Two novel susceptibility loci for non‑small cell lung cancer map to low‑density lipoprotein receptor‑related protein 5
- Authors:
- Published online on: August 5, 2016 https://doi.org/10.3892/ol.2016.4954
- Pages: 2307-2318
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Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related mortality worldwide. In 2008, lung cancer accounted for 1.6 million of new cases and 1.4 million cancer-related mortalities worldwide (1). Non-small cell lung cancer (NSCLC) is the most common histological subtype, accounting for ~85% of all lung cancers (2). NSCLC is also one of the few cancers for which there has been no substantial progress in early detection and treatment options (3). Tobacco smoke is the single most significant risk factor for lung cancer, and outdoor air pollution (4) and genetic factors are also notable factors (5–7). A previous study demonstrated that a heritable component or a gene-environment interaction leads to lung cancer development (5); for example, not all tobacco smokers suffer from lung cancer. Thus, it is crucial to identify these genes and their genetic variations to clarify their association with lung cancer risk (6,7).
Low-density lipoprotein-related receptor 5 (LRP5) is a member of the family of lipoprotein receptor-related proteins (LRPs), a small group of single-pass transmembrane proteins (8). LRP5 was originally identified based on its homology to the low-density lipoprotein (LDL) receptor (9); it contains large extracellular domains, including four β-propeller motifs followed by three type 1 LDL ligand-binding domains (10), and is a transmembrane cell-surface receptor involved in receptor-mediated endocytosis of lipoprotein and protein ligands (11). Functionally, LRP5 plays a significant role in Wnt/β-catenin signaling, and the latter is integral to developmental biology. For example, when Wnt ligands bind to a member of the Frizzled family and LRP5, it allows β-catenin to shuttle into the nucleus and bind to T-cell factor/lymphocyte-enhancing factor proteins to activate the canonical Wnt/β-catenin signaling cascade (10). In the absence of the Wnt ligand binding to Frizzled receptors, the canonical Wnt pathway is turned off, which leads to β-catenin degradation. The non-canonical pathway may lead to proliferation of lung cancer cells (12). Signaling by the Wnt family of secreted glycolipoproteins is known to play a key role in the embryonic development of organisms ranging from nematodes to mammals, and is also implicated in several types of human cancer (13–15). In lung cancer, it has been reported that lipoprotein receptor-related protein is inactivated in more than 40% of cases (16). Another study has demonstrated that chromosome 11q is frequently altered in NSCLC (17), where LRP5 is localized. Thus, in this study, we hypothesized that LRP5 polymorphisms may play a role in susceptibility to NSCLC.
Patients and methods
Study population
A total of 500 NSCLC patients and 500 unrelated healthy controls were recruited from Zhejiang Cancer Hospital, Hangzhou, China, between March 2011 and April 2012. All cases and controls were of Chinese Han origin and lived in the same geographic region (Zhejiang province, China). Exclusion criteria included a history of previous primary cancer other than lung cancer. The controls were free of lung-related disease to avoid any probable interference from overlapping genes. The control subjects were matched to patients for gender and age. A regular smoker was defined as someone who had smoked more than one pack per year, and a current smoker or former smoker was defined as a regular smoker who still smoked in the year of the interview or in the previous year (18). This study was approved by the Ethics Committee of Zhejiang Cancer Hospital, and all of the studied subjects provided informed consent.
SNP selection and genotyping
Tagging SNPs of LRP5 were selected based on pairwise r2 values (≥0.8) among all common SNPs with a minor allele frequency (MAF) ≥0.1 using the Tagger program implemented in Haploview version 4.1 (http://www.broad.mit.edu/mpg/haploview). The Chinese HapMap database [population=Han Chinese, Beijing (CHB)] was used to select LRP5 SNPs in this study (http://www.hapmap.org).
