Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer.

Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

Prognostic relevance of DNA copy number changes in colorectal cancer.

In a study of 109 colorectal cancers, DNA copy number aberrations were identified by comparative genomic hybridization using a DNA microarray covering the entire genome at an average interval of less than 1 Mbase. Four patterns were revealed by unsupervised clustering analysis, one of them associated with significantly better prognosis than the others. This group contained tumours with short, dispersed, and relatively few regions of copy number gain or loss. The good prognosis of this group was not attributable to the presence of tumours showing microsatellite instability (MSI-H). Supervised methods were employed to determine those genomic regions where copy number alterations correlate significantly with multiple indices of aggressive growth (lymphatic spread, recurrence, and early death). Multivariate analysis identified DNA copy number loss at 18q12.2, harbouring a single gene, BRUNOL4 that encodes the Bruno-like 4 splicing factor, as an independent prognostic indicator. The data show that the different patterns of DNA copy number alterations in primary tumours reveal prognostic information and can aid identification of novel prognosis-associated genes.

BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.

Colorectal cancer is believed to progress through an adenoma-carcinoma sequence. However, recent evidence increasingly supports the existence of an alternative route for colorectal carcinogenesis through serrated polyps, a group that encompasses a morphological spectrum, including hyperplastic polyp (HP), admixed hyperplastic polyp/adenoma (HP/AD), and serrated adenoma (SA; the latter two manifest epithelial dysplasia). We have studied a large series of serrated polyps for BRAF and KRAS mutations. BRAF mutations were detected in 18 of 50 (36%) HPs, 2 of 10 (20%) HP/ADs, and 9 of 9 (100%) SAs. Twenty-six of 29 mutations caused amino acid substitutions at valine 599, the known hotspot. KRAS mutations were detected in 9 of 50 (18%) HPs, 6 of 10 (60%) HP/ADs, and 0 of 9 (0%) SAs. BRAF and KRAS mutations are mutually exclusive (P = 0.001). The associations of BRAF mutations with SAs (P < 0.001) and KRAS mutations with HP/ADs (P = 0.005) are statistically significant. A majority (90%) of the serrated polyps showing dysplasia had mutations in either BRAF or KRAS, significantly different from those without dysplasia (54%; P = 0.014). Our data highlight the important role of activation of the RAS-RAF-mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase-mitogen-activated protein kinase pathway in the initiation and progression of serrated neoplasms. Acquisition of a BRAF mutation appears to be associated with the progression of HP to SA, whereas progression to HP/AD is predominantly associated with acquisition of a KRAS mutation. The high incidence of BRAF mutations in HPs and SAs is consistent with the notion that the group of colorectal cancers carrying BRAF mutations may harbor most that have progressed through the HP-SA-carcinoma pathway.

Variation in gene expression patterns in human gastric cancers.

Gastric cancer is the world's second most common cause of cancer death. We analyzed gene expression patterns in 90 primary gastric cancers, 14 metastatic gastric cancers, and 22 nonneoplastic gastric tissues, using cDNA microarrays representing approximately 30,300 genes. Gastric cancers were distinguished from nonneoplastic gastric tissues by characteristic differences in their gene expression patterns. We found a diversity of gene expression patterns in gastric cancer, reflecting variation in intrinsic properties of tumor and normal cells and variation in the cellular composition of these complex tissues. We identified several genes whose expression levels were significantly correlated with patient survival. The variations in gene expression patterns among cancers in different patients suggest differences in pathogenetic pathways and potential therapeutic strategies.

Involvement of bcl-2 and caspase-3 in apoptosis induced by cigarette smoke extract in the gastric epithelial cell.

Previous studies have shown that short-term passive cigarette smoking can increase apoptosis in rat gastric mucosa. However, the mechanism is not yet defined. Chloroform and ethanol extracts were used to investigate whether cigarette smoke could induce apoptosis in a human gastric epithelial cell line (AGS) as well as the roles of bcl-2, caspase-3, and cytochrome c in this process. AGS cell lines were treated with either chloroform extract (CE) or ethanol extract (EE) for 5 hours, and the level of bcl-2, the activity of caspase-3, and the level of cytosolic cytochrome c in these cells were determined. Time course studies on the effects of cigarette smoke extracts (CSEs) on DNA fragmentation and cytochrome c relocalization were also performed. Data showed that only CE induced apoptosis in a dose- and time-dependent manner in AGS cells, along with a decrease of bcl-2 and an increase of caspase-3 activity. Pretreatment with Z-DEVD-FMK (specific inhibitor of caspase-3) dose-dependently blocked the DNA fragmentation induced by the CE. Moreover, CE could time- and dose-dependently increase the level of cytochrome c in the cytoplasm, which might activate caspase-3. In conclusion, CSE triggers apoptosis in AGS cells through the inhibition of bcl-2 and the activation of a mitochondria-related pathway.

Phospholipase A2 group IIA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis.

We analyzed gene expression patterns in human gastric cancers by using cDNA microarrays representing approximately equal 30,300 genes. Expression of PLA2G2A, a gene previously implicated as a modifier of the Apc(Min/+) (multiple intestinal neoplasia 1) mutant phenotype in the mouse, was significantly correlated with patient survival. We confirmed this observation in an independent set of patient samples by using quantitative RT-PCR. Beyond its potential diagnostic and prognostic significance, this result suggests the intriguing possibility that the activity of PLA2G2A may suppress progression or metastasis of human gastric cancer.

Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.

Activation of the RAS/RAF/extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by RAS mutations is commonly found in human cancers. Recently, we reported that mutation of BRAF provides an alternative route for activation of this signaling pathway and can be found in melanomas, colorectal cancers, and ovarian tumors. Here we perform an extensive characterization of BRAF mutations in a large series of colorectal tumors in various stages of neoplastic transformation. BRAF mutations were found in 11 of 215 (5.1%) colorectal adenocarcinomas, 3 of 108 (2.8%) sporadic adenomas, 1 of 63 (1.6%) adenomas from familial adenomatous polyposis (FAP) patients, and 1 of 3 (33%) hyperplastic polyps. KRAS mutations were detected in 34% of carcinomas, 31% of sporadic adenomas, 9% of FAP adenomas, and no hyperplastic polyps. Eight of 16 BRAF mutations were V599E, the previously described hotspot, and none of these was associated with a KRAS mutation in the same lesion. The remaining eight mutations involve other conserved amino acids in the kinase domain, and 62.5% have a KRAS mutation in the same tumor. Our data suggest that BRAF mutations are, to some extent, biologically similar to RAS mutations in colorectal cancer because both occur at approximately the same stage of the adenoma-carcinoma sequence, both are associated with villous morphology, and both are less common in adenomas from FAP cases. By contrast, colorectal adenocarcinomas with BRAF mutations are associated with early Dukes' tumor stages (P = 0.006) and no such relationship was observed for KRAS mutations. The presence in some colorectal neoplasms of mutations in both BRAF and KRAS suggests that modulation of the RAS-RAF-extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway may occur by mutation of multiple components.

Mutations of the BRAF gene in human cancer.

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.