DNA AND CELL BIOLOGY Volume 33, Number 11, 2014 ª Mary Ann Liebert, Inc. Pp. 787–792 DOI: 10.1089/dna.2014.2490
Lysyl Oxidase Genetic Variants Affect Gene Expression in Cervical Cancer Meimei Bu,1,* Li Li,2,* Yong Zhang,1 Ying Xu,1 Shizhi An,1 Fang Hou,1 and Xuemei Jie1
Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the homeostasis of tumors. The aim of this study was to investigate the association between LOX polymorphisms and cervical cancer, and the effect of these polymorphisms on gene expression. We evaluated two polymorphisms of LOX, rs1800449G/A (G473A) and rs2278226C/G, in 262 cervical cancer cases and 298 healthy controls in the Chinese population. Results showed that the prevalence of rs1800449AA genotype was significantly increased in cases than in controls ( p = 0.004). Individuals who carried the rs1800449A allele had a 1.56-fold increased risk for cervical cancer than those with the rs1800449G allele ( p = 0.003). The rs2278226CG genotype also revealed a significantly higher proportion in cases (20.6%) than in controls (7.7%, p < 0.001). Interestingly, when analyzing these two polymorphisms with the serum level of LOX, we identified that cervical cancer patients carrying the rs2278226CG genotype had a significantly elevated level of LOX than those with rs2278226CC wild type, whereas the same phenomenon was not observed in controls. The rs1800449 polymorphism did not affect the LOX serum level in either controls or patients. These results suggest that the polymorphisms in the LOX gene may be involved in the development of cervical cancer through various mechanisms.
Introduction
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ervical cancer is the second most common cancer in women worldwide (Tota et al., 2014). The human papilloma virus (HPV) appears to be a necessary factor in the development of almost all cases of cervical cancer (Olesen et al., 2014). However, only a small fraction of HPV-infected individuals develop cervical cancer, suggesting that additional environmental, genetic, or immunological factors contribute to the pathogenesis of the disease (Caffarel and Coleman, 2014). Etiology of cancer is complicated and has not been elucidated completely. Recent studies have suggested that genetic variants may greatly involve in the disease (Tota et al., 2014). Lysyl oxidase (LOX), a copper-dependent amine oxidase, initiates the process of collagen and elastin crosslinking by oxidatively deaminating specific lysine and hydroxylysine residues located in the telopeptide domains, thus stabilizing the fibrous deposits of these proteins in the extracellular matrix (ECM) (Nishioka et al., 2012). LOX is expressed as a 46-kDa preproenzyme and a glycosylated 50 kDa proenzyme (Nishioka et al., 2012). Following secretion into the ECM, the 50-kDa proLOX is further processed by procollagen proteases into the 32-kDa mature species (Nishioka
et al., 2012; Qu et al., 2013). The normal enzymatic activity of LOX plays a critical role in morphogenesis and tissue structural integrity. An abnormal LOX activity can lead to connective tissue disorders such as emphysema (Mizuno et al., 2012), osteoporosis (Viguet-Carrin et al., 2006), cutis laxa (Debret et al., 2010), Menkes syndrome (Price et al., 2007), organ fibrosis (Correa-Costa et al., 2014), atherogenesis (Ovchinnikova et al., 2014), and cardiovascular diseases (Ishiwata et al., 2014). Perturbation of LOX expression is also involved in carcinogenesis and cancer progression (Cox and Erler, 2013). Markedly, LOX displays a dual role as a tumor suppressor as well as a metastasis promoter in tumorigenesis in its biological functions (Cano et al., 2012; Nishioka et al., 2012; Dudakova and Jirsova, 2013). LOX antagonizes tumorigenesis. Expression of LOX in ras-transformed fibroblasts decreases the activity of NFkB inhibiting cell growth and neoplastic transformation (Goto et al., 2013). Reduced LOX expression was found in several tumor cell lines. LOX propeptide reverses the invasive phenotype of Her-2/neu-driven breast cancer (Min et al., 2007). On the other hand, LOX promoted tumor progression. Increased expression of LOX in some tumors was associated with poor prognosis (Han et al., 2014). LOXmediated collagen and elastin crosslinking functions as a
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The Maternal and Child Health Hospital of Jinan City, Jinan, Shandong, China. Department of Gynecology, Affiliated Tumor Hospital, Xinjiang Medical University, Urumqi, China. *These authors contributed equally to this work.
