Abstract
Background
Smoking cessation lowers lung cancer (LC) risk, but the magnitude of risk reduction varies across individuals. FOXO3, a key regulator of cellular stress-response and longevity pathways, may influence susceptibility to LC after smoking cessation. The aim of the present study was to investigate whether FOXO3 genotype modifies the association between smoking cessation and incident LC.
Methods
We examined 4,339 ever-smoking American men of Japanese ancestry who were free of LC at baseline in the Kuakini Japan-Hawaii Cancer Study (1971–1975; mean age, 60 years). Participants were genotyped for FOXO3 rs2802292 and classified as FOXO3-G (TG/GG; longevity-associated G-allele carriers) or FOXO3-TT (TT, common allele homozygotes). Smoking status (former or current), cumulative exposure (pack-years), and duration of smoking cessation (years; current smokers assigned 0) were assessed. Incident LC cases through 1999 were identified. Cox proportional hazards models adjusted for potential confounders were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs), and test interactions.
Results
During 28 years of follow-up, 180 LC cases occurred, including 117 non–small cell lung cancer (NSCLC) cases. Significant interactions were observed between smoking status and FOXO3 genotype for LC (P=0.02) and NSCLC (P=0.003). Among former smokers, FOXO3-G carriers had a lower risk than FOXO3-TT carriers (LC: HR, 0.42; 95% CI, 0.23–0.79; NSCLC: HR, 0.23; 95% CI, 0.09–0.60). In contrast, no genotype-related differences were observed among current smokers. Among FOXO3-G carriers, each additional year of smoking cessation was associated with a reduced risk of LC (HR, 0.87; 95% CI, 0.81–0.94) and NSCLC (HR, 0.72; 95% CI, 0.58–0.89), whereas only weaker associations were observed among FOXO3-TT carriers (LC: HR, 0.97; 95% CI, 0.94–1.00; NSCLC: HR, 0.98; 95% CI, 0.94–1.01).
Conclusions
FOXO3 genotype may modify LC risk associated with smoking cessation. Compared with carriers of the longevity-associated FOXO3 G allele, individuals with the FOXO3-TT genotype had a substantially elevated risk of LC after smoking cessation; this association was not observed among current smokers. These findings support the potential for genotype-informed precision prevention, pending replication in larger and more diverse cohorts.
Keywords: Smoking cessation, FOXO3 genotype, lung cancer (LC), non-small cell lung cancer (NSCLC), gene-environment interaction
Highlight box.
Key findings
• The longevity-associated FOXO3 genotype is associated with reduced lung cancer risk after smoking cessation, but not among current smokers.
What is known and what is new?
• Smoking cessation reduces lung cancer risk, although residual risk persists among former smokers compared with never smokers.
• However, carriers of the FOXO3-G allele experience a greater reduction in lung cancer risk after smoking cessation compared with individuals with the FOXO3-TT genotype, suggesting a potential gene-smoking interaction influencing post-cessation risk.
What is the implication, and what should change now?
• Residual lung cancer risk following smoking cessation may differ by genetic background, highlighting potential heterogeneity in post-cessation risk trajectories.
• After smoking cessation, the FOXO3 G allele may suppress lung tumor development.
• These findings raise the possibility that genetically informed smoking cessation counseling and post-cessation lung cancer surveillance strategies could be explored, pending validation in larger and more diverse populations.
Introduction
Lung cancer ranks as the third most commonly diagnosed cancer in the United States (1). A recent UK study focusing on men aged 35–69 years reported that although lung cancer incidence declined from 109.3 per 100,000 in 1993–1996 to 64.2 per 100,000 in 2016–2018, largely due to reduced smoking rates, it remains the third most common cancer in men, following prostate and colorectal cancers (2). Moreover, lung cancer continues to be the leading cause of cancer-related deaths in both men and women (3). Although the number of current smokers in the United States decreased from 46.5 million to 28.7 million between 2000 and 2022, the number of former smokers remained high, increasing from 44.3 million to 55.5 million during the same period (4). Lung cancer prevention among former smokers warrants continued attention.
The FOXO3 gene, a part of the FOXO transcription factor family, contains a single nucleotide polymorphism (SNP rs2802292) whose minor G allele enhances FOXO3 gene expression under oxidative stress (5,6). FOXO3 is well recognized for its association with human longevity (7,8). Specifically, the minor G allele of SNP rs2802292—known as the FOXO3 longevity genotype—has been associated with reduced risks of all-cause mortality (9), coronary heart disease (9,10), Alzheimer’s disease (11), intracerebral hemorrhage (12), and cerebral microinfarcts (13). The protective effects of the FOXO3 G allele may be attributed to its function as a ‘damage controller,’ regulating cellular stress-response pathways that mitigate the harmful effects of physiological stressors, particularly oxidative stress associated with hypertension.
FOXO3 plays a complex role in cancer biology. It frequently acts as a tumor suppressor by promoting apoptosis, cell-cycle arrest, reactive oxygen species (ROS) inactivation, and DNA repair through expression of its target genes (14-19). However, its effects are context-dependent; in FOXO3-enhanced human embryonic stem cell-derived vascular cells, FOXO3-activated vascular cells exhibited delayed aging and increased resistance to oxidative injury compared with wild-type cells. When tested in a therapeutic context, FOXO3-enhanced vascular cells promoted vascular regeneration in a mouse model of ischemic injury and were resistant to tumorigenic transformation both in vitro and in vivo (20).
