Journal of

Cancer Therapeutics & Research

ISSN 2049-7962

Special Section | Epidemiolgy, Genetics and Genomics | Research

Open Access

Apoptosis-related single nucleotide polymorphisms and the risk

of non-small cell lung cancer in women
Anand Pathak1*, Angela S. Wenzlaff2, Paula L. Hyland3, Michele L. Cote2, Greg R. Keele2, Susan Land4, Matthew L. Boulton5 and Ann G.
Schwartz2
*Correspondence: pathaka@mail.nih.gov

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Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 9609 Medical Center Drive,
Rockville, MD, USA.
2
Population Studies and Disparities Research Program, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA.
3
Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 9609 Medical Center
Drive, Rockville, MD, USA.
4
Applied Genomics Technology Center and Department of Obstetrics & Gynecology, Wayne State University School of Medicine,
CS Mott Center. Detroit MI, USA.
5
Preventive Medicine Residency, Department of Epidemiology, University of Michigan School of Public Health, MI, USA.
1

Abstract

Background: Germline apoptosis-related single nucleotide polymorphisms (SNPs) have been shown to contribute to the risk of
developing non-small cell lung cancer (NSCLC). However, very few studies have looked specifically at apoptosis-related SNPs in a
racially-stratified analysis of white and African-American women.
Methods: We examined the risk of developing NSCLC associated with 98 germline SNPs in 32 apoptosis-related genes among women
in a population-based case-control study from the Detroit metropolitan area. We examined 453 cases of NSCLC and 478 control
subjects. We used an unconditional logistic regression with a dominant model, stratified by race, and adjusted for age, pack-years
smoked, ever/never smoking status, family history of lung cancer, history of COPD, BMI and education.
Results: Our logistic regression identified 3 significant apoptosis-related SNPs in whites (APAF-1, rs1007573; CD40 rs3765459, and
CD40 rs1535045), and 7 significant SNPs (ATM, rs1801516; BAK1, rs513349; TNF, rs1800629; TP63, rs6790167; TP63, rs7613791,
TP63, rs35592567 and TP63, rs3856775) in African-Americans. In a downstream analysis, these SNPs were further prioritized
utilizing the False Positive Report Percentage (FPRP) methodology and backwards elimination. In whites, APAF-1 (rs1007573),
CD40 (rs3765459) and CD40 (rs1535045) were all found to be significant by FPRP. In African-Americans, TP63 SNPs rs6790167 and
rs7613791 were found to have a significant FPRP. In parallel, a backward elimination procedure was used on the 3 significant SNPs
in whites and 7 significant SNPs in African-Americans. This procedure identified APAF-1 rs1007573 (OR=1.86, 95% CI:1.17-2.95)
and CD40 rs1535045 (OR=0.58, 95% CI:0.40-0.84) as significant independent predictors of risk among whites, and ATM rs1801516
(OR=24.15, 95% CI:3.50-166.55), TNF rs1800629 (OR=0.42, 95% CI:0.18-0.99) and TP63 rs6790167 (OR: 2.85, 95% CI:1.33-6.09) as
significant, independent predictors in African-Americans.
Conclusion: In whites, only SNPs APAF-1 rs1007573 and CD40 rs1535045 were significant by both FPRP and backwards elimination,
while in African-Americans, only TP63 rs6790167 was significant by both methodologies. Thus, we have identified three promising
variants associated with increased risk of NSCLC that warrant additional investigation in future studies.
Keywords: Non-small cell lung cancer (NSCLC), single nucleotide polymorphisms, apoptosis, females, APAF-1, CD40, ATM, TNF,
TP63

