Christopher R. Herzog

Expression of Cell Cycle Proteins in 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone-Induced Mouse Lung Tumors

Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined by immunohistochemistry in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mutations in p16INK4a were not found by SSCP analysis. Immunohistochemical analyses of normal lung tissues showed intense staining for Rb protein in alveolar epithelial cells and in other lung cell types; however, in the lung tumors the staining intensity was reduced and the distribution was altered. Expression of Rb was detected in normal lung tissues but was barely detectable by Northern blot hybridization in lung tumors. Western blot analysis indicated the presence of both hypophosphorylated and hyperphosphorylated Rb protein in lung tumors and in normal lung tissues. These results suggest that alterations in the cell cycle proteins, cyclin D1, cdk4, p16INK4a, and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the importance of immunohistochemical studies to examine expression in tissues that contain multiple cell types, such as the lung, and in tumors that by nature are heterogeneous.

FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

The FOXO family of transcription factors elicits cell cycle arrest, apoptosis, and resistance to various physiologic and pathologic stresses relevant to sporadic cancer, such as DNA damage and oxidative stress. Although implicated as tumor suppressors, FOXO genetic inactivation has not been observed in human cancer. In an investigation of the two major types of non-small cell lung cancer, here, we identify the FOXO3 gene as a novel target of deletion in human lung adenocarcinoma (LAC). Biallelic or homozygous deletion (HD) of FOXO3 was detected in 8 of 33 (24.2%) mostly early-stage LAC of smokers. Another 60.6% of these tumors had losses of FOXO3 not reaching the level of HD (hereafter referred to as sub-HD). In contrast, no HD of FOXO3 was observed in 19 lung squamous cell carcinoma. Consistent with the deletion of FOXO3 were corresponding decreases in its mRNA and protein levels in LAC. The potential role of FOXO3 loss in LAC was also investigated. The carcinogen (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is strongly implicated as a cause of human lung cancer. Here, we show that FOXO3a is functionally activated and augments the level of caspase-dependent apoptosis in cells exposed to this DNA-damaging carcinogen. These results implicate FOXO3 as a suppressor of LAC carcinogenesis, a role frequently lost through gene deletion.

Carcinogen-specific targeting of chromosome 12 for loss of heterozygosity in mouse lung adenocarcinomas: implications for chromosome instability and tumor progression.

Genotoxic carcinogens exert their tumorigenic effects in part by inducing genomic instability. We recently showed that loss of heterozygosity (LOH) on chromosome 12 associates significantly with the induction of chromosome instability (CIN) by the likely human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate (VC) during mouse lung carcinogenesis. Here, we demonstrate the carcinogen specificity of this event and its effect on lung tumor evolution. LOH on chromosome 12 was observed in 45% of NNK-induced, 59% of VC-induced, 58% of aflatoxin B1 (AFB1)-induced, 14% of N-ethyl-N-nitrosourea (ENU)-induced and 12% of spontaneous lung adenocarcinomas. The frequency of LOH in each of the carcinogen-induced groups, except ENU, was significantly higher than in the spontaneous group (P<0.001). Deletion mapping revealed four potential candidate regions of 1–4 centiMorgans suspected to contain targeted tumor suppressor genes, with at least one expected to have a role in CIN. The relationship between LOH on chromosome 12 and additional chromosomal alterations occurring during lung tumor progression was also examined. LOH on chromosomes 1 and 14 were moderately frequent during malignant progression in tumors from all treatment groups, occurring in 21–35 and 18–33% of tumors. However, these alterations showed significant concurrence with LOH on chromosome 12 in VC-, NNK- and AFB1-induced tumors (P<0.05). The results suggest that a carcinogen-selective mechanism of lung cancer induction involves the frequent inactivation of genes on chromosome 12, including a stability gene that evidently promotes the evolutionary selection of additional chromosomal alterations during malignant progression.