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contributor to tumor ECM stiffening (Cox and Erler, 2013). ECM remodeling and stiffening are characteristics of these tumors, promoting tumor motility, migration, and rigidity disrupting tissue morphogenesis (Cox and Erler, 2013). Matrix remodeling by LOX provides the basis for cell movement important for tumor invasion and migration (Ahn et al., 2013). LOX is known to be essential for hypoxia-induced metastasis in breast cancer (Ahn et al., 2013). Thus, a further understanding of the mechanism(s) by which LOX regulates tumor progression has a potential benefit for novel cancer therapy. LOX may also play critical roles in the development of cervical cancer. Recent study has shown that inactivation of LOX by b-aminopropionitrile can inhibit hypoxia-induced invasion and migration of cervical cancer cells (Yang et al., 2013). Single-nucleotide polymorphisms (SNPs) can affect the development of different diseases (Wu et al., 2012; Zhang et al., 2012; Yu et al., 2013). However, no literature reported LOX polymorphism and cervical cancer. Also, the functional study of LOX SNPs remains limited. In this study, we investigated rs1800449G/A (G473A) and rs2278226C/G polymorphisms in cervical cancer and assessed their effects on the serum level of LOX. Materials and Methods Patients and controls
The study population and collection of subjects have been previously described elsewhere. In brief, this was a hospital-based case–control study, including 262 cervical cancer cases and 298 cancer-free controls. All the patients were newly diagnosed and confirmed by histopathologic examinations. Subjects with a minimum 15 pack-year smoking history were defined as smokers. The controls were recruited from people coming to the hospital for general health examinations. The exclusion criteria for the control subjects included a self-reported history of cancers or constant medication on other severe diseases. All of the control subjects were frequency matched to the cases on age, sex, and residence area (urban or rural). Each participant was interviewed in person with a structured questionnaire that elicited information on demographic factors and health characteristics. Venous blood was collected from each of the study participants. DNA genotyping
Detection of rs1800449G/A and rs2278226C/G SNPs was based on previously described methods (Zhang et al., 2012; Han et al., 2014). In brief, rs1800449G/A was genotyped using a polymerase chain reaction (PCR)–restriction fragment length polymorphism method. The PCR primers are shown in Table 1. The PCRs were performed in a total volume of 35 mL reaction mixture containing 100 ng genomic DNA, 12.5 pmol of each primer, 0.1 mM of each dNTP, 1 · PCR buffer, 1.0 mM MgCl2, and 1.5 U of Taq DNA polymerase (Fermentas). Reactions were conducted using the thermal cycler (Biometra) under the following conditions: an initial incubation at 95C for 10 min, 30 cycles of 95C for 30 s, 57C for 30 s, 72C for 30 s, and a final extension at 72C for 7 min. The efficiency of the PCR was confirmed by gel electrophoresis on a 1.5% agarose gel.
BU ET AL.
Table 1. Primers Used for Detecting the Lysyl Oxidase Single-Nucleotide Polymorphisms Primers rs1800449 (G473A) For PCR F: 5¢-CTCACAGTACCAGCCTCAGCG-3¢ R: 5¢-CCAGGTCTGGGCCTTTCATA-3¢ rs2278226 C/G For PCR F: 5¢-CTGCTCGGGCCTTTGCAGCT-3¢ R: 5¢-ACTCCAGATGAGCCGGCCGT-3¢ PCR, polymerase chain reaction.
After DNA amplification, the PCR products were digested overnight at 37C with 10 U of the specific restriction endonuclease PstI (Fermentas), which cuts the allele A. The digestion products were then resolved and separated on a 2% agarose gel stained with ethidium bromide for visualization under ultraviolet light. After electrophoresis, homozygous A alleles were represented by DNA bands with sizes at 291 and 114 bp; an uncut fragment of 405 bp indicated the homozygous G alleles, while the heterozygous genotype was displayed as a combination of 405, 291, and 114 bp. The detection of the other polymorphism, rs2278226C/G, was performed by similar methods. In brief, the polymorphism amplified by PCR and determined by the enzyme restriction method using SacII (Fermentas). In addition, more than 5% of PCR-amplified DNA samples were examined by DNA sequencing to confirm the genotyping results. Results from these two methods were concordant. Serum LOX measurement
Serum samples were diluted with an equal volume of phosphate-buffered saline, and duplicate diluted samples were assayed for serum LOX concentrations using an ELISA kit (USCN Life Science, Inc.). ELISAs were conducted at the same time by three individuals who were blinded to all subject and sample details. Linearity of the assay has been demonstrated over a concentration range of 0.156– 10 ng/mL with a sensitivity of 0.053 ng/mL. Statistical analysis
Statistical analysis was performed using SPSS 13.0 software. The chi-square test was used to assess the association between LOX polymorphisms and susceptibility to cervical cancer. Hardy–Weinberg equilibrium (HWE) was tested by comparing the observed and expected genotype frequencies using the chi-square test. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. Student’s t-test was used to compare the sera level of LOX between different genotypes. p < 0.05 was considered as statistically significant. Results Clinical characteristics of the study subjects
A total of 262 cervical cancer cases and 298 cancer-free controls were recruited for the present study. All subjects were of Chinese ethnicity. Demographic and other selected characteristics of the cases and controls are presented (Table 2).
LYSYL OXIDASE POLYMORPHISMS AND CERVICAL CANCER
Table 2. Characteristics of the Subjects Characteristics Age (years) Range Smoking status Yes No HPV infection Yes No Clinical stage 0 I II III IV
Patients (n = 262)
Controls (n = 298)
p-Value
38.2 – 9.2 21–73
41.5 – 9.8 18–79
> 0.05
51 (24.2) 211 (75.8)
20 (6.7) 278 (93.3)
195 (74.4) 67 (25.6)
32 (10.7) 266 (89.3)
> 0.05 < 0.001
56 115 45 36 10
(21.4) (43.9) (17.2) (13.7) (3.8)
HPV, human papilloma virus.