Introduction

between a limited number of apoptosis-related single nucleotide

Lung cancer is one of the leading causes of cancer in the United polymorphisms (SNPs) and lung cancer. Specifically, some
States with 159,480 deaths and 228,190 new cases estimated focused studies have implicated SNPs in CASP8 [2], MDM2 [3], and
in 2013 [1]. While smoking is the principal risk factor for lung TGFB1 [4] as associated with altered risk of developing noncancer, a genetic component is also known to contribute to risk. small cell lung cancer (NSCLC).
A better understanding of germline mutations that alter lung
Genome wide association studies (GWAS) have permitted
cancer risk may add to the future diagnosis and prevention of agnostic interrogation of the entire genome for the association
this disease. We hypothesize that attenuation of apoptosis- between SNPs and NSCLC. A GWAS study performed on 2331
related pathways could contribute to uncontrolled cellular lung cancer cases and 3,077 controls in China revealed significant
proliferation and the development of lung cancer. To date, a associations of SNPs in apoptosis-related genes (TP63 and
number of targeted studies have examined the relationship TNFRSF19) and NSCLC [5]. In a study of 1,952 European cases
2014 Pathak et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pathak et al. Journal of Cancer Therapeutics & Research 2014,

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doi: 10.7243/2049-7962-3-1

and 1,438 European controls, a SNP in the apoptosis-related Blood collection and genotyping
gene BAT3 (BCL2-associated athanogene 6) was found to be After blood was drawn into EDTA containing Vacutainer
significantly associated with lung cancer [6,7]. The remainder Plus tubes, DNA was purified from whole blood using
of the lung cancer GWAS studies did not implicate any other manufacturers protocols for Qiagen AutoPure LS Genomic DNA
apoptosis-related gene, though nicotinic acid receptor genes Purification System (Gentra Systems). After purification, 250 ng
[8], inflammatory genes such as IL1RAP and CRP [9] and a of genomic DNA was submitted to the Applied Genomics
telomerase associated gene TERT [5,10,11] all were significantly Technology Core at Wayne State University. The Illumina Golden
associated with lung cancer. However, none of these GWAS Gate assay Cancer SNP Panel (Catalog # GT-17-211) consisting
studies included a focus on either African-Americans or women. of 1421 SNPs in 400 genes selected from the National Cancer
To evaluate the genetic contributions to lung cancer risk in Institutes Cancer Genome Anatomy SNP500 Cancer Database,
African-American and white women, we examined 98 SNPs in was used. Data were analyzed using Illuminas BeadStudio
32 apoptosis-related genes, with the a priori hypothesis that software. Of the 1421 SNPs, 8 SNPs had more than 5% missing,
variations in apoptosis-related genes may have a significant thus 99.4% of the SNPs were called with greater than 95% of
effect on the risk of NSCLC in women. The current state of the samples genotyped.
the literature supports a prominent role for apoptosis in
lung cancer and provides the rationale behind this apoptosis Statistical analysis
focused study.
Cases were first compared with controls by the Students t-test
stratified by race for age, pack-years smoked and body mass
Methods
index (BMI). Differences in categorical variables (current
Study participants
smoker/never smoker, family history of lung cancer/no family
A population-based case-control study was conducted history, COPD/no COPD, and education) were determined
with the cases identified through the Metropolitan Detroit using a Chi-Square test. Hardy Weinberg Equilibrium (HWE)
