Search

Barrett’s esophagus (BE) is a metaplastic condition caused by chronic gastroesophageal reflux which represents an early step in the development of esophageal adenocarcinoma (EAC). Single-nucleotide polymorphism microarray (SNP-chip) analysis is a novel, precise, high-throughput approach to examining genomic alterations in neoplasia. Using 250K SNP-chips, we examined the neoplastic progression of BE to EAC, studying 11 matched sample sets: 6 sets of normal esophagus [NE], BE and EAC, 4 of NE and BE, and 1 of NE and EAC. Six (60%) of 10 total BE samples and 4 (57%) of 7 total EAC samples exhibited one or more genomic abnormalities comprising deletions, duplications, amplifications, and copy-number-neutral loss of heterozygosity (CNN-LOH). Several shared abnormalities were identified, including chromosome 9p CNN-LOH (2 BE samples [20%]), deletion of CDKN2A (4 BE samples [40%]), and amplification of 17q12–21.2 involving the ERBB2, RARA and TOP2A genes (3.1Mb, 2 EAC [29%]). Interestingly, one BE sample contained a homozygous deletion spanning 9p22.3-p22.2 (1.2 Mb): this region harbors only one known gene, basonuclin 2 (BNC2). Real-time PCR analysis confirmed deletion of this gene and decreased expression of BNC2 mRNA in the BE sample. Furthermore, transfection and stable expression of BNC2 caused growth arrest of OE33 EAC cells, suggesting that BNC2 functions as a tumor suppressor gene in the esophagus, and that deletion of this gene occurs during the development of EAC. Thus, this SNP-chip analysis has identified several early cytogenetic events and novel candidate cancer-related genes that are potentially involved in the evolution of BE to EAC.

Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPalpha, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPalpha was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPalpha was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPalpha markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPalpha and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPalpha localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPalpha causes dysregulation of its function, suggesting that C/EBPalpha is a novel candidate tumor suppressor gene in pancreatic cancer cells.

Tumor suppressor genes are often silenced in human cancer; this can occur by transcriptional repression by deacetylation in the promoter regions, mediated by histone deacetylase (HDAC). HDAC inhibitors can block cancer cell growth by restoring expression of tumor suppressor genes. In this study, we investigated the effects of a HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) on pancreatic cancer cells. SAHA inhibited the growth of 6 pancreatic cancer cell lines in a dose-dependent manner as measured by MTT and clonogenic assays (ED(50) approximately 10(-6) M) associated with induction of apoptosis, G2 cell cycle arrest and also induced differentiation as indicated by morphology and increased expression of cytokeratin 7. It increased expression of p21(WAF1) (independent of the mutational status of p53), C/EBPalpha, RARalpha and E-cadherin; these genes have been associated with decreased proliferation in other cancers. SAHA decreased cyclin B1 expression; this cyclin normally promotes progression through G2 of the cell cycle. SAHA mediated acetylation of histone H3 globally, as well as, associated with the p21(WAF1) promoter, as measured by chromatin immunoprecipitation. SAHA also decreased levels of c-myc and cyclin D1, independent of an active beta-catenin pathway. In further studies, the combination of SAHA and an inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine, had an enhanced antiproliferative effect on pancreatic cancer cells. In summary, SAHA inhibited the growth of human pancreatic cancer cells by inducing apoptosis, differentiation and cell cycle arrest, as well as increase in the expression of several tumor suppressor genes. SAHA is a novel, promising therapeutic agent for human pancreatic cancers.