Cases and controls did not reveal any statistical significance with regard to age ( p > 0.05). Patients showed significantly higher smoking status and HPV infection (Table 2). Association between LOX polymorphisms and cervical cancer
The genotype and allele frequencies of the LOX rs1800449G/A and rs2278226C/G SNPs in cervical cancer cases and controls are summarized in Table 3. The genotype distributions of these two polymorphisms among the controls were in agreement with the HWE ( p > 0.05). The G and A allele frequencies of rs1800449 polymorphism were 75.2% and 24.8% among the cases and 82.6% and 17.4% among the controls. Prevalence of the rs1800449AA geno-
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type and A allele showed significant differences between patients and controls ( p = 0.004 and p = 0.003, respectively). As for the LOX rs2278226C/G polymorphism, the prevalence of C and G alleles were, respectively, 89.7% and 10.3% in patients and 96.1% and 3.9% in controls. We did not identify the rs2278226GG genotype in our study population. The CG genotype and G allele showed significantly increased frequencies in patients than in controls (OR = 3.10, 95% CI: 1.85–5.22, p < 0.001 and OR = 2.86, 95% CI: 1.73–4.73, p < 0.001, respectively). We also analyzed the status of linkage disequilibrium between the two SNPs, and data showed that they were not linked with each other (D¢ < 0.08). In addition, four haplotypes were identified in our study population, in which GG, AC, and AG haplotypes had significantly higher numbers in patients than in controls ( p < 0.001, p = 0.003, and p = 0.009, respectively, Table 3). These data suggested that LOX rs1800449G/A and rs2278226C/G polymorphisms were associated with increased susceptibility to cervical cancer in the Chinese population. Effect of rs1800449G/A and rs2278226C/G on serum level of LOX
To understand the potential mechanism of the association between rs1800449G/A and rs2278226C/G and risk of cervical cancer, we investigated whether these polymorphisms could affect gene expression by examining serum levels of LOX. As for the rs1800449G/A SNP, a total of 45 healthy controls and 45 patients were included, in which 15 controls or patients carried GG genotype, 15 controls or patients carried GA genotype, and 15 controls or patients carried AA genotype. The serum level of LOX did not reveal clear differences among these subjects (Fig. 1A). Similarly, patients carrying different rs1800449 genotypes did not present significant variations in LOX serum levels (Fig. 1B). As for the rs2278226C/G SNP, a total of 40 healthy controls and 40
Table 3. Lysyl Oxidase Polymorphisms in Cervical Cancer Patients and Controls Polymorphisms rs1800449 (G473A) Genotype GG GA AA Allele G A rs2278226 C/G Genotype CC CG Allele C G Haplotypes GC GG AC AG a
Cases (n = 262)
Controls (n = 298)
OR (95% CI)
163 (62.2) 68 (26.0) 31 (11.8)
210 (70.5) 72 (24.2) 16 (5.3)
Referent 1.22 (0.82–1.80) 2.50 (1.32–4.72)
0.323 0.004a
394 (75.2) 130 (24.8)
492 (82.6) 104 (17.4)
Referent 1.56 (1.17–2.09)
0.003a
208 (79.4) 54 (20.6)
275 (92.3) 23 (7.7)
Referent 3.10 (1.85–5.22)
< 0.001a
470 (89.7) 54 (10.3)
573 (96.1) 23 (3.9)
Referent 2.86 (1.73–4.73)
< 0.001a
355 39 115 15
475 17 98 6
Referent 3.07 (1.71–5.52) 1.57 (1.16–2.13) 3.35 (1.29–8.71)
< 0.001a 0.003a 0.009a
(67.7) (7.4) (21.9) (3.0)
p < 0.05. CI, confidence interval; OR, odds ratio.
(79.7) (2.9) (16.4) (1.0)
p-Value
790
FIG. 1. Serum level of lysyl oxidase (LOX) in healthy controls (A) and cervical cancer patients (B) carrying rs1800449GG, GA, and AA genotypes. A total of 45 healthy controls and 45 patients were included, in which 15 controls or patients carried GG genotype, 15 controls or patients carried GA genotype, and 15 controls or patients carried AA genotype. patients were included, in which 20 controls or patients carried CC genotype and 20 controls or patients carried CG genotype. We did not observe significantly different levels of LOX between controls carrying CC genotype and CG genotype (Fig. 2A). However, cervical cancer patients with the polymorphic CG genotype showed clearly increased levels of LOX than those with CC genotype (7.5 – 1.8 ng/mL vs. 5.2 – 0.5 ng/mL, p < 0.01, Fig. 2B). In addition, we compared the sera levels of LOX between cervical cancer patients and healthy controls, and no significant difference was identified (data not shown). Discussion
In the current study, we identified that LOX rs1800449G/ A and rs2278226C/G SNPs were associated with increased susceptibility to cervical cancer. Also, our data showed that
BU ET AL.