Experimental evidence suggests that FOXO3 may influence the fidelity of immune elimination of precancerous lung epithelial clones along the recognized progression from atypical adenomatous hyperplasia (AAH) to adenocarcinoma in situ (AIS) and ultimately invasive lung adenocarcinoma (LUAD). FOXO3 appears to act at two complementary checkpoints: (I) maintaining an “on-target” immune response characterized by effective antigen-specific priming and cytotoxic activity rather than chronic, tumor-promoting inflammation or myeloid-driven immune tolerance; and (II) increasing the susceptibility of emerging transformed epithelial cells to immune clearance through activation of apoptosis and cellular stress response pathways, thereby limiting stem-like escape. Because early LUAD develops along this AAH → AIS continuum, immune surveillance is operating in a setting where the airway microenvironment can become chronically inflamed and tumor-promoting if not tightly regulated (21).
Under certain contexts, however, FOXO3 may facilitate tumor progression. In hepatocellular carcinoma, for example, FOXO3 activation has been shown to drive oxidative damage and tumorigenesis through regulation of ROS and survival pathways (22). Similarly, experimental studies demonstrate that cigarette smoke suppresses FOXO3 expression while inducing epithelial-mesenchymal transition and increased cell migration, thereby promoting tumor progression and cell migration in lung cancer (23). Together, these findings suggest that the tumor-suppressive effects of FOXO3 may be overridden by environmental exposures such as cigarette smoke, providing a biological rationale for examining FOXO3 genotype in human lung cancer risk.
Smoking cessation is the most effective strategy for reducing lung cancer incidence (24). However, whether FOXO3 genotype modifies the association between smoking cessation and lung cancer risk—particularly among former smokers no longer exposed to cigarette smoke—remains unknown.
This study aimed to investigate, in a male cohort, (I) whether the tumor-suppressive effect of the FOXO3 longevity-associated FOXO3-G genotype on lung cancer incidence is attenuated by continued smoking, and (II) whether the reduction in lung cancer risk associated with smoking cessation differs by FOXO3 genotype. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0267/rc).
Methods
Study cohort
This prospective cohort study used data from the Kuakini Japan-Hawaii Cancer Study (Kuakini-JHCS), which was derived from the Kuakini Honolulu Heart Program (Kuakini-HHP) cohort. The Kuakini-HHP was initiated between 1965 and 1968, enrolling 8,006 American men of Japanese ancestry, aged 45 to 68 years, all residing on the island of O‘ahu, Hawai‘i. Detailed information on participant selection has been reported previously (25). The Kuakini-JHCS began during the third examination cycle of the Kuakini-HHP cohort, conducted from 1971 to 1975, which included 6,860 participants aged 51 to 75 years, coinciding with the start of the cancer surveillance program (26,27). The Kuakini-JHCS surveillance program concluded on December 31, 1999. Participants who attended the third examination of the Kuakini-HHP and reported being former or current smokers, who had FOXO3 genotype data available, and who had no prevalent lung cancer were eligible for the present study. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Kuakini Medical Center (#18-02). Written informed consent was obtained from participants for all clinical examinations, study procedures, and access to medical records for disease surveillance. When participants were unable to provide consent, written consent was obtained from their families or caregivers. All participants also provided written consent for publication of their de-identified data during each Kuakini-HHP examination.
Risk factors
At baseline (the third examination of the Kuakini-HHP, 1971–1975), self-reported cigarette smoking status was categorized as never smoker, former smoker (those who had quit smoking), or current smoker. Age at smoking initiation and age at cessation were recorded, and duration of smoking cessation was calculated for former smokers. Pack-years of cigarette smoking were calculated as packs per day multiplied by years smoked for former and current smokers. In analyses, duration of smoking cessation for current smokers was defined as 0 years. Duration of smoking cessation was analyzed as both a continuous and a categorical variable (grouped by years since cessation).
Potential confounders
Age was calculated using birth date and baseline examination date. Body mass index (BMI) was computed as weight in kilograms divided by height in meters squared. Alcohol intake (oz/month) was calculated based on self-reported usual monthly consumption of beer, wine, and spirits (including whiskey, gin, brandy, or other liquor) among current drinkers. Prevalent chronic lung disease was self-reported.
Additional covariates, including physical activity index (PAI), alcohol intake, dietary saturated fat intake, and years of education, were collected from the Kuakini-HHP Examination 1 (1965-68). PAI was quantified as metabolic output during a typical 24-hour period by multiplying a weighting factor by the reported number of hours spent in 5 activity levels (weights: no activity = 1.0, sedentary = 1.1, slight = 1.5, moderate = 2.4, and heavy = 5.0) (28). Years of education were self-reported. Percent of calories as saturated fat (%SFAT) was calculated using a 24-hour dietary recall as described previously (29).
Genotyping
The SNP rs2802292 in FOXO3 was genotyped in all participants who provided blood samples during the Kuakini-HHP Examination 4 (1991–1994). FOXO3 genotype refers to the rs2802292 polymorphism, categorized as carriers of the G allele (TG or GG; FOXO3-G) versus TT homozygotes (FOXO3-TT). In 2020, rs2802292—along with nine additional SNPs in other longevity-related genes—was additionally genotyped using archived biospecimens in participants who attended Kuakini-HHP Examination 3 (1971–1975) but did not participate in Examination 4. DNA for Examination 4 participants was extracted from the buffy coat of blood samples. For participants who did not attend Examination 4, DNA was isolated from archived blood serum using the QIAamp cell-free DNA kit and amplified using REPLI-g Single-Cell WGA & WTA amplification (QIAGEN Sciences, Germantown, MD, USA). Genotyping was conducted using a TaqMan assay on an Applied Biosystems QuantStudio 12K Flex system (Thermo Fisher Scientific, Waltham, MA, USA). Quality control comparing genotypes derived from the two DNA sources demonstrated 95% consistency.
