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.

FOXO3a elicits a pro-apoptotic transcription program and cellular response to human lung carcinogen nicotine-derived nitrosaminoketone (NNK).

Long-term carcinogen exposure exerts continuous pressure on key mechanisms that repair or eliminate carcinogen-damaged cells giving rise to selective failures that contribute to lung cancer. FOXO3a is a transcription factor that elicits a protective response to diverse cellular stresses. Although implicated as a tumor suppressor, its role in sporadic cancer is uncertain. We recently observed that FOXO3a gene inactivation occurs frequently in carcinogen-induced lung adenocarcinoma (LAC). This suggests that FOXO3a may play a role in LAC suppression by eliciting a protective response to carcinogenic stress. Here we investigated this possibility by examining the role of FOXO3a in the cellular response to nicotine-derived nitrosaminoketone (NNK), a lung carcinogen implicated as a cause of human LAC. We show that restoration of FOXO3a in FOXO3a-deficient LAC cells increases sensitivity to apoptosis caused by a DNA-damaging intermediate of NNK. Prior to this cellular outcome, FOXO3a is functionally activated and mediates a large-scale transcription program in response to this damage involving a significant modulation of 440 genes. Genes most significantly represented in this program are those with roles in cell growth and proliferation>protein synthesis>gene expression>cell death>cell cycle. The results of this study show that FOXO3a directs an anti-carcinogenic transcription program that culminates in the elimination of carcinogen-damaged cells. This suggests that FOXO3a is a potential suppressor of carcinogenic damage in LAC.

FoxO3a gene is a target of deletion in mouse lung adenocarcinoma.

Lung adenocarcinomas (LAC) of smokers and never-smokers differ from one another in epidemiology, and clinical and molecular characteristics. The pathogenetic differences between these tumors are potential biomarkers and therapeutic targets. Mouse carcinogenesis models of human LAC are proven tools applicable for the identification of these molecular changes. Allelic loss frequency on human chromosome 6q is higher in LAC of smokers compared with never smokers. We analyzed the orthologous region on mouse chromosome 10 and found this region similarly was a more frequent site of allelic loss in carcinogen-induced LAC compared with non-induced or spontaneous LAC. We then conducted high resolution quantitative PCR-based deletion mapping of this region and identified the FoxO3a gene as the focus of bi-allelic or homozygous deletion (HD). HDs were detected in 5 out of 15 (33.3%) LAC cell lines and in 6 out of 75 (8%) carcinogen-induced primary LAC. FoxO3a was exclusively affected by HD in 7 of the samples examined, as loss of both alleles did extend to the nearest flanking genes of FoxO3a. Deletion of FoxO3a, either by HD or subclonal loss was detected in 38 out of 75 (50.7%) of carcinogen-induced LAC in contrast to only 1 out of 10 (10%) of LAC of untreated mice. Several of the samples also were subjected to direct sequence analysis; however, no intragenic mutations were detected. These results implicate FoxO3a as a selective target of deletion in mouse LAC. Significant association with carcinogenic induction suggests that deletion of FoxO3a contributes to the development of carcinogen-initiated tumors.

Adenylosuccinate synthetase 1 gene is a novel target of deletion in lung adenocarcinoma.

Tobacco smoke consists of numerous carcinogens whose effect on lung tumor development includes the induction of mutations in key genes as well as the induction of chromosome instability (CIN). Consequently, carcinogen-induced mouse lung adenocarcinomas (LAC) display many more recurrent site- and chromosome-specific changes in DNA copy number compared with noninduced LAC. Here we identified the Adenylosuccinate synthetase 1 (Adss1) gene located on distal chromosome 12q as a focus of bi-allelic or homozygous deletion (HD) in LAC. HDs of Adss1 were detected in 10 out of 84 carcinogen-induced mouse primary LAC and mouse LAC cell lines. In only four of these cases did the deletions affect either Siva1 or Inverted-formin 2 (Inf2), which immediately flank the Adss1 locus, indicating that Adss1 is a selective target of deletion in LAC. Losses of Adss1 not meeting the quantitative threshold of HD were detected in 36 out of 84 (42.9%) of the mouse tumors and cell lines. A similar frequency of ADSS1 deletion was observed in human LAC cell lines, suggesting relevance in human lung cancer. Adss1 losses were also found to be significantly associated with a more extensive CIN phenotype in the primary mouse tumors. These results implicate ADSS1 inactivation as a novel somatic alteration in lung carcinogenesis, and suggest that its selective deletion in LAC may be triggered by CIN.