FIG. 2. Serum level of LOX in healthy controls (A) and cervical cancer patients (B) carrying rs2278226CC and CG genotypes. A total of 40 healthy controls and 40 patients were included, in which 20 controls or patients carried CC genotype and 20 controls or patients carried CG genotype.
the rs2278226CG genotype could specifically increase serum levels of LOX in cervical cancer patients, but not in healthy controls. Meanwhile, the other SNP, rs1800449G/A, did not show any effects on serum levels of LOX. Recent reports have indicated that the mature LOX enzyme mediates tumor progression both in a tumor cell autonomous fashion and in the tumor microenvironment. An increase in LOX or LOXL expression, as measured by mRNA, protein, and/or enzymatic activity, is associated with tumor progression and poorer survival in patients with breast, esophageal, prostate, renal cell, and head and neck cancers (Min et al., 2007; Nishioka et al., 2012; Zhang et al., 2012). Inhibition of LOX under hypoxic conditions was reported to reduce tumor invasion and metastases without any change in primary tumor growth in a murine model of breast cancer (Nishioka et al., 2012). In addition to
LYSYL OXIDASE POLYMORPHISMS AND CERVICAL CANCER
local effects on invasion, secreted LOX can circulate to distant sites and contribute to the formation of the premetastatic niche by stimulating collagen crosslinking in the ECM of a target organ and promoting the adherence and invasion of circulating tumor cells and bone marrow-derived cells that stimulate angiogenesis (Goto et al., 2013). Furthermore, a study has shown that LOX may act as a biomarker of invasion of lung adenocarcinoma (Shi et al., 2012; Wei et al., 2012). It has been reported the LOX is upregulated in anaplastic astrocytoma cells (Laczko et al., 2007). Also, a study has shown that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behavior. These researches indicate that functional changes in LOX may affect the development of various cancers. Our data demonstrated that LOX polymorphisms were associated with increased risk of cervical cancer (Table 3), suggesting the involvement of LOX in the development of this disease. Interestingly, it seemed that rs2278226C/G polymorphism had a stronger correlation with cervical cancer than rs1800449G/A SNP (Table 3). The LOX rs1800449G/A polymorphism, which causes an Arg158Gln substitution in a highly conserved region within LOX-PP, may reduce the ability of LOX-PP to suppress Ras signaling and therefore may result in an increased tumor risk (Ma et al., 2011; Liu et al., 2012; Wang et al., 2012). The rs2278226 polymorphism is located in the exon of LOX gene (Zhang et al., 2012). However, it is a silent SNP and does not cause substitution of amino acid. Mechanism of rs2278226 on the susceptibility to cervical cancer remains unclear. Our results revealed that rs2278226C/G polymorphism did not affect serum levels of LOX in healthy controls. However, cervical cancer patients with rs2278226CG genotype presented significantly increased levels of LOX than those with CC genotype (Fig. 2), suggesting that the SNP may specifically affect cervical cancer by upregulating LOX. The rs1800449G/A polymorphism did not influence the LOX level, suggesting that the two SNPs may use different pathways in affecting cervical cancer. In conclusion, this case–control study provided evidence that LOX polymorphisms were associated with increased susceptibility to cervical cancer, in which rs2278226 could increase serum levels of LOX in cervical cancer patients. Our results shed light on the etiology of cervical cancer and identify a potential target for developing novel therapies for this malignancy. Disclosure Statement
No competing financial interests exist. References
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lesions. J Intern Med [Epub ahead of print]; DOI: 10.1111/ joim.12228. Price, D.J., Ravindranath, T., and Kaler, S.G. (2007). Internal jugular phlebectasia in Menkes disease. Int J Pediatr Otorhinolaryngol 71, 1145–1148. Qu, Y., Dang, S., and Hou, P. (2013). Gene methylation in gastric cancer. Clin Chim Acta 424, 53–65. Shi, W., Yang, B., Li, X., Sun, S., Wang, L., and Jiao, S. (2012). The effect of lysyl oxidase polymorphism on susceptibility and prognosis of nonsmall cell lung cancer. Tumour Biol 33, 2379–2383. Tota, J.E., Ramana-Kumar, A.V., El-Khatib, Z., and Franco, E.L. (2014). The road ahead for cervical cancer prevention and control. Curr Oncol 21, e255–e264. Viguet-Carrin, S., Garnero, P., and Delmas, P.D. (2006). The role of collagen in bone strength. Osteoporos Int 17, 319–336. Wang, X., Cong, J.L., Qu, L.Y., Jiang, L., and Wang, Y. (2012). Association between lysyl oxidase G473A polymorphism and ovarian cancer in the Han Chinese population. J Int Med Res 40, 917–923. Wei, L., Song, X.R., Sun, J.J., Wang, X.W., Xie, L., and Lv, L.Y. (2012). Lysyl oxidase may play a critical role in hypoxia-induced NSCLC cells invasion and migration. Cancer Biother Radiopharm 27, 672–677. Wu, J., Cai, C., Tong, D., and Hou, H. (2012). Lysyl oxidase G473A polymorphism is associated with increased risk of ovarian cancer. Genet Test Mol Biomarkers 16, 915–919.