Cancer Surveillance System, part of the NCIs Surveillance, was tested in whites and African-Americans separately for
Epidemiology and End Results (SEER) program, as described 105 SNPs. Seven SNPs not in HWE were excluded from further
previously [12]. Eligible cases were women between 18 to analysis; a total of 98 SNPs were examined in this study. Next,
74 years of age, diagnosed with histologically confirmed we excluded any SNP that was not called in at least 90% of
NSCLC between November 1, 2001 and October 31, 2005, and subjects. We then reran the clustering algorithm in BeadStudio
residing in Wayne, Oakland or Macomb counties. A total of before exporting the data. In addition, we calculated linkage
453 cases were used for this study. In total, 54.5% of eligible disequilibrium R2 values for all combinations of SNPs.
cases completed the in-person interview. Fifteen cases who
Controlling for age, smoking pack-years, ever/never
reported race other than African-American or white were smoking status, COPD history, family history of lung cancer,
excluded from further analysis. Age (5-year intervals) and BMI and education, a dominant mode of transmission
race-matched population-based controls living in the same was modeled utilizing unconditional logistic regression in
geographic area were identified by random digit dialing. The SAS version 9.1.3 (SAS Institute). The odds ratios obtained
majority (70%) of those who were eligible for screening agreed reflected the risk in the heterozygote and variant genotype
to participate in the same interview administered to the cases. group versus the homozygote wild-type genotype. OverEleven controls reported a race other than African-American fitting was assessed utilizing 1- [12,13]. The association
or white and were excluded from further analysis. Data from between significant genes and ever/never smoking status
a total of 478 control subjects were utilized in the study.
was assessed by stratification by ever and never smoking
status among whites only, as there were insufficient samples
Questionnaire data collection
within African-Americans to support such stratification. Next,
Questionnaire data collection was carried out as described in a for the significant SNPs, we utilized backwards elimination, to
previous report from this study [12]. A detailed questionnaire discern which SNPs were significantly associated with NSCLC,
covering personal health history, exposure history and while accounting for all significant SNPs (p<0.05) among
family history was administered to consenting participants. whites and African-Americans, respectively.
Never-smokers were defined as those who had smoked <100
The False Positivity Report Probability (FPRP) was determined
cigarettes in their lifetime. The diagnoses of COPD, chronic as described previously [14]. For this analysis, the a priori
bronchitis and emphysema were combined to obtain a recoded likelihood for the SNPs in CD40 and APAF-1 was set at 0.1
composite COPD variable. Incident COPD diagnoses within because of a lack of previous work suggesting association
the year prior to lung cancer diagnosis were not included. in SNPs in these genes and lung cancer. For TP63, there were
The COPD and BMI variables were by self-report. In addition, several GWAS studies [5,10,15-17] that have shown strong
the education variable was categorized into less than 12 evidence for association between TP63 and lung cancer, so
years, 12 to 16 years and greater than 16 years, to facilitate we judged it appropriate to set a strong a priori hypothesis
appropriate statistical analysis.
of significance for this gene (0.25). A FPRP of <20% was

