The EPHB6 receptor tyrosine kinase is a metastasis suppressor that is frequently silenced by promoter DNA hypermethylation in non-small cell lung cancer.

PURPOSE: Loss of EPHB6 receptor tyrosine kinase expression in early-stage non-small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. EXPERIMENTAL DESIGN: The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. Metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. RESULTS: EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. CONCLUSIONS: Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC.

A gene-alteration profile of human lung cancer cell lines.

Aberrant proteins encoded from genes altered in tumors drive cancer development and may also be therapeutic targets. Here we derived a comprehensive gene-alteration profile of lung cancer cell lines. We tested 17 genes in a panel of 88 lung cancer cell lines and found the rates of alteration to be higher than previously thought. Nearly all cells feature inactivation at TP53 and CDKN2A or RB1, whereas BRAF, MET, ERBB2, and NRAS alterations were infrequent. A preferential accumulation of alterations among histopathological types and a mutually exclusive occurrence of alterations of CDKN2A and RB1 as well as of KRAS, epidermal growth factor receptor (EGFR), NRAS, and ERBB2 were seen. Moreover, in non-small-cell lung cancer (NSCLC), concomitant activation of signal transduction pathways known to converge in mammalian target of rapamycin (mTOR) was common. Cells with single activation of ERBB2, PTEN, or MET signaling showed greater sensitivity to cell-growth inhibition induced by erlotinib, LY294002, and PHA665752, respectively, than did cells featuring simultaneous activation of these pathways, underlining the need for combined therapeutic strategies in targeted cancer treatments. In conclusion, our gene-alteration landscape of lung cancer cell lines provides insights into how gene alterations accumulate and biological pathways interact in cancer.

Frequent epigenetic inactivation of chromosome 3p candidate tumor suppressor genes in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a highly malignant neoplasm that represents the leading cause of death for cancer in Chilean females. There is limited information about the molecular abnormalities involved in its pathogenesis. We have identified a number of molecular changes in GBC, including frequent allelic losses at chromosome 3p regions. Four distinct 3p sites (3p12, 3p14.2, 3p21.3 and 3p22-24) with frequent and early allelic losses in the sequential pathogenesis of this neoplasm have been detected. We investigated epigenetic and genetic abnormalities in GBC affecting 6 candidate tumor suppressor genes (TSG) located in chromosome 3p, including DUTT1 (3p12), FHIT (3p14.2), BLU, RASSF1A, SEMA3B and hMLH1 (3p21.3). DNA extracted from frozen tissue obtained from 50 surgical resected GBCs was examined for gene promoter methylation using MSP (methylation-specific PCR) technique after bisulfite treatment in all 6 genes. In addition, we performed PCR-based mutation examination using SSCP in FHIT and RASSF1A genes and loss of heterozygosity (LOH) analysis using microdissected tissue in a subset of tumors for the 3p21.3 region with 8 microsatellite markers. A very high frequency of GBC methylation was detected in SEMA3B (46/50, 92%) and FHIT (33/50, 66%), intermediate incidences in BLU (13/50, 26%) and DUTT1 (11/50, 22%) and very low frequencies in RASSF1A (4/50, 8%) and hMLH1 (2/50, 4%). Allelic loss at 3p21.3 was found in nearly half of the GBCs examined. We conclude that epigenetic inactivation by abnormal promoter methylation is a frequent event in chromosome 3p candidate TSGs in GBC pathogenesis, especially affecting genes SEMA3B (3p21.3) and FHIT (3p14.2).

Wnt signaling promoter hypermethylation distinguishes lung primary adenocarcinomas from colorectal metastasis to the lung.

Promoter hypermethylation is responsible for gene inactivation during carcinogenesis. It has been proposed that there is some degree of specificity in the set of genes that become altered by this mechanism in distinct tumor types. To understand whether promoter hypermethylation may differentiate the site of origin, 49 lung adenocarcinomas from 31 lung primaries and 18 metastases from colorectal primaries, respectively, were tested for the presence of this alteration in the APC, CDH1, DAPK, GSTP1, MLH1, MGMT, P14, P16, RARbeta2, RASSF1, sFRP1 and WIF-1 genes. A distinct profile was apparent for the 2 groups of lung tumors and the frequencies of promoter hypermethylation at sFRP1 and WIF-1, 2 genes involved in Wnt signaling, and at CDH1 were significantly higher in colorectal metastases than in lung primaries, whereas methylation of the APC promoter was significantly more common in lung primary adenocarcinomas. Some tumors showed concomitant APC, sFRP1 and WIF-1 gene inactivation, indicating that multiple DNA methylation events must have occurred to definitively down-regulate the signaling through Wnt. However, promoter hypermethylation at the APC and CDH1 genes tended to be mutually exclusive (Fisher's exact test, p = 0.006), suggesting a similar role in carcinogenesis. In conclusion, we propose that inactivation by promoter hypermethylation at the APC, CDH1, sFRP1 and WIF-1 genes may contribute to the discrimination of lung primary adenocarcinomas from colorectal metastasis to the lung, and report the simultaneous presence of methylation at the promoters of multiple genes involved in the Wnt signaling. This may have biological consequences for carcinogenesis.

