Marshall W. Anderson

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Proliferative Lesions of the Mouse Lung: Progression Studies in Strain A Mice

The progression of pulmonary neoplasia was examined in strain A/J male mice treated with a single dose of vinyl carbamate (60 mg/kg, i.p.) 6 weeks after birth. Interim sacrifices were performed at 7, 8, 10, 12, or 14 months. Proliferative lesions of the lung were divided into four categories: hyperplasias, adenomas, carcinomas arising within adenomas, and carcinomas. Grossly visible surface tumor counts, histologic diagnoses, and morphometric measurements of histologic lesions were used to evaluate progression. Vinyl carbamate-treated mice showed increased mean surface tumor counts at all time points. Diagnostic evaluation suggested that as a function of time, the relative frequency of hyperplasias decreased and the relative frequency of adenomas increased. The relative frequency of adenomas subsequently decreased, whereas the relative frequency of carcinomas increased. At all time points, carcinomas arising within adenomas were present. As time progressed, the number of carcinomas arising within adenomas decreased, whereas the number of pure carcinomas increased. Morphometric analysis of lesions indicated hyperplasias to be small, that adenomas were larger than hyperplasias, and carcinomas were larger than adenomas and hyperplasias, suggesting that few adenomas or carcinomas arise de novo. Collectively, these data suggest that the majority of pulmonary tumors in A/J mice treated with vinyl carbamate arise as hyperplasias, progress to adenomas, and ultimately result in carcinomas.

Metabolism of benzo (a) pyrene to reactive intermediate (S) via prostaglandin biosynthesis

Abstract We have examined the oxidation of benzo (a) pyrene (BP) to electrophilic metabolites during the formation of prostaglandins (PG) and thromboxanes (TX) from arachidonic acid (AA) by guinea pig lung microsomal protein. In the presence of NADPH or AA, electrophilic metabolites of [14C]-BP were generated which were non-extractable from microsomal protein and thus assumed to be covalently bound. The total amount of BP metabolized in the presence of NADPH was 2–2 1 2 times the amount of BP metabolized in the presence of AA. Only 4–5% of BP metabolized by the NADPH mediated mixed-function oxidase system was covalently bound, whereas 12–15% of the BP metabolized in the presence of AA was covalently bound to tissue protein and DNA. Quinones were the major metabolites produced by the AA dependent system, while dihydrodiols were the major metabolites formed by the NADPH dependent system. 7, 12-Dimethyl-benzanthracene, and 7,8-BP-dihydrodiol, but not 3 hydroxy-BP were also oxidized by PG synthetase to reactive metabolites.

Prostaglandin removal and metabolism by isolated perfused rat lung.

We have investigated the mechanism(s) involved in the removal of prostaglandins (PG) from the pulmonary circulation by the lung. Unidirectional fluxes of PG from the circulation into the lung are measured in an isolated perfused rat lung preparation. Evidence is presented which suggests that a transport system for PG exists in lung tissue. This transport system is responsible for the removal of some PG from the circulation by the lung. PGE1 and PGF2alpha are substrates for this system, whereas PGB1, PGA1, and 15-keto-PGF2alpha are not. Since PGA1 is a substrate for the intracellular PG dehydrogenase, the selectivity of the lungs metabolism system for circulating PG is probably due to the selectivity of the transport system for PG. It is shown that the percentage of the pulmonary arterial concentration (CA) of PGE1 or PGF2alpha that is metabolized on passage through the pulmonary circulation decreases rapidly as CA increases. When the lungs were perfused with PGE1(PGF2alpha), the metabolites detected in the venous effluent were 15-keto-PGE1 (PGF2alpha) and 15-keto-13, 14-dihydro-PGE1 (PGF2alpha). The time course pattern of the appearance of metabolites in the venous effluent after the initiation of a constant CA, and the relative concentrations of the metabolites in the venous effluent, were examined as a function of CA.