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Yang, X., Li, S., Li, W., Chen, J., Xiao, X., Wang, Y., Yan, G., and Chen, L. (2013). Inactivation of lysyl oxidase by betaaminopropionitrile inhibits hypoxia-induced invasion and migration of cervical cancer cells. Oncol Rep 29, 541–548. Yu, H., Li, T., Zou, X., Yuan, L., Hu, J., Xu, Z., Peng, L., Zhang, C., and Zou, Y. (2013). Effects of lysyl oxidase genetic variants on the susceptibility to rhegmatogenous retinal detachment and proliferative vitreoretinopathy. Inflammation 36, 839–844. Zhang, H.F., Zhao, K.J., Xu, Y., Hong, B., Zhao, W.Y., Liu, J.M., and Huang, Q.H. (2012). Lysyl oxidase polymorphisms and ischemic stroke—a case control study. Mol Biol Rep 39, 9391–9397.
Address correspondence to: Xuemei Jie, MD The Maternal and Child Health Hospital of Jinan City No. 2 Jian Guo Xiao Jing San Road Jinan 250001 Shandong China E-mail: [email protected] Received for publication April 26, 2014; received in revised form May 2, 2014; accepted May 23, 2014.
Lysyl Oxidase Genetic Variants Affect Gene Expression in Cervical Cancer Meimei Bu,1,* Li Li,2,* Yong Zhang,1 Ying Xu,1 Shizhi An,1 Fang Hou,1 and Xuemei Jie1
Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the homeostasis of tumors. The aim of this study was to investigate the association between LOX polymorphisms and cervical cancer, and the effect of these polymorphisms on gene expression. We evaluated two polymorphisms of LOX, rs1800449G/A (G473A) and rs2278226C/G, in 262 cervical cancer cases and 298 healthy controls in the Chinese population. Results showed that the prevalence of rs1800449AA genotype was significantly increased in cases than in controls ( p = 0.004). Individuals who carried the rs1800449A allele had a 1.56-fold increased risk for cervical cancer than those with the rs1800449G allele ( p = 0.003). The rs2278226CG genotype also revealed a significantly higher proportion in cases (20.6%) than in controls (7.7%, p < 0.001). Interestingly, when analyzing these two polymorphisms with the serum level of LOX, we identified that cervical cancer patients carrying the rs2278226CG genotype had a significantly elevated level of LOX than those with rs2278226CC wild type, whereas the same phenomenon was not observed in controls. The rs1800449 polymorphism did not affect the LOX serum level in either controls or patients. These results suggest that the polymorphisms in the LOX gene may be involved in the development of cervical cancer through various mechanisms.
Introduction
C
ervical cancer is the second most common cancer in women worldwide (Tota et al., 2014). The human papilloma virus (HPV) appears to be a necessary factor in the development of almost all cases of cervical cancer (Olesen et al., 2014). However, only a small fraction of HPV-infected individuals develop cervical cancer, suggesting that additional environmental, genetic, or immunological factors contribute to the pathogenesis of the disease (Caffarel and Coleman, 2014). Etiology of cancer is complicated and has not been elucidated completely. Recent studies have suggested that genetic variants may greatly involve in the disease (Tota et al., 2014). Lysyl oxidase (LOX), a copper-dependent amine oxidase, initiates the process of collagen and elastin crosslinking by oxidatively deaminating specific lysine and hydroxylysine residues located in the telopeptide domains, thus stabilizing the fibrous deposits of these proteins in the extracellular matrix (ECM) (Nishioka et al., 2012). LOX is expressed as a 46-kDa preproenzyme and a glycosylated 50 kDa proenzyme (Nishioka et al., 2012). Following secretion into the ECM, the 50-kDa proLOX is further processed by procollagen proteases into the 32-kDa mature species (Nishioka
et al., 2012; Qu et al., 2013). The normal enzymatic activity of LOX plays a critical role in morphogenesis and tissue structural integrity. An abnormal LOX activity can lead to connective tissue disorders such as emphysema (Mizuno et al., 2012), osteoporosis (Viguet-Carrin et al., 2006), cutis laxa (Debret et al., 2010), Menkes syndrome (Price et al., 2007), organ fibrosis (Correa-Costa et al., 2014), atherogenesis (Ovchinnikova et al., 2014), and cardiovascular diseases (Ishiwata et al., 2014). Perturbation of LOX expression is also involved in carcinogenesis and cancer progression (Cox and Erler, 2013). Markedly, LOX displays a dual role as a tumor suppressor as well as a metastasis promoter in tumorigenesis in its biological functions (Cano et al., 2012; Nishioka et al., 2012; Dudakova and Jirsova, 2013). LOX antagonizes tumorigenesis. Expression of LOX in ras-transformed fibroblasts decreases the activity of NFkB inhibiting cell growth and neoplastic transformation (Goto et al., 2013). Reduced LOX expression was found in several tumor cell lines. LOX propeptide reverses the invasive phenotype of Her-2/neu-driven breast cancer (Min et al., 2007). On the other hand, LOX promoted tumor progression. Increased expression of LOX in some tumors was associated with poor prognosis (Han et al., 2014). LOXmediated collagen and elastin crosslinking functions as a
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The Maternal and Child Health Hospital of Jinan City, Jinan, Shandong, China. Department of Gynecology, Affiliated Tumor Hospital, Xinjiang Medical University, Urumqi, China. *These authors contributed equally to this work.