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doi: 10.7243/2049-7962-3-1

considered to be significant.

and education. Supplement Table S1 list the odds ratios and

p-values for all SNPs, among whites and African-Americans,
SNP function annotation
respectively. The FPRP [14] was assessed for significant SNPs
To explore whether any of the significant SNPs identified might in whites and African-Americans. Among whites, SNPs in
have potential regulatory functions in lung and other tissues/ CD40 (rs1535045 and rs3765459) and APAF-1 (rs1007573)
cells, we used custom tracks on the UCSC Genome browser were significant with an a priori hypothesis of 0.1, having
(http://genome.ucsc.edu) to screen NIH Roadmap and ENCODE FPRP values <20%. Among African-Americans, two of the
data of the implicated SNP regions for evidence for regulatory four TP63 SNPs (rs6790167 and rs7613791), were found to
relevance [18,19], such as overlapping with chromatin marks have acceptable FPRP (less than 20%) for noteworthiness,
and interactions, CpG-site methylation, protein binding and given the strong a priori hypothesis (0.25) from the abundant
transcription factor binding motifs. We also used the online literature implicating TP63 in lung cancer. These TP63 SNPs
tools HaploReg (http://www.broadinstitute.org/mammals/ were ~12 kb apart at the 3 end of TP63 gene, with an R2 of
haploreg/haploreg.php) and RegulomeDB (http://regulome. 0.77 and a Lewontins D of 0.908. The CD40 SNPs rs1535045
stanford.edu) as a complementary analysis and to confirm the and rs3765459 were also in linkage disequilibrium (R2=0.87);
location of each SNP in relation to annotated protein-coding they were ~9.3 kb apart in an intronic region of CD40.
genes and/or non-coding RNA genes.
In parallel, we also conducted backwards elimination for
all significant SNPs. In whites, APAF-1 SNP rs1007573 and
Informed consent
CD40 SNP rs1535045 remained significant in the multivariable
The study was approved by the Wayne State University model. For African-Americans, the only SNPs that remained
institutional review board and each participant provided significant in the model after this selection procedure were
written informed consent.
ATM rs1801516, TNF rs1800629 and TP63 rs6790167.
In summary, only two SNPs in whites were significant by
Results
both FPRP and backward elimination (APAF-1 rs1007573 and
Participant characteristics
CD40 rs1535045), and only one SNP (TP63 rs6790167) was
Key participant characteristics stratified by race are provided significant by both methodologies in African-Americans.
in Table 1. There were no significant differences in age These three SNPs appear to be the most promising NSCLC
between the cases and controls for both whites and African- risk-modifying apoptosis-related SNPs in this study.
Americans. Approximately 90% of the cases were current
smokers among both whites and African-Americans; mean Stratification by ever/never smoking status
pack-years smoked was significantly greater in white and To investigate whether there might be interaction between
African-American cases relative to controls (46.17 vs. 12.49, smoking status and the contribution of a SNP to the
p<0.001 and 27.03 vs. 12.1, p<0.0001, respectively). Cases development of NSCLC, we built separate logistic regression
were significantly more likely to report a family history of lung models for never-smokers and ever-smokers among white
cancer relative to controls for both whites (24% versus 11.7 %, subjects. Basic characteristics of this white population, stratified
p<0.0001), and African-Americans (28.7% versus 10.8%, by smoking status, are listed in Table 3. An interaction was
p<0.0015). In addition, cases were more than twice as likely detected between smoking status and APAF-1. Among neveras controls to report a COPD diagnosis in whites (33.98% vs. smokers, the association between carrying the heterozygote
14.10%, p<0.0001) and African-Americans (23.40% cases vs. or variant homozygote genotype (rs1007573) and lung cancer
12.75% controls, p<.05). Controls had greater BMI than cases was OR=0.72 (95% CI:0.28-1.88), while the OR for this SNP
(p<0.0001) among both whites and African-Americans. Finally, among smokers was 2.86 (95% CI:1.58-5.16; p=0.01), after
for the categorical education variable defined, white controls adjustment. We found very similar associations for the CD40
were significantly different from cases (2=45.06 p<0.0001). SNPs in both never-smokers and smokers; however, these
However, this trend was not statistically significant among findings were only significant for smokers. The OR was nonAfrican-Americans (2=5.0357, p=0.08).
significant in CD40 rs3765459 among never-smokers 0.62
(95% CI:0.29-1.33) and significantly protective for smokers
Apoptosis-related SNPs and risk for NSCLC by race
(OR=0.58; 95% CI:0.37-0.89). Analogous results were found
We stratified our analysis by race since the allele frequency for the CD40 SNP rs1535045 (OR=0.66; 95% CI:0.311.40) for
distribution for 74.3% of the SNPs we differed significantly non-smokers and smokers (OR=0.52; 95% CI:0.33-0.80).
between African-American and white controls, as measured
by 2 analysis. Table 2 summarizes significant SNPs for whites Functional annotation of SNPs
and African-Americans as determined by our stratified Based on ENCODE data, rs228652 (TP63), rs100753 (APAF1)
multivariate logistic regression model, after adjustment for and rs1535035 (CD40) overlapped with open chromatin in
the covariates of age, pack-years smoked, ever/never smoking a number of ENCODE cell lines including A459 lung cancer
status, family history of lung cancer, history of COPD, BMI cells (Supplement Table S2 and Figures 1 and 2). Furthermore,

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Table 1. Characteristics of Participants.

