Functional Polymorphisms in Monocyte Chemoattractant Protein-1 Are Associated with Increased Susceptibility to Ovarian Cancer

Abstract

Ovarian cancer ranks fifth in cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. Monocyte chemoattractant protein-1 (MCP-1) is highly expressed in various malignancies and promotes carcinogenesis. The aim of the study was to investigate the association between MCP-1 genetic polymorphisms and the susceptibility to ovarian cancer. MCP-1 rs1024611A/G and rs3760396C/G polymorphisms were examined in 257 ovarian cancer patients and 273 healthy controls. We found that distributions of rs1024611GG genotype and rs3760396GG genotype were clearly increased in ovarian cancer cases compared to healthy donors (odds ratio [OR]=1.93, 95% confidence interval [CI]: 1.13–3.29, p=0.015; OR=3.89, 95% CI: 1.63–9.33, p=0.001). Stratification analyses revealed that patients with serous papillary type had further increased percentage of rs3760396GG genotype than those with other types (OR=3.89, 95% CI: 1.11–13.66, p=0.024). In addition, we evaluated the possible effect of MCP-1 polymorphisms on gene expression by examining the serum level of MCP-1 in patients and controls. Data revealed that subjects carrying rs1024611AG and GG genotypes had a significantly higher serum level of MCP-1 than those with AA genotype. These data suggest that MCP-1 rs1024611A/G and rs3760396C/G polymorphisms are associated with increased susceptibility to ovarian cancer, in which rs1024611A/G may increase serum level of MCP-1 in the Chinese population.

Introduction

Ovarian cancer remains a clinically challenging disease that affects more than 190,000 women worldwide each year (Kligerman and White, 2011; Bai et al., 2013). Most patients present with advanced disease that is initially highly responsive to surgery and chemotherapy, but the majority of patients succumb to recurrent disease that is resistant to further treatment (Kligerman and White, 2011). Early detection could theoretically improve outcomes, but this remains a challenging problem due to the relative rarity of the disease in the general population. Understanding the pathogenesis of ovarian cancer can greatly improve the diagnosis and treatment of the disease. Studies have suggested that human genetic variations may be one of the key factors in the development of this malignancy (Li et al., 2012; Shi et al., 2012).

Monocyte chemotactic protein-1 (MCP-1), also known as CC chemokine ligand 2 (CCL2), is a key member of the CCβ chemokine superfamily (Leonard et al., 1991; Yoshimura and Leonard, 1991). MCP-1 exerts its biologic effect by binding to CC chemokine receptor (CCR2), a G-protein-coupled receptor. MCP-1-CCR2 signaling elicits directional migration and phenotypic polarization of monocytes, promotes cancer-related inflammation, angiogenesis, and metastasis (Kalinowska and Losy, 2008; Proudfoot et al., 2008). MCP-1-mediated recruitment of tumor-associated monocytes indirectly promotes angiogenesis, tumor growth, invasion, and chemotactic homing to metastatic sites (Hasegawa and Sato, 2008). Moreover, MCP-1 mediates osteoclastogenesis and osteoblastic bone metastasis (Lu et al., 2007; Wintges et al., 2013). MCP-1 also directly impacts tumor proliferation, migration, and survival through phosphatidylinositol 3-kinase/protein kinase B-dependent signaling, mammalian target of rapamycin kinase activation, and enhanced survivin expression (Tanaka et al., 2010). MCP-1 is secreted by stromal elements (e.g., bone marrow endothelial cells and osteoblasts) and expressed at high levels in numerous tumors, including breast, prostate, lung, esophageal, ovarian, and pancreatic cancers (Kudo-Saito et al., 2013; Sanford et al., 2013). Elevated MCP-1 levels correlate with vascular endothelial growth factor secretion, active angiogenesis, enhanced tumor proliferation, poor prognosis, and early disease relapse (Moriya et al., 2014).

Studies about MCP-1 and ovarian cancer remain controversial. Reports have shown that the levels of MCP-1 in the ascites or serum of patients with ovarian cancer are higher than those of healthy people or patients with benign tumors (Su et al., 2010; Furukawa et al., 2013). In addition, expression of MCP-1 is higher in cancerous ovarian tissues than in healthy ovarian tissues (Furukawa et al., 2013). However, Arnold et al. (2005) demonstrated that MCP-1 expression was significantly silenced or downregulated in primary ovarian adenocarcinomas when compared to normal epithelial ovarian tissue, and Wojnarowicz et al. (2012) confirmed this finding in a microarray study of serous ovarian cancer cell lines and primary malignant tumors.