Assignment of a locus for mouse lung tumor susceptibility to proximal Chromosome 19

Previous studies have hypothesized that at least three genetic loci contribute to differences in pulmonary adenoma susceptibility between mouse strains A/J and C57BL/6J. One gene that may confer susceptibility to lung tumorigenesis is the Kras protooncogene. To identify other relevant loci involved in this polygenic trait, we determined tumor multiplicity in 56 randomly chosen N-ethyl-N-nitrosourea-treated (A/J×C57BL/6J) N1×C57BL/6 backcross (AB6N2) progeny and correlated it with genotypes at 77 microsatellite markers spanning the genome. A correlation of lung tumor multiplicity phenotypes with genotypes of microsatellite markers on distal Chromosome (Chr) 6 in the Kras region (Pas1) was confirmed, and a new region on Chr 19 (designated Pas3) was identified that also contributes to susceptibility. Linkage analysis on Chr 19 with 270 AB6N2 mice localized the region flanked by D19Mit42 and D19Mit19 that is most closely associated with lung tumor susceptibility. The Pas3 locus may be an enhancer of the susceptibility locus on Chr 6.

Isolation and quantitation of DNA-bound benzo(a)pyrene metabolites: Comparison of hydroxyapatite and precipitation procedures

Abstract The hydroxyapatite and precipitation procedures are two commonly used methods for isolating DNA, and both have been employed in studies on chemical carcinogenesis. A comparison was made of the specific activity (pmol/mg DNA) of benzo(a)pyrene metabolite-DNA adducts in DNA isolated by the two procedures from the lung, liver, and forestomach of A/HeJ mice given oral benzo(a)pyrene. Total DNA-associated radioactivity per milligram DNA was higher with the precipitation procedure than with the hydroxyapatite method. This was partly due to large amounts of early eluting peaks in the high-pressure liquid chromatography analyses of digests of DNA isolated by the precipitation procedure. However, the specific activities obtained for binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide isomers to DNA isolated by the two methods were in close agreement. The hydroxyapatite procedure resulted in the isolation of purer DNA and required less time and sample handling than the precipitation method. For isolation of DNA from whole tissue for carcinogen-DNA adduct analysis, these properties make the hydroxyapatite procedure superior to the precipitation method. Our findings have important implications when comparing the results of DNA-binding studies which employ different DNA isolation procedures.

Role of Clara Cells and Type II Cells in the Development of Pulmonary Tumors in Rats and Mice Following Exposure to a Tobacco-Specific Nitrosamine

The role of the Clara and type II cell in the development of pulmonary tumors in the A/J mouse and Fischer rat was investigated by determining the relationship of DNA methylation and repair in pulmonary cells to oncogene activation and by characterizing the morphology of pulmonary tumors induced by treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Marked differences in the formation of the promutagenic adduct O6-methylguanine (O6MG) were observed in pulmonary cells following treatment of rats with NNK. Concentrations of this adduct in Clara cells greatly exceeded (3- to 30-fold) those detected in type II cells and whole lung with doses of NNK ranging from 0.1 to 50 mg/kg. In addition, very low rates of repair of this adduct were detected in Clara cells, whereas efficient adduct removal occurred in type II cells. The importance of this adduct and the role of cell specificity was suggested by the fact that a strong correlation was observed between the concentration of O6MG in Clara cells and tumor incidence in the Fischer rat with doses of NNK ranging from 0.03-50 mg/kg. In contrast, no differences in adduct concentration between type II and Clara cells from A/J mice were observed under conditions resulting in pulmonary tumor formation. Activation of the K-ras gene was detected in lung tumors from A/J mice. This gene was activated by a mutation in codon 12 involving a GC to AT transition (GGT to GAT) and is consistent with base mispairing produced by the formation of O6MG. Activation of this gene was not associated with lung tumor formation in the Fischer rat. DNA from rat lung tumors did induce tumors in the nude mouse carcinogenicity assay. In addition, rat repetitive sequences were detected in DNA isolated from these nude mouse tumors. In spite of the cell selectivity for DNA methylation in Clara cells from rat and the relationship between O6MG formation and tumorigenicity, early proliferative lesions observed in both mice and rats involved the alveolar areas. Ultrastructural examination of these lesions and adenomas revealed morphologic features characteristic of the type II cell. Thus the lack of agreement between biochemical and morphological findings makes it difficult to hypothesize a cell of origin for the pulmonary neoplasms induced by NNK. However, these studies indicate that the concentration of O6MG in Clara cells is an excellent indicator of the carcinogenic potency of NNK in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

