Abstract
Background/Aim: Numerous studies have reported the over-expression of the radiation-sensitive protein 51 (RAD51) in various types of cancer. However, the role of RAD51 genotypes in lung cancer remains largely unknown. This study aimed to assess the impact of the common variant RAD51 rs1801320 (G-135C) genotypes on the risk of lung cancer in Taiwan. Materials and Methods: The contribution of RAD51 rs1801320 genotypes to lung cancer risk was investigated in a cohort comprising 358 lung cancer patients and 716 age- and sex-matched healthy controls, utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Results: The analysis revealed that among the control subjects, the percentages of GG, CG, and CC genotypes of RAD51 rs1801320 were 73.2%, 24.3%, and 2.5%, respectively. Among the lung cancer patients, these percentages were 71.0%, 25.1%, and 3.9%, respectively (p for trend=0.4075). Allelic frequency distributions showed no significant association between the C allele of RAD51 rs1801320 and lung cancer risk determination (p=0.2987). Specifically, the RAD51 rs1801320 CC genotypes were associated with an elevated risk of lung cancer among males [adjusted odds ratio (aOR)=2.28, 95% confidence interval (95%CI)=1.03-4.87] and smokers (aOR=2.93, 95%CI=1.23-5.87), but not among females and non-smokers. Conclusion: The RAD51 rs1801320 CC genotype was identified as a risk factor for elevated lung cancer risk in males and smokers. This genotype may serve as a molecular biomarker at the DNA level for early detection and prediction of lung cancer in Taiwan.
Lung cancer stands as the foremost cause of global cancer mortality, and projections anticipate that the number of lung cancer-related deaths will escalate to 3 million by 2035 (1-3). Advancements in biomarker-driven targeted therapies are swiftly transforming the landscape of lung cancer treatment (4). From an epidemiological standpoint, the most prominent factor implicated in lung carcinogenesis is the enduring individual habit of tobacco consumption, which also proves valuable in predicting lung cancer prognosis (5, 6). Cigarettes contain a diverse array of carcinogens that elevate reactive oxygen species, leading to DNA adducts and strand breaks in lung tissues and cells. This complexity renders individual susceptibility to the various carcinogens in cigarette smoke intricate and unpredictable (7, 8). Accumulated studies in the literature report that specific genotypes exert differential effects on lung cancer risk among both cigarette smokers and non-smokers (9-17). These epidemiological studies, elucidating the interactions between genetic factors and smoking behaviors in lung cancer etiology, may offer a viable predictive model for personalized understanding of lung cancer origins.
From a molecular perspective, DNA repair pathways are activated to safeguard genetic stability and integrity when cells are exposed to various endogenous or exogenous DNA-damaging agents. Failure to correct these damages may lead to genomic instability and a gradual accumulation of mutations, constituting one of the hallmarks of cancer (18, 19). Among the diverse forms of DNA damage, DNA double-strand breaks (DSBs) pose a particular threat to cells. Two primary pathways, namely non-homologous end joining (NHEJ) and homologous recombination (HR), are responsible for repairing DSBs (20, 21). In general, NHEJ is an error-prone repair mechanism that brings together the broken ends, while HR is a precise and error-free repair process (22, 23).
From a molecular standpoint, the DSB repair protein RAD51 homolog 1, encoded by RAD51 and located at chromosome position 15q15.1 (24), plays a pivotal role in maintaining genetic stability and integrity when cells are exposed to various DNA-damaging agents. This region, known for exhibiting loss of heterozygosity in tumors, such as breast, colorectal, and lung cancers, is significant in understanding cancer progression (25). RAD51, a 339-amino acid protein in humans, is crucial for HR during DSB repair (26, 27). Over-expression of RAD51 has been reported in various cancers, including breast (28-33), pancreatic (34, 35), head and neck (36), prostate (37), soft tissue sarcoma (38), and esophageal cancer (39). Notably, RAD51 is found to be over-expressed in non-small cell lung cancer (40). The only exception observed so far is its under-expression in renal cell carcinoma (41). In genomic studies, one of the most investigated polymorphisms is the G to C polymorphism in RAD51’s promoter region, rs1801320 (G-135C) (42). Literature reports associate RAD51 rs1801320 genotypes with susceptibility to various cancers, including breast (43-49), laryngeal (50), colorectal (51, 52), prostate (42), ovarian (46, 53, 54), cervical (55), endometrial (56, 57), and glioblastoma (58).
