Assessment of susceptibility to mTOR rs2295080 gene polymorphism in Guangxi Zhuang lung cancer population.

Introduction
Lung cancer ranks as the most prevalent malignancy with the highest mortality rate worldwide, and its global incidence and mortality have both risen steadily in recent years (1). The pathogenesis of lung cancer is a multifaceted process involving the interplay of numerous factors, including genetics, environment, and metabolism (2). Genetic alterations have been identified as a major contributor to the development of lung cancer, with numerous specific variants being shown to disrupt normal growth regulation, disturb the balance between cell proliferation and apoptosis, and ultimately lead to lung cancer (3,4). It is important to note that alterations in key signaling pathways are central to this process. For instance, mutations and polymorphisms in the epidermal growth factor receptor (EGFR) gene have been identified as significant contributors to lung tumor proliferation, survival, and therapeutic response, thereby establishing them as a fundamental basis for targeted therapeutic interventions (5). Concurrently, the mammalian target of rapamycin (mTOR) signaling pathway represents another critical intracellular regulatory network that plays a pivotal role in various biological processes, including cell growth, proliferation, metabolism, and autophagy (6-8). The interplay and convergence of these pathways, including EGFR and mTOR signaling, fundamentally shape the biology of lung cancer.
A plethora of studies have demonstrated that aberrant activation of the mTOR signaling pathway is closely associated with the development of various tumors (9,10). Furthermore, mTOR gene polymorphisms have been linked to an increased susceptibility to multiple tumor types, including gastric (11,12), breast (13,14), bladder (15), and colorectal (16) cancers. The mTOR gene is located on the short arm of human chromosome 1 (1p36.22) (17), and is primarily expressed in distinct complexes: mTORC1 and mTORC2. In cancer, both mTORC1 and mTORC2 can be phosphorylated and activated (18). This activation has been shown to inhibit the pro-apoptotic protein Bad (19) and activate downstream effector molecules such as S6K and 4EBP1 (20), thereby enhancing proliferative and invasive potential. In addition, it has been demonstrated that this activation promotes tumor angiogenesis and immune evasion by regulating the expression of growth factors [vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β)] and programmed death-ligand 1 (PD-L1) (21-23).
Given its crucial role, genetic variation within the mTOR gene itself is a plausible modifier of lung cancer susceptibility. The mTOR gene contains multiple single-nucleotide polymorphisms (SNPs), which can affect an individual’s risk by regulating the gene’s transcriptional, translational, or protein functions (12). However, existing studies (10,24,25) on the association between mTOR gene polymorphisms and lung cancer risk have yielded inconsistent results and are often limited in scope to a single pathological subtype.
In the present study, an investigation was conducted into the association of the mTOR promoter polymorphism rs2295080 with susceptibility to overall lung cancer, as well as across its major pathological subtypes. Furthermore, we explored potential underlying mechanisms by integrating immunohistochemical analysis of mTOR protein expression. This comprehensive approach aims to provide new insights into the genetic etiology of lung cancer and its potential implications for prevention and early intervention. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-aw-2375/rc).

Methods

Study subjects and ethics
A total of 377 participants of Zhuang ethnicity were included in this study, comprising 241 lung cancer patients (cases) and 136 healthy individuals (controls). All participants were enrolled from The First Affiliated Hospital of Guilin Medical University and its Physical Examination Center between August 2020 and April 2023. The sample size was determined based on the availability of eligible patients and matched controls during the study period, aiming for a case-to-control ratio of approximately 1.8:1 to maximize statistical power within the constraints of a single-center study.
To minimize selection bias, consecutive eligible lung cancer patients meeting the inclusion criteria were recruited. Healthy controls were frequency-matched to cases by age (±5 years) and sex, drawn from the same geographical region during the same period. All study participants were unrelated, independent individuals. The diagnostic criteria for lung cancer followed the Chinese Medical Association Guidelines for the Clinical Diagnosis and Treatment of Lung Cancer (2022 edition). All patients in the lung cancer group had a pathological diagnosis of lung cancer, with other malignancies excluded. Healthy controls had no personal history of any cancer.
For subsequent protein expression analysis, differences in protein levels between tumor and adjacent non-cancerous tissues in LUAD and LUSC patients were preliminarily assessed using the Clinical Proteomics Tumor Analysis Consortium (CPTAC) database (https://proteomics.cancer.gov/programs/cptac). Additionally, for immunohistochemistry (IHC) validation, three tumor samples per genotype were randomly selected from the available formalin-fixed, paraffin-embedded tissue archive, based on criteria of sufficient tumor content and consistent staining conditions.
This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments, and was approved by the Ethics Committee of The First Affiliated Hospital of Guilin Medical University (No. 2023QTLL-36). Written informed consent was obtained from all individual participants.

