Faculty Bibliography

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis.

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.

LKB1, a tumor-suppressor gene that codifies for a serine/threonine kinase, is mutated in the germ-line of patients affected with the Peutz-Jeghers syndrome (PJS), which have an increased incidence of several cancers including gastrointestinal, pancreatic and lung carcinomas. Regarding tumors arising in non-PJS patients, we recently observed that at least one-third of lung adenocarcinomas (LADs) harbor somatic LKB1 gene mutations, supporting a role for LKB1 in the origin of some sporadic tumors. To characterize the pattern of LKB1 mutations in LADs further, we first screened for LKB1 gene alterations (gene mutations, promoter hypermethylation and homozygous deletions) in 19 LADs and, in agreement with our previous data, five of them (26%) were shown to harbor mutations, all of which gave rise to a truncated protein. Recent reports demonstrate that LKB1 is able to suppress cell growth, but little is known about the specific mechanism by which it functions. To further our understanding of LKB1 function, we analysed global expression in lung primary tumors using cDNA microarrays to identify LKB1-specific variations in gene expression. In all, 34 transcripts, 24 of which corresponded to known genes, differed significantly between tumors with and without LKB1 gene alterations. Among the most remarkable findings was deregulation of transcripts involved in signal transduction (e.g. FRAP1/mTOR, ARAF1 and ROCK2), cytoskeleton (e.g. MPP1), transcription factors (e.g. MEIS2, ATF5), metabolism of AMP (AMPD3 and APRT) and ubiquitinization (e.g. USP16 and UBE2L3). Real-time quantitative RT-PCR on 15 tumors confirmed the upregulation of the homeobox MEIS2 and of the AMP-metabolism AMPD3 transcripts in LKB1-mutant tumors. In addition, immunohistochemistry in 10 of the lung tumors showed the absence of phosphorylated FRAP1/mTOR protein in LKB1-mutant tumors, indicating that LKB1 mutations do not lead to FRAP1/mTOR protein kinase activation. In conclusion, our results reveal that several important factors contribute to LKB1-mediated carcinogenesis in LADs, confirming previous observations and identifying new putative pathways that should help to elucidate the biological role of LKB1.

The transcription factor activator protein-2alpha (AP-2alpha) has recently been implicated as a tumor suppressor protein that can be lost during tumor progression and that exhibits growth-inhibitory properties when overexpressed in cancer cell lines. We now demonstrate that hypermethylation of a discrete 5' region within a promoter CpG island of the gene is associated in breast cancer with the loss of AP-2alpha expression. Multiple CpG sites within the island become hypermethylated during breast cancer evolution. However, only hypermethylation of the most CpG-rich region, a small, approximately 300-bp area at the 3' end of exon 1, fully distinguishes neoplastic from normal breast tissue and correlates with transcriptional silencing. In cell culture, silenced AP-2alpha, associated with exon 1 hypermethylation, is re-expressed by 5-aza-2'deoxycytidine resulting in the restoration of a functional DNA sequence-specific binding protein. In vivo, as detected by a very sensitive nested PCR approach, methylation of the discrete AP-2alpha exon 1 region does not occur in normal breast epithelium and occurs in only 3 (16%) of 19 ductal carcinoma in situ (DCIS) lesions, but is present in 12 (75%) of 16 invasive breast tumors (P < 0.001; DCIS versus invasive cancers). Tumors unmethylated for this region expressed AP-2alpha protein throughout, whereas tumors with hypermethylation showed large areas of loss. Our studies then determine that hypermethylation of a small region of a CpG island correlates with silencing of AP-2alpha in breast cancer and suggest that inactivation of this gene could be a factor in, and a useful marker for, the progression of DCIS lesions.

Microsatellite instability (MSI) defines a specific type of genetic instability. Although consensus diagnostic criteria for MSI definition in colorectal cancer have been established, their utility in other tumor types remain to be proven. Previously we developed a mathematical model for MSI definition in colorectal cancer. The aim of this study was to establish diagnostic criteria for MSI evaluation in human gastric cancer. We designed an algorithm for the efficient characterization of MSI and used it to analyze data on 7 microsatellite markers in 35 gastric carcinomas. Theoretical models considering 1, 2, or 3 populations were tested against the data collected. Also, hypermethylation of hMLH1 gene promoter and hMLH1 protein expression were studied. The observed frequencies of MSI in our series of samples best fit a 2-population model: stable and unstable, defined by instability in 2 or more of a minimum of 7 markers analyzed. MSI was observed in 29% of the tumors. Misclassification rate was <4% when any 7 loci were analyzed. MSI(+) tumors inversely associated with p53 protein overexpression. A good correlation between hMLH1 status (either protein or promoter hypermethylation) and MSI classification was observed. We have developed a simple, sensitive, and specific approach to assess the presence of MSI in gastric cancer that may have clinical applications.

