Retinoic acid-related orphan receptor alpha ( RORA ) variants and risk of breast cancer

Abstract

BACKGROUND:

Breast cancer is the most common type of cancer and the second leading cause of cancer death in females. Despite numerous studies in this field, the etiology and clinical behavior of breast tumors have not been understood yet. Retinoid orphan nuclear receptor alpha (RORA) is a member of the orphan nuclear factor family involved in the regulation of lipid and steroid metabolism, immune response and circadian rhythms. Recent evidences support its role as a tumor suppressor gene.

OBJECTIVES:

To find the associations between RORA polymorphisms and breast cancer.

METHODS:

In the present study, we evaluated the association between two functional polymorphisms in RORA (rs11639084 and rs4774388) and breast cancer risk in a population of 122 Iranian breast cancer patients as well as 200 healthy subjects by means of tetra primer-amplification refractory mutation system-PCR (4P-ARMS-PCR) method.

RESULTS:

The rs4774388 has been shown to be associated with breast cancer risk in recessive inheritance model (OR (95% C I ) = 0.51 (0.26–0.97) and P = 0.041). However, the allele and genotype frequencies of rs11639084 were not different in patients and control (P > 0.05). Haplotype analysis revealed no significant association of any estimated block of rs11639084/rs4774388 in breast cancer patients versus healthy controls.

CONCLUSIONS:

The results of this study support a putative role for RORA in breast cancer pathogenesis.

Introduction

Breast cancer is the most common type of cancer and the second leading cause of cancer death in females [1]. Various studies have aimed at identification of susceptibility genes [2] as well as putative cancer biomarker [3–6] for this kind of human malignancy. Despite numerous studies in this field, the etiology and clinical behavior of breast tumors have not been understood yet. Regardless of identification of several environmental risk factors, linkage and familial association studies have led to the assumption that breast cancer is a complex polygenic disease in which genetic factors have crucial participation [7]. Considering the complex linkage disequilibrium patterns and different environmental exposures in diverse populations, identification of variants associated with breast cancer in each population would help in understanding the underlying causes of mammary carcinogenesis [7].

Retinoid orphan nuclear receptor (ROR) is a subfamily of the orphan nuclear factor family with significant homologies to the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) [8]. RORs are involved in the regulation of lipid and steroid metabolism, immune response and circadian rhythms [9]. ROR alpha (RORA) as the first recognized member of ROR subfamily has been shown to have a tumor suppressor function in various tumor types including breast cancer. RORA regulates a range of cellular activities, such as proliferation, invasion and cell polarization in cancer cells via activation of nuclear receptor pathways as well as alteration of post-translational modifications and interaction [8]. In addition, RORA gene is located in the middle of a common fragile site. In spite of its expression in normal breast, prostate and ovarian epithelium, it is commonly inactivated in cancers originated from these organs. It participates in the cellular response to hypoxia as well as a variety of different cellular stresses [10]. Its tumor suppressor effect in colon cancer has been revealed by the observation that phosphorylated RORA reduces transcriptional activity of β -catenin and inhibits anchorage-independent growth of colon cancer cells [11]. Besides, RORA attenuates breast tumor invasiveness by modulating cell microenvironment which has been shown in both 3D culture and in the xenograft model [12]. Two SNPs in RORA (rs1482057 and rs12914272) have been shown to be associated with breast cancer in a population of French women [13]. Consequently, in the current study, we evaluated the frequency of two functional polymorphisms in RORA (rs11639084 and rs4774388) in a population of Iranian breast cancer patients as well as healthy subjects.

Material and methods

Subjects

This case-control study has been approved by the local ethical committee. The study included 122 unrelated breast cancer patients diagnosed at the department of surgery, Hamadan University Hospital between 2014 and 2016. The breast cancer diagnosis has been confirmed by pathologic study. None of the patients received chemotherapy or radiotherapy before sampling. The control group comprised 200 normal females who were referred to a routine health survey during 2015. Informed consent was obtained from all participants.

Genotyping of rs11639084 and rs4774388

Peripheral blood samples were collected from the study participants in EDTA tubes and genomic DNA was extracted by using the salting out method. The tetra primer-amplification refractory mutation system-PCR (4P-ARMS-PCR) method was used for genotyping the rs11639084 and rs4774388 variants of the RORA gene as previously described [14]. In brief, the rs11639084 C allele produced a 162 bp band; the T allele produced a 215 bp band; and a common 321 bp band was amplified by outer primers. In the case of rs4774388, the C allele was recognized by a 197 bp band; the T allele was recognized a 273 bp band; and a common 412 bp band was amplified by outer primers. Ten percent of the samples were sequenced by using ABI 3730xl DNA analyzer (Macrogen, Korea) to confirm the results.