Case ascertainment
All incident cancer cases between 1971 and December 1999 were identified through the Kuakini-JHCS surveillance program. Cases occurring before the start of the Kuakini-JHCS examination period (1971–1975), when the systematic cancer surveillance program began, were ascertained retrospectively (26,27) to determine prevalent cancer cases at baseline. Incident lung cancer cases were determined by a physician consensus panel based on hospital records and the Hawai‘i tumor registry, and were confirmed by histological evidence (26,27). Lung cancer subtypes included non-small cell lung cancer (NSCLC), encompassing squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and other non-small cell types, as well as small cell carcinoma. Cases with insufficient diagnostic information were classified as lung cancer, type unknown.
Addressing potential sources of bias
Selection bias was minimized by utilizing a well-characterized prospective cohort with high participation rates and standardized examination procedures across study cycles. Misclassification of cancer outcomes was minimized through a rigorous surveillance program. Information bias was reduced through standardized data collection protocols during cohort examinations and the use of previously validated measures of key exposures and covariates. Smoking exposure was characterized using multiple metrics (smoking status, pack-years, age at initiation, and duration of cessation) to reduce exposure misclassification.
To assess potential bias from reverse causation, we compared the proportion of participants who transitioned from current smoking at Examination 1 to former smoking at Examination 3 between those with the FOXO3-TT genotype (23.7%) and those carrying the FOXO3-G allele (22.1%). No significant difference was observed (P=0.34).
Potential confounding was addressed through multivariable adjustment for established lung cancer risk factors and lifestyle variables, including age, BMI, alcohol intake, PAI, dietary saturated fat intake, and years of education. In addition, stratified analyses by smoking status were conducted to evaluate potential effect modification and reduce residual confounding related to smoking behavior.
Statistical analysis
Baseline characteristics were compared across participants by smoking status, by FOXO3 rs2802292 genotypes TG or GG (noted as FOXO3-G) versus the common homozygous genotype TT (FOXO3-TT), and by lung cancer status during the follow-up period. Student’s t-test was used to compare continuous variables, and χ2 tests were used to compare categorical variables. Age-adjusted lung cancer incidence rates (cases per 10,000 person-years) were estimated by the direct method. Crude Kaplan-Meier survival curves were generated to visualize lung cancer risk by smoking status overall and stratified by FOXO3 genotype. Cox proportional hazards models were used to assess the associations of FOXO3 genotype and smoking status with lung cancer risk. Variables including age, BMI, pack-years of smoking, alcohol intake, and PAI were modeled as continuous variables. Duration of smoking cessation was examined both as a continuous variable and as a categorical variable (0 years, 1–9 years, and ≥10 years since cessation) to assess potential non-linear associations.
Main effects [hazard ratios (HRs) and 95% confidence intervals (CIs)] of genotype and smoking status were estimated using a Cox proportional hazards model (main-effects model) that included FOXO3 genotype and smoking status, adjusting for age and other potential confounders. An interaction term between smoking status and FOXO3 genotype was added to the main-effects model to test for interaction. Stratified analyses by smoking status were conducted to evaluate the association between FOXO3 genotype and lung cancer and NSCLC risk within each smoking category. The association between smoking cessation duration and lung cancer risk was examined by modeling cessation duration as both continuous and categorical (current smokers [0 years], former smokers 1–9 years, and ≥10 years). Relative risks were also estimated across four joint gene-smoking exposure groups and compared using pairwise comparisons. The proportional hazards assumption was evaluated for stratified analyses and met. Missing data for confounding variables were excluded in multivariate analyses. All statistical analyses were performed using the Statistical Analysis System (SAS) version 9.4 (SAS Institute Inc., Cary, NC, USA; RRID: SCR_008567). Stata 16 (StataCorp LLC; RRID: SCR_012763) was used to generate survival curves.
Results
Participants
Of the 6,860 participants who attended the third examination of the Kuakini-HHP, 4,339 who reported being current or former smokers, had complete data on FOXO3 genotype, and were free of lung cancer at baseline were included in the analytic cohort. Specifically, 9 participants with prevalent lung cancer, 57 with missing smoking status, 2,183 never smokers, and 272 with missing FOXO3 genotype data were excluded. Participants were followed from baseline (1971–1975) until lung cancer diagnosis, death, or the end of surveillance on December 31, 1999. The cohort contributed 86,653 person-years of follow-up. During a median follow-up of 22.8 years [interquartile range (IQR): 14.7–26.3 years; maximum: 28.2 years], 180 incident lung cancer cases were diagnosed, including 117 cases of NSCLC. Thus, 65% of ascertained lung cancer cases were NSCLCs and 35% were small cell carcinoma or of unknown histologic type.
Table 1 shows the baseline characteristics of participants by smoking status, FOXO3 SNP rs2802292 genotype, and lung cancer status during follow-up. Among 4,339 ever smokers, 49.8% were former smokers, and 50.2% were current smokers. Mean age at baseline was 59.8±5.4 years, and mean BMI was 23.6±3.1 kg/m2. Current smokers were slightly younger and had lower BMI compared with former smokers (both P<0.001). As expected, cumulative smoking exposure, pack-years, increased among current smokers, 39.1±29.6 among former smokers and 44.2±22.8 among current smokers (P<0.001). No significant differences in baseline characteristics were observed between participants with the FOXO3-G and FOXO3-TT genotypes (P=0.18–0.96). During follow-up, 180 participants developed lung cancer. Compared with those who remained lung cancer-free, men who developed lung cancer had lower BMI (23.0±3.2 vs. 23.6±3.1 kg/m2; P=0.01), greater cumulative smoking exposure (55.9±25.4 vs. 41.0±26.4 pack-years; P<0.0001), and shorter duration of smoking cessation (2.8±5.8 vs. 6.4±9.7 years; P<0.0001). Of note, among those who developed lung cancer, current smokers outnumbered former smokers by more than 2:1. Age at baseline did not differ significantly between those with and without lung cancer (P=0.98).