BACKGROUND AND OBJECTIVES/OBJECTIVE:Aberrant promoter methylation targets CpG islands causing gene silencing. We explored aberrant promoter methylation of genes potentially involved in B-cell malignancies and encoding proteins implicated in DNA repair (O6-methylguanine-DNA methyltransferase, MGMT), detoxification of environmental xenobiotics (glutathione S-transferase P1, GSTP1), apoptosis regulation (death associated protein kinase, DAP-k and caspase 8, CASP8) and cell cycle control (p73). DESIGN AND METHODS/METHODS:Three hundred and seventeen B-cell malignancies were investigated by methylation-specific polymerase chain reaction (MSP) of MGMT, GSTP1, DAP-k, CASP8 and p73 genes. In selected cases, MSP results were matched to protein expression studies by immunohistochemistry or Western blotting. RESULTS:DAP-k promoter methylation occurred at highest frequency in follicular lymphoma (85.0%) and MALT-lymphoma (72.2%). MGMT methylation targeted both precursor B-cell neoplasia (23.8%) and mature B-cell tumors (27.6%). GSTP1 methylation was commonest in hairy cell leukemia (75.0%), follicular lymphoma (55.5%), Burkitt s lymphoma (52.0%), and MALT lymphoma (50.0%). Methylation of p73 and CASP8 was rare or absent. DAP-k and MGMT methylation caused absent protein expression. INTERPRETATION AND CONCLUSIONS/CONCLUSIONS:Methylation of MGMT, DAP-k and GSTP1 represents a major pathogenetic event in several B-cell malignancies. In follicular lymphoma and MALT lymphoma, frequent inactivation of the apoptosis extrinsic pathway through DAP-k methylation may reinforce the survival advantage already conferred by deregulation of the intrinsic apoptotic pathway. Inactivation of GSTP1 in gastric MALT lymphoma represents an additional mechanism favoring accumulation of reactive oxygen species and lymphomagenesis. Finally, the frequency of GSTP1 aberrant methylation in diffuse large B-cell lymphoma prompts studies aimed at verifying the prognostic impact of this epigenetic lesion in these lymphomas.

The absence of functional p53 has complex consequences on the cellular responses to cytotoxic drugs. Here, we have examined the role of p53 in the response to 5-aza-2'-deoxycytidine (5-aza-dC or decitabine). Primary mouse embryonic fibroblasts deficient for p53 undergo apoptosis after treatment with 5-aza-dC. When compared with other demethylating drugs or chemotherapeutic treatments, 5-aza-dC showed the highest selectivity ratio for triggering apoptosis in p53-deficient cells relative to wild-type cells. Moreover, the apoptotic efficacy of 5-aza-dC is proprietary of p53-deficient cells, not being observed in cells lacking other cell-cycle regulators, such as p19ARF, p16INK4a, p21(CIP1/WAF1), E2F-1, or E2F-2. Interestingly, treatment with 5-aza-dC results in the same degree of global genomic hypomethylation in wild-type and p53-null cells. However, wild-type cells activate p53 and arrest at G2/M, whereas p53-null cells accumulate severe chromosomal aberrations and undergo apoptosis. Significantly, the impact of p53-deficiency on the response to 5-aza-dC is not exclusive of primary non-neoplastic cells, but it is also present in neoplastically transformed cells. Finally, treatment of mice bearing genetically defined tumors with nontoxic doses of 5-aza-dC results in therapeutical responses only on tumors lacking p53, but not on tumors lacking p19ARF. Together, our results put forward the hypothesis that the absence of p53 may determine a higher chemotherapeutic index for 5-aza-dC.

O(6)-methylguanine DNA methyltransferase (MGMT) is a key enzyme in the DNA repair network. MGMT removes mutagenic and cytotoxic adducts from O(6)-guanine in DNA, the preferred point of attack of many carcinogens (i.e. methylnitrosourea) and alkylating chemotherapeutic agents (i.e. BCNU, temozolamide, etc.). Hypermethylation of the CpG island located in the promoter region of MGMT is primarily responsible for the loss of MGMT function in many tumor types. The methylation-mediated silencing of MGMT has two consequences for cancer. First, tumors with MGMT methylation have a new mutator phenotype characterized by the generation of transition point mutations in genes involved in cancer etiology, such as the tumor suppressor p53 and the oncogene K-ras. Second, MGMT hypermethylation demonstrates the possibility of pharmacoepigenomics: methylated tumors are more sensitive to the killing effects of alkylating drugs used in chemotherapy. These recent results underscore the importance of MGMT in basic and translational cancer research.