White

African Americans

Variable

Cases (n=359)

Controls (n=376)

Age (Mean, SD)

60.65 (9.4)

59.60 (9.2)

Pack Years Smoked (Mean, SD)

46.17 (31.9)

Ever Smoker (n, %)

324 (90.3%)

Family History of Lung Cancer (n, %)

History of COPD* (n, %)

P-value

Cases (n=95)

Controls (n=102)

0.127

57.71 (9.3)

58.08 (9.4)

P-value
0.7856

12.49 (20.4)

<0.0001

27.03 (21.8)

12.1 (20.6)

<0.0001

185 (40.2%)

<0.0001

86 (91.5%)

54 (52.9%)

<0.0001

86 (24.0%)

44 (11.7%)

<0.0001

27 (28.7%)

11 (10.8%)

0.0015

122 (34.0%)

53 (14.1%)

<0.0001

22 (23.4%)

13 (12.8%)

0.0516

BMI (Mean, SD)

26.24 (5.7)

28.71 (6.7)

<0.0001

26.73 (6.7)

32.31 (8.2)

<0.0001

Education (Mean, SD)

12.89 (2.3)

14.3 (2.7)

<0.0001

12.33 (2.7)

13.5 (2.6)

0.0303

*COPD: Chronic Obstructive Pulmonary Disease including diagnosis of emphysema and chronic bronchitis.

Table 2. Evaluation of SNPs significantly associated with non-small cell lung cancer with the False Discovery
Reporting Percentage Method.
Race

Gene

Refseq

OR*** (95% CI)

0.1 A Priori FPRP (OR 2.0)

0.25 A Priori FPRP (OR 2.0)

Whites:

APAF-1

rs1007573

1.86 (1.17-2.95)

0.108*

0.0399**

CD40

rs3765459

0.62 (0.42-0.90)

0.105*

0.038**

CD40

rs1535045

0.58 (0.40-0.84)

0.045*

0.016**

African Americans:

ATM

rs1801516

24.15 (3.50-166.55)

0.659

0.392

BAK1

rs513349

0.35 (0.12-0.98)

0.629

0.361

TNF

rs1800629

0.42 (0.18-0.99)

0.548

0.288

P63

rs6790167

2.85 (1.33-6.09)

0.254

0.102 **

P63

rs7613791

2.70 (1.26-5.82)

0.31

0.13**

P63

rs35592567

0.44 (0.20-0.95)

0.471

0.235

P63

rs3856775

2.13 (1.03-4.43)

0.467

0.226

*Significant with an a priori hypothesis that the SNP may be associated with lung cancer.
**Significant with an a priori hypothesis that the SNP has a high likelihood of being associated with lung cancer.
***Adjusted for age, pack years smoked, ever/never smoking status, family history of lung cancer, history of COPD,
BMI and education.
Table 3. Characteristics of White Participants Stratified by
Smoking Status.
White
Varible

Smokers (n=509)

Non-Smokers

P-value

Age (Mean, SD)

60.28 (9.3)

59.7 (9.2)

0.463

Family history of Lung

Cancer (n, %)

95 (18.7%)

35 (15.5%)

0.298

History of COPD* (n,%)

152 (29.9%)

23 (10.9%)

<0.0001

BMI (Mean, SD)

26.89 (5.9)

28.89 (7.0)

<0.0001

*COPD: Chronic Obstructive Pulmonary Disease including

diagnosis of emphysema and chronic bronchitis.

the DNA regions containing rs100753 (APAF1) and rs1535035

(CD40) overlapped with weak enhancer and/or promoter
regions, respectively in normal fetal lung cells. Chromatin
interaction paired-end tags (ChiA-PET) data can define two
different DNA regions genomically far from each other or

on different chromosomes, but spatially close to each other

in the nucleus due to interaction for regulatory functions.
The DNA region containing rs100753 was enriched for CTCF
binding in MCF-7 and K562 cells and also has the potential
to form independent chromatin interactions/loops with the
3UTR and the gene body of the downstream gene ANKS1B,
albeit in K562 cells only. The chromatin interaction regions in
K562 cells can be observed as open chromatin sites (DNaseI
sites and FAIRE data) in A549 lung cancer cells (Figure 1). SNP
rs1535035 (CD40) also mapped to a DNA region enriched for
Pol2 enzyme in K562 and HCT-116 cells and characterized a
strong chromatin interaction with the upstream promoter
region of NCOA5 (Figure 2). A summary of the analyses for
each of the SNPs is presented in Supplement Table 2.