Single-nucleotide polymorphisms (SNPs) may be associated with various diseases (Rong et al., 2013; Han et al., 2014). MCP-1 rs1024611A/G (MCP-1-2518A/G) and rs3760396C/G polymorphisms have been identified in the Chinese population. However, correlations between these polymorphisms and ovarian cancer remain unknown. In the current study, we investigated whether MCP-1 rs1024611A/G and rs3760396C/G polymorphisms were associated with susceptibility to ovarian cancer. We also evaluated the possible effect of MCP-1 polymorphisms on gene expression by examining the serum level of MCP-1 in patients and controls.

Materials and Methods

Patients and controls

The study group included 257 ovarian cancer patients and 273 controls recruited from the Liaocheng People's Hospital. All the patients had epithelial ovarian cancer. The diagnosis was confirmed by a histopathologic examination. The control population was recruited from people who came for general health examinations from the same hospital and were confirmed to be without any malignant diseases. All subjects were of Han Chinese ethnicity and unrelated to each other. 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. The study was performed upon the approval of the Review Board of the Liaocheng People's Hospital.

DNA extraction and genotyping

DNA extraction and genotyping were conducted based on a previously described method (Bai et al., 2012). In brief, genomic DNA was extracted from 5 mL frozen whole blood, using the DNA Extraction Kit (Fastagen) according to the manufacturer's protocol. Genotyping for the MCP-1 rs1024611A/G polymorphism was performed by using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. Primers for PCR amplification were 5′-TCTCTCACGCCAGCACTGACC-3′ and 5′-GAG TGTTCACATAGGCTTCTG-3′. The PCR was performed in a total volume of 50 μL, which included 0.5 μg of genomic DNA, 25 pmol of each primer, 1.5 mM of MgCl₂, 10× buffer with KCl, 200 μM of dNTPs, and 1 U of Taq DNA polymerase. The following amplification protocol was used: denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min 30 s. Amplified fragments were digested using PvuII at 37°C overnight. The substitution of an adenine by a guanine at position −2518 of the MCP-1 gene resulted in the appearance of a novel PvuII restriction site. The mutant allele yielded 159-bp and 75-bp fragments, whereas the wild-type allele remained undigested (234 bp). Detection of rs3760396C/G polymorphism was performed by direct sequencing (Perkin-Elmer ABI model 377; Applied Biosystems). Primers used were 5′-GTGTCATACTCACA GAGTGC-3′ or 5′-GAGGAAATCTAAGGCACAAC-3′.

Serum level of MCP-1

Serum samples were collected and immediately stored at −80°C. Serum MCP-1 was assessed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems). The standard range is 31.3–2000 pg/mL and the sensitivity is 10 pg/mL.

Statistical analysis

The SPSS statistical software package ver.19.0 (SPSS, Inc.) was used for statistical analysis. The polymorphisms were tested for deviation from the Hardy–Weinberg equilibrium by comparing the observed and expected genotype frequencies using the chi-square test. For SNP analysis, genotype and allele frequencies were compared between groups using the chi-square test, and odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. The serum level of MCP-1 was compared by the Student's t-test. p-Values less than 0.05 were considered significant.

Results

MCP-1 polymorphisms among the patients and controls

Selected characteristics of the patients and controls are shown in Table 1. Age did not reveal significant differences between the ovarian cancer cases and controls. As shown in Table 2, the percentage of rs1024611GG genotype was significantly higher in patients than in controls (OR=1.93, 95% CI: 1.13–3.29, p=0.015). For the MCP-1 rs3760396C/G polymorphism, the percentage of polymorphic GG genotypes was significantly increased in cases than controls (OR=3.89, 95% CI: 1.63–9.33, p=0.001). Also, the prevalence of rs1024611G allele and rs3760396G allele was significantly higher in patients compared to controls (p=0.008 and p<0.001). In addition, we analyzed the linkage status of the two SNPs and no linkage disequilibrium was found. These data suggest that the MCP-1 rs1024611 and rs3760396 SNPs are associated with increased susceptibility to ovarian cancer in the Chinese population.

Table 1.

General Characteristics of the Subjects

	Ovarian cancer (n=257) (%)	Healthy control (n=273) (%)	p-Value
Age			>0.05
≤50	118 (45.9)	129 (47.3)
>50	139 (54.1)	144 (52.7)
Mean age	54.6±12.2	53.2±11.7	>0.05
Histology
Serous papillary	170 (66.1)
Endometrioid	24 (9.3)
Clear cell	21 (8.2)
Mucinous	18 (7.1)
Mixed and other	24 (9.3)
FIGO stage
I	35 (13.6)
II	42 (16.3)
III	122 (47.5)
IV	58 (22.6)

Table 2.

