Characterization of the gene structure, functional significance, and clinical application of RNF180, a novel gene in gastric cancer.

BACKGROUND: By using genome-wide methylation screening, the authors identified ring finger protein 180 (RNF180) as preferentially methylated in cancer. This study was undertaken to clarify its structure and functional role in gastric cancer. METHODS: The transcription start site and core functional promoter region of RNF180 were revealed by 5' rapid amplification of cDNA ends and luciferase activity assays. Promoter methylation was detected by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell growth was detected by colony formation assay, apoptosis by annexin V assay, and RNF180 target genes by cDNA microarray. RESULTS: The authors revealed the transcription start site of RNF180 gene and identified the functional core promoter region (-202/+372) in the CpG island, which could be silenced by in vitro methylation assay. RNF180 was silenced in 6 of 7 gastric cancer cell lines and significantly down-regulated in primary gastric cancers compared with adjacent normal tissues (P = .001). Loss of gene expression was associated with promoter methylation. Re-expression of RNF180 suppressed cell growth (P < .001) and induced apoptosis (P < .05), which were mediated by up-regulating the antiproliferation regulators MTSS1 and CDKN2A and the proapoptotic mediator TIMP3. Promoter methylation of RNF180 was detected in 76% (150 of 198) of primary gastric cancers and 55% (11 of 20) of intestinal metaplasia, but in none of 23 normal gastric tissues. Methylated RNF180 DNA was detected in the plasma of 56% of gastric cancer patients, but not in healthy controls (P = .003). Patients with low or loss of RNF180 expression had significantly poorer overall survival. CONCLUSIONS: RNF180 is a novel potential tumor suppressor in gastric carcinogenesis and has potential clinical utility as a biomarker for gastric cancer patients.

Inhibitory role of peroxisome proliferator-activated receptor gamma in hepatocarcinogenesis in mice and in vitro.

UNLABELLED: Although peroxisome proliferator-activated receptor gamma (PPARgamma) agonist have been shown to inhibit hepatocellular carcinoma (HCC) development, the role of PPARgamma in hepatocarcinogenesis remains unclear. We investigated the therapeutic efficacy of PPARgamma against HCC. PPARgamma-deficient (PPARgamma(+/-)) and wild-type (PPARgamma(+/+)) littermates were used in a diethylnitrosamine (DEN)-induced HCC model and treated with PPARgamma agonist (rosiglitazone) or the vehicle alone for 8 months. The effects of PPARgamma on HCC cell growth and apoptosis were examined using PPARgamma-expressing adenovirus (Ad-PPARgamma). PPARgamma(+/-) mice were more susceptible to DEN-induced HCC than PPARgamma(+/+) mice (94% versus 62%, P < 0.05), and rosiglitazone significantly reduced the incidence of HCC in PPARgamma(+/+) mice (vehicle 62% versus treatment 24%, P < 0.01), but not in PPARgamma(+/-) mice, indicating that PPARgamma suppresses hepatocellular carcinogenesis. A pronounced expression of PPARgamma was observed in a HCC cell line (Hep3B) infected with Ad-PPARgamma. Such induction markedly suppressed HCC cell viability (P < 0.01). Further, Hep3B infection with Ad-PPARgamma revealed a decreased proportion of cells in S-phase (12.92% versus 11.58%, P < 0.05), with arrest at G(2)/M phase (38.2% versus 55.68%, P < 0.001), and there was concomitant phosphorylation of the key G(2)/M phase inhibitors cdc25C and cdc2. PPARgamma overexpression increased cell apoptosis (21.47% versus 35.02%, P < 0.01), mediated by both extrinsic (Fas and tumor necrosis factor-alpha) and intrinsic (caspase-9, caspase-3, caspase-7, and poly[ADP-ribose] polymerase) pathways. Moreover, PPARgamma directly induced a putative tumor suppressor gene, growth differentiation factor-15. CONCLUSION: Loss of one PPARgamma allele is sufficient to enhance susceptibility to HCC. PPARgamma suppresses tumor cell growth through reducing cell proliferation and inducing G(2)/M phase arrest, apoptosis, and up-regulating growth differentiation factor-15. Thus, PPARgamma acts as a tumor-suppressor gene in the liver.