Discussion

The results of candidate gene SNP analysis [2-4] and largescale GWAS studies [5-7,10] have both strongly pointed to
apoptosis-related variations as key modifiers of lung cancer

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Figure 1. UCSC Genome Browser track display of ENCODE and Roadmap data from specific cells and tissues.
UCSC Genome Browser image of 3 region of APAF-1 and gene body of ANKS1B on human assembly hg19 showing
chr12q23.1region, NHGRI SNPs, RefSeq Genes, CpG island annotation, Roadmap Chromatin State Segmentation by a Hidden
Markov Model (HMM) for Roadmap fetal lung cells, ChIA-PET interactions and enrichment for CTCF in K562 and MCF-7 cells and
open chromatin (peaks (Pk) and signals (DS and Sg) for DNaseI-seq and FAIRE data) in A549 lung cancer cells. The HMM utilizes
computationally integrated ChIP-seq data for histone marks with associated segment color indicating a weak enhancer annotation
for the overlapping SNP region in fetal lung cells/tissue. The overlapping SNP region is also marked by open chromatin in A549 cells
and is enriched for CTCF binding and chromatin interactions in MCF-7 cells. Note all MCF-CTCF ChIA-PET interactions are not
shown in full for this genomic region or the overlapping SNP region and the data segment has been adapted for the purpose of this
illustration to show the relevant interactions only (bars connected by a line; intensity indicative of strength of interaction). The SNP
rs1007573 is colored red.

Pathak et al. Journal of Cancer Therapeutics & Research 2014,

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doi: 10.7243/2049-7962-3-1

Figure 2. UCSC Genome Browser track display of ENCODE and Roadmap data from specific cells and tissues.
UCSC genome Browser image of the CD40 gene and the 5 region of NCOA5 on human assembly hg19 showing 20q13.12 region,
RefSeq genes, Roadmap Chromatin State Segmentation by a HMM for Roadmap normal fetal lung cells, CpG islands, ChIA-PET
interactions and enrichment for Pol2 in K562 cells as well as binding of Pol2 in HCT-116 cells, and open chromatin (peaks (Pk),
signals (DS) and overlapping signals (OS) for DNaseI-seq and FAIRE data) in A549 lung cancer cells. The SNP rs1535045 is colored
red. The rs1535045 containing region also overlaps with open chromatin in A549 cells and is enriched for POL2 binding and POL2mediated chromatin interactions in K562 cells.

risk. In this study, we assessed the significance of 98 SNPs

in 32 apoptosis-related genes selected from the 400 genes
on the Illumina GoldenGate Assay Cancer SNP Panel. Given
that allele frequencies differed for 74% of the SNPs initially
analyzed between African-Americans and whites, we stratified

analysis of our candidate SNPs by race.

Associations differed significantly by race. In total, only
five SNPs in three genes reached a level of significance, as
determined by the False Positivity Report Probability Method.
One SNP in APAF-1 and two SNPs in CD40 were found to be

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significant by FPRP among whites and two SNPs in TP63 were