Table 1. Baseline characteristics by smoking status, FOXO3 longevity genotype, and incident lung cancer status.
| Variables | All | Smoking status | FOXO3 genotype | Lung cancer | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Former | Current | P for trend | TT | G | P | No | Yes | P | ||||
| N | 4,339 | 2,159 | 2,180 | 2,337 | 2,002 | 4,159 | 180 | |||||
| Age (years) | 59.8±5.4 | 60.2±5.6 | 59.3±5.2 | <0.0001 | 59.8±5.5 | 59.7±5.4 | 0.70 | 59.8±5.5 | 59.8±5.1 | 0.98 | ||
| Body mass index (kg/m2) | 23.6±3.1 | 24.0±3.0 | 23.1±3.2 | <0.0001 | 23.6±3.2 | 23.6±3.1 | 0.95 | 23.6±3.1 | 23.0±3.2 | 0.01 | ||
| Smoking (pack-years) | 41.6±26.5 | 39.1±29.6 | 44.2±22.8 | <0.0001 | 41.7±26.7 | 41.6±26.3 | 0.87 | 41.0±26.4 | 55.9±25.4 | <0.0001 | ||
| Alcohol intake (oz/month) | 16.4±27.1 | 13.4±23.1 | 19.3±30.4 | <0.0001 | 17.0±27.8 | 15.7±26.3 | 0.18 | 16.0±26.1 | 25.0±43.6 | <0.0001 | ||
| Physical activity index (score) | 32.9±4.6 | 32.7±4.4 | 33.1±4.7 | 0.006 | 32.9±4.7 | 32.8±4.5 | 0.34 | 32.9±4.6 | 32.7±4.3 | 0.61 | ||
| Education (years) | 10.3±2.8 | 10.4±2.9 | 10.2±2.8 | 0.07 | 10.4±2.9 | 10.2±2.8 | 0.19 | 10.3±2.8 | 10.6±2.8 | 0.18 | ||
| % saturated fat intake | 12.2±4.0 | 12.3±4.0 | 12.1±4.0 | 0.09 | 12.2±4.0 | 12.3±4.0 | 0.80 | 12.2±4.0 | 12.3±4.2 | 0.85 | ||
| Chronic lung disease | <0.0001 | 0.005 | ||||||||||
| No | 4,040 (93.8) | 2,054 (95.5) | 1,986 (92.0) | 2,164 (93.4) | 1,876 (94.2) | 0.32 | 3,882 (94.0) | 158 (88.8) | ||||
| Yes | 268 (6.2) | 96 (4.5) | 172 (8.0) | 152 (6.6) | 116 (5.8) | 248 (6.0) | 20 (11.2) | |||||
| Missing | 31 | 9 | 22 | 21 | 10 | 29 | 2 | |||||
| Duration of cessation (years)† | 6.3±9.6 | 12.6±10.2 | 0 | – | 6.3±9.6 | 6.2±9.5 | 0.93 | 6.4±9.7 | 2.8±5.8 | <0.0001 | ||
| Categories of cessation | – | <0.0001 | ||||||||||
| Quit 0 years | 2,180 (50.3) | 0 | 2,180 (100.0) | 1,172 (50.2) | 1,008 (50.3) | 0.96 | 2,053 (49.4) | 127 (70.6) | ||||
| Quit 1–9 years | 1,004 (23.1) | 1,004 (46.5) | 0 | 544 (23.3) | 460 (23.0) | 977 (23.5) | 27 (15.0) | |||||
| Quit ≥10 years | 1,152 (26.6) | 1,152 (53.5) | 0 | 618 (26.5) | 534 (26.7) | 1,126 (27.1) | 26 (14.4) | |||||
| Duration of cessation, missing | 3 | 3 | 0 | 3 | 0 | 3 | 0 | |||||
| Smoking status | – | <0.0001 | ||||||||||
| Former smokers | 2,159 (49.8) | – | – | 1,165 (49.8) | 994 (49.7) | 0.89 | 2,106 (50.6) | 53 (29.4) | ||||
| Current smokers | 2,180 (50.2) | – | – | – | 1,172 (50.2) | 1,008 (50.3) | 2,053 (49.4) | 127 (70.6) | ||||
| Genotype | 0.89 | 0.17 | ||||||||||
| FOXO3-TT | 2,337 (53.9) | 1,165 (54.0) | 1,172 (53.8) | – | – | – | 2,231 (53.6) | 106 (58.9) | ||||
| FOXO3-G | 2,002 (46.1) | 994 (46.0) | 1,008 (46.2) | – | – | – | 1,928 (46.4) | 74 (41.1) | ||||
Data are presented as mean ± standard deviation, n (%), or n. †, three participants with missing duration of smoking cessation were excluded from the analysis.
Descriptive data for lung cancer cases
For descriptive purposes, participants were categorized into four gene-smoking exposure groups based on FOXO3 genotype and smoking status: FOXO3-TT-Former, FOXO3-TT-Current, FOXO3-G-Former, and FOXO3-G-Current.
Table 2 presents the number at risk, the number of incident cases, and the age-adjusted incidence rates (cases per 10,000 person-years) for incident lung cancer and NSCLC by smoking status, for the entire cohort and for the four gene-smoking exposure groups. Notably, among former smokers, men with the FOXO3-G genotype had lower incidence rates of both lung cancer and NSCLC (7.2 and 2.4, per 10,000 person-years, respectively) compared to those with the FOXO3-TT genotype (16.2 and 10.1, per 10,000 person-years, respectively). However, this pattern was not observed among current smokers.