For the genotyping of LRP5 SNPs, genomic DNA was extracted from whole blood using the AxyPrep Blood Genomic DNA Miniprep kit (Axygen Biosciences, Union City, CA, USA) and subjected to genotyping of LRP5 SNPs with the Sequenom MassARRAY matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform (Sequenom, San Diego, CA, USA). Primers for polymerase chain reaction (PCR) and single base extension were designed using Assay Designer software version 3.0 (Sequenom) and synthesized by Sangon Biotech (Shanghai, China; Table I).
 | Table I.Polymerase chain reaction primers and extensions used in the genotyping of LRP5 single-nucleotide polymorphisms. |
Multiplex PCR was performed in 5 µl volumes containing 0.1 units of HotStarTaq polymerase (Qiagen, Hilden, Germany), 10 ng whole-genome-amplified genomic DNA, 2.5 pmol of each PCR primer and 2.5 µmol deoxynucleotides (dNTP; Qiagen). Thermocycling was performed at 94°C for 15 min followed by 45 cycles at 94°C for 20 sec, 56°C for 30 sec, and 72°C for 1 min, and a final incubation at 72°C for 3 min. Unincorporated dNTPs were deactivated using 0.3 units of shrimp alkaline phosphatase (Sequenom) followed by primer extension using 5.4 pmol of each primer extension probe, 50 µmol of the appropriate ddNTP combination, and 0.5 units of iPLEX enzyme (Sequenom). The extension reactions were carried out at 94°C for 30 sec and then 94°C for 5 sec, followed by 5 cycles at 52°C for 5 sec and 80°C for 5 sec for a total of 40 cycles, and then 72°C for 3 min. A cation exchange resin was used to remove residual salt from the reactions. Purified primer extension reaction products were spotted onto a 384-well spectroCHIP using the MassARRAY Nanodispenser (Sequenom) and determined by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Sequenom). Genotype calling was performed in real time with MassARRAY RT software version 3.0.0.4 (Sequenom) and analyzed by using the MassARRAY Typer software version 3.4 (Sequenom).
Statistical analysis
All statistical calculations were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). Hardy-Weinberg equilibrium (HWE) testing was carried out for all SNPs using the χ2 test, and P<0.001 was considered to indicate a statistically significant difference between cases and controls. The χ2 test was also used to assess frequencies of the selected allele and genotype between the cases and controls. The association between SNPs and NSCLC risk was evaluated by computing the odds ratio (OR) and 95% confidence interval (CI) from multivariate unconditional logistic regression analysis. Haploview software version 4.1 was used to analyze the association between haplotypes and the disease. All P-values were two-sided, and P<0.05 was considered to indicate a statistically significant difference.
Results
Study population description and clinical characteristics
A total of 500 patients (350 males and 150 females) and 500 healthy controls (259 males and 240 females; the gender information for one control subject was not available) of Chinese Han origin were included in this study. A total of 331 patients had adenocarcinoma (ADC), while 169 had squamous cell carcinoma (SCC). There were 280 male and 21 female patients who were smokers or former smokers, and 189 male and 14 female controls who were smokers or former smokers. All patients and controls were subjected to genotyping of 11 LRP5 tag SNPs (i.e., rs4930573, rs312009, rs312014, rs3781590, rs312015, rs491347, rs1784235, rs648438, rs3736228, rs624947 and rs607887). The selection was based on the following criteria: HapMap CHB, pairwise r2 ≥0.8, and MAF ≥0.1. The cases and controls were within the HWE for these 11 Tag SNPs, and the P-values are shown in Table II.
| Table II.Hardy-Weinberg equilibrium of LRP5 single-nucleotide polymorphisms between cases and controls. |
LRP5 polymorphisms in cases and controls
The allele frequencies of LRP5 SNPs in the cases and controls were analyzed. The frequency of the rs3736228 T allele was 18.9% and that of the C allele was 81.1% in the male NSCLC patients, whereas they were 23.9% and 76.1%, respectively, for the male controls (P=0.03). Logistic regression analysis revealed that the T allele is associated with a lower risk of developing NSCLC (OR=0.74; 95% CI, 0.56–0.67). In contrast, the frequency of rs3736228 was similar between NSCLC patients and controls and between female NSCLC patients and female controls (P>0.05). The differences in allele frequencies in the other 10 Tag SNPs (i.e., rs4930573, rs312009, rs312014, rs3781590, rs312015, rs491347, rs1784235, rs648438, rs624947 and rs607887) between cases and controls, male patients and male controls, as well as female patients and female controls were not statistically significant (Table III).
