Survival of cells undergoing DNA damage from genomic instability or genotoxic stress leads to the accumulation of genetic lesions characterizing human tumors. Abrogation of cell-cycle checkpoint and apoptotic safeguards against replication of damaged DNA is a hallmark of cancer. The result of protection against DNA damage induced apoptosis includes both vulnerability to unchecked somatic mutation and decreased sensitivity to radiotherapy and chemotherapy. The previously referenced study by Zienolddinny et al. suggests that CLPTM1L polymorphisms may affect DNA damage accumulation 
. Based on our observations, it is plausible that protection from apoptosis by CLPTM1L contributes to accumulation of DNA damage, thereby conferring susceptibility to tumorigenesis. An alternative hypothesis is that CLPTM1L plays a role in recognition or repair of DNA damage, affecting the accumulation of such damage. Another is that CLPTM1L directly influences the amount of DNA damage that is incurred by genotoxic agents. Our data (Figure S4
) suggests that CLPTM1L expression does not directly affect levels of acute DNA damage incurred by cisplatin or NNK. Moreover, the current study demonstrates that CLPTM1L has an apoptotic role downstream of DNA damage and through regulation of Bcl-xL expression, which is likely to affect accumulation of DNA damage. The observation of differential accumulation of Bcl-xL upon modulation of CLPTM1L expression, along with reconstitution of an apoptosis resistant phenotype with expression of exogenous Bcl-xL, provide evidence that CLPTM1L acts upstream of Bcl-xL to confer resistance to genotoxic stress induced apoptosis. In exogenous Bcl-xL expression experiments, it appears that less Bcl-xL is expressed in vector containing cells than was observed in previous experiments, although decreased accumulation in cells with CLPTM1L knockdown remains evident. Concurrently, apoptotic killing is slightly more robust in vector containing cells observed in previous experiments, but the trend of increased sensitivity upon loss of CLPTM1L and Bcl-xL remains. Importantly, this study shows that the anti-apoptotic function of CLPTM1L is not exclusive to cisplatin sensitivity, but is a general inhibition of the mitochondrial pathway of apoptosis.
Common overexpression of CLPTM1L in tumors supports the notion of an oncogenic or tumor promoting role for CLPTM1L in lung cancers. Protein expression studies performed and annotated by the Human Protein Atlas project 
confirm our findings. Immunohistochemistry on human normal alveolar cells and lung tumors demonstrated negative expression of CLPTM1L in normal tissues, while expression in 12 lung tumors averaged a score of 1.91 on a scale of 0–3 (Figure S5
). A score of 0 is negative, 1 is “weak”, 2 is “moderate” and 3 is “strong”. Six of these patients were diagnosed with squamous cell carcinoma and six were diagnosed with adenocarcinoma. No significant difference in expression between the two pathologies was observed. A total of 66 normal tissues from various organs scored an average of 0.98 (weak). None of the lung tumors tested were negative. Lung tumor cell lines A549 (NSCLC) and SCLC-21H (small cell) showed strong staining and moderate staining, respectively.
Microarray data from tumor and immortalized cell lines demonstrates that mechanisms other than copy number gain result in increased expression in a subset of tumors. In genome wide associations, the 5
p15.33 locus accounts for the greatest contribution to lung cancer risk of three known loci in humans 
. The 5
p locus is also implicated in cutaneous squamous cell carcinoma and melanoma 
, ovarian cancer 
, testicular germ cell cancer 
and cervical cancer by copy number gain 
. In the cervical cancer study, CLPTM1L emerged from the 5
p locus as having expression patterns that correlated with copy number gain. Another recent study of copy number and expression changes in cervical cancer cell lines revealed that with copy number gain across 5
p, CLPTM1L expression was increased approximately 5 fold over normal cervical epithelial cells, while expression of the other genes at 5
p15.33 was not changed 
. The TERT gene is also within this genetic region. In analysis of SNPs in the 5
p region, we have found that lung cancer associated SNPs are not associated with telomere length (data not shown), in agreement with two other studies 
. In contrast, a study by Rafnar et al. showed an association (p
0.017 and 0.027, respectively) between 5
p variants (rs401681 and rs2736098) and telomere length, although this effect was only seen when women older than 75 years with homozygous genotypes were included 
. To our knowledge, no common coding mutations in either CLPTM1L or TERT have been identified. It is plausible that both TERT and CLPTM1L contribute to susceptibility at this locus, having a co-founder effect. A recent study 
provides multiple lines of evidence that the lung cancer association of rs31489 in the CLPTM1L gene is an independent observation from the association of rs2376100 in the TERT gene. The most recent dense genotyping study of 5
p15.33 found multiple associations at 5p15.33, with the region of association being centered over CLPTM1L 
. Our previous analysis shows that rs31489 is the variant most strongly associated with familial lung cancer at this locus. The current study indicates that CLPTM1L also plays an important functional role in relation to cancer, and that this gene may to be at least partially responsible for the association of variants in the 5p15.33 region with lung cancer. Continued efforts to further define the genetic variation driving lung cancer susceptibility in this genetic region are an important next step in evaluating the relationship of CLPTM1L and other genes in the region with lung tumor susceptibility.
Common overexpression of CLPTM1L in lung tumors and a functional role in genotoxic stress induced apoptosis identify CLPTM1L as an important factor influencing survival of DNA damaged tumor cells and potentially lung cancer susceptibility. Targeting CLPTM1L as well as Bcl-xL may prove to be useful approaches to chemoprevention and lung cancer therapy. Targeting these anti-apoptotic proteins may also have potential for sensitization of tumors to traditional chemotherapies and radiotherapies.