Estrogen Receptor Alpha Gene Expression in Breast Cancer Tissues from the Iranian Population-a Pilot Study

Previous studies suggest that during breast cancer development alterations occur in estrogen signaling pathways, mainly estrogen receptor α (ER-α) (MedinaJaime et al., 2014). Role of estrogen receptors and its modulators in breast cancer have been widely studied in western population (Buzdar, 2013). Recently, there were Few studies have been reported recently on the genetic variance of ER-α in the Iranian population (Abbasi et al., 2009; 2012; Izadi et al., 2012). Abbasi et al. (2012) reported that single nuclear pleomorphism (SNP) s in estrogen receptor α and β have additive effects in increasing risk for developing breast cancer among Iranian breast cancer patients (Abbasi et al., 2012; Rahimzadeh et al., 2014). Also, another study reported the incidence of hypermethylation in the promoter promoter region of ER in Iranian population (Izadi et al., 2012). To the best of our knowledge, there was no study has been reported so far to determine the expression patterns of ERα mRNA or protein in breast cancer tissues due to the lack of access to human tissue samples. In this study, we aimed to measure ERα gene expression in breast cancer tissues obtained from Iranian population at both mRNA and protein. Also, some clinopathological parameters from these patients


Introduction
Previous studies suggest that during breast cancer development alterations occur in estrogen signaling pathways, mainly estrogen receptor α (ER-α) (Medina-Jaime et al., 2014).Role of estrogen receptors and its modulators in breast cancer have been widely studied in western population (Buzdar, 2013).Recently, there were Few studies have been reported recently on the genetic variance of ER-α in the Iranian population (Abbasi et al., 2009;2012;Izadi et al., 2012).Abbasi et al. (2012) reported that single nuclear pleomorphism (SNP) s in estrogen receptor α and β have additive effects in increasing risk for developing breast cancer among Iranian breast cancer patients (Abbasi et al., 2012;Rahimzadeh et al., 2014).Also, another study reported the incidence of hypermethylation in the promoter promoter region of ER in Iranian population (Izadi et al., 2012).To the best of our knowledge, there was no study has been reported so far to determine the expression patterns of ERα mRNA or protein in breast cancer tissues due to the lack of access to human tissue samples.In this study, we aimed to measure ERα gene expression in breast cancer tissues obtained from Iranian population at both mRNA and protein.Also, some clinopathological parameters from these patients

Selection of patients
Formalin fixed paraffin tissues from 19 female patients diagnosed with breast carcinoma and 10 non-neoplastic (control) breast tissues were recruited from Moayyed laboratory, Mashhad, Iran.All breast cancers selected in this study were ductal adenocarcinomas and they were recruited retrospectively with no selection bias.Histopathological analysis was confirmed a hospital pathologist.

Total RNA extraction and quantitative RT-PCR
Total RNA was extracted using similar methods we published previously (Lam et al., 2011;Gopalan et al., 2014).Reverse transcription of the mRNA into cDNA was carried out using RevertAid™ H Minus Reverse Transcriptase kit (Fermentas, Burlington, USA).Details of the primers and amplicon size are illustrated in Table 1.A quantitative RT-PCR was used for the determination of ERα gene expression levels in the breast specimens on an ABI Prism 7300 Thermal Cycler (Applied Biosystems, Foster city, USA) using GAPDH as a ubiquitous control.

Immunohistochemical determination of ERα protein expression
Immunohistochemical (IHC) staining for ERα protein was performed by the Pathology Department, Moayyed lab following routine IHC procedures.Primary monoclonal ERα antibody (ER-6F11, Novocastra, Newcastle, UK) was used at 1:50 dilution.Counterstaining was performed using 3,3-Diaminobenzidine (DAB) and Mayer's hematoxylin.Cutoff for positivity was determined at % of tumor cells staining positively for ER (i.e<1% of cells in the tumor stained was considered negative for ERα).

