Revealing the association between vitamin D metabolic pathway gene variants and lung cancer risk: a systematic review and meta-analysis

Lung cancer is a crucial global issue, with more than one million deaths annually. While smoking is considered the main etiology of the disease, several genetic variants are associated with it. Alterations in vitamin D pathway genes have also been studied in regards to lung cancer, but the findings have been inconclusive. We here present a systematic review and meta-analysis of seven genes in this pathway: CYP2R1, CYP27B1, CYP24A1, CYP3A4, CYP3A5, GC, and VDR. Four databases (PubMed, Scopus, Cochrane Library, and Web of Science (WOS) databases) were searched. From these, 16 eligible case–control studies comprising 6,206 lung cancer cases and 7,272 health controls were obtained. These studies were subjected to comprehensive data extraction and quality scoring, and the pooled odds ratio with a 95% confidence interval was calculated to estimate the effect of each variant along with heterogeneity analysis and a risk of bias assessment. Our meta-analysis revealed an association between CYP3A4 (rs2740574) and lung cancer in the allelic, heterozygous, and dominant models. In addition, both VDR (Fok1: rs2228570) and VDR (Cdx-2: rs11568820) displayed a protective role in lung cancer development in the heterozygous and dominant models. Furthermore, VDR (Taq1: rs731236) showed a decreased risk of lung cancer in the allelic, homozygous, and recessive models. Similarly, VDR (BsmI: rs1544410) had a positive effect on lung cancer risk when subjected to allelic and recessive models. Our meta-analysis revealed the lack of association of CYP2R1 (rs10741657), CYP27B1 (rs3782130), CYP27B1 (rs10877012), CYP24A1 (rs6068816), CYP24A1 (rs4809960), CYP3A5 (rs776746), GC (rs7041), GC (rs4588), and VDR (ApaI: rs7975232) with lung cancer. Our work revealed that CYP3A4 (rs2740574) can represent an independent risk factor for lung cancer. This conclusion can aid better personalized medicine for lung cancer management, while further assessment for genetic variants of CYP3A4, CYP27B1, CYP24A1, GC, and VDR is still required to address more robust evidence.