| Table III.Allele frequency of LRP5 single-nucleotide polymorphisms in non-small cell lung cancer patients and healthy controls. |
The genotype distribution of LRP5 SNPs was also analyzed. For rs3736228 polymorphisms, logistic regression analysis revealed that the C/C major allele homozygote was associated with an increased risk of NSCLC in the male population (C/C frequencies were 64.9% and 56.4% in male NSCLC patients and controls, respectively; OR=1.43; 95% CI, 0.33–1.99; P=0.03; Table IV). For rs64843 polymorphisms, the T/T major allele homozygote had a 1.77-fold greater risk of developing SCC compared with the C/C and C/T genotypes (95% CI, 1.02–3.04; P=0.04). The T/T frequencies were 87.0% and 79.2% in male SCC patients and controls, respectively (Table V).
| Table IV.Genotypes of LRP5 single-nucleotide polymorphisms in non-small cell lung cancer patients and controls. |
| Table V.Genotypes of LRP5 single-nucleotide polymorphisms in squamous cell carcinoma patients and controls. |
Haploview software identified two blocks among NSCLCs, ADCs and controls, and male NSCLCs, male ADCs and male controls: the haplotypes were CG, TC and CC in Block 1 (the length of one block was 6 kb, including rs312009 and 3120015), and TTC, CCT and TTT in Block 2 (the length of the other block was 27 kb, including rs491347, rs1784235 and rs607887). Two blocks were identified among female NSCLCs, female ADCs and female controls: the haplotypes were CG, TC and CC in Block 1 (the length of one block was 6 kb, including rs312009 and 3120015), and TTCC, CCTT, TTTC and TTTT in Block 3 (the length of the other block was 31 kb, including rs491347, rs1784235, rs607887 and rs3736228). Two blocks were identified among SCCs and controls, and male SCCs and male controls: the haplotypes were CGC, TCG, CCG and CGG in Block 4 (the length of one block was 7 kb, including rs312009, rs3120014 and rs3120015), and TTC, CCT and TTT in Block 2 (the length of the other block was 27 kb, including rs491347, rs1784235 and rs607887). Two blocks were identified among female SCCs and female controls: the haplotypes were CGC, TCG, CCG, CGG and CCC in Block 4 (the length of one block was 7 kb, including rs312009, rs3120015 and rs3120014), and TTCC, CCTT, TTTC and TTTT in Block 3 (the length of the other block was 31 kb, including rs491347, rs1784235, rs607887 and rs3736228). Among these results, only the frequency of haplotypes rs312009/rs3120015/rs3120014 CCC were significantly higher in female SCC patients vs. female controls (0.064 vs. 0.009; P=0.04; Table VI).
| Table VI.Haplotype distribution and formation between patients and controls for association of haplotype blocks with non-small cell lung cancer risk. |
Discussion
In the current study, we investigated the association between LRP5 polymorphisms and NSCLC risk. We observed that LRP5 rs3736228 and rs648438 polymorphisms were strongly associated with the risk of NSCLC and lung SCC. Based on this finding, rs3736228 and rs648438 are two novel susceptibility loci that were associated with an increased risk of developing NSCLC in this Chinese male population. To the best of our knowledge, this is the first study describing the association of rs3736228 and rs648438 SNPs with NSCLC risk in humans.