MCP-1 Polymorphisms in Ovarian Cancer Patients and Controls

Polymorphisms	Patients (%) n=257	Controls (%) n=273	OR (95% CI)	p-Value
rs1024611
Genotype
AA	30 (11.7)	47 (17.2)	1.00
AG	115 (44.7)	135 (49.5)	1.34 (0.79–2.25)	0.277
GG	112 (43.6)	91 (33.3)	1.93 (1.13–3.29)	0.015^a
Allele
A	175 (34.0)	229 (41.9)	1.00
G	339 (66.0)	317 (58.1)	1.40 (1.09–1.80)	0.008^a
rs3760396
Genotype
CC	176 (68.5)	218 (79.9)	1.00
CG	59 (23.0)	48 (17.5)	1.52 (0.99–2.34)	0.054
GG	22 (8.5)	7 (2.6)	3.89 (1.63–9.33)	0.001^a
Allele
C	411 (80.0)	484 (88.6)	1.00
G	103 (20.0)	62 (11.4)	1.96 (1.39–2.75)	<0.001^a

p<0.05.

OR, odds ratio; CI, confidence interval; MCP-1, monocyte chemoattractant protein-1.

MCP-1 polymorphisms and histological types of ovarian cancer cases

Ovarian cancer has different histological types, including serous papillary, endometrioid, and clear cell, in which serous papillary is the major type. Our patient group included 170 (66.1%) serous papillary, 24 (9.3%) endometrioid, 21 (8.2%) clear cell, 18 (7.1%) mucinous, and 24 (9.3%) mixed type (Table 1). We investigated whether the MCP-1 rs1024611 and rs3760396 SNPs were associated with histological types in ovarian cancer patients (Table 3). Data revealed that the rs1024611 SNP did not reveal any significant differences between serous papillary and other types (Table 3). However, the percentage of MCP-1 rs3760396GG genotype was significantly increased in patients with serous papillary type than those with other types (OR=3.89, 95% CI: 1.11–13.66, p=0.024).

Table 3.

MCP-1 Polymorphisms and Histological Types of the Patients

Polymorphisms	Serous papillary n=170	Other types n=87	OR (95% CI)	p-Value
rs1024611
Genotype
AA	19 (11.2)	11 (12.6)	1.00
AG	75 (44.1)	40 (46.0)	1.09 (0.47–2.51)	0.847
GG	76 (44.7)	36 (41.4)	1.22 (1.53–2.84)	0.640
Allele
A	113 (33.2)	62 (35.6)	1.00
G	227 (66.8)	112 (64.4)	1.11 (1.76–1.63)	0.587
rs3760396
Genotype
CC	109 (64.1)	67 (77.0)	1.00
CG	42 (24.7)	17 (19.5)	1.52 (0.80–2.88)	0.199
GG	19 (11.2)	3 (3.5)	3.89 (1.11–13.66)	0.024^a
Allele
C	260 (76.5)	151 (86.8)	1.00
G	80 (23.5)	23 (13.2)	2.02 (1.22–3.35)	0.006^a

p<0.05.

MCP-1 polymorphisms and gene expression

The function of MCP-1 rs1024611 SNP remains controversial. Moreover, no literature has reported its effect on MCP-1 expression in the Chinese population. In this study, we evaluated the possible function of MCP-1 polymorphisms on gene expression by examining the serum level of MCP-1 in patients and controls. To reach this purpose, we evaluated the serum level of MCP-1 in 88 healthy controls, in which 30 subjects carrying rs1024611AA genotype, 30 subjects carrying rs1024611AG genotype, and 28 subjects carrying rs1024611GG genotype. As shown in Figure 1A, healthy controls carrying rs1024611AG and GG genotypes presented a significantly higher serum level of MCP-1 than those with AA genotype (p<0.05 and p<0.01, respectively), in which subjects with rs1024611GG genotype showed the highest level of MCP-1 (p>0.05). To learn whether the polymorphism has the same effect in ovarian cancer patients, we conducted a similar experiment in 96 ovarian cancer patients, in which 29 patients carrying rs1024611AA genotype, 32 patients carrying rs1024611AG genotype, and 35 patients carrying rs1024611GG genotype. Consistent to the controls, patients with rs1024611AG and GG genotypes presented clearly upregulated levels of MCP-1 than those with AA genotype (p<0.05 and p<0.05, respectively) (Fig. 1B). We also investigated the effect of rs3760396 SNP on MCP-1. However, serum levels of MCP-1 remained consistent among subjects with different genotypes. This phenomenon was observed in both controls (Fig. 1C) and ovarian patients (Fig. 1D).

FIG. 1.

