Krüppel-like factor 12 plays a significant role in poorly differentiated gastric cancer progression.

Gastric cancer is the second common malignant neoplasia in Japan, and its poorly differentiated form is a deadly disease. To identify novel candidate oncogenes contributing to its genesis, we examined copy-number alterations in 50 primary poorly differentiated gastric cancers using an array-based comparative genomic hybridization (array-CGH). Many genetic changes were identified, including a novel amplification of the 13q22 locus. Several genes are located in this locus, and selective knockdown of one for the Krüppel-like factor 12 (KLF12) induced significant growth-arrest in the HGC27 gastric cancer cell line. Microarray analysis also demonstrated that genes associated with cell proliferation were mostly changed by KLF12 knockdown. To explore the oncogenic function of KLF12, we introduced a full length of human KLF12 cDNA into NIH3T3 and AZ-521 cell lines and found that overexpression significantly enhanced their invasive potential. In clinical samples, KLF12 mRNA in cancer tissue was increased in 11 of 28 cases (39%) when compared with normal gastric epithelium. Clinicopathological analysis further demonstrated a significant correlation between KLF12mRNA levels and tumor size (p = 0.038). These data suggest that the KLF12 gene plays an important role in poorly differentiated gastric cancer progression and is a potential target of therapeutic measures.

Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

RUNX1 permits E4orf6-directed nuclear localization of the adenovirus E1B-55K protein and associates with centers of viral DNA and RNA synthesis.

The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-DeltaN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1.

Genome-wide array-based comparative genomic hybridization analysis of pancreatic adenocarcinoma: identification of genetic indicators that predict patient outcome.

We analyzed the subchromosomal numerical aberrations of 44 surgically resected pancreatic adenocarcinomas by array-based comparative genomic hybridization. The aberration profile ranged widely between cases, suggesting the presence of multiple or complementary mechanisms of evolution in pancreatic cancer, and was associated with lymph node metastasis and venous or serosal invasion. A large number of small loci, previously uncharacterized in pancreatic cancer, showed non-random loss or gain. Frequent losses at 1p36, 4p16, 7q36, 9q34, 11p15, 11q13, 14q32-33, 16p13, 17p11-13, 17q11-25, 18q21-tel, 19p13, 21q22 and 22q11-12, and gains at 1q25, 2p16, 2q21-37, 3q25, 5p14, 5q11-13, 7q21, 7p22, 8p22, 8q21-23, 10q21, 12p13, 13q22, 15q13-22 and 18q11 were identified. Sixteen loci were amplified recurrently. We identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Gain of LUNX, HCK, E2F1 and DNMT3b at 20q11, loss of p73 at 1p36 and gain of PPM1D at 17q23 independently predicted patient outcome. Expression profiling of amplified genes identified Smurf1 and TRRAP at 7q22.1, BCAS1 at 20q13.2-3, and VCL at 10q22.1 as potential novel oncogenes. Our results contribute to a complete description of genomic structural aberrations and the identification of potential therapeutic targets and genetic indicators that predict patient outcome in pancreatic adenocarcinoma.

The SYT-SSX fusion protein down-regulates the cell proliferation regulator COM1 in t(x;18) synovial sarcoma.

Chromosomal translocations are frequently associated with soft-tissue sarcomas. Fusion proteins generated by such translocations often play critical roles in tumorigenesis. Therefore, it is important to understand the function of the fusion protein to develop therapeutic interventions. The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in a fusion between the SYT gene on chromosome 18 and an SSX gene on the X chromosome. Although SYT-SSX fusion proteins appear to trigger synovial sarcoma development, little is known about the downstream targets of SYT-SSX. We found that the SYT-SSX fusion protein produces a dominant-negative function for SYT, which is a transcriptional coactivator. We then analyzed the gene expression profiles of SYT-SSX1-expressing HeLa cells using oligonucleotide microarrays and found that the SYT-SSX1 fusion protein directly down-regulated the expression of COM1, a regulator of cell proliferation. COM1 was found to be expressed at relatively low levels in synovial sarcoma tissues and cell lines. We then investigated the impact of conditional COM1 expression in the synovial sarcoma cell line. Increased COM1 expression resulted in induced apoptosis and in reduced cell growth and colony formation activity. Our results suggested that restoration of COM1 expression may be of therapeutic benefit in synovial sarcoma.

Expression status of RUNX1/AML1 in normal gastric epithelium and its mutational analysis in microdissected gastric cancer cells.

Although Runt-related transcription factors RUNXs (RUNX1-3) have a high similarity in their structure, only RUNX3 is known to be involved in gastric carcinogenesis. First, we examined mRNA expression of these three RUNX genes in the gastric mucosa, and, finding only RUNX2 was not expressed there, we further investigated RUNX1 and RUNX3 expression in three regions including the pit, isthmus/neck, and gland regions of the human normal stomach and whether RUNX1 is involved in gastric carcinogenesis. The mRNA expression of RUNX1 and RUNX3 was examined by use of the three regions isolated by laser-captured microdissection (LCM) and by use of primary gastric cancer tissues. Furthermore, RUNX1 mutational analysis was performed in the cancer cells, which also were isolated from 44 paraffin-embedded gastric cancer tissues by LCM. RUNX1 was co-expressed with RUNX3 in the pit region, and has cell growth-inhibition activity similar to RUNX3. RUNX3 has been reported to be suppressed by DNA methylation in a subset of gastric cancers; however, the expression of RUNX1 mRNA was observed in all of the gastric cancer cell lines and gastric cancer tissues that we examined. No RUNX1 mutation was found in the 44 gastric cancer patients. Although RUNX1 is similar to RUNX3 in both the expression pattern in the stomach and its cell growth-inhibition activity, RUNX1 is not involved in most cases of gastric cancers. These results suggest that the transcriptional target genes are different between these two family genes.

