Association of CCR2 ( + 190 G/A) Gene Variants and Ovarian Cancer Severity

Abstract

Aim:

Chemokines and their receptors play an important role in tumor progression. In the current study, we aimed to determine the association between the CCR2 gene (+190 G/A) polymorphism and ovarian cancer severity.

Methods:

CCR2 (+190 G/A) genotyping was performed using real-time polymerase chain reaction for DNA isolated from blood samples from a cohort of patients with ovarian cancer (n = 44) and a control group (n = 45).

Results:

The CCR2 (+190 G/A) GG genotype frequencies for patients were significantly higher in the stage III-IV cancer group (p = 0.036), and A allele carriers were significantly higher in the stage I-II ovarian cancer group.

Conclusion:

The CCR2 (+190 G/A) GG genotype may be a potential risk factor for the severe forms of ovarian cancer and the A allele may be a risk-reducing factor for severe ovarian cancer.

Introduction

Different tumor types express various chemokines and their corresponding receptors. Chemokines and their receptors have been demonstrated to play important roles in malignant tumor progression. In addition, they have a crucial role in regulating interactions between tumor and stroma (Kulbe et al., 2004; Jöhrer et al., 2008). Changes in chemokine receptor activity are involved in mechanisms that activate tumor-promoting signals. Thus, chemokines and their receptors can influence disease severity, survival, cell proliferation, and tumor growth, as well as cancer metastasis (Rossi and Zlotnik, 2000). Chemokine receptors interact with mitogen-activated protein kinases and complex mechanisms are involved in ligand-dependent activation of growth factors such as the GTP binding protein (Salazar et al., 2013).

Ovarian cancer is one of the most common tumors in women, and affects more than 190,000 women worldwide each year (Rosa et al., 2013). Expression levels of inflammatory chemokines are associated with ovarian cancer, and several chemokines have been documented to play a role in the progression and development of ovarian cancer (Sica et al., 2000).

The CCR2 + 190 G/A (rs1799864) single-nucleotide polymorphism (SNP), located in exon 1 of C-C chemokine receptor type 2 (CCR2) within the regulatory and coding regions, has been associated with the pathogenesis of particular diseases (Navratilova, 2006). The G to A transition causes a valine-to-isoleucine substitution at position 64 in the first transmembrane domain of the CCR2 protein that enhances protein stability and cell surface expression of CCR2A isoforms (Wong et al., 1997). Studies that determined the frequency of the CCR2 (+190 G/A) polymorphism indicated that CCR2 gene variants are associated with the risk of gynecologic cancers (Chatterjee et al., 2010).

In this study, we aimed to determine the association between CCR2 + 190 G/A gene polymorphism and susceptibility to ovarian cancer in a Turkish population.

Patients and Methods

Study group

The study group comprised 89 women who were admitted to the Gynecology Clinic of Yeditepe University Hospital, Department of Obstetrics and Gynecology. Individuals in the control group visited the hospital for gynecologic evaluation as part of routine checkups. The diagnosis of ovarian cancer was determined through histopathologic examination performed at the Department of Clinical Pathology, Yeditepe University. Ovarian cancer stage was assigned according to the International Federation of Gynecology and Obstetrics (FIGO) classification (revised, 2014). Local Ethics Committee approval was obtained for the study. The study protocol was consistent with the World Medical Association Declaration of Helsinki (Ethical Principles for Medical Research Involving Human Subjects).

DNA extraction

Blood samples from all participants were collected in tubes containing ethylenediaminetetraacetic acid. Genomic DNA was extracted from 350 μL peripheral whole blood using an Invitrogen iPrep PureLink gDNA blood isolation kit with an iPrep Purification Instrument (Invitrogen, Life Technologies, Carlsbad, CA). The isolation procedure took 45 min and was performed in a closed system. The procedure yielded 100 μL of DNA per sample. Sample DNA concentrations and optical density ratios (at 260/280 nm) were measured using a Nanodrop 2000 (Thermoscientific, Waltham, MA). Isolated DNA samples were preserved at 4°C until genotyping assessments were conducted.

Genotyping

Analysis of CCR2 (+190 G/A) genotype was performed in a LightCycler 4800 real-time polymerase chain reaction (Roche Diagnostics, Mannheim, Germany) with a CCR2 (rs1799864) lightsnip kit (Roche Diagnostics, TIB MOLBIOL GmbH, Berlin, Germany).