found to be significant by FPRP among African-Americans.
Fewer SNPs were significant by both FPRP and backwards
eliminationfor lung cancer risk: two SNPs in whites (APAF-1
rs1007573 and CD40 rs1535045) and one SNP in AfricanAmericans (TP63 rs6790167).
We observed an association between SNPs in TP63
(rs6790167: OR=2.85; 95% CI:1.33-6.09; rs7613791: OR=2.70;
95% CI:1.26-5.82) and NSCLC in African-Americans. These SNPs
were located within introns at the 3 end of TP63. Previous
GWAS studies have shown an association of TP63 SNP
rs4488809 in intron 1 and an increased risk of lung cancer
[5,10]. TP63 is a member of the p53 tumor suppressor gene
family and is involved in response to cellular stress [10] and is
known to be induced by DNA damage [20,21]. TP63 activation
may be an important part of the response mechanism to DNA
damage caused by cigarette smoking.
The observed association between APAF-1 SNP rs1007573
and NSCLC was significant by FPRP in white females. APAF-1
plays a critical role in the apoptosome by activating cleavage
of CASP9 in the apoptosis pathway. APAF-1 levels have been
reported to be decreased [22,23] and increased [24] in NSCLC.
Nuclear localization of APAF-1 has been associated with
improved 5-year survival and lower stage at diagnosis [25].
In addition, APAF-1 has been implicated in activation of CHK1
and cell cycle arrest under conditions of DNA damage [26],
implicating a non-apoptosome related function for APAF-1.
Taken together, APAF-1 appears to be functionally important in
the cellular response to stress; polymorphisms that decrease
APAF-1 levels and APAF-1 nuclear localization may lead to
more aggressive tumor formation. From this study, APAF-1
SNP rs1007573, was only significant among smokers; thus,
it is possible that the smoking associated mutagenic effects
are further potentiated by aberrations in the APAF-1 signaling
axis. In K562 and MCF-7 cells, rs1007573 was shown to overlap
with a region enriched for CTCF binding, which can act as a
transcriptional repressor or insulator. The SNP overlapping
region also has the potential to form a weak CTCF-mediated
chromatin interaction with the proximal 3UTR of ankyrin
repeat and sterile alpha motif domain containing 1B (ANKS1B),
as well as two stronger interactions with the gene body of
ANKS1B or upstream of four of its variant mRNA transcripts. A
recent paper has demonstarted a protective effect between
SNPs in ANKS1B and lung cancer risk in Caucasions [27].
The protective effect we observed of CD40 SNPs rs3765459
and rs1535045 against NSCLC has not been previously reported.
In this study we observed that rs1535045 overlaps with an
open chromatin site in A459 lung cancer cells. In other ENCODE
cell lines this SNP region also overlapped with a repressive
H3K27me3 mark and a H3K4me3 mark suggesting a cell-typespecific bivalent chromatin domain that is silenced, but poised
for activation. Notably, the rs1535045 containing region is
enriched for POL2 binding in both MCF-7 and HCT-116 cells,
and in MCF-7 cells has the potential to form a POL2-mediated

doi: 10.7243/2049-7962-3-1
chromatin loop with the promoter region of the nearby nuclear
receptor coactivator 5 (NCOA5) gene. CD40 is a member of
the tumor necrosis factor (TNF) receptor superfamily (TNFRSF).
Studies have shown that CD40 is expressed in NSCLC and in
lung cancer cell lines [28]. In a study of 129 surgical biopsy
samples, increased CD40 expression was associated with a
poorer prognosis [28]. Other studies, however, have shown that
gene transfer of CD40 in NSCLC produces a direct inhibition of
growth [29] and suppression of tumor proliferation [30]. This
discrepancy regarding the function of CD40 in NSCLC should
be investigated in future studies. It is important to note that
the protective effect of the CD40 SNPs were only significant
among smokers. The direction and magnitude of the effect
was similar in never-smokers but numbers in this group were
too small to make a definitive statement.