The potential role of DFNA5, a hearing impairment gene, in p53-mediated cellular response to DNA damage.

The tumor suppressor p53 plays a crucial role in the cellular response to DNA damage by transcriptional activation of numerous downstream genes. Although a considerable number of p53 target genes have been reported, the precise mechanism of p53-regulated tumor suppression still remains to be elucidated. Here, we report a novel role of the DFNA5 gene in p53-mediated etoposide-induced cell death. The DFNA5 gene has been previously reported to be responsible for autosomal-dominant, nonsyndromic hearing impairment. The expression of the DFNA5 gene was strongly induced by exogenous and endogenous p53. The chromatin immunoprecipitation assay indicated that a potential p53-binding sequence is located in intron 1 of the DFNA5 gene. Furthermore, the reporter gene assay revealed that the sequence displays p53-dependent transcriptional activity. The ectopic expression of DFNA5 enhanced etoposide-induced cell death in the presence of p53; however, it was inhibited in the absence of p53. Finally, the expression of DFNA5 mRNA was remarkably induced by gamma-ray irradiation in the colon of p53(+/+) mice but not in that of p53(-/-) mice. These results suggest that DFNA5 plays a role in the p53-regulated cellular response to genotoxic stress probably by cooperating with p53.

Association of polymorphisms in the MTH1 gene with small cell lung carcinoma risk.

Fifty single-nucleotide polymorphisms (SNPs) associated with amino acid changes in 36 genes involved in diverse DNA repair pathways were assessed for associations with risk for small cell lung carcinoma (SCLC) by a case-control study consisting of 211 SCLC cases and 685 controls. An SNP, Val83Met, in the MTH1 (microtT homolog 1) gene encoding a triphosphatase that hydrolyzes pro-mutagenic oxidized nucleoside triphosphates, such as 8-hydroxy-dGTP and 2-hydroxy-dATP, showed the strongest and a significant association with SCLC risk [odds ratio (OR)=1.6, 95% confidence interval (CI): 1.2-2.2, P=0.004], while three other SNPs in the TP53, BLM and SNM1 genes, respectively, also showed marginal associations (0.05

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.

Methylation-associated silencing of the nuclear receptor 1I2 gene in advanced-type neuroblastomas, identified by bacterial artificial chromosome array-based methylated CpG island amplification.

To identify genes whose expression patterns are altered by methylation of DNA, we established a method for scanning human genomes for methylated DNA sequences, namely bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA). In the course of a program using BAMCA to screen neuroblastoma cell lines for aberrant DNA methylation compared with stage I primary neuroblastoma tumors, we identified CpG methylation-dependent silencing of the nuclear receptor 1I2 (NR1I2) gene. NR1I2 was methylated in a subset of neuroblastoma cell lines and also in advanced-stage primary tumors with amplification of MYCN. Its methylation status was inversely associated with gene expression. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored NR1I2 transcription in neuroblastoma cell lines lacking endogenous expression of this gene. A CpG island located around exon 3 of NR1I2 showed promoter activity, and its methylation status was clearly and inversely correlated with NR1I2 expression status. The gene product, NR1I2, has a known function in regulating response to xenobiotic agents but it also suppressed growth of neuroblastoma cells in our experiments. We identified some possible transcriptional targets of NR1I2 by expression array analysis. The high prevalence of NR1I2 silencing by methylation in aggressive neuroblastomas, together with the growth-suppressive activity of NR1I2, suggests that this molecule could serve as a diagnostic marker to predict prognosis for neuroblastomas.

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 profile of hepatocellular carcinoma revealed by array-based comparative genomic hybridization: identification of genetic indicators to predict patient outcome.

BACKGROUND/AIMS: We conducted an analysis of chromosomal numerical aberrations and their clinical significance in hepatocellular carcinoma. METHODS: We analyzed 87 hepatocellular carcinomas by array-based comparative genomic hybridization with an array containing 800 bacterial artificial chromosome clones. RESULTS: Frequent (>30%) chromosomal losses on 1p36.1, 4q21-25, 4q34-35.1, 8p23.3b-11.1, 13q14.1-14.3, 16p13.3, 16q22.1-24.3b, 17p13.3-13.1 and 17p13.3-11, and gains on 1q21-44f, 2q21.2, 2q34, 3q11.2, 5p14.2, 5q13.2-14, 7p22, 7p14.2, 7q21.1, 7q22.3, 7q34, 8q12-24.3 and 17q23, were observed. Recurrent (>5%) amplifications were detected on 1q25, 8q11 and 11q11, and we discovered a novel homozygous deletion at 14q32.11. The extent of chromosomal aberrations correlated significantly with various clinicopathological characteristics of the tumors, and increased in a stepwise manner with the progression of hepatocellular carcinoma. We also identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Multivariate analysis revealed that both chromosomal loss on 17p13.3 and gain on 8q11 are independent prognostic indicators. CONCLUSIONS: Our results contribute to a complete description of genomic structural aberrations in relation to hepatocarcinogenesis and provide a valuable basis from which we can begin to understand the characteristics of tumors, predict patient outcomes and discover novel therapeutic targets for hepatocellular carcinoma.

