Lung tumor development is believed to occur through a step-wise series of molecular changes that influence cell growth and survival. This process can be facilitated by tobacco smoke, which contains numerous carcinogens that are known to induce mutations and chromosomal instability (CIN). The FOXO family of transcription factors has been shown to function in cell cycle arrest, apoptosis, and response to various physiologic and pathologic stresses, such as DNA damage and oxidative stress. Because these transcription factors have been implicated as tumor suppressors, genetic inactivation of these genes could contribute to the development of cancer. Here, I examined the status of the FOXO3a gene in human lung tumors from patients with early stage non-small cell lung cancer (NSCLC). Using qPCR, allelic loss of 80% or higher was detected in 8 out of 33 (24.4%) mostly-early stage lung adenocarcinoma (LAC) samples in smokers suggesting bi-allelic or homozygous deletion (HD) of FOXO3a . The remaining 60.6% of these tumors had losses of FOXO3a not reaching the level of HD, a loss of between 40–79% (hereafter referred to as sub-HD). Consistent with the status of the FOXO3a gene, there were corresponding decreases in its mRNA and protein levels in LAC samples. Squamous cell lung carcinomas (LSqCC) were also analyzed for allelic loss of FOXO3a. No HDs were detected in these samples, but 47% contained sub-HD loss of FOXO3a. Although a broader analysis of FOXO3a’s activity (either through mutations, protein expression, or cellular localization) in all samples is needed in order more fully assess FOXO3a’s inactivation in both LSqCC and LAC, our results suggest that FOXO3a inactivation occurs more frequently in LAC and that it’s inactivation plays a role in the development of LAC tumors.
FOXO3a’s ability to stimulate apoptosis or cell cycle arrest in response to carcinogens was investigated in H1299 and A549 LAC cell lines as a potential role in suppressing lung carcinogenesis. Here we have demonstrated that FOXO3a is functionally activated and augments caspase-dependent apoptosis in cells exposed to (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a DNA-damaging carcinogen present at high concentrations in tobacco smoke. Consistent with this result was the FOXO3a-dependent upregulation of pro-apoptotic effectors BIM, BNIP3 and FASL with treatment. These results implicate FOXO3a in the elimination of carcinogen-damaged cells, a role consistent with the suppression of LAC carcinogenesis, and one frequently lost through gene deletion in LAC development.
Docetaxel and vinorelbine are common anti-mitotic drugs used in second line therapy of several cancer types, including lung adenocarcinoma. We investigated the role of FOXO3a in the cellular response to anti-mitotic agents in LAC cells, as this function would be lost in LAC as a consequence of gene deletion. MTS assays revealed that FOXO3a transfection causes a significant decrease in the number of LAC cells (both A549 and H460 cell lines) upon treatment with either of two different anti-mitotics, docetaxel and vinorelbine. These results indicate a potential pro-therapeutic response by FOXO3a in response to anti-mitotics. Unlike treatment with BPDE, annexin-PE and western blot analysis of various caspases showed only a modest stimulation of apoptosis in response to anti-mitotics in LAC cells expressing exogenous FOXO3a. Consistent with this result, RT-PCR expression analysis of known pro-apoptotic effectors of FOXO3a (BIM, BNIP and FASL) showed no increase in mRNA levels following treatment. Interestingly, cell cycle analysis of A549 and H460 LAC cell lines treated with anti-mitotic agents showed a pronounced reduction in G2/M fraction in cells expressing FOXO3a exogenously compared with cells that did not express FOXO3a. RT-PCR analysis of known FOXO3a cell cycle effectors showed no change in Cyclin-D2 or p27Kip1 expression levels in response to treatment. However, analysis of CDC14A mRNA levels in treated cells show evidence of a pronounced FOXO3a-dependent derepression of this gene’s expression following treatment. CDC14A is known to stimulate exit from mitosis. This potentially accounts for the observed decrease in the G2/M fraction in FOXO3a transfectants treated with anti-mitotic agents. These results suggest a potential pro-therapeutic role for FOXO3a in LAC, which involves the stimulation of mitotic exit of tumor cells with therapeutically induced mitotic spindle defects. Together these results suggest that the loss of the FOXO3a gene in LAC is likely to contribute to lung carcinogenesis and lessen the response to anti-mitotics used to threat these tumors.