Statistical analyses

The distribution of CCR2 (+190 G/A) genotype and allele frequencies in the study groups were in agreement with the theoretically expected values derived according to the Hardy-Weinberg equilibrium. Statistical analyses were performed using SPSS version 23 (SPSS, Inc., Chicago, IL). Values are given as mean ± standard deviation (SD). Student's t-test was used to examine the significance of differences between the two groups and χ² and Fisher's exact tests were used to compare demographic information with expression. Multivariate logistic regression analysis was used to study the effect of each genotype on the risk of ovarian cancer. The relative risk at a 95% confidence interval (CI) was calculated as the odds ratio (OR). Individuals homozygous for the common genotype were used as a reference to test for any association of genotype with ovarian cancer by employing a logistic regression model to calculate the OR with 95% CI; p-values <0.05 denoted statistical significance.

Results

The study group consisted of patients with ovarian cancer (n = 44) and a control group (n = 45) for which demographic information was collected (Table 1). For the ovarian cancer and control group, the age and SD was 55.87 ± 11.72 and 51.84 ± 13.39 years, respectively (p = 0.108). Body mass index (BMI) and SD were 29.49 ± 5.76 and 22.52 ± 3.19 kg/m² for the ovarian cancer and control group, respectively, and BMI was significantly higher in patients with ovarian cancer (p > 0.001). Body surface area and SD was 1.78 ± 0.17 m² for patients with ovarian cancer and 1.64 ± 0.13 m² for control groups. Fasting blood glucose plus SD for the ovarian cancer group was 107.39 ± 34.09 mg/dL, which was notably higher compared with the control group (86.24 ± 7.12 mg/dL). For all these parameters, the p-values were statistically significant at p < 0.001. For smoking status, 6 and 23 individuals in the patient group and control group, respectively, were smokers. One cancer patient and 19 individuals in the control group drank alcohol. p-Values were calculated as <0.001 for both smoking and alcohol parameters. Regarding the revised FIGO pathologic classification, 43.2% (n = 19) of patients were stage I-II and 56.8% (n = 25) were stage III-IV (n = 44). Metastasis and relapse were observed in 75% (n = 33) and 50% (n = 22) of the stage I-II and III-IV patients, respectively (Table 1).

Table 1.

Demographic Characteristics of the Study Population

Parameter	Ovarian cancer (n = 44)	Control (n = 45)	p
Age (years), mean ± SD	55.87 ± 11.72	51.84 ± 13.39	0.108
Body surface area (m²), mean ± SD	1.78 ± 0.17	1.64 ± 0.13	<0.001^a
Body mass index (kg/m), mean ± SD	29.49 ± 5.76	22.52 ± 3.19	<0.001^a
Fasting blood glucose (kg/m), mean ± SD	107.39 ± 34.09	86.24 ± 7.12	<0.001^a
Smoker
Yes	13.6%	51.1%	<0.001^a
No	86.4%	48.9%
Alcohol use
Yes	2.27%	42.2%	<0.001^a
No	97.73%	57.8%
Diabetes
Yes	24.5%	9.6%	0.043^a
No	75.5%	90.4%
Pathological FIGO stage
I-II	43.2%	—	—
III-IV	56.8%	—	—
Metastasis
Yes	75%	—	—
No	25%	—	—
Relapse
Yes	50%	—	—
No	50%	—	—

Statistically significant difference.

FIGO, International Federation of Gynecology and Obstetrics; n, number of individuals; SD, standard deviation.

Genotype frequency distributions of CCR2 were in agreement with the Hardy-Weinberg equilibrium. There were no statistically significant differences between the groups regarding frequency of either CCR2 (+190 G/A) genotype (χ² = 2.347; p = 0.309) or alleles (p > 0.05) (Table 2). CCR2 (+190 G/A) GG, AG, and AA genotype frequencies of individuals who had stage I-II were 26.3%, 68.4%, and 5.3%, respectively, and patients who had stage III-IV were 58.3%, 33.3%, and 8.3%, respectively (Table 3). We found that CCR2 (+190 G/A) GG genotype frequencies for patients and controls were significantly higher in the stage III-IV group relative to the other groups (χ² = 4.408; p = 0.036, OR = 3.920, 95% CI = [1.064-14.446]). Furthermore, CCR2 (+190 G/A) AG genotype frequencies were significantly higher in patients who had stage I-II ovarian cancer (χ² = 5.225; p = 0.022, OR = 0.231, 95% CI = [1.064-14.446]). In addition, the number of A allele carriers was significantly higher in the stage I-II ovarian cancer group than among patients who had stage III-IV cancer (χ² = 4.408, p = 0.036; OR = 0.255, 95% CI = [0.069-0.940]).

Table 2.