Introduction
Lung cancer is the most robust lethal carcinoma worldwide, accounting for 1.76 million deaths annually (Bade and Dela Cruz, 2020).Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) are two primary histological subtypes of lung cancer.NSCLC accounts for 80%-85% of lung cancer cases and includes adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma (Ben et al., 2021).Smoking has been identified as the leading factor contributing to the incidence of lung cancer.Other risk factors include viral infections, exposure to heavy metals, radiation, asbestos, and air pollution (Li et al., 2019).However, these etiologies rarely account for lung cancer, suggesting that genetic factors play a significant role in its incidence (Zhu et al., 2021).Genome-wide association studies (GWASs) in populations from Europe and Asia have conclusively proven the relationship between genetic polymorphisms and the risk of developing lung cancer (Zhou et al., 2020).Therefore, the identification of vulnerable genes and high-risk populations is one of the main goals of lung cancer research for achieving early prevention and treatment (Wei et al., 2021).
Vitamin D is a seco-steroidal prohormone which is synthesized and metabolized by a series of reactions catalyzed by several enzymes.First, pro-vitamin D, absorbed from food or produced in the skin after exposure to sunlight, is converted by the vitamin D 25-hydroxylase enzyme CYP2R1 in the liver (Janoušek et al., 2022).A summary of this process is shown in Figure 1.The 25(OH) D produced is then converted into 1,25-dihydroxyvitamin D [1.25(OH) 2 D 3 ] by the 25hydroxyvitamin D-1α-hydroxylase enzyme that is encoded by the CYP27B1 gene in the kidney.Additionally, 25(OH)D 3 can also be converted into 24.25(OH) 2 D 3 by hydroxylation at C-24 through the mitochondrial inner membrane enzyme 1,25-dihydroxyvitamin D (3) 24-hydroxylase encoded by the CYP24A1 gene (Christakos et al., 2015).
Additionally, 1,25-dihydroxyvitamin D (3) 24-hydroxylase encoded by the CYP24A1 gene is considered a factor in the vitamin D transformation process and participates in the degradation process of both 25-OH-D3 and 1,25-(OH) 2 D 3 into 24-hydroxylated products in a tissue-dependent manner (Jones et al., 2012;Wang et al., 2013).In fact, it has been suggested that alterations in the vitamin D metabolism result mainly from the induction of hepatic P450 enzymes, including CYP3A4 and CYP24A1 (Wang et al., 2013).Furthermore, in the case of vitamin D toxicity, the liver microsomal enzymes CYP3A4 and CYP3A5 and the extrarenal enzyme CYP24A1 promote the hydroxylation of vitamin D3 into inactive metabolites (Klyushova et al., 2022;Kasarla et al., 2022b).Vitamin D-binding proteins encoded by the GC gene transport the active metabolite 1.25-(OH)2D3 to target tissues where it can bind to the vitamin D receptor gene VDR and regulate physiological genes (Kasarla et al., 2022b).
Vitamin D is involved in a variety of cellular processes such as proliferation, differentiation, metastasis, angiogenesis, and apoptosis (Wei et al., 2018).Vitamin D levels have been linked to the risk of developing several cancers, including breast (Kim and Je, 2014), colorectal (Garland and Gorham, 2017), and prostate (Gilbert et al., 2011).Additionally, a previous meta-analysis has reported an inverse association between serum vitamin D levels and lung cancer risk (Zhang et al., 2015a).Furthermore, it has been found that vitamin D could suppress the metastatic growth of lung cancer cells in animal models (Nakagawa et al., 2005;Zhang et al., 2015b).Therefore, results from previous reports have revealed the potentially preventive role of vitamin D against lung cancer.Genetic variations in vitamin D genes are potential modulators of their enzymatic functions, levels of expression, and subsequent roles in the susceptibility, progression, and prognosis of lung cancer (Pineda Lancheros et al., 2021;Pineda Lancheros et al., 2022).CYP2R1 gene polymorphisms are linked to an increased risk of NSCLC mortality, particularly in elderly NSCLC patients who have not received treatment.Furthermore, the prognosis of NSCLC may be impacted by several genetic variants connected to the Vitamin D pathway (Kong et al., 2020).Furthermore, the activity of CYP27B1 has been found to be attenuated among some small cell and nonsmall cell cancer cell lines (Hansdottir et al., 2008).CYP3A4 and CYP3A5 activities as part of liver machinery P450 protein content in the liver account for the metabolism of more than 50% of all drugs and exogenous carcinogens (Ingelman-Sundberg et al., 2007).CYP3A4 and CYP3A5 affect the activation of benzo [a]pyrene (B [a]P), N9-nitrosonornicotine (NNN), aflatoxin B1, stergmatocystin, alpha-hydroxytamoxifen, and procarcinogens present in tobacco smoke (Islam et al., 2014).Altered levels of these toxins are thought to be involved in lung cancer pathogenesis.Increased VDR expression in lung cancer has been correlated with improved survival (Salama et al., 2022).Genetic variants within the VDR gene might potentially influence the binding of 1,25(OH) 2 D and subsequently alter vitamin D levels and lung cancer pathogenesis (Haznadar et al., 2018).Nevertheless, despite research into the association between vitamin D gene pathway variants and lung cancer, much is still unknown due to a lack of conclusive findings.So this work extensively investigated the genetic variations in the vitamin D pathway, specifically the polymorphisms of CYP2R1, CYP27B1, CYP24A1, CYP3A4, CYP3A5, GC, and VDR genes, to determine their connection with increased risk of developing lung cancer.

Inclusion and exclusion criteria
The following inclusion criteria were used to identify the eligible studies for our meta-analysis: 1) case-control studies evaluated the potential association between the CYP2R1, CYP27B1, CYP24A1, CYP3A4, CYP3A5, GC, or VDR polymorphisms and lung risk; 2) studies with sufficient data to calculate an odds ratio (OR) with 95% confidence interval (95% CI); 3) original full-text articles.Otherwise, studies were excluded if they were 1) review articles, meta-analyses, letters, case reports, or articles with abstract only; 2) articles without controls; 3) non-human studies; 4) had duplicated or republished data.