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.

ADAM23, a possible tumor suppressor gene, is frequently silenced in gastric cancers by homozygous deletion or aberrant promoter hypermethylation.

Array-based comparative genomic hybridization (CGH-array) has a powerful potential for high-throughput identification of genetic aberrations in cell genomes. We identified a homozygous loss of ADAM23 (2q33.3) in the course of a program to screen a panel of gastric cancer (GC) cell lines (1/32, 3.1%) for genomic copy-number aberrations using our custom-made CGH-array. Infrequent homozygous deletion of ADAM23 was also seen in primary gastric tumors (1/39, 2.6%). ADAM23 mRNA was expressed in normal stomach tissue, but not in the majority of GC cell lines without homozygous deletion of this gene. Expression of ADAM23 mRNA was restored to gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. The methylation status of the ADAM23 CpG island, which showed promoter activity, correlated inversely with its expression. Methylation of this CpG island was observed both in GC cell lines and in primary GC tissues; in primary tumors with a hypermethylated CpG island, expression of ADAM23 was lower than in adjacent noncancerous tissues. Moreover, restoration of ADAM23 in GC cells reduced their numbers in colony-formation assays. These results suggest that genetic or epigenetic silencing by hypermethylation of the ADAM23 CpG-rich promoter region leads to loss of ADAM23 function, which may be a factor in gastric carcinogenesis.

Physical and functional link of the leukemia-associated factors AML1 and PML.

The AML1-CBFbeta transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The promyelocytic leukemia (PML) gene is also frequently involved in AML-associated translocation. Here we report that a specific isoform PML I forms a complex with AML1. PML I was able to recruit AML1 and coactivator p300 in PML nuclear bodies and enhance the AML1-mediated transcription in the presence of p300. A specific C-terminal region of PML I and a C-terminal region of AML1 were found to be required for both their association and colocalization in the nuclear bodies. Overexpression of PML I stimulates myeloid cells to differentiate. These results suggest that PML I could act as a mediator for AML1 and its coactivator p300/CBP to assemble into functional complexes and, consequently, activate AML1-dependent transcription and myeloid cell differentiation.

Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk.

Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (associated with amino acid changes) SNPs identified were assessed for associations with lung cancer risk by a case-control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest association with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double-strand break repair protein, also showed associations (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, respectively). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was associated with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages < 61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, respectively). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.

First case of aplastic anemia in a Japanese child with a homozygous missense mutation in the NBS1 gene (I171V) associated with genomic instability.

The NBS1 gene is strongly linked to several factors involved in genome integrity. Functional disruption of NBS1 could therefore induce genomic instability and carcinogenesis. Four children with acute lymphoblastic leukemia have been reported to be heterozygous for a germline and/or somatic missense mutation in NBS1, leading to the I171V substitution. We screened healthy controls and pediatric patients with hematological malignancies and aplastic anemia (AA) for the presence of I171V. Of the 62 patients, one individual with AA was confirmed to harbor a homozygous I171V mutation. Genetic analysis of NBS1 in this patient and her healthy parents indicated that she inherited the germline I171V mutation from her father and the wild-type allele from her mother, and that the second I171V hit occurred on the wild-type allele early in embryonic development. Furthermore, cytogenetic analysis of lymphoblastic cell lines from the patient indicated a remarkable increase in numerical and structural chromosomal aberrations in the absence of clastogens, suggesting that she potentially carried genomic instability. This is the first report of AA with a homozygous I171V mutation. We hypothesize that NBS1 may play an important role in the pathogenesis of AA.

Transcriptional co-activator activity of SYT is negatively regulated by BRM and Brg1.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to the SSX gene on chromosome X. Although the SYT-SSX fusion proteins may trigger synovial sarcoma development, the biological functions of SYT, SSX and SYT-SSX genes are unclear. Transfections of Gal4 DNA binding domain fusion protein constructs demonstrate that SYT protein acts as a transcriptional co-activator at the C-terminal domain and that the activity is repressed through the N-terminus. The N-terminal 70 amino acids of SYT bind not only to BRM, but also to Brg1, both of which are subunits of SWI/SNF chromatin remodelling complexes. Here, we have investigated the functions of BRM and Brg1 on the repression of SYT activity. The negative regulation of SYT transcriptional co-activator activity is dependent on the ATP-hydrolysis of BRM and Brg1 in the protein complexes. This indicates that the SWI/SNF protein complexes regulate SYT activity using the chromatin remodelling activity.