IHC
The IHC procedure is as follows: firstly, the sample is prepared, and the tissue is fixed, embedded, and cut into thin slices, which are adhered to slides. The slides were then dewaxed and hydrated using xylene to remove the wax, and then passed through an alcohol gradient into water. The slides were subsequently immersed in hydrogen peroxide to block endogenous peroxidase and antigen repair by high-pressure heating. It is then closed with serum to reduce non-specific binding. This is followed by sequential dropwise incubation with primary and secondary antibodies to allow specific binding of the antibody to the antigen. Then the color was developed with 3,3'-diaminobenzidine (DAB) (Beyotime Biotechnology, Shanghai, China) chromogen. Finally, hematoxylin is used to re-stain the nuclei of the cells, which can be observed under the microscope after dehydration and transparency, and the slices are sealed with neutral dendrimer. Primary antibody was used: anti-mTOR (ZM-0097, ZSGB-BIO, Beijing, China; dilution 1:200). Calculate the index of damage (IOD)/area ratio using ImageJ (26).

Clinical study data
Clinical baseline information was collected from the medical records of all study participants, including sex, age, common tumor markers [carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH)], liver function indicators, and renal function indicators. The blood biochemical index was tested in the laboratory department of The First Affiliated Hospital of Guilin Medical University, and all tests passed the ISO15189 certification (No. ML00036). All tumor markers and biochemical markers were measured using Roche Cobas E701 and E801 (Roche Diagnostics, Shanghai, China) analyzer.

mTOR polymorphism analysis

Specimen collection
Fasting venous blood (3 mL) was collected into an ethylenediaminetetraacetic acid dipotassium salt (EDTA-K2) anticoagulant tube, then stratified by low-speed centrifugation and separated into serum, tunica albuginea, and blood cell layers. The tunica albuginea layer was used for genomic DNA extraction, while the serum was used to detect various clinical biochemical indices. All specimens were stored at −80 ℃.

DNA separation
Extraction was performed on 200 µL of blood samples from the leucomatous layer using a commercial extraction kit (Tiangen Biotech, Beijing, China). The absorbance values (A260 nm/A280 nm) of all DNA samples were detected by a ultraviolet (UV) spectrophotometer (Furi Technology, Shanghai, China), and qualified samples with ratios between 1.7 and 2.0, concentrations between 30 and 50 ng/µL, and total amounts of 600 ng or more were used for genotyping.

Primer design
Primers for the rs2295080G/T locus were designed and synthesized by Genesky Biotechnologies Inc. (Shanghai, China). Polymerase chain reaction (PCR) primer sequence information is as follows: rs2295080 upstream primer sequence: 5'-CGAGGGAAGGAGGGTTCCAAG-3'; downstream primer sequence: 5'-CGAGGGAAGGAGGGTTCCGAT-3'.

Genotyping
Genotyping of the rs2295080 locus was performed using the SNPscan™ genotyping kit (Tianhao Biology, Shanghai, China) according to the manufacturer’s instructions. Briefly, multiplex PCR was performed, followed by ligation of allele-specific oligonucleotides. The ligation products were amplified by fluorescent PCR. The final PCR products were separated by capillary electrophoresis on an ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). Raw data were collected, and genotype calls were made using GeneMapper 4.1 software (Applied Biosystems). This method has been widely used for SNP genotyping (27). The genotypes of rs2295080 were TT, GT, and GG.

Statistical analysis
All statistical analyses were conducted using IBM SPSS Statistics software (version 26.0; IBM Corp., Armonk, NY, USA). A two-sided P value of less than 0.05 was considered statistically significant. Continuous variables were tested for normality using the Kolmogorov-Smirnov test and for homogeneity of variance. Data conforming to a normal distribution are presented as mean ± standard deviation (SD). Comparisons between two groups were performed using the independent samples t-test, while comparisons among three or more groups were conducted using one-way analysis of variance (ANOVA). For non-normally distributed data, results are presented as median with interquartile range (IQR), and group comparisons were performed using the Mann-Whitney U test or the Kruskal-Wallis H test, respectively. Categorical variables are expressed as numbers and percentages [n (%)] and were compared using the Chi-squared (χ2) test. The Hardy-Weinberg equilibrium (HWE) for genotype distribution in the control group was also assessed using the χ2 test. To evaluate the association between the mTOR rs2295080 polymorphism and lung cancer risk, binary logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). To control for potential confounding, all reported confounders and 95% CIs were derived from models adjusted for age and sex. Participants with missing genotype data or key clinical variables (age, sex) were excluded from the final analysis. The rate of missing data was less than 5% for all analyzed variables.