Table 2. Age-adjusted incidence rates of lung cancer and NSCLC (number of cases per 10,000 person-years) in the entire cohort and by gene-smoking exposure group.
| Outcome | Smoking status |
Entire cohort | FOXO3-TT | FOXO3-G | |||||
|---|---|---|---|---|---|---|---|---|---|
| N (cases) | Incidence rates | N (cases) | Incidence rates | N (cases) | Incidence rates | ||||
| Lung cancer | Former | 2,159 (53) | 12.0 | 1,165 (38) | 16.2 | 994 (15) | 7.2 | ||
| Current | 2,180 (127) | 31.3 | 1,172 (68) | 31.2 | 1,008 (59) | 31.9 | |||
| NSCLC | Former | 2,159 (29) | 6.5 | 1,165 (24) | 10.1 | 994 (5) | 2.4 | ||
| Current | 2,180 (88) | 21.7 | 1,172 (45) | 20.3 | 1,008 (43) | 23.6 | |||
NSCLC, non-small cell lung cancer.
Figure 1 presents the crude Kaplan-Meier curves for lung cancer-free survival by smoking status, shown overall and stratified by FOXO3 genotype. In the overall analysis combining both FOXO3 genotypes, former smokers exhibited better lung cancer-free survival than current smokers. When stratified by genotype, however, distinct patterns emerged. Among individuals with the FOXO3-TT genotype, the survival curve for former smokers shifted toward that of current smokers. In contrast, among carriers of the FOXO3-G, the survival curve for former smokers was further separated from that of current smokers. This pattern is consistent with a gene-smoking interaction influencing lung cancer risk.
Figure 1.
Crude Kaplan-Meier curves for lung cancer-free survival by smoking status, stratified by FOXO3 genotype. Log-rank test P values: entire cohort, P=1.2×10–9; subjects with FOXO3-TT, P=9.2×10–4; subjects with FOXO3-G, P=3.7×10–8.
Main and interaction effects of FOXO3 genotype and smoking status on lung cancer and NSCLC risk
Table 3 presents the main and interaction effects of the FOXO3 genotype and smoking status on lung cancer risk, estimated using multivariate Cox proportional hazards models. Both FOXO3 genotype and smoking status were modeled as categorical variables, with adjustment for age and other potential confounders.
Table 3. Main and interaction effects of smoking status and FOXO3 genotype on lung cancer and NSCLC.
| Model | Variable | Lung cancer | NSCLC | |||
|---|---|---|---|---|---|---|
| HR (95% CI) | P | HR (95% CI) | P | |||
| Main effect model | Current smokers | 1.00 (ref) | 1.00 (ref) | |||
| Former smokers | 0.46 (0.29–0.75) | 0.002 | 0.37 (0.20–0.69) | 0.002 | ||
| FOXO3-TT | 1.00 (ref) | 1.00 (ref) | ||||
| FOXO3-G | 0.82 (0.61–1.11) | 0.20 | 0.82 (0.57–1.19) | 0.30 | ||
| Full model (main effects + interaction) | Smoking status × FOXO3 genotype | 0.02 | 0.003 | |||
Main effect of smoking status and FOXO3 genotype were estimated as HRs with 95% CIs, using a Cox proportional hazards model adjusted for age, BMI, education, alcohol consumption, physical activity, percentage of saturated fat intake, chronic lung disease, smoking pack-years, and duration of smoking cessation. FOXO3-G and smoking status were modeled as categorical variables. The global effects of the interaction between FOXO3 genotype and smoking status were obtained from the Type 3 test of the full Cox model with GLM parameterization. The terms “main effect” and “interaction effect” refer to statistical associations and are not intended to imply causality. CI, confidence interval; HR, hazard ratio; NSCLC, non-small cell lung cancer.
As shown in Table 3, in the main-effects models, former smokers had a lower risk of lung cancer and NSCLC than current smokers. No overall association was observed between the FOXO3 genotype and the incidence of lung cancer or NSCLC. In the full models including an interaction term, the interaction between smoking status and FOXO3 genotype was statistically significant for LC (P=0.02) and NSCLC (P=0.003), and both P values were below the multiple-testing adjusted significance threshold of 0.025. These findings suggest that FOXO3 genotype modifies the association between smoking status, including smoking cessation, and lung cancer and NSCLC risk.
Figure 2 presents the relative risks (RRs) for four gene-smoking exposure groups, estimated using a multivariable Cox proportional hazards model. The reference group was individuals with the FOXO3-TT genotype who were former smokers (TT-Former). Pairwise comparisons were restricted to: (I) evaluating whether the relative risks (relative to TT-Former) differed between FOXO3 genotypes within each smoking category, and (II) evaluating whether the relative risks differed between former and current smokers, within each genotype. Notably, among former smokers, men with longevity-associated FOXO3-G had substantially lower risk compared to those with FOXO3-TT genotype.
Figure 2.
Relative risks were estimated using a Cox proportional hazards model adjusted for age, BMI, education, alcohol consumption, physical activity, percentage of saturated fat intake, and smoking pack-years. Subjects with the FOXO3-TT genotype who were former smokers (TT-Former) served as the reference group. P values for pairwise comparisons were obtained using Least Squares Means with a GLM parameterization. BMI, body mass index; GLM, generalized linear model.
FOXO3 genotype and lung cancer risk stratified by smoking status
Table 4 shows stratified analyses by smoking status. HRs for incident lung cancer and NSCLC of FOXO3-G comparing with FOXO3-TT genotype were estimated using Cox proportional hazards models adjusted for potential confounders. Among current smokers, no significant differences in HRs for incident lung cancer or NSCLC were observed between FOXO3 genotypes. In contrast, among former smokers, individuals carrying the FOXO3-G genotype exhibited significantly lower risks of both incident lung cancer (HR: 0.42; 95% CI, 0.23–0.79; P=0.007) and NSCLC (HR: 0.23; 95% CI, 0.09–0.60; P=0.003) compared with individuals with the FOXO3-TT genotype.
