Frequent silencing of the candidate tumor suppressor PCDH20 by epigenetic mechanism in non-small-cell lung cancers

Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction. We identified a homozygous loss of protocadherin 20 (PCDH20, 13q21.2) in the course of a program to screen a panel of non-small-cell lung cancer (NSCLC) cell lines (1 of 20 lines) for genomic copy number aberrations using an in-house array-based comparative genomic hybridization. PCDH20 mRNA was expressed in normal lung tissue but was not expressed in the majority of NSCLC cell lines without a homozygous deletion of this gene (10 of 19 lines, 52.6%). Expression of PCDH20 mRNA was restored in gene-silenced NSCLC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the PCDH20 CpG-rich region correlated inversely with the expression of the gene and a putative target region for methylation showed clear promoter activity in vitro. Methylation of this PCDH20 promoter was frequently observed in primary NSCLC tissues (32 of 59 tumors, 54.2%). Among our primary NSCLC cases, the methylated PCDH20 seemed to be associated with a shorter overall survival (P = 0.0140 and 0.0211 in all and stage I tumors, respectively; log-rank test), and a multivariate analysis showed that the PCDH20 methylation status was an independent prognosticator. Moreover, restoration of PCDH20 expression in NSCLC cells reduced cell numbers in colony formation and anchorage-independent assays. These results suggest that epigenetic silencing by hypermethylation of the CpG-rich promoter region of PCDH20 leads to loss of PCDH20 function, which may be a factor in the carcinogenesis of NSCLC.     

Nuclear expression of cIAP-1, an apoptosis inhibiting protein, predicts lymph node metastasis and poor patient prognosis in head and neck squamous cell carcinomas

cIAP-1, an apoptosis inhibiting protein, has been suggested to play important roles in the development of cervical and esophageal squamous cell carcinomas (SCCs). In order to clarify the subcellular localization of cIAP-1 and to investigate its clinicopathological significance in head and neck SCCs (HNSCCs), we examined cIAP-1 expression in four oral SCC cell lines by immunocytochemistry and Western blot. Expressions of nuclear and cytoplasmic cIAP-1, caspase-3, and Smac/DIABLO were also examined immunohistochemically in 57 cases of the HNSCCs. cIAP-1 expression was detected in HSC-2, HSC-3, and HSC-4 cells by immunohistochemistry and Western blot. In HSC-2 and HSC-4 cells, cIAP-1 was detected in both the nuclear and cytoplasmic fractions. Nuclear cIAP-1 expression was positive in 17 (30%) of HNSCCs, was correlated with lymph node metastasis (P=0.020) and advanced disease stage (P=0.032), and tended to be correlated with poor patient prognosis (P=0.059). Cytoplasmic cIAP-1 expression showed similar but weaker clinicopathological correlations. Nuclear cIAP-1 expression was inversely correlated with caspase-3 expression, but was correlated with Smac/DIABLO expression. Nuclear cIAP-1 expression appears to be a useful marker for predicting poor patient prognosis in HNSCCs, and may play roles in HNSCCs through the signaling pathway mediated by Smac/DIABLO and caspase-3.     

Array-based comparative genomic hybridization analysis of high-grade neuroendocrine tumors of the lung

We examined a large number of primary high-grade neuroendocrine tumors of the lung (10 small cell lung carcinomas and 31 large cell neuroendocrine carcinomas) by using array-based comparative genomic hybridization using microarrays spotted with 800 bacterial artificial chromosome clones containing tumor-related genes from throughout the human genome. We identified the genome-wide copy number alteration profiles of these tumors, including recurrent amplifications located at 2q21.2, 3q21-27, 3q26, 3q27-29, 5p14.2, 5p13, 7q21.1, 8q21, and 8q24 and homozygous deletions at 1p36, 4p16, 4p16.3, 9p21.3, 9p21, 19p13.3, and 20q13. Our results revealed that small cell lung carcinomas and large cell neuroendocrine carcinomas have multiple characteristic chromosomal alterations in common, but that distinctive alterations also exist between the two subtypes. Moreover, we found that the two subtypes undergo different processes of accumulating these genetic alterations during tumor development. By comparing the genetic profiles with the clinicopathological features, we discovered many chromosomal loci whose alterations were significantly associated with clinical stage and patient prognosis. These results will be valuable for evaluating clinical status, including patient prognosis, and for identifying novel molecular targets for effective therapies.     