Results

Study subjects’ clinical and biochemical traits
The study included 136 healthy controls and 241 lung cancer cases, with average ages of 56 and 59 years, respectively. There was no significant gender difference between the control and case groups (P=0.08), but a significant age difference was observed (P<0.001). The case group comprised 145 lung adenocarcinoma (LUAD) cases, 48 lung squamous cell carcinoma (LUSC) cases, 44 small cell lung cancer (SCLC) cases, and 4 other lung cancer cases. All liver and kidney function indicators in lung cancer patients differed significantly from those in normal controls, suggesting potential impairment of liver and kidney function in these patients. Additionally, LDH levels were significantly higher in lung cancer patients compared to controls (P<0.001), consistent with its role as a nonspecific tumor marker. Detailed data are presented in Table 1.

Genotype distribution and lung cancer susceptibility
The mTOR genotype and allele frequency distributions, as determined by capillary electrophoresis, are shown in Figure 1 and Table 2. The genotype frequency of rs2295080G/T (TT vs. GT vs. GG: χ2=6.873, P=0.03) and allele frequency (T vs. G: χ2=5.332, P=0.02) showed significant associations between the groups. All genotype frequencies of rs2295080G/T were observed in healthy controls and were consistent with HWE (P>0.05). Table 3 presents the distribution of rs2295080G/T genotypes and alleles across different lung cancer types. Among these types, the genotype frequency distribution was not significantly different (TT vs. GT vs. GG: χ2=7.984, P=0.09), whereas the allele frequency distribution (T vs. G: χ2=8.254, P=0.02) showed a significant correlation. The associations of the mTOR SNP with lung cancer, LUAD, LUSC, and SCLC are summarized in Table 4. After adjusting for age and sex, rs2295080 was significantly associated with the risk of lung cancer, LUAD, and SCLC. Specifically, the adjusted GT genotype was associated with a reduced risk of lung cancer and LUAD compared to the TT genotype (OR =0.549, 95% CI: 0.348–0.865; OR =0.513, 95% CI: 0.300–0.877). Additionally, the G allele was associated with a reduced risk of lung cancer and SCLC compared to the T allele (OR =0.648, 95% CI: 0.453–0.926; OR =0.377, 95% CI: 0.185–0.769).

The relationship between mTOR protein expression levels and polymorphisms in lung cancer tissues
Our analysis of mTOR protein expression using the CPTAC database revealed that, compared to normal tissues, protein levels were significantly decreased in both LUSC and LUAD (Figure 2A,2B). Additionally, we performed IHC on three lung cancer tissue samples selected for each genotype based on genotype classification across different pathological types of lung cancer. The results demonstrated that the protein expression level in the rs2295080-GT genotype was the lowest in both adenocarcinoma and squamous cell carcinoma, while the expression level in the rs2295080-TT genotype was significantly higher than that of the GT genotype (GT vs. TT: P<0.05; Figure 2C-2F). In SCLC, no significant difference in protein expression was observed between the rs2295080-GT and rs2295080-TT genotypes; however, the GT genotype still exhibited lower protein levels than the TT genotype (GT vs. TT: P>0.05; Figure S1A,S1B). Since the GG genotype was absent in SCLC samples in this study, no corresponding IHC data were available.