Methylation of protocadherin 10, a novel tumor suppressor, is associated with poor prognosis in patients with gastric cancer.

BACKGROUND & AIMS: By using methylation-sensitive representational difference analysis, we identified protocadherin 10 (PCDH10), a gene that encodes a protocadherin and is silenced in a tumor-specific manner. We analyzed its epigenetic inactivation, biological effects, and prognostic significance in gastric cancer. METHODS: Methylation status was evaluated by combined bisulfite restriction analysis and bisulfite sequencing. The effects of PCDH10 re-expression were determined in growth, apoptosis, proliferation, and invasion assays. PCDH10 target genes were identified by complementary DNA microarray analysis. RESULTS: PCDH10 was silenced or down-regulated in 94% (16 of 17) of gastric cancer cell lines; expression levels were restored by exposure to demethylating agents. Re-expression of PCDH10 in MKN45 gastric cancer cells reduced colony formation in vitro and tumor growth in mice; it also inhibited cell proliferation (P < .01), induced cell apoptosis (P < .001), and repressed cell invasion (P < .05), up-regulating the pro-apoptosis genes Fas, Caspase 8, Jun, and CDKN1A; the antiproliferation gene FGFR; and the anti-invasion gene HTATIP2. PCDH10 methylation was detected in 82% (85 of 104) of gastric tumors compared with 37% (38 of 104) of paired nontumor tissues (P < .0001). In the latter, PCDH10 methylation was higher in precancerous lesions (27 of 45; 60%) than in chronic gastritis samples (11 of 59; 19%) (P < .0001). After a median follow-up period of 16.8 months, multivariate analysis revealed that patients with PCDH10 methylation in adjacent nontumor areas had a significant decrease in overall survival. Kaplan-Meier survival curves showed that PCDH10 methylation was associated significantly with shortened survival in stage I-III gastric cancer patients. CONCLUSIONS: PCDH10 is a gastric tumor suppressor; its methylation at early stages of gastric carcinogenesis is an independent prognostic factor.

Identification of a novel homozygous deletion region at 6q23.1 in medulloblastomas using high-resolution array comparative genomic hybridization analysis.

PURPOSE: The aim of this study is to comprehensively characterize genome copy number aberrations in medulloblastomas using high-resolution array comparative genomic hybridization. EXPERIMENTAL DESIGN: High-density genomic arrays containing 1,803 BAC clones were used to define recurrent chromosomal regions of gains or losses throughout the whole genome of medulloblastoma. A series of 3 medulloblastoma cell lines and 16 primary tumors were investigated. RESULTS: The detected consistent chromosomal aberrations included gains of 1q21.3-q23.1 (36.8%), 1q32.1 (47.4%), 2p23.1-p25.3 (52.6%), 7 (57.9%), 9q34.13-q34.3 (47.4%), 17p11.2-q25.3 (89.5%), and 20q13.31-q13.33 (42.1%), as well as losses of 3q26.1 (57.9%), 4q31.23-q32.3 (42.1%), 6q23.1-25.3 (57.9%), 8p22-23.3 (79%), 10q24.32-26.2 (57.9%), and 16q23.2-q24.3 (63.2%). One of the most notable aberrations was a homozygous deletion on chromosome 6q23 in the cell line DAOY, and single copy loss on 30.3% primary tumors. Further analyses defined a 0.887 Mbp minimal region of homozygous deletion at 6q23.1 flanked by markers SHGC-14149 (6q22.33) and SHGC-110551 (6q23.1). Quantitative reverse transcription-PCR analysis showed complete loss of expression of two genes located at 6q23.1, AK091351 (hypothetical protein FLJ34032) and KIAA1913, in the cell line DAOY. mRNA levels of these genes was reduced in cell lines D283 and D384, and in 50% and 70% of primary tumors, respectively. CONCLUSION: Current array comparative genomic hybridization analysis generates a comprehensive pattern of chromosomal aberrations in medulloblastomas. This information will lead to a better understanding of medulloblastoma tumorigenesis. The delineated regions of gains or losses will indicate locations of medulloblastoma-associated genes. A 0.887 Mbp homozygous deletion region was newly identified at 6q23.1. Frequent detection of reduced expression of AK091351 and KIAA1913 genes implicates them as suppressors of medulloblastoma tumorigenesis.