(A) Serum levels of monocyte chemoattractant protein-1 (MCP-1) from healthy controls carrying different rs1024611 genotypes. (B) Serum levels of MCP-1 from ovarian cancer patients carrying different rs1024611 genotypes. (C) Serum levels of MCP-1 from healthy controls carrying different rs3760396 genotypes. (D) Serum levels of MCP-1 from ovarian cancer patients carrying different rs3760396 genotypes.

Discussion

In this case–control study, we investigated the correlation between MCP-1 polymorphisms and susceptibility to ovarian cancer, and found for the first time that rs1024611 and rs3760396 polymorphisms were risk factors for this disease. In addition, our data revealed that MCP-1 rs3760396 SNP was specifically correlated with serous papillary type of ovarian cancer, whereas rs1024611 SNP could increase serum levels of MCP-1. These results indicate that the MCP-1 may play critical roles in the development of ovarian cancer.

MCP-1 and its receptor, CCR2, plays critical roles in modulating inflammatory responses, tumor proliferation, angiogenesis, and metastasis. The rs1024611 polymorphism has been reported to be involved in various diseases. For instance, it has been shown that G allele of the MCP-1 rs1024611 SNP is a risk factor for ischemic stroke (Arakelyan et al., 2014). Liu et al. (2013) have presented that renal cell carcinoma patients with MCP-1 rs1024611GG genotype revealed significantly shorter survival time compared to cases with MCP-1 rs1024611AA and AG genotypes. Bai et al. (2012) have shown that individuals who had the GG genotype had a 1.59-fold increased risk of chronic obstructive pulmonary disease (p=0.036). Our data demonstrated that MCP-1 rs1024611GG genotype and G allele were associated with increased susceptibility to ovarian cancer (Table 2).

The rs1024611 polymorphism is located at 2518-bp upstream of MCP-1 translation start site. The −2518 G variant in the regulatory region of MCP-1 gene is known to increase the expression of MCP-1. Several MCP-1 promoter reporter assays have shown that the construct with the G allele has a higher activity than its counterpart A allele. The polymorphism can influence the transcriptional activity since cells with GG genotype produce more MCP-1 protein than cells with AA genotype. Moreover, the serum MCP-1 concentration is higher in G-allele carriers than in AA homozygote (Palmieri et al., 2010; Pham et al., 2012). In addition, immunohistochemistry has revealed that the MCP-1 expression level is higher in nasopharyngeal carcinoma tumor cells from GG carriers compared with those from AA carriers (Tse et al., 2007). However, some studies have reported that the rs1024611 SNP cannot affect serum levels of MCP-1 (Grzegorzewska et al., 2014). Our data for the first time identified that rs1024611GG genotype could upregulate serum levels of MCP-1 in the Chinese population (Fig. 1). Our results also observed increased levels of MCP-1 in ovarian cancer. Therefore, it is possible that the rs1024611 SNP affects susceptibility to ovarian cancer by elevating gene expression of MCP-1. The prevalence of rs1024611G/A SNP is quite different in different populations. Rs1024611A allele was the minor allele in our population, which is consistent with other studies about the polymorphism in the Han Chinese population (Bai et al., 2012). However, the rs1024611A allele is the major allele in many other ethnic groups such African and European.

The rs3760396 SNP can be detected in the Chinese population. Study has shown that variant genotypes of rs3760396 are associated with a significantly decreased risk of death for nonsmall-cell lung cancer (Ma et al., 2011). In this study, we found that the percentage of GG genotype was significantly increased in ovarian patients. The CG genotype also showed a nonsignificant increase in patients than in controls (OR=1.52, 95% CI: 0.99–2.34, p=0.054). These data suggest that rs3760396 SNP may have biological functions. However, we did not identify any correlation between the polymorphism and serum level of MCP-1, suggesting that rs1024611 and rs3760396 may have different mechanisms. Rs3760396, but not rs1024611, specifically associated with the serous papillary type also implied this point. Further research about the function of rs3760396 is required.

In summary, our study demonstrated that MCP-1 rs1024611 and rs3760396 polymorphisms were associated with increased susceptibility to ovarian cancer in the Chinese population. We also identified that MCP-1 rs3760396 SNP was specifically correlated with the serous papillary type of ovarian cancer, whereas rs1024611 SNP could increase serum levels of MCP-1. Our results provided important insights for understanding the genetics of ovarian cancer and would be helpful for the development of MCP-1 as a possible therapeutic approach to this disease. Since MCP-1 can be detected in different cell types, it would be important and very interesting to study the effect of these SNPs on the expression of MCP-1 in ovarian tissues. Further study on this field is needed.

Footnotes

Disclosure Statement

No competing financial interests exist.

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