Metastatic disease is the primary cause of death in cutaneous malignant melanoma (CMM) patients. To understand the mechanisms of CMM metastasis and identify potential predictive markers, we analyzed gene-expression profiles of 34 vertical growth phase melanoma cases using cDNA microarrays. All patients had a minimum follow-up of 36 months. Twenty-one cases developed nodal metastatic disease and 13 did not. Comparison of gene expression profiling of metastatic and nonmetastatic melanoma cases identified 243 genes with a >2-fold differential expression ratio and a false discovery rate of <0.2 (206 up-regulated and 37 down-regulated). This set of genes included molecules involved in cell cycle and apoptosis regulation, epithelial-mesenchymal transition (EMT), signal transduction, nucleic acid binding and transcription, protein synthesis and degradation, metabolism, and a specific group of melanoma-and neural-related proteins. Validation of these expression data in an independent series of melanomas using tissue microarrays confirmed that the expression of a set of proteins included in the EMT group (N-cadherin, osteopontin, and SPARC/osteonectin) were significantly associated with metastasis development. Our results suggest that EMT-related genes contribute to the promotion of the metastatic phenotype in primary CMM by supporting specific adhesive, invasive, and migratory properties. These data give a better understanding of the biology of this aggressive tumor and may provide new prognostic and patient stratification markers in addition to potential therapeutic targets. [Cancer Res 2007;67(7):3450-60]

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 tumorsuppressor 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 methylationdependent 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. DNA methylationW erner syndrome (WS) is an autosomal recessive disease characterized by premature aging and a high incidence of malignant neoplasms (1, 2). Mutations in the WS gene (WRN) are found in patients exhibiting the clinical symptoms of WS (3-5). The vast majority of WRN mutations result in loss of function of the WRN protein (6). The WRN protein has been demonstrated to possess helicase and exonuclease activities (7-9), and cultures of WS cells show increased chromosomal instability, with abundant deletions, reciprocal translocations, and inversions (10, 11).WRN belong to the RecQ family of helicases, which are highly conserved from bacteria to human, and whose members are thought to be essential caretakers of the genome (11,12). In addition to WRN, germline mutations of two other RecQ helicases, BLM in Bloom syndrome and RECQL4 in Rothmund-Thomson syndrome, are also associated with an elevated incidence of cancer (12). Because patients with WRN germline mutations develop a broad spectrum of epithelial and mesenchymal tumors, which is one of the main causes of their death before the age of 50, a tumorsuppressor function for WRN has been proposed. This putative role is also supported by a very high rate of loss of heterozygosity at the chrom...

The epsilon4 allele of the apolipoprotein E gene (APOE) has been associated with an increased risk of developing Alzheimer's disease (AD; refs 1,2). However, it is apparent that the APOEepsilon4 allele alone is neither necessary nor sufficient to cause the disease. We have recently found three new polymorphisms within the APOE transcriptional regulatory region (M.J.A. et al., manuscript submitted) and now establish an association between one of these polymorphisms (-491A/T) and dementia as observed in Alzheimer's disease, in two independent clinical populations. The results suggest that homozygosity of a common variant (-491A) is associated with increased risk for AD, and that this association is independent of APOEepsilon4 status. In vitro studies suggest that the -491A/T polymorphism may increase risk for AD by altering the level of ApoE protein expression.

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides 31017 to +406. Upon a population study, three new polymorphic sites (3491, 3427 and 3219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites 3491 and 3219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.z 1998 Federation of European Biochemical Societies.

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

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.

While the straightepsilon4 allele of apolipoprotein E ( APOE, gene; ApoE, protein) is widely accepted as a major genetic risk factor for the late onset form of Alzheimer's disease (AD), recent evidence points to variations in ApoE levels as another important factor. We have previously reported that a common variant in the regulatory region of APOE (-491A) is associated with risk for late onset AD. In this report we analyze the association of another APOE promoter polymorphism (-427T/C) with AD in two case-control clinical samples and demonstrate a correlation between APOE promoter transcriptional activity and risk for AD. The association studies show that the allelic variant (-427C) and the haplotype [-491A-427C] of the APOE promoter are associated with increased risk for AD. Study of the transcriptional activity of the common haplotypes defined by combination of the -491 and -427 alleles indicated that the risk for late onset AD positively correlates with transcriptional activity of the APOE gene, suggesting that increases in the local expression of ApoE could be responsible for the association of APOE promoter polymorphism with AD.

The -491 and -219 APOE promoter polymorphisms incur risk for AD in addition to risk associated with the APOE-epsilon4 allele, with age accentuating the effect of the -219 TT genotype. Because these polymorphisms appear to influence apoE levels, these results suggest that APOE expression is an important determinant of AD pathogenesis.