Table 4. Hazard ratios and 95% confidence intervals for lung cancer and NSCLC according to FOXO3 genotype, stratified by smoking status.
| Outcome | Variable | Former smoker (n=2,159)† | Current smoker (n=2,180)‡ | |||
|---|---|---|---|---|---|---|
| HR (95% CI) | P | HR (95% CI) | P | |||
| Lung cancer | FOXO3-TT (ref) | 1 | 1 | |||
| FOXO3-G | 0.42 (0.23–0.79) | 0.007 | 1.03 (0.72–1.47) | 0.86 | ||
| NSCLC | FOXO3-TT (ref) | 1 | 1 | |||
| FOXO3-G | 0.23 (0.09–0.60) | 0.003 | 1.16 (0.76–1.77) | 0.50 | ||
†, HRs and 95% CIs were adjusted for age, BMI, education, alcohol consumption, physical activity, percentage of saturated fat intake, self-reported chronic lung disease, smoking pack-years and duration of cessation. ‡, HRs and 95% CIs were adjusted for age, BMI, education, alcohol consumption, physical activity, percentage of saturated fat intake, self-reported chronic lung disease and smoking pack-years. BMI, body mass index; CI, confidence interval; HR, hazard ratio; NSCLC, non-small cell lung cancer.
Smoking cessation duration and lung cancer risk stratified by FOXO3 genotype
To explore the association between duration of smoking cessation and lung cancer incidence by FOXO3 genotype, Table 5 shows the association between duration of smoking cessation and lung cancer incidence by FOXO3 genotype. Each additional year of smoking cessation was associated with a modest risk reduction of lung cancer (~6%; HR: 0.94; 95% CI, 0.91–0.97; P<0.0001) among all ever smokers. Among individuals with the FOXO3-TT genotype, each additional year of smoking cessation was associated with a small reduction (~3%; HR: 0.97; 95% CI, 0.94–1.00; P=0.07). In contrast, among individuals with the FOXO3-G genotype, each additional year of smoking cessation was associated with a significantly greater risk reduction (13%; HR: 0.87; 95% CI, 0.81–0.94; P=0.0003). Similar results were observed for NSCLC.
Table 5. Association of duration of smoking cessation with risk of lung cancer.
| Variable | Ever smokers (n=4,336) | FOXO3-TT (n=2,334) | FOXO3-G (n=2,002) | |||||
|---|---|---|---|---|---|---|---|---|
| HR (95% CI) | P | HR (95% CI) | P | HR (95% CI) | P | |||
| Duration of smoking cessation (per year) | ||||||||
| Outcome: lung cancer | 0.94 (0.92–0.97) | <0.0001 | 0.97 (0.94–1.00) | 0.07 | 0.87 (0.81–0.94) | 0.0003 | ||
| Outcome: NSCLC | 0.94 (0.90–0.97) | 0.0006 | 0.98 (0.94–1.01) | 0.23 | 0.72 (0.59–0.89) | 0.002 | ||
| Categories of duration of smoking cessation (outcome: lung cancer only) | ||||||||
| Current smokers | 1 | 1 | 1 | |||||
| Duration 1–9 years | 0.37 (0.24–0.57) | <0.0001 | 0.40 (0.23–0.71) | 0.002 | 0.33 (0.17–0.65) | 0.001 | ||
| Duration ≥10 years | 0.44 (0.28–0.70) | 0.0005 | 0.75 (0.44–1.27) | 0.29 | 0.11 (0.04–0.37) | 0.0003 | ||
| P for trend | 0.0005 | 0.29 | 0.0003 | |||||
HRs and 95% CIs were estimated using Cox proportional hazard models adjusted for age, BMI, education, alcohol consumption, physical activity, percentage of saturated fat intake, self-reported chronic lung disease, and smoking pack-years. Three participants with missing duration of smoking cessation were excluded. Because of the limited number of NSCLC cases across cessation categories, NSCLC-specific analyses were not conducted. BMI, body mass index; CI, confidence interval; HR, hazard ratio; NSCLC, non-small cell lung cancer.
Among FOXO3-G carriers, lung cancer risk declined monotonically with increasing years since cessation when cessation duration was analyzed as a categorical variable (Table 5). However, this trend was not observed among FOXO3-TT carriers. Compared with current smokers who never quit, former smokers who had quit 1–9 years earlier had a 60% reduction in risk (HR: 0.40; 95% CI, 0.23–0.71; P=0.002). In contrast, among those who had quit ≥10 years earlier, the reduction was attenuated to 25% and was no longer statistically significant (HR: 0.75; 95% CI, 0.44–1.27; P=0.29). Results from the fully adjusted Cox proportional hazards models are presented in Table S1. Because of the limited number of NSCLC cases across cessation categories, NSCLC-specific analyses were not conducted.
Discussion
In this population-based prospective cohort study, we observed that smoking cessation was associated with a reduced risk of lung cancer in men who quit smoking compared with men who currently smoke, regardless of the FOXO3 genotype, consistent with findings from a large population-based study (24). However, the magnitude of risk reduction after smoking cessation varied by FOXO3 genotype. Among former smokers, carriers of the FOXO3-G allele experienced a greater reduction in lung cancer risk (HR: 0.42) and NSCLC risk (HR: 0.23) compared with carriers of the FOXO3-TT genotype; these differences were not seen among current smokers. Moreover, unlike FOXO3-TT carriers, FOXO3-G carriers showed a linear decline in lung cancer risk with longer duration of smoking cessation. Together, these findings suggest that FOXO3 genotype may be associated with heterogeneity in lung cancer risk reduction following smoking cessation.
No statistical evidence of interaction was observed between smoking pack-years (a measure of cumulative smoking exposure) and FOXO3 genotype on lung cancer incidence (data not shown). This suggests that the observed effect modification by FOXO3-G genotype may be more related to smoking status (current versus former) or to the post-cessation environment, rather than to cumulative lifetime smoking exposure alone.
