented in which changes in DNA methylation, in particular, global hypomethylation related to methyltransferase Dnmt1 downregulation, and specific hypermethylation related to methyltransferase Dnmt3b upregulation as seen during cellular aging, could be responsible for the inactivation of replication origins or foci and the subsequent documented reduction in DNA replication capacity and increased mutations that are observed in senescent cells. Thus, Werner syndrome cells could be mimicking what is observed in normal aging in an accelerated form.

Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer.

Werner syndrome (WS) is an inherited disorder characterized by premature onset of aging, genomic instability, and increased cancer incidence. The disease is caused by loss of function mutations of the WRN gene, a RecQ family member with both helicase and exonuclease activities. However, despite its putative tumor-suppressor function, little is known about the contribution of WRN to human sporadic malignancies. Here, we report that WRN function is abrogated in human cancer cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also show that, at the biochemical and cellular levels, the epigenetic inactivation of WRN leads to the loss of WRN-associated exonuclease activity and increased chromosomal instability and apoptosis induced by topoisomerase inhibitors. The described phenotype is reversed by the use of a DNA-demethylating agent or by the reintroduction of WRN into cancer cells displaying methylation-dependent silencing of WRN. Furthermore, the restoration of WRN expression induces tumor-suppressor-like features, such as reduced colony formation density and inhibition of tumor growth in nude mouse xenograft models. Screening a large collection of human primary tumors (n = 630) from different cell types revealed that WRN CpG island hypermethylation was a common event in epithelial and mesenchymal tumorigenesis. Most importantly, WRN hypermethylation in colorectal tumors was a predictor of good clinical response to the camptothecin analogue irinotecan, a topoisomerase inhibitor commonly used in the clinical setting for the treatment of this tumor type. These findings highlight the importance of WRN epigenetic inactivation in human cancer, leading to enhanced chromosomal instability and hypersensitivity to chemotherapeutic drugs.

Cyclooxygenase 2 expression in colorectal cancer with DNA mismatch repair deficiency.

BACKGROUND: Cyclooxygenase 2 (COX-2) overexpression is a frequent but not universal event in colorectal cancer. It has been suggested that COX-2 protein expression is reduced in colorectal cancer with a defective mismatch repair (MMR) system, a phenomenon commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC) but also present in up to 15% of sporadic tumors. AIM: To assess COX-2 expression in a large series of fully characterized colorectal cancer patients with respect to the MMR system and to dissect the mechanisms responsible for altered COX-2 expression in this setting. PATIENTS AND METHODS: MMR-deficient colorectal cancer were identified in a nationwide, prospective, multicenter study (EPICOLON project). Control MMR-proficient colorectal cancer patients were randomly selected. COX-2 expression was evaluated by immunohistochemistry. Personal and familial characteristics, as well as MSH2/MLH1 expression and germ line mutations, were evaluated. RESULTS: One hundred fifty-three patients, 46 with MMR deficiency and 107 with MMR proficiency, were included in the analysis. Overall, tumor COX-2 overexpression was observed in 107 patients (70%). COX-2 overexpression was observed in 85 patients (79%) with a MMR-proficient system, but only in 22 patients (48%) with a MMR-deficient colorectal cancer (P < 0.001). The lack of COX-2 overexpression was independently associated with a MMR-deficient system (odds ratio, 3.89; 95% confidence interval, 1.78-8.51; P = 0.001) and a poor degree of differentiation (OR, 3.83; 95% CI, 1.30-11.31; P = 0.015). In the subset of patients with a MMR-deficient colorectal cancer, lack of COX-2 overexpression correlated with a poor degree of differentiation, no fulfillment of Amsterdam II criteria, absence of MSH2/MLH1 germ line mutations, presence of tumor MSH2 expression, and lack of tumor MLH1 expression. CpG island promoter hypermethylation of COX2 was observed in 6 of 18 (33%) tumors lacking COX-2 expression in comparison with 2 of 28 (7%) tumors expressing this protein (P = 0.04). CONCLUSIONS: Up to half of MMR-deficient colorectal cancer do not show COX-2 overexpression, a fact observed almost exclusively in patients with sporadic forms. COX2 hypermethylation seems to be responsible for gene silencing in one third of them. These results suggest the potential utility of nonsteroidal anti-inflammatory drugs in HNPCC chemoprevention and may explain the lack of response of this approach in some sporadic tumors.

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.

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.

Detection of lymph node micrometastases by gene promoter hypermethylation in samples obtained by endosonography- guided fine-needle aspiration biopsy.

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has become a fundamental procedure for gastrointestinal and lung cancer staging. However, there is growing evidence that micrometastases are present in lymph nodes, which cannot be detected with standard pathological methods. The aim of this study was to evaluate whether hypermethylation gene promoter analysis was feasible on samples obtained by EUS-FNA from lymph nodes, as well as to establish the usefulness of this strategy for the detection of micrometastases in patients with gastrointestinal and non-small cell lung cancer. Suspicious lymph nodes based on EUS findings from consecutive patients with esophageal, gastric, rectal, and non-small cell lung cancer were sampled by EUS-FNA. Hypermethylation analysis of the MGMT, p16(INK4a), and p14(ARF) gene promoter CpG islands were performed by methylation-specific PCR. Effectiveness of conventional cytology, methylation analysis, and their combination were established with respect to the definitive diagnosis. Twenty-seven patients were included, thus representing a total of 42 lymph nodes (esophageal cancer, n = 11; rectal cancer, n = 7; gastric cancer, n = 3; and lung cancer, n = 21). According to definitive diagnosis, 21 (50%) corresponded to metastatic lymph nodes. Sensitivity, specificity, and overall accuracy of conventional cytology were 76%, 100%, and 88%, respectively, whereas the corresponding values for the methylation analysis were 81%, 67%, and 74%, respectively. Combination of both techniques increased sensitivity (90%) but decreased specificity (67%) with respect to conventional cytology. In conclusion, it is feasible to detect occult neoplastic cells in EUS-FNA samples by hypermethylation gene promoter analysis. Moreover, addition of methylation analysis to conventional cytology may increase its sensitivity at the expenses of a decrease in its specificity.