LRP5 is a co-receptor for canonical Wnt-mediated signaling (8). In transgenic mice, loss of LRP5 expression markedly reduces the formation of mammary tumors (19). In osteosarcoma tissue, the expression of LRP5 mRNA has been correlated with metastatic disease and a poorer event-free survival in patients (20). Moreover, dominant-negative LRP5 inhibits the growth and metastasis of osteosarcoma in animal models and reduces the expression of cancer cell invasion-associated markers (including N-cadherin, Snail and matrix metalloprotease-2) (21). Thus, these data suggest that LRP5 may function as an oncogene. In the current study, we analyzed rs3736228 and rs648438 SNPs, which are localized in the region of the LRP5 gene on 11q13.2. A previous study has revealed that chromosome 11q is a susceptibility region for NSCLC (17). The present study confirmed these data, suggesting that LRP5 may be a candidate susceptibility gene for NSCLC.
By selecting the Tag SNPs across the LRP5 gene from the HapMap CHB database using the approach of Carlson et al (22), the criteria for the selection of Tag SNPs enabled us to maximize the power to detect SNPs (the statistical power of our study was >80%); thus, 11 SNPs were selected and two SNPs (rs3736228 and rs648438) were associated with NSCLC risk, which indicated that this methodology is useful in identifying susceptibility loci for NSCLC.
Furthermore, rs3736228 is a SNP in LRP5, which is also known as Ala1330Val or A1330V; the more common C allele encodes Ala, while the rarer T allele encodes Val, and the latter is the risk allele. In the LRP5 gene, a C→T transition at rs3736228 results in a substitution of Val for Ala, and this transition significantly decreases the response to canonical Wnt signaling (23). Published data have identified that polymorphisms of rs3736228 are associated with a decrease in bone mineral density in postmenopausal Maya-mestizo females (24), Mexican females (25), healthy fertile French females (26), Japanese male workers (27), Chinese patients (28), and Chinese patients with osteoporosis (29,30). However, to date, there are no studies describing an association between rs3736228 polymorphisms and lung cancer. In the present study, we noted that individuals with the rs3736228 C allele had a lower risk of developing NSCLC compared with those carrying the T allele. Compared with the C/C homozygote, other genotypes (C/T and T/T) had a greater risk of developing NSCLC in the male population, but not in the female population. The reason for this discrepancy is not clear, but it may be due to the fact that a high percentage of males in China smoke cigarettes (over 90% of the males in our study were smokers, compared with less than 10% of the females). Polymorphisms of rs3736228 have a combined effect with cigarette smoking; people that smoke cigarettes (current and former smokers) with rs3736228 polymorphisms have a 4.1-fold greater (95% CI, 1.6–10.2) risk of having metabolic syndrome (31). In addition, tobacco smokers have a much greater chance of developing lung cancer (32). Our results suggest that, among smokers, East Asian males with the rs3736228 SNP have a higher susceptibility to develop NSCLC.
Another potential susceptibility locus for NSLCL risk was determined to be rs648438 in our current study. Rs648438 is located in the intron region of LRP5, which has not been reported to be associated with any diseases. The present study reveals that an rs648438 polymorphism was associated with lung SCC development in males carrying at least one C allele (C/C and C/T) compared with those carrying the T/T homozygote. In this study, there were 549 male smokers among the 609 male subjects (including cases and controls), whereas there were only 35 female smokers among the 490 female subjects. Tobacco smoking is a strong risk factor for all types of lung cancer, and among male smokers SCC is the predominant subtype: the greater the amount smoked, the greater the proportion of SCC cases relative to ADC cases (18). The current data indicated that among smokers, East Asian males with rs648438 had a higher susceptibility of developing SCC.
Although these two potential susceptibility loci are novel and were associated with an increased risk of NSCLC, the present study does have certain limitations, For example, our eligible population was living in Zhejiang province, China. Gene polymorphisms are known to be influenced by ethnicity, location and environment. Therefore, further investigation is required to confirm our data using other ethnicities.
Acknowledgements
The authors thank Xiaohong Xu and Yejiang Bao for their assistance in control screening. They also thank Hailong Liu for his technical support. The abstract of this paper was previously published at the 6th Asian Oncology Summit and 10th Annual Conference of the Organisation for Oncology and Translational Research, 11 April 2014–13 April 2014: www.ejcancer.com/article/S0959-8049(14)00306-2/abstract.
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