Quality assessment and data extraction
Two authors independently reviewed and extracted the following baseline information for all included studies: first author's name, year of publication, country of origin, geographical distribution, source of control [hospital-based (HB) or population-based (PB)], mean/median age of cases and controls, sample size of cases and control, classification of lung cancer, genotyping method, allelic/genotype frequencies of cases and controls, matching criteria, and quality control of genotyping.Potential conflicts were resolved by discussion with two other authors to reach a final consensus.Each study was given quality scores based on certain criteria (Supplementary Table S1) (Xu et al., 2015).

Statistical analysis
First, the allelic and genotypic frequencies of eligible studies were extracted and calculated from the selected records.HWE within cancer-free controls was calculated by the chi-squared method with a p-value >0.05 representing equilibrium between subjects (Elsalahaty et al., 2023).Next, the crude odds ratios (ORs) and their 95% confidence intervals (CI) were processed to assess the association of included variants with the risk of lung cancer.Different genetic association models were performed, including the allelic, homozygous, heterozygous, dominant, and recessive models (Elshazli et al., 2018).Heterogeneity testing amongst eligible reports was performed using the Q-statistic test along with the I 2 index, with a p-value for the Q-test ≤0.10 and I 2 index >50%, which signified strong heterogeneity between studies; hence, the random-effects model was chosen (Higgins et al., 2003;El Awady et al., 2022).Publication bias was evaluated using Egger's regression method and Begg's funnel plot to ensure a symmetric fashion; the p-value <0.05 was considered significant.We conducted a sensitivity analysis by excluding any included case-control study at a time and then recalculated the significance of the results to detect whether our outcomes were significantly impacted by the presence of each individual study.The present meta-analysis was performed using Comprehensive Meta-analysis version 3.0 (Brüggemann and Rajguru, 2022).

Sensitivity analysis and publication bias
Sensitivity analysis was subsequently executed to evaluate whether our results were substantially affected by one individual study.Each time, we removed one included study to recalculate the significance of the results, and the obtained results showed that the ORs were not significantly changed.The graphs of Begg's funnel were symmetrically executed, guaranteeing the absence of publication bias among the pooled studies in our meta-analysis (Supplementary Figure S2).Additionally, Egger's regression test showed a p-value of >0.05 within included studies under all genetic models, addressing the lack of publication bias across our present study.

Genetic association models of the onestudy variants with risk of lung cancer within included studies among CYP27B1, CYP24A1, CYP3A4, GC, and VDR with lung cancer polymorphisms in lung cancer risk
As depicted in Table 3, we subjected data of one-study variants to five heredity models to assess their association with lung cancer.Of the CYP24A1-included variants, CYP24A1 (rs2585439) Association between CYP3A4 (rs2740574) and lung cancer risk under (A) allelic, (B) heterozygous, and (C) dominant models.Association between VDR (Fok1: rs2228570) and decreased lung cancer risk under (D) heterozygous and (E) dominant models.Association between VDR (Cdx-2: rs11568820) and decreased lung cancer risk under (F) heterozygous and (G) dominant models.Association between VDR (Taq1: rs731236) and decreased lung cancer risk under (H) allelic, (I) homozygous, and (J) recessive models.Association between VDR (BsmI: rs1544410) and decreased lung cancer risk under (K) allelic and (L) recessive models.
rs6022999) indicated significance with lung cancer susceptibility under allelic and homozygous models (Table 3).