Strength and limitations

Our case-control study has several key strengths. The

population-based in-person interview design, plus the collection
of detailed data on exposure history, comorbidities and family
history, permitted us to account for possible confounders
that could obscure the relationship between candidate SNPs
and NSCLC risk. In addition, we had a sizeable number of
African-Americans in this study, permitting us to stratify our
analysis by race. Furthermore, this study is unique in that it
focuses primarily at women, and identifies germline NSCLC
risk modifiers in this select population.
Our study has a number of limitations. First of all, in
comparison to the GWAS studies, our sample size was relatively
small, reducing our power to detect significant SNPs of small
effect or low frequency alleles. Secondly, our interview
response rates among the cases was relatively modest at 54.5%
only. Third, we were only able to carry out stratified analysis
by ever/never smoking status in whites because of limited
sample size in African-Americans. Future large-scale studies
should address the possible interaction between smoking and
the presence of the TP63 SNPs. The majority of our cases were
lung adenocarcinomas and the results of our study may not
be generalizable to other histologic subtypes of NSCLC [31].
It is important to note that none of the p-values adjusted for
multiple comparisons in our study were significant. However,
as noted by Wacholder et al., [14], the a priori hypothesis of a
SNPs importance needs to be factored in when determining
signifcance in a study with multiple comparisons. Thus, we
utilized the Wacholder method, using existing evidence in
the literature to find SNPs significant by FPRP.
Also, while we observe open chromatin domains in the
UCSC tracks for A459 lung cancer cells, which coincided
with the CHIA-PET protein binding and interactions data
described for APAF-1 rs1007573 and CD40 rs1535045 from
other cancer cell types; the potential effect of the variant G
allele of rs1007573 or the T allele of rs1535045 on CTCF or
POL2 binding, respectively and/or chromatin interactions
across the specific genomic region in lung tissue is not known.

Pathak et al. Journal of Cancer Therapeutics & Research 2014,

http://www.hoajonline.com/journals/pdf/2049-7962-3-1.pdf

doi: 10.7243/2049-7962-3-1

Testing these hypotheses and the tissue-specific nature of a

potential regulatory function for these SNPs remain interesting
biological questions for the future. Also, detailed mechanistic
work examining the effect of these SNPs under conditions of
cellular stress is needed.

Conclusions

Our investigation of apoptosis-related SNPs in this casecontrol study of African-American and white women from
the Detroit Metropolitan area has identified three promising
variants associated with increased risk of NSCLC (whites:
APAF-1 rs1007573 and CD40 rs1535045; African-Americans:
TP63 rs6790167). These variants are significant by the FPRP
methodology and also remained in the model as independent
risk factor upon backwards elimination. Replication of our
findings in larger, additional well-powered studies will be
required to validate that these variants truly modify lung
cancer risk. Like this study, future association studies should
also account for important covariates that influence lung
cancer risk. In summary, we have carried out a study that has
rigorously assessed the lung cancer risk-modifying effects of
apoptosis-related SNPs, while accounting for critical covariates,
identifying 3 promising SNPs with potential regulatory functions
that warrant further investigation in larger and more representative
populations.
List of Abbreviations

SNPs: Single nucleotide polymorphisms

NSCLC: Non-small cell lung cancer
FPRP: False Positive Report Percentage
GWAS: Genome Wide Association Study

Additional files
Supplement Table S1
Supplement Table S2

Competing interests

The authors declare that they have no competing interests.

Authors contributions
Authors
contributions

AP

ASW

PLH

MLC

GRK

Research concept
and design

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Collection and/or
assembly of data

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Data analysis and

interpretation

--

--

Writing the article

--

Critical revision of
the article

Final approval of
article
Statistical analysis

MLB

AGS

--

--

--

--

--

--

--

--

--

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Acknowledgement and funding

SL

This work was supported by the National Institutes of Health

[R01-CA87895 to A.G.S. and contracts N01-PC35145 to A.G.S. and
P30CA22453]. This research was also supported in part by the

Intramural Research Program of the Division of Cancer Epidemiology

and Genetics of the NIH and NCI.

Publication history

EIC: G.J. Peters, VU University Medical Center, Netherlands.

Received: 31-Aug-2013 Revised: 06-Jan-2014
Accepted: 17-Jan-2014 Published: 31-Jan-2014

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Citation:
Pathak A, Wenzlaff AS, Hyland PL, Cote ML,
Keele GR, Land S, Boulton ML and Schwartz
AG. Apoptosis-related single nucleotide
polymorphisms and the risk of non-small cell
lung cancer in women. J Cancer Ther Res. 2014;
3:1.
http://dx.doi.org/10.7243/2049-7962-3-1