Discussion
The present study investigated the association between the mTOR promoter polymorphism rs2295080 and lung cancer susceptibility. Furthermore, it linked genetic findings to MTOR protein expression levels for the first time in this context. The primary results demonstrated that the rs2295080-GT genotype and G allele were significantly associated with a reduced risk of overall lung cancer, particularly in the LUAD and SCLC subtypes. The observed trend of lower mTOR protein expression in tumor tissues from GT genotype carriers of LUAD and LUSC provides preliminary biological plausibility for the protective genetic association, suggesting that this polymorphism may influence susceptibility by modulating gene or protein expression.
The mTOR signaling pathway is a central regulator of cell growth, proliferation, and metabolism, and its dysregulation is a hallmark of many cancers (6). Although the pathway is generally regarded as oncogenic when activated, an observation derived from the CPTAC database reveals a paradox. This is evidenced by the finding that total mTOR protein levels are lower in LUAD and LUSC tumors in comparison to normal tissues. This finding highlights a significant distinction: the oncogenic activity of the mTOR pathway is primarily governed by its phosphorylation state and downstream signaling flux (e.g., through p-S6K, p-4EBP1), rather than solely by the abundance of the core mTOR protein alone (20). A reduction in total protein levels may be indicative of tumor-specific feedback mechanisms, alterations in protein degradation, or selective pressures in particular cancer subtypes. Consequently, the protective effect of the rs2295080 variant, which is potentially associated with reduced mTOR expression, may be achieved through the precise regulation of pathway activity, thereby circumventing the potentially deleterious consequences of both hyperactivation and excessive suppression. Furthermore, the elevated levels of LDH in the case group provide additional evidence supporting systemic metabolic alterations associated with lung cancer (28).
Our genetic findings align with a substantial body of evidence linking the mTOR rs2295080 polymorphism to cancer risk. The GT genotype and G allele have been consistently reported as protective factors in several cancers among Chinese and Iranian populations, including gastric (29), thyroid (30), colorectal (31), breast (32), and urological malignancies (8,16). The observed consistency across different cancer types suggests a fundamental role for this locus in oncogenesis. In contrast, studies conducted on multi-ethnic cohorts or other cancers, such as acute leukemia, have yielded contradictory results (33,34). This discrepancy may be attributed to tumor heterogeneity and population-specific genetic backgrounds. Regarding lung cancer specifically, direct evidence is limited and somewhat inconsistent. Although certain meta-analyses have demonstrated inconsistent correlations between rs2295080 and tumor development outcomes (9,25), a study on SCLC revealed that a related microRNA-SNP exhibited a positive association with prognosis (24), thereby indirectly corroborating the hypothesis that genetic variation within the mTOR regulatory network exerts an influence on the manifestation of lung cancer. The present study contributes to the existing body of knowledge by offering a high degree of clarity by demonstrating a significant protective association specifically for LUAD and SCLC in a Chinese population.
Beyond the mTOR gene itself, polymorphisms in other components of the PI3K-AKT-mTOR pathway have been implicated in lung cancer outcomes, further underscoring the pathway’s clinical relevance. It has been established that genetic variants in the AKT1 and PIK3CA loci are associated with an elevated risk of brain metastasis (35,36), disease progression subsequent to chemotherapy (37), chemotherapy toxicity, and distant metastasis (38). The polymorphism in rapamycin-insensitive companion of mTOR (RICTOR), a key component of mTORC2, has been demonstrated to influence the efficacy of chemotherapy in non-SCLC (NSCLC) (39). The findings of these studies, when considered in conjunction with the evidence that mTOR activity is influenced by genomic alterations such as copy number variation (17), establish a strong precedent for the functional importance of genetic variation within this pathway. The present study investigates the mTOR promoter polymorphism rs2295080, a hitherto unexplored aspect of this field. The preliminary IHC data, demonstrating the lowest mTOR protein levels in tumor tissues from GT genotype carriers, offer a plausible mechanism that the protective effect is mediated through reduced expression. However, it should be noted that this process necessitates rigorous functional validation.
From a translational perspective, the findings of this study may hold significant clinical implications. The identification of a genetic variant associated with a reduced risk of lung cancer, particularly in major subtypes such as LUAD, could contribute to the refinement of risk stratification models. If this hypothesis is validated in larger, independent cohorts, the rs2295080-G allele or GT genotype could be incorporated into polygenic risk scores to identify individuals at differential risk. Furthermore, a comprehensive understanding of the mechanisms by which germline genetics regulates baseline mTOR expression may provide novel insights into the causes of inter-individual variability in response to therapies targeting the PI3K/AKT/mTOR pathway, a field currently undergoing rapid therapeutic development.
It is imperative to acknowledge the limitations of this study. First, the IHC analysis was performed on a very limited number of samples per genotype. While the observed trend is hypothesis-generating and consistent with the genetic data, the findings related to protein expression require validation in a larger, independent set of tumor tissues. Second, the absence of the GG genotype in our SCLC subset prevented a comprehensive evaluation of genotype-protein expression relationships in this aggressive subtype. Third, the present study focused on a single polymorphic locus; future research should investigate haplotype blocks or incorporate other functional SNPs in the mTOR gene and its regulatory regions. Finally, the lack of detailed data on environmental and behavioral risk factors (e.g., smoking history and exposure details) precluded analysis of their interaction with genetic susceptibility. It is recommended that future studies employ functional assays, such as luciferase reporter gene experiments, to directly assess the impact of this polymorphism on promoter activity. Large-scale prospective studies integrating genetic, environmental, and detailed clinical data are necessary to confirm these associations and fully explore their potential in precision prevention and oncology.

Conclusions
This study provides evidence that the mTOR rs2295080 polymorphism is associated with a reduced risk of lung cancer, particularly LUAD and SCLC, in a Chinese population. The concurrent observation of decreased mTOR protein expression in tumors from individuals with the protective genotype suggests a potential underlying mechanism.

Supplementary
The article’s supplementary files as