Discussion
Lung cancer is a critical problem for human health and is associated with a high rate of mortality.Therefore, its rapid and personalized management is an urgent priority.The well-established roles of vitamin D and genetic variants in the pathogenesis of several types of cancer, including lung cancer, are multifactorial and involve the alteration of transcription, translation, expression, and protein function (Shaw, 2013;Wang et al., 2017;Elsalahaty et al., 2023).Vitamin D has been attributed a favorable role in pulmonary inflammation, and its alteration was thought to be linked to lung cancer progression by modulating the tumor microenvironment and immune function (Prietl et al., 2013).Furthermore, vitamin D metabolites have been found to possess anticancer potency in preclinical models of lung cancer (Shaurova et al., 2018).
While numerous clinical trials have investigated the association of vitamin D gene variants with lung cancer, their findings were not conclusive and were not comprehensively analyzed through different genetic models.Additionally, the most recent studies have not been included.To the best of our knowledge, this is the first systematic review to extract all available data of vitamin D gene variants and subject them to genetic analyses.Our meta-analysis evidence is based on a sum of 6,206 lung cancer cases together with 7,272 healthy controls included in 16 case-control studies among seven genes of the vitamin D pathway (CYP2R1, CYP27B1, CYP24A1, CYP3A4, CYP3A5, GC, and VDR) to assess their association with lung cancer.
Our analysis showed a significant association of CYP24A1 (rs4809957) with an increased risk of lung cancer in the homozygous model and a protective impact within the heterozygous comparison.However, this analysis is limited because of its dependence on data from two available studies.Altered levels of toxins manipulated through the CYP450 machinery, including CYP3A4 and CYP3A5, are thought to participate in the pathogenesis of lung cancer.Our analysis revealed that CYP3A4 (rs2740574) has a significant association with increased lung cancer risk, while CYP3A5 (rs776746) was not correlated with lung cancer risk.
Vitamin D-binding protein encoded by the GC gene is the main transport protein for 25(OH)D and vitamin D metabolites (Speeckaert et al., 2006;Anic et al., 2014).The potential implementation of GC in diseases is thought to be linked to the altered expression of 25(OH)D, actin scavenging, and immunomodulatory roles in macrophage activation and neutrophil chemotaxis (Chun, 2012).Our analysis could not reject the null hypothesis regarding GC (rs7041) and GC (rs4588), and no correlation was observed with the risk of lung cancer in all genetic models.These findings were consistent with those of a previous meta-analysis (Duan et al., 2020).
VDR is tightly linked to vitamin D levels attributed to the modulation of the risk of lung cancer due to the antiproliferative impact of vitamin D. VDR (Apa1: rs7975232) did not reveal a significant impact on lung cancer susceptibility.The same findings were confirmed in a recent meta-analysis (Li et al., 2019;Duan et al., 2020).However, VDR (Fok1: rs2228570) and VDR (Cdx-2: rs11568820) showed significant protection in heterozygous and dominant models.Similarly, Li et al. (2019) obtained a protective impact of Cdx-2: rs11568820 in heterozygous and dominant models relying on two reports.In addition, VDR (Taq1: rs731236) indicated a significant association with a decreased risk of lung cancer in allelic, homozygous, and recessive models.Lastly, VDR (BsmI: rs1544410) indicated protection against lung cancer in the allelic and recessive models.
Our comprehensive systematic review and data extraction for all included variants, gathered data for one-study variants that were subjected to genetic models.As a result, CYP24A1 (rs2762937), CYP24A1 (rs2762940), and CYP24A1 (rs2209314) showed a correlation with lung cancer susceptibility.In contrast, CYP24A1 (rs6022993), CYP24A1 (rs8120563), and CYP24A1 (rs6068816) revealed a protective role for lung cancer in all genetic models.Our evidence-based study suggests that CYP3A4 (rs2740574) contributes to the development of lung cancer by modulating vitamin D metabolism.It could therefore represent an independent risk factor for developing lung cancer.Genetic testing for this variant in lung cancer patients may be a useful tool for addressing which patients are characterized by higher aggressive characteristics and poorer outcomes, allowing clinicians to choose more personalized and robust treatments early in the course of the disease when treatment is most likely to be effective.

FIGURE 2
FIGURE 2 Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocols (PRISMA) flow diagram of the search strategy.

TABLE 1
Main characteristics of the 16 records included in this meta-analysis.

Table 1 .
Additionally, comprehensive data extraction of genotypic and allelic frequencies, along with quality scores for the 16 studies, is summarized in Supplementary TableS2.

TABLE 1 (
Continued) Main characteristics of the 16 records included in this meta-analysis.
. Source (HB or PB).Control subjects' source, where HB indicates hospital-based control and PB indicates population-based control.b NA: not applicable.c Mixed: unidentified sub-classification of lung cancer.d NSCLC: non-small-cell lung cancer.e PCR-RFLP: polymerase chain reaction-restriction fragment length polymorphism.a No.: no. of studies involved.b−ΔΔCT .: control subjects.c OR: odds ratio.d 95% CI: 95% confidence interval.e R: random-effects model.f F: fixed-effects model; bold values indicate significance.

TABLE 3
Genetic association models of one-study variants with the risk of lung cancer within the included studies.

TABLE 3 (
Continued) Genetic association models of one-study variants with the risk of lung cancer within the included studies.