Genetic classification of lung adenocarcinoma based on array-based comparative genomic hybridization analysis: its association with clinicopathologic features.

The array-based comparative genomic hybridization using microarrayed bacterial artificial chromosome clones allows high-resolution analysis of genome-wide copy number changes in tumors. To analyze the genetic alterations of primary lung adenocarcinoma in a high-throughput way, we used laser-capture microdissection of cancer cells and array comparative genomic hybridization focusing on 800 chromosomal loci containing cancer-related genes. We identified a large number of chromosomal numerical alterations, including frequent amplifications on 7p12, 11q13, 12q14-15, and 17q21, and two homozygous deletions on 9p21 and one on 8p23. Unsupervised hierarchical clustering analysis of multiple alterations revealed three subgroups of lung adenocarcinoma that were characterized by the accumulation of distinct genetic alterations and associated with smoking history and gender. The mutation status of the epidermal growth factor receptor (EGFR) gene was significantly associated with specific genetic alterations and supervised clustering analysis based on EGFR gene mutations elucidated a subgroup including all EGFR gene mutated tumors, which showed significantly shorter disease-free survival. Our results suggest that there exist multiple molecular carcinogenesis pathways in lung adenocarcinoma that may associate with smoking habits and gender, and that genetic cancer profiling will reveal previously uncharacterized genetic heterogeneity of cancer and be beneficial in estimating patient prognosis and discovering novel cancer-related genes including therapeutic targets.     

PPM1D is a potential target for 17q gain in neuroblastoma

Neuroblastomas (NBs) show complex patterns of genetic abnormalities, which may include amplification of the MYCN gene, deletion of 1p, or a gain of DNA at 17q, the last being the most frequent observation in NB tumors. However, the specific genes and the molecular mechanisms responsible for development and progression of NB remain poorly understood. We investigated aberrations of DNA copy number in 25 NB cell lines using comparative genomic hybridization and identified a minimal common region of gain at 17q23. Although gain of distal 17q is the most powerful genetic predictor of adverse outcome currently available for patients with NB, thus far, no potential target genes have been reported for that region. Therefore, we defined the 17q23 amplicon in detail and determined expression levels of 15 genes located within the smallest region of overlap observed among our NB cell lines to identify the most likely target gene(s). Among them, seven (CLTC, VMP1, delta-tubulin, RPS6KB1, FLJ22087, APPBP2, and PPM1D) were consistently overexpressed through increases in regional copy number. Analysis of expression levels of those seven genes in 32 primary NB tumors revealed a significant correlation between higher expression and poorer clinical outcome only with respect to PPM1D. Moreover, down-regulation of PPM1D by transfection of an antisense oligonucleotide suppressed the growth of NB cell lines to a remarkable degree, at least partly by participating in a process leading to apoptotic cell death. Taken together, our results indicate that PPM1D is the most likely target of the 17q23 gain/amplification in NB tumors and may have an important role in the pathogenesis of this disease.     