ADPase activity of isolated perfused rat lung

Abstract More than 90% of 3H-ADP was metabolized following bolus injection into rat isolated perfused lungs. The major metabolite was inosine, with lesser amounts of adenosine and AMP. The mean pulmonary transit time for the radioactivity associated with ADP and its metabolites was the same as that for the vascular marker 14C-dextran, indicating that ADP is metabolized by enzymes in the pulmonary vessel walls. The metabolism of 3H-ADP was apparently unaffected by the simultaneous injection of prostacyclin or by continuous infusion of indomethacin or aspirin. 3H-ADP was similarly metabolized by the lung following continuous infusion, although relatively higher amounts of adenosine were observed. The metabolism of ADP by the lung represents biological inactivation since over 95% of the platelet-aggregatory activity of ADP was lost on passage through the lung.

Role of DNA methylation in the activation of proto-oncogenes and the induction of pulmonary neoplasia by nitrosamines

The relationships between DNA methylation and repair induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to the activation of proto-oncogenes and the induction of pulmonary neoplasia by this carcinogen is described. The formation of the O6-methylguanine (O6MG) adduct following metabolic activation of NNK appears to be a major factor in the induction of lung tumors in both rats and mice and in the activation of the K-ras oncogene in lung tumors from A/J mouse. The potent carcinogenicity of NNK in the rat lung correlated strongly with cell specificity for formation and persistence of the O6MG adduct in the Clara cells. This conclusion was supported by studies with nitrosodimethylamine (NDMA), a weak carcinogen in the rodent lung. Treatment with NDMA was not associated with any pulmonary cell specificity for DNA methylation. The high affinity for activation of NNK compared to NDMA was ascribed to a difference in cytochrome P-450 isozymes involved in the activation of these two nitrosamines. In the A/J mouse, the induction of pulmonary tumorigenesis involved direct genotoxic activation of the K-ras proto-oncogene as a result of the base mispairing produced by formation of the O6MG adduct. In contrast, the induction of pulmonary tumors in the rat by NNK does not appear to involve the ras pathway. It is apparent that different molecular mechanisms are involved in the development of pulmonary tumors by NNK in the mouse and rat. The studies described in this paper illustrate the utility of performing dose-response experiments and the quantitation of DNA methylation and repair in not only target tissues but also target cell types. The fundamental knowledge gained from unraveling the mechanism of carcinogenesis by NNK could lead ultimately to the identification of factors important in the development of human lung cancer.

Structural requirements for, and the effects of chemicals on, the rat pulmonary inactivation of prostaglandins

We have investigated the removal of prostaglandins (PGs) from the pulmonary circulation using the isolated perfused rat lung in order to determine which parts of the PG molecule were essential for transport into the pulmonary tissue. From these studies we propse that three functional groups of the PG molecule are necessary for transport into the lung tissue: the carboxylic acid group at carbon 1, hydroxyl group at carbon 15, and an oxygen group at carbon 11. The geometrical relationship between these groups is important for transport since reduction of the 13, 14-double bond reduced transport, and changing the C-15 hydroxyl from an S to R configuration abolished transport. Various chemicals, drugs, and PG antagonists were tested for their ability to inhibit the transport system resposible for PG removal from the circulation. Diphloretin phosphate and polyphloretin phosphate were effective inhibitors, whereas dexamethasone, bromocresol green N-ethyl maleimide and imipramine were moderately effective inhibitors. The PG antagonist, SC-19220, 7-oxo-13-prostynoic acid, and hydrocortisone were ineffective.