Cigarette smoke contributes to lung carcinogenesis through multiple stages including initiation, promotion, and progression. During initiation, carcinogens such as polycyclic aromatic hydrocarbons (PAHs), nitrosamines (NNK), and ROS induce DNA damage and impair repair (30,31). Persistent exposure promotes chronic inflammation and epigenetic alterations that silence tumor suppressor genes, enabling clonal expansion of genetically unstable epithelial cells (32,33). Progression involves mutations in oncogenes such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and myelocytomatosis oncogene (MYC), facilitating invasive carcinoma within an immunosuppressive microenvironment that drives angiogenesis and immune evasion (34-36).
Under conditions of oxidative stress, the FOXO3-G allele has been associated with higher FOXO3 expression (5,6), suggesting an enhanced tumor-suppressive “damage-control” function. In contrast, experimental studies indicate that exposure to cigarette smoke suppresses FOXO3 expression (23), potentially diminishing genotype-related differences during ongoing exposure. This framework is consistent with our observation that FOXO3 genotype was not associated with lung cancer risk among current smokers, in whom persistent smoke exposure may overwhelm any genotype-dependent protective mechanisms.
Even after smoking cessation, residual molecular damage and microenvironmental changes sustain the risk of lung carcinogenesis. Carcinogens in cigarette smoke, including PAHs and tobacco-specific NNK, and ROS, induce persistent DNA damage and mutations in bronchial epithelial cells, affecting oncogenes such as KRAS and tumor suppressors such as TP53 with RB1 commonly inactivated along with TP53 in smoking-related small-cell lung cancer (31). These initiated cells can persist in the lungs and subsequently undergo malignant expansion. In addition, smoking produces widespread genetic and epigenetic alterations across the airway epithelium, termed ‘field cancerization,’ which remain detectable years after cessation and provide fertile ground for independent tumor development (37,38). The lung tissue microenvironment also exhibits long-lasting sequelae of smoking, including chronic inflammation, fibrosis, and impaired immune surveillance, which further promote malignant transformation of pre-damaged cells (39). Epigenetic ‘memory’ of tobacco exposure, such as persistent DNA methylation and microRNA dysregulation, can silence DNA repair genes or tumor suppressors, thereby maintaining a pro-carcinogenic state even in the absence of continued exposure (40,41). Finally, premalignant clones that survive cessation may gradually expand through age-related or environmental pressures, contributing to tumor promotion without further smoke exposure (42). Thus, former smokers continue to face an elevated risk of lung cancer for decades after cessation.
In our study, residual lung cancer risk after smoking cessation was most pronounced among men with the FOXO3-TT genotype (Figure 2). Among FOXO3-G carriers, risk declined progressively with increasing years since quitting (Table 5), whereas no clear temporal gradient was observed among FOXO3-TT carriers. Notably, FOXO3-TT carriers who had quit ≥10 years earlier did not experience greater risk reduction than those who had quit for <10 years, suggesting attenuation of long-term cessation benefit.
Accumulating evidence has shown the paradoxical intrinsic role of the Forkhead box O (FOXO) family of transcription factors in cancer, which can act as a tumor repressor while also maintaining cancer stem cells (43,44). We believe that the FOXO3-G carriers have greater control between growth (i.e., cancer) vs quiescence (i.e., stem cell maintenance) and that the FOXO3-TT participants did not benefit from this control.
The finding among FOXO3-G carriers suggests that removal of ongoing smoke exposure may restore FOXO3-mediated protective functions, with the G allele potentially enhancing antioxidative and DNA repair pathways under oxidative stress. Such activity may mitigate malignant progression and suppress tumor development to a level that does not manifest clinically as detectable lung cancer. Cancer cells arise from stem cells. FOXO3 often supports long-term maintenance by promoting quiescence, oxidative stress defenses, and genomic stability (43,44). In LUAD, FOXO3 has been described as a target of genomic deletion (i.e., reduced gene dosage), consistent with a classic tumor-suppressor axis in at least a subset of tumors (45). Blood DNA methylation at FOXO3 has been reported to be associated with lung cancer survival, suggesting that FOXO3 regulation (whatever the upstream driver—genotype, exposures, inflammation) can be clinically meaningful (46). Further investigation of this potential protective mechanism, using animal models and cell-line experiments, is warranted.
One key implication of our findings is that residual lung cancer risk among former smokers may not be uniform and may vary by genetic background. Without stratification by FOXO3 genotype, the risk of lung cancer may be underestimated among former smokers with the FOXO3-TT genotype and overestimated among FOXO3-G carriers.
Emerging evidence suggests that genotype-informed feedback tools are acceptable and feasible among smokers, providing a foundation to test tailored messaging alongside guideline-concordant cessation programs (47). Consistent with meta-analyses showing that DNA-based risk communication alone does not reliably increase quitting, genotype disclosure should augment, not replace, proven interventions (48). In this context, integrating FOXO3 (and other) genotype feedback into evidence-based treatment and supportive policy environments may help target messaging or follow-up intensity without displacing standard care. Our results support prioritizing cessation access for all, with genotype-guided tailoring where warranted, for example, more intensive lung cancer screening for FOXO3-TT former smokers who appear to retain higher residual risk (49).
However, because this is an observational study, clinical translation of genotype-based risk stratification and screening strategies should be considered hypothesis-generating. Independent replication, prospective validation, and evaluation of predictive performance within established clinical frameworks are needed before any clinical implementation can be considered.
FOXO3 has been explored as a potential therapeutic target in lung cancer biology (50). Prior work shows that elevated genetic susceptibility to smoking-related lung cancer (e.g., CHRNA5) can be substantially mitigated by smoking cessation, highlighting the primacy of cessation even among genetically high-risk individuals (51). Our findings suggest that strategies aimed at enhancing FOXO3 activity or downstream pathways may be most relevant for former smokers with the FOXO3-TT genotype; however, these hypotheses require additional prospective and mechanistic evaluation.
