In the current study, we determined the mRNA expression of 3 key components of the DNA DSB repair pathway in patients with NSCLC and observed significantly higher expression in tumor tissues compared with normal tissues for ATM and DNA-PKcs but not Ku80. The high T/N expression ratios of ATM and DNA-PKcs, but not Ku80, were associated significantly with increased risks of death and notably shorter MST compared with the low T/N expression ratios. To our knowledge, this is the first study to measure mRNA expression of these 3 genes in NSCLC tissue and to report that ATM and DNA-PKcs may be useful prognostic markers in NSCLC.
There have been some studies evaluating the expression of these genes in other cancer tissues. Consistent with our results, Hosoi et al.20
observed an elevated mRNA levels of DNA-PKcs
in colorectal cancer tumor tissues compared with normal tissues. Studies of ATM
expression in tumor tissues have yielded inconsistent findings across different cancer types, with some reporting higher expression, some reporting lower expression, and others reporting no changes.12,19,20,25-27
The inconsistency in ATM
regulation in different tumor types suggests a remarkable difference on the molecular alterations related to the progression of cancer at different sites. In addition, the methodology of measuring mRNA expression and the selection of control genes also may contribute to the inconsistency of results. In this study, we used the geometric mean of the expression of 3 housekeeping genes (GAPDH, 18S rRNA
, and β-actin
) as internal controls to normalize target gene expression. Vandesompele et al.28
provided compelling evidence to support the finding that geometric averaging of multiple internal control genes offers more accurate normalization of real-time quantitative RT-PCR data than single internal control genes. This method can eliminate the expression variability of a single control gene in response to various factors. In the current study, we also compared the use of single and multiple housekeeping genes and observed that the variations were much smaller when the data were normalized to the geometric mean of 3 housekeeping genes than any individual gene (data not shown). Most previous studies used a single gene as an internal control; therefore, it is difficult to compare the results across different studies.
The mechanisms of ATM
, and Ku80
regulation remain to be elucidated. However, exogenous and endogenous factors may play a role in their regulations. Jiang et al.25
observed that the benzo (a)pyrene diol epoxide in tobacco smoke was responsible for increased ATM
expression levels in premalignant and malignant esophageal tissues, and Hosoi et al.20
demonstrated that overexpression of the transcription factor SP1 contributes to elevated levels of DNA-PKcs
. A correlation between Ku80
expression has been reported in patients with colorectal cancer and head and neck cancer.20,29
The promoter regions of both Ku80
contain consensus SP1 recognition elements; thus, DNA-PKcs
may be regulated transcriptionally by SP1. A connection also may exist between the levels of DNA-PKcs
Peng et al.31
demonstrated that the amount or activity of DNA-PKcs
expression. In our study, the mRNA levels of ATM
, and Ku80
were correlated with one another. These findings suggest that there are highly coordinated efforts of the cellular defense system in repairing DNA DSBs.
Most noteworthy, we observed that high expression of ATM
was associated with poor overall survival in patients with NSCLC. Safar et al.32
reported that ATM
hypermethylation was associated with good overall survival in patients with NSCLC, and Bosken et al.33
observed that patients with NSCLC who had effective systemic nucleotide excision repair had poorer survival than patients who had suboptimal nucleotide excision repair. Those results are consistent with our findings. However, there also have been some inconsistent results in terms of the prognostic value of these 3 genes in other cancer types. For example, low ATM
expression has been associated with an increased risk of death in colorectal and breast cancers.12,14
Again, these inconsistencies may be the results of many factors, including different cancer types, different histology and tumor characteristics, and accuracy in measuring mRNA expression. In addition, all of the literature on the prognostic value of ATM
, and Ku80
discussed above were based on gene expression in tumor tissues. However, in our study, we attempted to assess the possibility of using the T/N expression ratios of 3 DNA repair genes as prognostic markers. We compared the prognostic values between T/N expression ratios and tumor expression levels of the 3 genes by using a multivariate Cox proportional-hazards model. Patients with a high T/N expression ratios of ATM
exhibited significantly increased risk of death, as discussed above. However, no significant associations were observed between survival and tumor expression levels of ATM
(HR, 0.92; 95% CI, 0.53–1.60; P
= .766), Ku80
(HR, 1.48; 95% CI, 0.90–2.43; P
= .120), or DNA-PKcs
(HR, 1.17; 95% CI, 0.71–1.92; P
= .530). Therefore, we believe that, for patients with cancer, the T/N expression ratio, which reflects alteration of the gene expression level, may be a better predictor of prognosis, although the requirement of having normal tissue available for analysis is a limitation for measurement of the T/N expression ratio.
The molecular mechanism of cell survival modulation by ATM
remains to be elucidated. Harima et al.22
suggested that tumor cells with a greater ability to repair damaged DNA are more likely to survive and proliferate. In a comprehensive review, Kaina also reported that cells that exhibit a defect in various DNA repair pathways are liable to suffer from DNA damage-triggered apoptosis.34
Therefore, we speculate that high expression of ATM
leads to increased DNA DSB repair capacity in tumor cells and that tumor cells with higher DSB repair capacity would be more likely to survive, proliferate, and metastasize. In addition, the expression of ATM
modulates patients’ survival by triggering growth factor-mediated pathways35
; for example, ATM
may increase the transcriptional expression of the insulin-like growth factor-1 receptor, a cell surface molecule with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and apoptosis inhibition. Other functional roles of ATM
in survival modulation are under investigation.
In the current study, no significant correlation was observed between the mRNA expression of ATM, Ku80, and DNA-PKcs in tumor tissues and patients’ age, sex, tumor grade, stage, and histologic type, further suggesting that the effect of the 3 DNA repair genes on patient survival is independent of these clinical characteristics. In addition, the results of our stratified analyses indicate that the prognostic values of ATM and DNA-PKcs are significant only in younger patients (aged <66 years), in women, in light smokers, and in patients with adenocarcinoma, suggesting that age, sex, smoking, and histologic type may play critical roles in modifying the prognostic importance of ATM and DNA-PKcs expression in patients with NSCLC. In addition, adjuvant therapy also appeared to have different modifying effects on the prognostic value of ATM and DNA-PKcs expression. This result may have been obtained because of the different functions of ATM and DNA-PKcs in DNA repair and cell proliferation. However, the results from these stratified analyses should be interpreted with caution given the small sample size in each stratum. Large studies are warranted to confirm these findings.
In summary, we observed that the expression levels of ATM and DNA-PKcs were higher in NSCLC tumor tissues than in normal tissues and that high T/N expression ratios of ATM and DNA-PKcs were associated significantly with poor survival in patients with NSCLC. The current results indicate that ATM and DNA-PKcs may be useful in predicting the clinical outcome of patients with NSCLC, thus helping physicians to determine optimal therapies for individual patients.