Concerning lung cancer, several reports have investigated the contribution of RAD51 rs1801320 to susceptibility, yielding inconsistent results (59-62). In this study, we aimed to investigate the contribution of RAD51 rs1801320 genotypes, a single-nucleotide polymorphic (SNP) site, to the risk of lung cancer and subsequently examine the combined effect of smoking habits and sex with RAD51 rs1801320 genotypes on lung cancer risk in Taiwan.
Materials and Methods
Investigated population. Three hundred and fifty-eight cases diagnosed with lung cancer were recruited in China Medical University Hospital. The clinical characteristics were established and recorded by expert doctors. Individuals with a history of any other cancer or pulmonary diseases, such as chronic obstructive pulmonary disease, pneumothorax, and asthma, were excluded. Participants were required to complete a questionnaire, inform consent, and provided 5-ml of their blood for DNA extraction. At the same time, twice the number (716) of non-cancer healthy volunteers were selected to serve as controls from the Health Examination Cohort of our hospital. The exclusion criteria for controls included any malignancy or metastasized cancer from other origin, or any known genetic familial diseases. The study was approved by the Institutional Review Board of our hospital (DMR100-IRB-284), adhering to the principles of the Helsinki Declaration.
RAD51 rs1801320 genotyping procedures. Genomic DNA extracted from peripheral blood leukocytes of both patients and controls was prepared using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) and processed as previously outlined (63-66). The polymerase chain reaction (PCR) cycling programs for RAD51 rs1801320 genotypes were as follows: an initial cycle at 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. The forward and reverse primers for RAD51 rs1801320 were 5′-CAGGATCAAGCTCTCGAGCT-3′ and 5′-GGTGTTGCCTATAAAGGCTC-3′, respectively. The PCR products were then subjected to digestion by the restriction enzyme PspG I (New England BioLabs, Ipswich, MA, USA). The G-allele contigs were cleaved into 333- and 281-base pair fragments, while the C-allele contigs remained intact at 614-base pair products. The genotypic process was carried out independently and blindly by laboratory researchers, and repeated data showed 100% concordance.
Statistical methodology. The deviation of RAD51 rs1801320 genotype frequencies in the controls from those expected under Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s Chi-square test with Yates’ correction, or Fisher’s exact test was employed to compare the distribution of RAD51 rs1801320 genotypes between lung cancer cases and controls. Associations between RAD51 rs1801320 genotypes and lung cancer risk were further validated by calculating odds ratios (ORs) and their corresponding 95% confidence intervals (95%CIs) through logistic regression analysis. A significance threshold of p<0.05 was applied, considering values below this threshold as statistically significant.
Results
Table I presents the distributions of age, sex, and smoking status for the 358 lung cancer patients and 716 non-cancer controls. The mean age of the lung cancer patients was 64.0 years, which did not show a significant difference from that of the controls (64.8 years). Through the age and sex matching strategy during sampling, no significant disparities were observed between the control and lung cancer groups concerning age (64.8 versus 64.0 years) and sex (male to female ratios being 7:3 in each group) (Table I). Histologically, the lung cancer group exhibited percentages of adenocarcinoma, squamous cell carcinoma, and patients with other histologies at 60.9%, 29.6%, and 9.5%, respectively (Table I).
Selected demographic data for the lung cancer cases and controls in Taiwan.