Correlation between genetic alteration and long-term clinical outcome of patients with oligodendroglial tumors,with identification of a consistent region of deletion on chromosome arm 1p

BACKGROUND: In oligodendroglial tumors, allelic losses on chromosome arms 1p and 19q are not only diagnostic molecular markers but also statistically significant predictors of both chemosensitivity and longer recurrence-free survival. In the current study, the authors attempted to analyze 21 patients genetically and clinically, with special emphasis on the correlation between genetic alterations and long-term therapeutic results. METHODS: The authors reviewed the clinical cases of 21 patients who had undergone surgery for oligodendroglial tumors (13 oligodendrogliomas, World Health Organization [WHO] Grade II; 3 anaplastic oligodendrogliomas, WHO Grade III; 3 oligoastrocytomas, WHO Grade II; and 2 anaplastic oligoastrocytomas, WHO Grade III). Genetic testing for 1p deletions was performed using fluorescence in situ hybridization, and testing for 1p, 17p, and 19q deletions was carried out by microsatellite analysis. Survival was analyzed with univariate and multivariate Cox regression models. In addition, a high-resolution deletion map of 1p, which led to the discovery of a new deleted region on 1p, was obtained. RESULTS: Statistical analysis revealed that both loss of 1p and loss of 19q independently and significantly predicted overall survival. A high-resolution deletion map, which displayed unusually narrow deletions, revealed a new region of deletion between D1S513 and D1S458 (1p34.3-36.11). CONCLUSIONS: One of the putative tumor suppressor loci exists more proximally than ever reported. Based on the observation that 1p and 19q deletions predicted survival, the authors suggest further use of diagnostic and prognostic genetic testing in the clinical setting.     

Bis, a Bcl-2-binding protein that synergizes with Bcl-2 in preventing cell death

Bcl-2 is the best characterized inhibitor of apoptosis, although the molecular basis of this action is not fully understood. Using a protein interaction cloning procedure, we identified a human gene designated as bis (mapped to chromosome 10q25) that encoded a novel Bcl-2-interacting protein. Bis protein showed no significant homology with Bcl-2 family proteins and had no prominent functional motif. Co-immunoprecipitation analysis confirmed that Bis interacted with Bcl-2 in vivo. DNA transfection experiments indicated that Bis itself exerted only weak anti-apoptotic activity, but was synergistic with Bcl-2 in preventing Bax-induced and Fas-mediated apoptosis. These results suggest that Bis is a novel modulator of cellular anti-apoptotic activity that functions through its interaction with Bcl-2.     

Non-incidental coamplification of Myc and ERBB2, and Myc and EGFR, in gastric adenocarcinomas.

This study was conducted to assess the frequencies of protein overexpression and gene amplification of Myc and to identify the mechanisms of Myc gene amplification, especially with regards to its possible coamplification with ERBB2 or EGFR in gastric adenocarcinomas. By immunohistochemical analysis of a total of 300 formalin-fixed and paraffin-embedded gastric adenocarcinomas, the nuclear overexpression of MYC was found in 47 tumors (16%). A fluorescence in situ hybridization (FISH) analysis revealed that nine (19%) of the 47 tumors with protein overexpression had cancer cells with high levels of Myc amplification, whereas only seven (6%) of the 122 tumors without protein overexpression showed high-level Myc gene amplification. Such Myc amplification was significantly correlated with positive nuclear protein overexpression. The coamplification of ERBB2 or EGFR with Myc that was found in six and four cases, respectively, is believed to be non-incidental because those frequencies were significantly higher than the individual frequencies observed for the total examined cases (ERBB2: 7%; EGFR: 4%). The high levels of gene amplification of these three genes, as visualized by FISH, could be broadly classified into two typical types, namely, 'multiple scattered signals' and 'large clustered signals'. Using two-color FISH, the coexistence of coamplified Myc and ERBB2, or Myc and EGFR, within single nuclei in various combinations of amplification types and copy numbers, could be ascertained in all nine cases, including one in which the synchronous 'multiple scattered type' coamplification of Myc and ERBB2 was observed. In three tumors, coamplification of ERBB2 and EGFR was found; however, ERBB2- and EGFR-amplified cell populations were separate and mutually exclusive. We propose that the non-incidental coamplification of Myc and either ERBB2 or EGFR occurred through translocation and subsequent rearrangement.