Faculty Bibliography

PURPOSE/OBJECTIVE:Polyamines are important regulators of cell growth and death. The polyamine modulated factor-1 (PMF-1) is involved in polyamine homeostasis. After identifying an enriched CpG island encompassing the PMF1 promoter, we aimed at evaluating the clinical relevance of PMF1 methylation in bladder cancer. EXPERIMENTAL DESIGN/METHODS:The epigenetic silencing of PMF1 by hypermethylation was tested in bladder cancer cells (n = 11) after azacytidine treatment. PMF1 methylation status was evaluated in 507 bladder tumors and 118 urinary specimens of bladder cancer patients and controls. PMF1 protein expression was analyzed by immunohistochemistry on tissue arrays containing bladder tumors for which PMF1 methylation was assessed (n = 218). RESULTS:PMF1 hypermethylation was associated with gene expression loss, being restored in vitro by a demethylating agent. An initial set of 101 primary frozen bladder tumors served to identify PMF1 hypermethylation in 88.1% of the cases. An independent set of 406 paraffin-embedded tumors also revealed a high PMF1 methylation rate (77.6%). PMF1 methylation was significantly associated with increasing stage (P = 0.025). Immunohistochemical analyses revealed that PMF1 methylation was associated with cytoplasmic PMF1 expression loss (P = 0.032). PMF1 protein expression patterns were significantly associated with stage (P < 0.001), grade (P < 0.001), and poor overall survival using univariate (P < 0.001) and multivariate (P = 0.011) analyses. Moreover, PMF1 methylation in urinary specimens distinguished bladder cancer patients from controls (area under the curve = 0.800). CONCLUSION/CONCLUSIONS:PMF1 was identified to be epigenetically modified in bladder cancer. The association of PMF1 methylation with tumor progression and its diagnostic ability using urinary specimens support including PMF1 assessment for the clinical management of bladder cancer patients.

Epigenetic deregulation has become a major topic in biomedical research. Most of the information currently available in the field comes from the study of epigenetic alterations in cancer, particularly in haematological malignancies. The recognition of the epigenetic component of autoimmune diseases in conjunction with the existence of common genes underpinning pathways that are crucial to the development of autoimmunity and haematological cancers, facilitates interaction between these two areas of research. Here, we examine what is presently known from epigenetic studies of autoimmune diseases, how epigenetic alterations in haematological malignancies can be used to better understand mechanisms of epigenetic deregulation and how they might be used in epigenetic therapy to treat autoimmune diseases.

Germline mutations in the tumour suppressor APC cause familial adenomatous polyposis (FAP), and somatic mutations are common in sporadic colorectal cancers (CRCs). Hypermethylation of APC promoter 1A has been reported in a substantial proportion of sporadic CRCs and may cause transcriptional silencing. Methylation has been proposed as an alternative to mutation or loss of heterozygosity as a mechanism of gene inactivation. However, the significance of APC methylation has remained unclear, because it has not previously been related to the presence of mono- or bi-allelic mutations at APC. We examined 103 FAP adenomas, 11 attenuated FAP adenomas, 31 sporadic CRCs and 30 CRC cell lines, all with known germline and/or somatic APC mutations. Overall, APC promoter 1A methylation was detected in 27-45% of colorectal tumours and cell lines, but generally not in histologically normal colorectum. In contrast to previous reports, methylation was detected in similar proportions of FAP/AFAP and sporadic CRCs. Importantly, methylation was independent of the presence, number and positions of APC mutations and was not associated with the CpG island methylator phenotype. Methylation resulted in a decrease or loss of 1A isoform mRNA and reduced total APC transcript levels, although expression was retained from promoter 1B. However, neither APC protein levels, nor transcription of a panel of Wnt target genes was associated with methylation status. Our data suggest that APC promoter 1A hypermethylation may influence APC expression levels in a subtle fashion, but methylation does not result in complete gene inactivation or act as a 'second hit'.

Hypomethylation of repeated elements in the genome is a common feature of human cancer, however, the direct consequences of this epigenetic defect for cancer biology are still largely unknown. Telomeres are specialized chromatin structures at the ends of eukaryotic chromosomes formed by tandem repeats of G-rich sequences and associated proteins, which have an essential role in chromosome end protection and genomic stability. Telomeric DNA repeats cannot be methylated, however, the adjacent subtelomeric DNA is heavily methylated in humans. Here, we show that the methylation status of subtelomeric DNA repeats negatively correlates with telomere length and telomere recombination in a large panel of human cancer cell lines. These findings suggest that tumor telomere length and integrity can be influenced by epigenetic factors. Finally, we show that treatment of human cancer cell lines with demethylating drugs results in hypomethylation of subtelomeric repeats and increased telomere recombination, which in turn may facilitate telomere elongation. All together, these findings suggest that tumor telomere length and integrity can be influenced by the epigenetic status of cancer cells.

Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

The importance of epigenetic processes in the development of cancer is clear. The study of epigenetics is therefore bound to contribute to the improvement of human health. Aberrations in DNA methylation, post-translational modifications of histones, chromatin remodeling and microRNAs patterns are the main epigenetic alterations, and these are associated with tumorigenesis. Epigenetic technologies in cancer studies are helping increase the number of cancer candidate genes and allow us to examine changes in 5-methylcytosine DNA and histone modifications at a genome-wide level. In fact, all the various cellular pathways contributing to the neoplastic phenotype are affected by epigenetic genes in cancer. They are being explored as biomarkers in clinical use for early detection of disease, tumor classification and response to treatment with classical chemotherapy agents, target compounds and epigenetic drugs. Encouraging results have been obtained with histone deacetylase and DNA methyltransferase inhibitors, leading the US Food and Drug Administration to approve several of them for the treatment of hematological malignancies and lymphoproliferative disorders, such as myelodysplastic syndrome and cutaneous lymphoma. However, many tasks remains to be done, such as the clinical validation of epigenetic biomarkers to allow the accurate prediction of the outcome of cancer patients and their potential chemosensitivity to current pharmacological treatments.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies (n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.