Table II illustrates the distribution of RAD51 rs1801320 genotypes among non-cancer controls and lung cancer patients. The RAD51 rs1801320 genotypes did not exhibit a differential distribution between the lung cancer and control groups (p for trend=0.4075) (Table II). Specifically, the RAD51 rs1801320 heterozygous variant CG and homozygous variant CC did not show a significant association with lung cancer risk (OR=1.07 and 1.60, 95%CI=0.79-1.43 and 0.79-3.28, p=0.7225 and 0.2642) (Table II). The negative association of RAD51 rs1801320 polymorphic variants with lung cancer risk was further confirmed (recessive model: OR=1.58, 95%CI=0.78-3.21, p=0.2807; dominant model: OR=1.12, 95%CI=0.84-1.48, p=0.4838; Table II).
Distribution of RAD51 rs1801320 genotypes among the lung cancer cases and controls in Taiwan.
To corroborate the observations presented in Table III, we conducted additional analysis on the allelic frequency distribution patterns of RAD51 rs1801320 among lung cancer patients and control subjects (Table III). Consistent with the earlier findings indicating no association between the heterozygous variant CG or homozygous variant CC genotype of RAD51 rs1801320 and altered lung cancer risk, the variant C allele showed comparable levels among lung cancer cases and control subjects (OR=1.15, 95%CI=0.90-1.47, p=0.2987, Table III).
Distribution of RAD51 rs1801320 allelic frequencies among the lung cancer cases and controls in Taiwan.
We are interested in exploring the combined effects of RAD51 rs1801320 with sex and smoking status. Concerning the former, lung cancer patients and controls were stratified based on their sexes (Table IV). The results revealed that males carrying the homozygous CC genotypes at RAD51 rs1801320 exhibited an increased risk of lung cancer after adjustment for age, smoking, alcohol drinking, and areca chewing habits (adjusted OR=2.28, 95%CI=1.03-4.87, p=0.0884). Additionally, there was a slight trend suggesting that individuals with CC genotypes had a higher risk than those with CG and GG genotypes (Table IV, left). Conversely, there was no significantly altered lung cancer risk for females with CG or CC genotypes at RAD51 rs1801320 (Table IV, right). Regarding the latter, lung cancer patients and controls were stratified based on their smoking status. The results indicated that ever smokers carrying the homozygous CC genotypes at RAD51 rs1801320 had an elevated risk of lung cancer after adjustment for age, sex, alcohol drinking, and areca chewing habits. A small trend suggested that individuals with CC genotypes had a higher risk than those with CG and GG genotypes (adjusted OR=2.93, 95%CI=1.23-5.87, p=0.0309) (Table V, right). Conversely, there was no significantly altered lung cancer risk for non-smokers with CG or CC genotypes at RAD51 rs1801320 (Table V, left).
Odds ratios for RAD51 rs1801320 genotype on lung cancer risk determination after stratified by sex.
Odds ratios for RAD51 rs1801320 genotype on lung cancer risk determination after stratified by smoking status.
Discussion
The RAD51 protein is known to play a central role in HR during the repair of DNA DSBs. Over-expression of RAD51 has been implicated in lung cancer etiology (40). However, the genomic contribution of RAD51 to lung cancer remains poorly understood. In this study, we first assessed the impact of RAD51 rs1801320 genotype, along with sex and smoking status, on lung cancer risk. Among non-cancer healthy subjects, the percentages of wild-type GG and variant CG and CC genotypes were 73.2%, 24.3%, and 2.5%, respectively, and conformed well to Hardy-Weinberg equilibrium (p=0.4371, Table II). The distribution of wild-type G and variant C alleles at RAD51 rs1801320 in our population was 85.3% and 14.7%, respectively (Table III). In the global 1000 Genomes Project, East Asians were found to have a distribution of 85.0% wild-type G and 15.0% variant C alleles at RAD51 rs1801320, based on a sample size of 1,170 subjects (67).