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contributor to tumor ECM stiffening (Cox and Erler, 2013). ECM remodeling and stiffening are characteristics of these tumors, promoting tumor motility, migration, and rigidity disrupting tissue morphogenesis (Cox and Erler, 2013). Matrix remodeling by LOX provides the basis for cell movement important for tumor invasion and migration (Ahn et al., 2013). LOX is known to be essential for hypoxia-induced metastasis in breast cancer (Ahn et al., 2013). Thus, a further understanding of the mechanism(s) by which LOX regulates tumor progression has a potential benefit for novel cancer therapy. LOX may also play critical roles in the development of cervical cancer. Recent study has shown that inactivation of LOX by b-aminopropionitrile can inhibit hypoxia-induced invasion and migration of cervical cancer cells (Yang et al., 2013). Single-nucleotide polymorphisms (SNPs) can affect the development of different diseases (Wu et al., 2012; Zhang et al., 2012; Yu et al., 2013). However, no literature reported LOX polymorphism and cervical cancer. Also, the functional study of LOX SNPs remains limited. In this study, we investigated rs1800449G/A (G473A) and rs2278226C/G polymorphisms in cervical cancer and assessed their effects on the serum level of LOX. Materials and Methods Patients and controls
The study population and collection of subjects have been previously described elsewhere. In brief, this was a hospital-based case–control study, including 262 cervical cancer cases and 298 cancer-free controls. All the patients were newly diagnosed and confirmed by histopathologic examinations. Subjects with a minimum 15 pack-year smoking history were defined as smokers. The controls were recruited from people coming to the hospital for general health examinations. The exclusion criteria for the control subjects included a self-reported history of cancers or constant medication on other severe diseases. All of the control subjects were frequency matched to the cases on age, sex, and residence area (urban or rural). Each participant was interviewed in person with a structured questionnaire that elicited information on demographic factors and health characteristics. Venous blood was collected from each of the study participants. DNA genotyping
Detection of rs1800449G/A and rs2278226C/G SNPs was based on previously described methods (Zhang et al., 2012; Han et al., 2014). In brief, rs1800449G/A was genotyped using a polymerase chain reaction (PCR)–restriction fragment length polymorphism method. The PCR primers are shown in Table 1. The PCRs were performed in a total volume of 35 mL reaction mixture containing 100 ng genomic DNA, 12.5 pmol of each primer, 0.1 mM of each dNTP, 1 · PCR buffer, 1.0 mM MgCl2, and 1.5 U of Taq DNA polymerase (Fermentas). Reactions were conducted using the thermal cycler (Biometra) under the following conditions: an initial incubation at 95C for 10 min, 30 cycles of 95C for 30 s, 57C for 30 s, 72C for 30 s, and a final extension at 72C for 7 min. The efficiency of the PCR was confirmed by gel electrophoresis on a 1.5% agarose gel.
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Table 1. Primers Used for Detecting the Lysyl Oxidase Single-Nucleotide Polymorphisms Primers rs1800449 (G473A) For PCR F: 5¢-CTCACAGTACCAGCCTCAGCG-3¢ R: 5¢-CCAGGTCTGGGCCTTTCATA-3¢ rs2278226 C/G For PCR F: 5¢-CTGCTCGGGCCTTTGCAGCT-3¢ R: 5¢-ACTCCAGATGAGCCGGCCGT-3¢ PCR, polymerase chain reaction.
After DNA amplification, the PCR products were digested overnight at 37C with 10 U of the specific restriction endonuclease PstI (Fermentas), which cuts the allele A. The digestion products were then resolved and separated on a 2% agarose gel stained with ethidium bromide for visualization under ultraviolet light. After electrophoresis, homozygous A alleles were represented by DNA bands with sizes at 291 and 114 bp; an uncut fragment of 405 bp indicated the homozygous G alleles, while the heterozygous genotype was displayed as a combination of 405, 291, and 114 bp. The detection of the other polymorphism, rs2278226C/G, was performed by similar methods. In brief, the polymorphism amplified by PCR and determined by the enzyme restriction method using SacII (Fermentas). In addition, more than 5% of PCR-amplified DNA samples were examined by DNA sequencing to confirm the genotyping results. Results from these two methods were concordant. Serum LOX measurement
Serum samples were diluted with an equal volume of phosphate-buffered saline, and duplicate diluted samples were assayed for serum LOX concentrations using an ELISA kit (USCN Life Science, Inc.). ELISAs were conducted at the same time by three individuals who were blinded to all subject and sample details. Linearity of the assay has been demonstrated over a concentration range of 0.156– 10 ng/mL with a sensitivity of 0.053 ng/mL. Statistical analysis
Statistical analysis was performed using SPSS 13.0 software. The chi-square test was used to assess the association between LOX polymorphisms and susceptibility to cervical cancer. Hardy–Weinberg equilibrium (HWE) was tested by comparing the observed and expected genotype frequencies using the chi-square test. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. Student’s t-test was used to compare the sera level of LOX between different genotypes. p < 0.05 was considered as statistically significant. Results Clinical characteristics of the study subjects
A total of 262 cervical cancer cases and 298 cancer-free controls were recruited for the present study. All subjects were of Chinese ethnicity. Demographic and other selected characteristics of the cases and controls are presented (Table 2).