PCR efficiency and data analysis
PCR efficiency and data analysis was performed using similar methods we published previously (Gopalan et al., 2010;Lam et al., 2011;Gopalan et al., 2014).Statistical analysis was performed using the Statistical Package for Social Sciences for Windows (version 20.0, SPSS Inc., Chicago, IL, USA).Significance level of the tests was taken at p<0.05.

High ERα mRNA expression in breast cancer tissues
The differences in ERα mRNA expression between the breast cancer and normal tissues were significant (Table 2).The mean inverse expression ratio between ERα and GAPDH (inverse) showed high ERα m RNA levels in the breast cancer tissues compared to the normal breast tissues (mean expression ratio, 0.612 versus 0.510, p=0.032) (Figure 1).In breast cancer tissues, 68% (n=13/19) had over expression of ERα mRNA, 21% showed reduced expression (n=4/19) and 11% (n=2/19) were within the normal range.

ERα protein expression in breast cancer tissues
The ERα protein expression was expressed in all      breast cancer tissues.ERα protein expression pattern in the remaining samples were noted as 0-30% stained cells in 11% (n=2) and 30-70% stained cells in 37% (n=5).

Correlation analysis of ERα mRNA and protein expression with clinicopathological parameters
All selected breast cancer tissues were clinically grouped as stage II tumours.ERα mRNA expression was noted to be high in breast cancers with bigger tumours compared to cancers with small tumours (89% over 50%, p=0.039).Also, no low expression of ERα mRNA was noted on cancers with high tumour sizes (Table 3).ERα protein expression was not correlated with any of these clinicopathological parameters.

Discussion
Detection and quantification of estrogen receptor is a useful tool in the diagnosis and prediction of hormone therapy response in breast cancer patients.(Clark et al., 1987;Nilsson et al., 2001;Hooshmand et al., 2014).Quantification of ERα mRNA and protein expression in breast cancer tissues using real time PCR assay and immunohistochemistry has been previous reported (Bieche et al., 2001;De Cremoux et al., 2002;Chuangsuwanich et al., 2014;Wang et al., 2014).In this study, we demonstrated altered ERα mRNA and protein expression for the first time in Iranian population.
Over expression of ERα plays a major role in breast cancer pathogenesis via promoting cell growth and proliferation.This study showed increased expression of ERα mRNA and protein in breast cancer tissues compared to normal breast tissues.Also, over expression of ERα was correlated with tumour size in breast carcinoma.These results support the previous findings that ERα over expression is a common event in breast cancer population (Holst et al., 2007).Also, this finding on Iranian population shows the significant use of this gene in the molecular diagnosis and screening for Iranian women.Furthermore, ERα expression changes can be useful in selecting patients for anti-estrogen therapy in Iranian population as similar to the breast cancer management plans in western countries.
In conclusion, we have identified changes in the expression of ERα in breast cancer and normal tissues from Iranian population.In breast adenocarcinomas, ERα over expression was often noted at both mRNA and protein level.The difference in expression of ERα between normal and cancerous tissue suggests that ERα expression may be a useful surrogate molecular marker in breast adenocarcinoma.Also, these results show that ERα gene can be used as biomarker for screening and diagnosis of breast cancer patients in Iran.Further studies into this gene should be performed to identify its role in cancer development in different population.

Figure 1 .
Figure 1.ERα mRNA Expression in Normal and Cancer Breast Tissues.The mean expression ratio (inverse ratio of ERα/GAPDH) showed high expression of ERα m RNA in breast cancer tissues compared to control samples (p=0.032)

Figure 2 .
Figure 2. ERα Protein Expression in Breast Cancer Tissues (A-D).A strong brown staining on the nucleus of breast ductal epithelium indicates the high protein expression of ERα
selected tissues with breast carcinoma.The ERα protein staining was located in the nuclei of the tumour cells (Figure2).Similar to mRNA expression changes, ERα protein also showed higher expression in breast cancer tissues compared to control samples.High ERα protein staining (> 70% of cells showing protein staining) was noted in almost half (53%, n=10/19) of the of the selected DOI