A strong candidate gene for the Papg1 locus on mouse chromosome 4 affecting lung tumor progression

Lung cancer is the leading cause of cancer death among both men and women, accounting for more than 28% of all cancer deaths. In fact, more people die of lung cancer than of colon, breast, and prostate cancers combined. Although lung cancer is largely induced by smoking, there is strong evidence for genetic susceptibility and gene-environment interactions in the development of lung cancer. Inbred mouse models offer an effective means of identifying candidate lung cancer susceptibility loci since genetic heterogeneity and enormous variation in exposure levels to environmental agents make it difficult to identify lung cancer susceptibility loci in humans. Papg-1 (pulmonary adenoma progression 1) was previously mapped to a region on mouse chromosome 4. This locus contains a candidate gene, Cdkn2a also referred to as Ink4a/Arf, which dually encodes two established tumor suppressors p16INK4a and ARF. Cdkn2a became a primary candidate for Papg-1 for two reasons: (1) two haplotypes of mouse Cdkn2a were found to segregate with differential genetic susceptibility to lung tumor progression in mice; and (2) in vitro studies showed that the p16INK4a allele from the BALB/cJ mouse had a significantly decreased ability to bind and inhibit CDK6 and to suppress cell growth when compared with the p16INK4a allele from the A/J mouse. Here, we report that mice with a heterozygous deficiency for the A/J Cdkn2a allele were significantly more susceptible to lung tumor progression than mice with a heterozygous deficiency for a BALB/cJ Cdkn2a allele, when compared to their respective wild type mice. These results offer strong evidence that naturally occurring variation of p16INK4a influences susceptibility to enhance lung tumor progression making it a strong candidate for the lung tumor progression locus, Papg-1.

INACTIVATION OF BOTH Rb AND p53 PATHWAYS IN MOUSE LUNG EPITHELIAL CELL LINES

Aberrant expression of key cell cycle regulatory genes is essential for the immortalization and transformation of cells in vitro. We examined 20 mouse lung epithelial cell lines (2 nontumorigenic, 5 nonmetastatic, and 13 metastatic)for mutations or alterations in the expression of key components of the Rb pathway (pRb and p16INK4a) and the p53 pathway (p53 and p19ARF). Seven cell lines had a mutation in exons 5 to 8 of p53. p19ARF was inactivated in the remaining 13 cell lines, primarily by homozygous deletion. Rb expression was present and unaltered in all cell lines, with both phosphorylated and unphosphorylated protein forms detectable. p16INK4a transcripts were undetectable in all cell lines tested except LM1. Loss of p16INK4a expression was a result of homozygous deletion in 11 out of 20 lung cell lines and promoter-exon 1 hypermethylation in 6 out of the remaining 8 cell lines. Other related components that were examined in this study included p21WAF1 and cyclin D1. Compared to normal lung tissue, p21WAF1 expression levels were reduced or undetectable in all cell lines, which did not correlate with loss of p53 function, but did correlate with inactivation of either p53 or p19ARF. Although cyclin D1 expression was variable between cell lines, transcript levels were decreased by at least 50% in the nontumorigenic lines C10 and E10 compared to the tumorigenic cell lines. These results demonstrate mutually exclusive relationships between p53 and p19ARF and between Rb and p16INK4a, but perhaps not between cyclin D1 and p16INK4a, and further describe the nature of involvement of both pathways in mouse lung tumorigenesis.Aberrant expression of key cell cycle regulatory genes is essential for the immortalization and transformation of cells in vitro. We examined 20 mouse lung epithelial cell lines (2 nontumorigenic, 5 nonmetastatic, and 13 metastatic) for mutations or alterations in the expression of key components of the Rb pathway (pRb and p16INK4a) and the p53 pathway (p53 and p19ARF). Seven cell lines had a mutation in exons 5 to 8 of p53. p19ARF was inactivated in the remaining 13 cell lines, primarily by homozygous deletion. Rb expression was present and unaltered in all cell lines, with both phosphorylated and unphosphorylated protein forms detectable. p16INK4a transcripts were undetectable in all cell lines tested except LM1. Loss of p16INK4a expression was a result of homozygous deletion in 11 out of 20 lung cell lines and promoter-exon 1 hypermethylation in 6 out of the remaining 8 cell lines. Other related components that were examined in this study included p21WAF1 and cyclin D1. Compared to normal lung tissue, p21WAF1 expression levels were reduced or undetectable in all cell lines, which did not correlate with loss of p53 function, but did correlate with inactivation of either p53 or p19ARF. Although cyclin D1 expression was variable between cell lines, transcript levels were decreased by at least 50% in the nontumorigenic lines C10 and E10 compared to the tumorigenic cell lines. These results demonstrate mutually exclusive relationships between p53 and p19ARF and between Rb and p16INK4a, but perhaps not between cyclin D1 and p16INK4a, and further describe the nature of involvement of both pathways in mouse lung tumorigenesis.