LYSYL OXIDASE POLYMORPHISMS AND CERVICAL CANCER
Table 2. Characteristics of the Subjects Characteristics Age (years) Range Smoking status Yes No HPV infection Yes No Clinical stage 0 I II III IV
Patients (n = 262)
Controls (n = 298)
p-Value
38.2 – 9.2 21–73
41.5 – 9.8 18–79
> 0.05
51 (24.2) 211 (75.8)
20 (6.7) 278 (93.3)
195 (74.4) 67 (25.6)
32 (10.7) 266 (89.3)
> 0.05 < 0.001
56 115 45 36 10
(21.4) (43.9) (17.2) (13.7) (3.8)
HPV, human papilloma virus.
Cases and controls did not reveal any statistical significance with regard to age ( p > 0.05). Patients showed significantly higher smoking status and HPV infection (Table 2). Association between LOX polymorphisms and cervical cancer
The genotype and allele frequencies of the LOX rs1800449G/A and rs2278226C/G SNPs in cervical cancer cases and controls are summarized in Table 3. The genotype distributions of these two polymorphisms among the controls were in agreement with the HWE ( p > 0.05). The G and A allele frequencies of rs1800449 polymorphism were 75.2% and 24.8% among the cases and 82.6% and 17.4% among the controls. Prevalence of the rs1800449AA geno-
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type and A allele showed significant differences between patients and controls ( p = 0.004 and p = 0.003, respectively). As for the LOX rs2278226C/G polymorphism, the prevalence of C and G alleles were, respectively, 89.7% and 10.3% in patients and 96.1% and 3.9% in controls. We did not identify the rs2278226GG genotype in our study population. The CG genotype and G allele showed significantly increased frequencies in patients than in controls (OR = 3.10, 95% CI: 1.85–5.22, p < 0.001 and OR = 2.86, 95% CI: 1.73–4.73, p < 0.001, respectively). We also analyzed the status of linkage disequilibrium between the two SNPs, and data showed that they were not linked with each other (D¢ < 0.08). In addition, four haplotypes were identified in our study population, in which GG, AC, and AG haplotypes had significantly higher numbers in patients than in controls ( p < 0.001, p = 0.003, and p = 0.009, respectively, Table 3). These data suggested that LOX rs1800449G/A and rs2278226C/G polymorphisms were associated with increased susceptibility to cervical cancer in the Chinese population. Effect of rs1800449G/A and rs2278226C/G on serum level of LOX
To understand the potential mechanism of the association between rs1800449G/A and rs2278226C/G and risk of cervical cancer, we investigated whether these polymorphisms could affect gene expression by examining serum levels of LOX. As for the rs1800449G/A SNP, a total of 45 healthy controls and 45 patients were included, in which 15 controls or patients carried GG genotype, 15 controls or patients carried GA genotype, and 15 controls or patients carried AA genotype. The serum level of LOX did not reveal clear differences among these subjects (Fig. 1A). Similarly, patients carrying different rs1800449 genotypes did not present significant variations in LOX serum levels (Fig. 1B). As for the rs2278226C/G SNP, a total of 40 healthy controls and 40
Table 3. Lysyl Oxidase Polymorphisms in Cervical Cancer Patients and Controls Polymorphisms rs1800449 (G473A) Genotype GG GA AA Allele G A rs2278226 C/G Genotype CC CG Allele C G Haplotypes GC GG AC AG a
Cases (n = 262)
Controls (n = 298)
OR (95% CI)
163 (62.2) 68 (26.0) 31 (11.8)
210 (70.5) 72 (24.2) 16 (5.3)
Referent 1.22 (0.82–1.80) 2.50 (1.32–4.72)
0.323 0.004a
394 (75.2) 130 (24.8)
492 (82.6) 104 (17.4)
Referent 1.56 (1.17–2.09)
0.003a
208 (79.4) 54 (20.6)
275 (92.3) 23 (7.7)
Referent 3.10 (1.85–5.22)
< 0.001a
470 (89.7) 54 (10.3)
573 (96.1) 23 (3.9)
Referent 2.86 (1.73–4.73)
< 0.001a
355 39 115 15
475 17 98 6
Referent 3.07 (1.71–5.52) 1.57 (1.16–2.13) 3.35 (1.29–8.71)
< 0.001a 0.003a 0.009a
(67.7) (7.4) (21.9) (3.0)
p < 0.05. CI, confidence interval; OR, odds ratio.