Statistical analysis

Alelle and genotype frequencies and the Hardy-Weinberg equilibrium were calculated by χ2 test using SNPStats [15]. The association of rs11639084 and rs4774388 polymorphisms with breast cancer risk was studied using additive, recessive, dominant, codominant and overdominant models. The strength of association between selected polymorphisms and susceptibility to breast cancer was evaluated by odds ratios (ORs) and 95% confidence intervals (CIs). Haplotype frequencies for RORA were calculated using SNPStats online programme (http://bioinfo.iconcologia.net/SNPstats) and Haploview 4.2 (http://www.broad.mit.edu/mpg/haploview/). The pairwise linkage disequilibrium (LD) between mentioned SNPs was calculated by describing D ^′ and r² values in Haploview software. D ^′ was defined as the ratio of the unstandardized coefficient to its maximal/minimal value. To decrease false positive results permutation tests were performed for multiple testing corrections of the haplotype analysis (n =10,000). P value of <0.05 was considered to be statistically significant.

Results

The random distribution of RORA alleles in both patients and control groups have been shown by their accordance with the assumption of Hardy-Weinberg equilibrium (P = 0.19 and 0.17 respectively). No significant difference has been shown in age between cases and controls (mean age of patients: 38.9 ± 2.1 and mean age of healthy controls: 39.1 ± 1.8). Other demographic data of patients and control group are summarized in Table 1. The genotyping results acquired by 4P-ARMS-PCR were in 100% accordance with sequencing results. The allele and genotype frequencies of the polymorphisms based on the statistical analyses are shown in Tables 2 and 3. The rs4774388 has been shown to be associated with breast cancer risk in recessive inheritance model (OR (95% C I = 0.51 (0.26–0.97)) and P = 0.041). However, the allele and genotype frequencies of rs11639084 were not different in patients and control (P > 0.05).

Linkage disequilibrium (LD) for the rs11639084 and rs4774388 in the RORA gene was calculated using Haploview version 4.2 software by describing D ^′ and r² value (D ^′ = 0.14, r² = 0.002). These two SNPs showed pair-wise linkage disequilibrium to each other. Haplotype analysis revealed no significant association of any estimated block of rs11639084/rs4774388 in breast cancer patients versus healthy controls. Four expected haplotype blocks originated from these two SNPs and their frequencies are provided in Table 4.

Discussion

Considering the high incidence and mortality rate for breast cancer, individual risk assessment based on genetic and environmental factors is an effective way of disease management. Consequently, identification of genetic variants contributing in breast cancer susceptibility is of practical significance. RORA is a putative candidate in this field regarding its role as a tumor suppressor gene and its location in a common fragile site. In addition, it is among genes involved in the regulation of the natural circadian rhythm whose disturbance due to exposure to light at night has been proposed to influence breast cancer risk [13]. In the present study we have detected an association between rs4774388 genotype and breast cancer risk in a population of Iranian breast cancer patients. A previous study has evaluated the association of two other SNPs in RORA (rs1482057 and rs12914272) with breast cancer in a population of French women [13]. The rs4774388 has been shown to be associated with bipolar disorder in the Han Chinese population [16]. In addition, both polymorphisms have been found to be associated with multiple sclerosis risk in Iranian population [14]. However, the present study is the first one to evaluate the associations of the rs4774388 and rs11639084 with the risk of breast cancer. Rs4774388 has been shown to change the affinity for binding of CEBPA1, NKX3.2, POU2F2 and TCF11 transcription factors that are involved in the regulation of oxidative stress response, differentiation of blood cells, and inflammatory response [14]. Notably, CEBPA has been regarded as a putative tumor suppressor in breast cancer [17] and has been shown to be down-regulated in breast cancer tissues [18]. Consequently, deregulation of its interaction with RORA is of clinical significance.

RORA participation in breast cancer pathogenesis has been shown in various studies. For instance, RORA has been demonstrated to increase the expression of aromatase in estrogen receptor (ER) α+ T47D and MCF7 breast cancer cells via its binding to the aromatase promoter [19]. In addition, it is among transcription factor partners participated in estrogen-mediated regulatory network in MCF7 cell line [20]. Further evidences support RORA participation in steroidogenesis [21]. Additionally, RORA expression has been shown to be decreased in breast cancer tumor cells compared to normal mammary tissue [10,22]. While overexpression of RORA in MCF12F breast cancer cells led to a decreased rate of cell proliferation [10], its knock down resulted in the disturbance of polarized acinar cells of mammary gland which is known to be an early event in breast cancer pathogenesis [12]. Furthermore, RORA participates in pathogenesis of breast cancer via suppression of Wnt/β catenin gene transcription as well as NF-κ β pathway [9]. Taken together, RORA is considered as a susceptibility locus for breast cancer. The non-coding polymorphisms in the RORA gene might change expression level or function of RORA and participate in breast cancer pathogenesis. Considering the role of RORA in steroidogenesis [21], it might be presented as a novel target for breast cancer treatment as well. However, future studies are needed for evaluation of the effect of functional RORA polymorphisms on the expression of this gene and their association with breast cancer risk or treatment response.