Limitations
Our study had several limitations. The study focused exclusively on American men of Japanese ancestry, limiting generalizability to women and other ethnic groups. As is common in longitudinal studies requiring repeated participation, individuals who took part in both the third and fourth Kuakini-HHP examinations (when blood samples were collected for genotyping) were generally healthier than those who did not, as discussed in a previous report (52). Former smokers who survived long enough to quit smoking and remain under long-term follow-up may represent a selected subgroup with greater baseline resilience, introducing potential survivor bias. FOXO3 is associated with longevity and cardiovascular outcomes, and competing risks of death may have influenced lung cancer incidence estimates, particularly at older ages. Information on second-hand smoke exposure and family history of lung cancer was not available in this cohort and therefore could not be included in the analyses. These factors may have contributed to residual confounding of lung cancer risk estimates, particularly among former smokers. For example, second-hand smoke exposure could have led to underestimation of smoking-related risk differences if exposure persisted after smoking cessation. Family history of lung cancer may also reflect underlying genetic or shared environmental susceptibility not fully captured in our models. However, it remains unclear whether these unmeasured factors differed systematically by FOXO3 genotype and therefore whether they would substantially bias the observed FOXO3-smoking interaction findings.
Smoking cessation duration and lung cancer risk stratified by FOXO3 genotype in smoking behavior over time could have introduced exposure misclassification. We evaluated changes in smoking behavior between Examination 3 (1971–1975), when participants reported being current smokers, and Examination 4 (1991–1994), when smoking status was reassessed. Among current smokers at Examination 3 who attended Examination 4 (attrition rate =46.8%), most had transitioned to former smokers. There was no significant difference in the proportion who remained current smokers between the FOXO3-TT group (23.8%) and the FOXO3-G group (21.6%; P=0.33). Therefore, changes in smoking status were unlikely to have substantially biased the observed interaction or association between genotype and lung cancer incidence among former smokers. However, smoking misclassification and changes in smoking behavior may have attenuated the observed difference in risk between former and current smokers, potentially leading to an underestimation of the risk reduction associated with smoking cessation.
In addition, the approximately 20-year interval between Examination 3 and Examination 4 complicated time-varying smoking assessment. Of the 180 lung cancer cases identified, 79% occurred during this period, reducing the utility of this time-dependent variable. Chronic lung disease was included as a covariate because it may also reflect underlying susceptibility to lung cancer independent of smoking exposure. Nevertheless, because chronic lung disease may partially lie on the causal pathway between smoking and lung cancer, some degree of overadjustment cannot be excluded.
Finally, because this is an observational study, clinical translation of genotype-based risk stratification and screening strategies should be considered hypothesis-generating. Independent replication, prospective validation, and evaluation of predictive performance within established clinical frameworks are needed before any clinical implementation can be considered.
Strengths
Despite these limitations, the study has several notable strengths. Uniform data collection: all participants completed standardized risk factor assessments and were followed using a consistent, well-established outcome surveillance protocol. The long duration of follow-up and large cohort size provided strong statistical power to evaluate gene-environment interactions and smoking cessation effects. The study population—men of Japanese ancestry—provided a unique genetic context, as Japanese individuals are generally more genetically homogeneous than many other racial groups (53). This homogeneity, together with the higher degree of linkage disequilibrium observed in Asian populations, can facilitate the detection of genotype-disease associations (54). Facilitated by ours being an island population, the follow-up was nearly complete (26), further strengthening the rigor of outcome surveillance. Finally, to our knowledge, this is the first prospective cohort study to investigate the association between FOXO3 genotype and smoking status (especially smoking cessation), with lung cancer risk, providing novel insights into genetic heterogeneity in post-cessation cancer risk.
Conclusions
The FOXO3 genotype may modify the association between smoking status and lung cancer risk. After smoking cessation, individuals with the longevity-associated FOXO3-G genotype experienced lower lung cancer risk compared with those carrying the FOXO3-TT genotype. In contrast, the FOXO3-G genotype did not appear to confer additional protection among current smokers. These findings are consistent with a possible gene-environment interaction between FOXO3 and smoking status. However, larger studies in diverse populations are needed to clarify the magnitude, consistency, and generalizability of this association.
Supplementary
The article’s supplementary files as
Acknowledgments
We thank all study participants and their families for their cooperation over many decades, the Hawaii State Department of Health for their help, Ayako Elliott and Eva Ardo for their assistance with genotyping, and Hiromi Nakada for monitoring the vital status of Kuakini-HHP participants.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Kuakini Medical Center (#18-02). Written informed consent was obtained from participants for all clinical examinations, study procedures, and access to medical records for disease surveillance. When participants were unable to provide consent, written consent was obtained from their families or caregivers. All participants also provided written consent for publication of their de-identified data during each Kuakini-HHP examination.
Footnotes
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0267/rc
Funding: This work was supported by the National Institute on Aging [Contract No. N01-AG-4-2149, Grants Nos. 5U01AG019349-05, 5R01AG027060, 2R01AG027060-13 (Kuakini Hawaii Lifespan Study), 5R01AG038707 (Kuakini Hawaii Healthspan Study), and 1P20GM125526-01A1, 5P20GM125526-05 (Kuakini HHP Center of Biomedical Research Excellence for Clinical and Translational Research on Aging)]; the National Heart, Lung, and Blood Institute (Contract No. N01-HC-05102); and the National Cancer Institute [Contracts Nos. N01-CP-33216, N01-CN-55424, N01-CA-15655, and N01-CP61060 (Kuakini Japan-Hawaii Cancer Study)]; and the Kuakini Medical Center.
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0267/coif). T.A.D. and B.J.W. hold a US patent 20130295566 entitled “Method of using FOXO3A polymorphisms and haplotypes to predict and promote healthy aging and longevity”. The other authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0267/dss
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