Molecular context of the EGFR mutations: evidence for the activation of mTOR/S6K signaling.

PURPOSE: Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in a small subset of lung adenocarcinomas. These mutations cluster in specific regions and confer sensitivity to inhibitors of the tyrosine kinase activity of EGFR. To further determine the genetic and molecular characteristics of tumors carrying EGFR gene mutations, we investigated the EGFR gene status in lung adenocarcinomas and evaluated its association with specific characteristics of the patients and tumors, such as mutations at KRAS and p53, EGFR and ErbB2 gene amplification, levels of EGFR and HER2 proteins, and levels of downstream effectors of EGFR, such as phospho-extracellular signal-regulated kinase and phospho-S6 proteins. EXPERIMENTAL DESIGN: The mutational status of EGFR was determined by direct sequencing in 86 primary lung adenocarcinomas and 12 lung cancer cell lines, and was correlated with a number of variables relating to the tumor and patient. A tissue microarray containing 37 lung tumors was constructed to determine, by fluorescence in situ hybridization analysis, the number of copies of EGFR and ErbB2 genes and, by immunohistochemistry, the levels of EGFR, HER2, phospho-ERK, and phospho-S6 proteins. RESULTS: EGFR gene mutations were identified in 13% of the primary tumors. The type and clustering of the mutations were identical to those previously reported. Amplification of the EGFR occurred in 14% of the tumors and could arise in tumors with EGFR mutations. Interestingly, mTOR activation, as measured indirectly by augmented levels of phospho-S6 protein, was more frequent in tumors with gene alterations in either EGFR or KRAS (P = 0.00005; Fisher's exact test) than in their wild-type counterparts. CONCLUSIONS: Our data agree with the accumulation of EGFR mutations in a subset of patients with lung cancer. Moreover, we report EGFR gene amplification in EGFR-mutant tumors and a positive correlation between EGFR or KRAS alterations and activation of mTOR signaling.

Mitochondrial DNA mutation at the D310 (displacement loop) mononucleotide sequence in the pathogenesis of gallbladder carcinoma.

PURPOSE: Mutations in the mitochondrial DNA (mtDNA) have been observed frequently in human neoplasia, in both coding and noncoding regions. A mononucleotide repeat (poly-C) between 303 and 315 nucleotides (D310) within the regulatory displacement loop has been identified recently as a frequent hot spot of deletion/insertion mutations in tumors. We investigated the frequency and pattern of D310 abnormalities in the pathogenesis of gallbladder carcinoma (GBC). EXPERIMENTAL DESIGN: DNA extracted from neoplastic and nonneoplastic archival gallbladder tissue including 123 tumors, 53 dysplastic areas, and 90 histologically normal epithelia adjacent to GBC, chronic cholecystitis, and 15 normal gallbladders were examined by PCR-based assay for D310 mutations, followed by sequencing in a subset of cases. RESULTS: D310 mutation was a relatively frequent (47 of 123; 38%) abnormality in GBC. A very high frequency of mutations were detected in dysplastic (8 of 14; 57%) and normal-appearing gallbladder epithelia (10 of 22; 46%) accompanying GBC, showing a clonal relationship compared with the corresponding tumors. D310 mutations were also detected in dysplastic (8 of 39; 21%) and normal (17 of 68; 25%) epithelia obtained from chronic cholecystitis. A single case of 15 normal gallbladders showed a D310 abnormality. Overall, deletions (67 of 91; 74%) at D310 were more frequent than insertions. CONCLUSIONS: D310 mutation at the mtDNA displacement loop is a relatively frequent and early event in the sequential pathogenesis of GBC, being detected in normal-appearing epithelium from chronic cholecystitis. Our findings suggest that mtDNA mutations should be additionally investigated in GBC pathogenesis, and D310 mononucleotide abnormalities could be included in a panel of molecular biomarkers for GBC early detection strategy.

Microsatellite analysis of synchronous and metachronous tumors: a tool for double primary tumor and metastasis assessment.

Despite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. In many cases, the classification of these lesions as either two separate primary tumors or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. To establish the origin of tumors, we assessed tumor cell clonality using PCR-based microsatellite analysis on microdissected archival tissues for loss of heterozygosity (LOH) and microsatellite instability (MSI) in a series of 19 paired synchronous and metachronous tumors from several organs. As a control group, 15 autopsy cases with an unequivocally recognizable primary tumor and associated metastases were also examined. Based on LOH and MSI findings, and using a panel of 4 to 12 (median 7) microsatellite markers, we were able to establish the clonal pattern of microsatellite changes in 17 out of 19 (89%) biopsy cases and thus determine if they were either double primary tumors (41%) or metastases (59%). Of interest, identical or similar pattern of microsatellite abnormalities were detected in 15 primary tumors and corresponding metastasis from autopsies. Our results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms and metastases in synchronous and metachronous tumors.