In the cohort comprising 358 lung cancer patients and 716 non-cancer healthy subjects, the genotyping results revealed that neither the heterozygous CG nor the homozygous CC genotypes of RAD51 rs1801320 were significantly associated with an increased risk of lung cancer (Table II). Allelic frequency analysis further supports the genotypic frequency findings, indicating that the C allele of RAD51 rs1801320 was not associated with the determination of lung cancer risk (Table III). While our results do not suggest RAD51 rs1801320 as a useful biomarker for early lung cancer detection, two independent studies proposed that RAD51 rs1801320 genotypes could serve as useful molecular markers for predicting the clinical outcome of lung cancer patients (59), and might influence lung cancer overall survival and pneumonitis after radiotherapy (60).
Sex has been identified as a significant risk factor for lung cancer, as proposed by Gasperino in 2011 (68). Recent cancer statistics from 2022 reveal that 117,910 males and 118,830 females in the USA were afflicted with lung and bronchus cancers. The estimated death toll stood at 68,820 for males and 61,360 for females (3). Analyzing data from the Taiwan National Health Insurance Research Database covering the period 2002 to 2008, it was observed that approximately two-thirds of the 33,919 recorded lung cancer cases were males (69). This sex distribution closely mirrors the findings of the present study. Despite extensive research, the underlying mechanism(s) responsible for the sex-dependent difference in lung cancer risk remains poorly understood. It is postulated that the endocrine system may play a crucial role in this phenomenon, as suggested by Gasperino and his colleagues in 2011 (68). Notably, Taiwan has witnessed a rising trend in lung cancer cases among females over the past decade, affecting both prevalence and mortality rates. Consequently, our study aimed to investigate whether the variant RAD51 rs1801320 genotypes contribute to the observed sex difference in lung cancer susceptibility. Upon sex stratification, we observed an uneven distribution of RAD51 rs1801320 genotypes among males, contrasting with a more even distribution among females (Table IV).
Smoking is identified as another significant risk factor for lung cancer (70, 71). Prolonged cigarette consumption is known to contribute to airway remodeling and is implicated in the etiology of lung cancer and various pulmonary diseases (72). Additionally, cigarette smoking has been linked to the induction of DNA lesions and deficiencies in the removal of DNA adducts caused by tobacco carcinogens (73, 74). Given the potential interplay between the genotype of RAD51 rs1801320 and the smoking status of participants, we conducted an analysis to explore this interaction further. The results revealed an association between the variant CC genotype of RAD51 rs1801320 and an increased risk of lung cancer among individuals with a history of smoking (Table V, right section). While the current data indicated no differential distribution among non-smokers (Table V, left section), it is important to note that the lack of significance may be influenced by the sample size, and further enlargement of the sample could potentially yield meaningful results.
Taken together, our study indicated that the variant CC genotype of RAD51 rs1801320 is associated with an increased lung cancer risk among males and smokers. Studies with larger samples or various ethnics are needed to confirm our findings.
Acknowledgements
The Authors are grateful to Tissue-bank of China Medical University Hospital and doctors/nurses under Prof. Hsia’s leadership for their excellent sample collection and technical assistance. This study was supported partially by research grants from Taichung Armed Forces General Hospital (TCAFGH-D-112008) to Wang, from Asia and China Medical University (CMU112-ASIA-02 and ASIA-109-CMUH-05) to Li and from Taichung Tzu Chi Hospital (TTCRD112-17) to Chiu.
Footnotes
Authors’ Contributions
Research design: Chiu KL, Wang SC, Li CH, Bau DT; patient and questionnaire summaries: Li CH, Shen TC, Shen YC, Hsia TC; experimental work: Wang SC, Chang WS, Tsai CW; statistical analysis: Li CH, Shen TC, Shen YC; manuscript writing: Chiu KL, Bau DT, Hsia TC; manuscript checking and discussing: Chiu KL, Wang SC, Li CH, Shen TC, Li CH, Shen YC, Chang WS, Tsai CW, Hsia TC, Bau DT.
Conflicts of Interest
All the Authors declare no conflicts of interest regarding this study.
- Received January 23, 2024.
- Revision received February 13, 2024.
- Accepted February 14, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