CCR2 Genotypic and Allelic Frequencies in Patients with Ovarian Cancer and the Control Group

	Ovarian cancer (n = 44)	Control (n = 45)	p	Chi square	Odds ratio (OR)	95% confidence interval (CI)
Genotype	n (%)	n (%)	0.309	2.347
GG	19 (44.2)	27 (60)	0.138	2.204	0.528	0.226-1.231
AG	21 (48.8)	15 (33.3)	0.139	2.186	1.909	0.807-4.517
AA	3 (7)	3 (6.7)	0.954	0.003	1.050	0.200-5.510
Allele	Allelic count (%)	Allelic count (%)
G	59 (46.1)	69 (53.9)	0.954	0.003	0.952	0.181-4.998
A	27 (56.2)	21 (43.8)	0.138	2.204	1.895	0.812-4.421

n, number of individuals.

Table 3.

CCR2 Genotypic and Allelic Frequencies in Patients with Ovarian Cancer and International Federation of Gynecology and Obstetrics Stage

	Stage I-II	Stage III-IV	p	Chi square	Odds ratio (OR)	95% confidence interval (CI)
Genotype	n (%)	n (%)	0.071	5.277
GG	5 (26.3)	14 (58.3)	0.036^a	4.408	3.920	1.064-14.446
AG	13 (68.4)	8 (33.3)	0.022^a	5.225	0.231	0.064-0.836
AA	1 (5.3)	2 (8.3)	0.695	0.154	1.636	0.137-19.540
Allele	Allele count (%)	Allele count (%)
G	23 (39)	36 (61)	0.695	0.154	0.611	0.051-7.297
A	15 (55.6)	12 (44.4)	0.036^a	4.408	0.255	0.069-0.940

Statistically significant difference.

n, number of individuals.

Discussion and Conclusion

The CCR2 gene is located on chromosome 3 (3p21), which is part of the chemokine receptor gene region (Daugherty and Springer, 1997). The CCR2 gene encodes two isoforms as CCR2A and CCR2B. The CCR2 (+190 G/A) polymorphism is located in exon 1 and this SNP results in an isoleucine substitution for valine in the transmembrane domain of the protein (Wong et al., 1997), although the physiological effect of this mutation is unclear. There are conflicting reports on the effects of CCR2 (+190 G/A) polymorphism on various cancers. The CCR2 (+190 G/A) polymorphism appears to enhance the stability of CCR2A, but does not affect CCR2B stability (Nakayama et al., 2004).

Several studies have examined the association between CCR2 (+190 G/A) polymorphism and cancer risk in different populations. Nakayama et al. (2004) reported a protective role for CCR2 (+190 G/A) GG and AG genotypes in AIDS, which, similar to some cancers, has inflammatory effects. Their data indicated that the molecular mechanism of CCR2 (+190 G/A) polymorphism is diverse among different phenotypes in humans (Nakayama et al., 2004). The relationship between CCR2 (+190 G/A) polymorphism and cancer was reported in a meta-analysis by Huang et al. (2013) who found that the CCR2 (+190 G/A) polymorphism was significantly associated with risk of various cancers.

Huang et al. asserted that AG and AA genotypes might be risk factors for several different cancers, which is in contrast to our results showing that the incidence of the A allele was significantly higher in the stage I-II ovarian cancer group than in the stage III-IV group (Table 3). Meanwhile, Zafiropoulos et al. (2004) determined that a mutated allele (A allele) (OR = 0.53, 95% CI = [0.33-0.84]) and a heterozygote GA genotype (OR = 0.54, 95% CI = [0.33-0.89]) were significantly more frequent in the control group than in a group of breast cancer patients, and suggested that the CCR2 (+190 G/A) A allele was a risk-reducing factor in breast cancer, but a risk factor in skin and bladder cancer.

The association of chemokines and their receptors with tumor malignancy has been proposed as a new therapeutic target for ovarian cancer treatment. Indeed, chemokine secretion by tumor cells could affect tumor growth, whereas chemokine-receptor interactions may play a crucial role in tumor progression (Muralidhar and Barbolina, 2013). Ivansson et al. (2007) investigated the relationship between CCR2 (+190 G/A) and cervical cancer susceptibility and found that genotype and allele frequencies were significantly different between the patient and control group. Based on these results, they proposed CCR2 as a candidate genetic factor in immunologic pathways associated with gynecologic cancers (Ivansson et al., 2007).

Although this study has some limitations, including a small sample size, the analysis of results indicate that the CCR2 (+190 G/A) GG genotype could be a potential risk factor. Furthermore, the presence of the CCR2 (+190 G/A) A allele might have a risk-reducing effect on ovarian cancer severity. Consequently, more studies are needed to gain further insight into the relationship between CCR2 (+190 G/A) polymorphism and ovarian cancer susceptibility.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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