(79.7) (2.9) (16.4) (1.0)
p-Value
790
FIG. 1. Serum level of lysyl oxidase (LOX) in healthy controls (A) and cervical cancer patients (B) carrying rs1800449GG, GA, and AA genotypes. A total of 45 healthy controls and 45 patients were included, in which 15 controls or patients carried GG genotype, 15 controls or patients carried GA genotype, and 15 controls or patients carried AA genotype. patients were included, in which 20 controls or patients carried CC genotype and 20 controls or patients carried CG genotype. We did not observe significantly different levels of LOX between controls carrying CC genotype and CG genotype (Fig. 2A). However, cervical cancer patients with the polymorphic CG genotype showed clearly increased levels of LOX than those with CC genotype (7.5 – 1.8 ng/mL vs. 5.2 – 0.5 ng/mL, p < 0.01, Fig. 2B). In addition, we compared the sera levels of LOX between cervical cancer patients and healthy controls, and no significant difference was identified (data not shown). Discussion
In the current study, we identified that LOX rs1800449G/ A and rs2278226C/G SNPs were associated with increased susceptibility to cervical cancer. Also, our data showed that
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FIG. 2. Serum level of LOX in healthy controls (A) and cervical cancer patients (B) carrying rs2278226CC and CG genotypes. A total of 40 healthy controls and 40 patients were included, in which 20 controls or patients carried CC genotype and 20 controls or patients carried CG genotype.
the rs2278226CG genotype could specifically increase serum levels of LOX in cervical cancer patients, but not in healthy controls. Meanwhile, the other SNP, rs1800449G/A, did not show any effects on serum levels of LOX. Recent reports have indicated that the mature LOX enzyme mediates tumor progression both in a tumor cell autonomous fashion and in the tumor microenvironment. An increase in LOX or LOXL expression, as measured by mRNA, protein, and/or enzymatic activity, is associated with tumor progression and poorer survival in patients with breast, esophageal, prostate, renal cell, and head and neck cancers (Min et al., 2007; Nishioka et al., 2012; Zhang et al., 2012). Inhibition of LOX under hypoxic conditions was reported to reduce tumor invasion and metastases without any change in primary tumor growth in a murine model of breast cancer (Nishioka et al., 2012). In addition to
LYSYL OXIDASE POLYMORPHISMS AND CERVICAL CANCER
local effects on invasion, secreted LOX can circulate to distant sites and contribute to the formation of the premetastatic niche by stimulating collagen crosslinking in the ECM of a target organ and promoting the adherence and invasion of circulating tumor cells and bone marrow-derived cells that stimulate angiogenesis (Goto et al., 2013). Furthermore, a study has shown that LOX may act as a biomarker of invasion of lung adenocarcinoma (Shi et al., 2012; Wei et al., 2012). It has been reported the LOX is upregulated in anaplastic astrocytoma cells (Laczko et al., 2007). Also, a study has shown that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behavior. These researches indicate that functional changes in LOX may affect the development of various cancers. Our data demonstrated that LOX polymorphisms were associated with increased risk of cervical cancer (Table 3), suggesting the involvement of LOX in the development of this disease. Interestingly, it seemed that rs2278226C/G polymorphism had a stronger correlation with cervical cancer than rs1800449G/A SNP (Table 3). The LOX rs1800449G/A polymorphism, which causes an Arg158Gln substitution in a highly conserved region within LOX-PP, may reduce the ability of LOX-PP to suppress Ras signaling and therefore may result in an increased tumor risk (Ma et al., 2011; Liu et al., 2012; Wang et al., 2012). The rs2278226 polymorphism is located in the exon of LOX gene (Zhang et al., 2012). However, it is a silent SNP and does not cause substitution of amino acid. Mechanism of rs2278226 on the susceptibility to cervical cancer remains unclear. Our results revealed that rs2278226C/G polymorphism did not affect serum levels of LOX in healthy controls. However, cervical cancer patients with rs2278226CG genotype presented significantly increased levels of LOX than those with CC genotype (Fig. 2), suggesting that the SNP may specifically affect cervical cancer by upregulating LOX. The rs1800449G/A polymorphism did not influence the LOX level, suggesting that the two SNPs may use different pathways in affecting cervical cancer. In conclusion, this case–control study provided evidence that LOX polymorphisms were associated with increased susceptibility to cervical cancer, in which rs2278226 could increase serum levels of LOX in cervical cancer patients. Our results shed light on the etiology of cervical cancer and identify a potential target for developing novel therapies for this malignancy. Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Xuemei Jie, MD The Maternal and Child Health Hospital of Jinan City No. 2 Jian Guo Xiao Jing San Road Jinan 250001 Shandong China E-mail: [email protected] Received for publication April 26, 2014; received in revised form May 2, 2014; accepted May 23, 2014.
