Background
Breast cancer (BC) is the second leading cause of deaths in women worldwide. The occurrence rate of male BC is rare; it is the most predominant cancer in women in United States (US) [
1]. It has been estimated that 2,52,710 new cases and 40,610 deaths are expected in women during the year 2017 in U.S alone [
2]. BC has been recently classified based on molecular patterns of gene expression into different subtypes [
3]. Luminal subtype which is characterized by the presence of estrogen receptor (ER) comprises ~ 60–70% of BC and responds better to endocrine therapy i.e.; tamoxifen [
4]. However, due to lack of therapy ER negative BC demands to identify molecular targets that might have therapeutic importance.
ERs are a group of nuclear receptors regulated by steroid hormone estrogen (E2). ERs are of three types; ERα, ERβ and ERγ [
5]. In the presence of E2, ERs either as a homodimer or heterodimer bind to estrogen response elements (ERE) present in the target gene promoter to regulate its transcriptional activity [
6‐
9]. ERα and ERβ expresses widely in different tissues including brain [
10]. Although ERα causes cell migration, division, tumor growth in response to E2 [
11,
12], ERβ inhibits migration, proliferation and invasion of breast cancer cells [
13‐
15]. Besides being a key molecule in breast cancer pathogenesis, ERα plays an anti-inflammatory role in brain [
16].
Estrogen related receptors (ERRs) are a group of nuclear receptors family having sequence homology with ERs and act as transcriptional regulators [
17]. Unlike ERs, ERRs are lesser known affected by steroid hormone estrogen. Since a decade after discovery, no natural ligand has been found for these receptors, hence called as orphan nuclear receptors [
18,
19]. Estrogen related receptors (ERRs) share target genes with ERs [
20,
21]. Estrogen related receptors (ERRs) are also of 3 types; ERRα, ERRβ and ERRγ [
22‐
25]. ERRs recognize a short sequence referred as ERR- responsive element (ERRE) on target gene promoter and regulate their transcriptional activity [
26‐
29]. The distribution of ERRs varies, although ERRα expresses in various tissues such as kidney, skeletal muscle, intestinal tract etc, but ERRγ restrict themselves mainly in heart and kidney [
30,
31]. ERRα mediates cell proliferation through pS2 [
21] and plays an important role in regulation of mitochondrial metabolism in breast cancer cells [
29,
32]. Knockdown of ERRα leads to cardiac arrest in mice [
33]. ERRβ expresses in early stages of mouse embryonic development [
34]. Mutation in ERRβ leads to autosomal recessive non syndromic hearing impairment in mice [
35]. ERRβ acts as tumor suppressor in prostate cancer by up-regulating p21
cip [
36]. Recent studies have demonstrated the abrogated expression of ERRβ in breast cancer cells [
37]. In this study we have demonstrated that ERα regulates the expression of ERRβ through estrogen in breast cancer. We demonstrated the elevated levels of ERRβ in normal breast tissues and ER + ve breast tumors compared to breast carcinoma and ER-ve breast tumors respectively. We also demonstrated that ectopic expression of ERRβ causes significant up-regulation of p18 and p21
cip in breast cancer cells and also arrest cell cycle in G0/G1 phase. Thus our data, suggest the tumor suppressor role of ERRβ which provide therapeutic potential to ERRβ signaling pathway.
Discussion
ERα plays an important role in breast cancer progression, metastasis and treatment [
50,
51]. DNA binding domain of ERα is highly conserved with ERRs hence can share target genes [
21,
22]. ERRs involve in cell proliferation and energy metabolism [
21,
29]. Expression of ERRβ was found to be constant throughout the menstrual cycle [
52]. ERRβ can regulate Nanog expression through interacting with Oct4 [
53] and acts as tumor suppressor in prostate cancer cells [
36]. A limited literature has addressed the role of ERRβ in breast cancer. We therefore studied the possible role of ERRβ in breast cancer. We found the relative expression of ERRβ is high in immortalized normal breast cells (MCF10A), in contrast to breast cancer cell lines (MCF7, T47D, MDA-MB231) and these findings were in agreement with the previous studies [
37]. Immunohistochemical staining with ERRβ showed a significant increased expression of ERRβ in normal breast tissues compared to breast carcinoma tissues. Breast cancer patients having high expression of ERRβ showed better survival [
47]. Both Immunohistochemical and western blot studies revealed high expression of ERRβ in ER + ve breast cancers and it is dependent on Estrogen receptor status. Furthermore, reduced ERRβ expression was observed in ERα depleted MCF7 cells. These results indicate the possible role of ERα in the regulation of ERRβ in breast cancer. Estrogen is required for the development of breast and ovaries in mammals [
54], acts as a ligand for ERs [
55], promotes cell proliferation and migration [
56]. In our study we attributed the role of estrogen in the regulation of ERRβ in breast cancer cells. We confirmed that the expression of ERRβ is highly elevated in the presence of estrogen in ER + ve breast cancer cells (MCF7). However, in competition studies ERRβ expression was reduced with tamoxifen treatment along with estrogen.
Since ERs and ERRs show sequence similarity, there is a possibility of sharing of target genes and cross-talk between these receptors. In this study we detected two half ERE sites in the upstream region of
ERRβ and proved the binding of ERα on those ERE sites both in-vitro and in-vivo. ERα interacts with various proteins such as Sp1 and Ap1 which can facilitate the binding of ERα on half ERE sites [
57]. Sp1 stabilizes ERα dimer and co-operate the binding of ERα on half EREs present on its target gene promoter [
58,
59]. Whereas, HMG1 interacts with ERα and stabilizes ERα-ERE binding through which it enhances the transcription activity [
60]. Since previous studies have suggested that ERα is an interacting partner of ERRβ [
48], therefore we hypothesize that ERRβ might be playing an important role in the regulation of its own promoter by acting as facilitator of ERα to bind to the half ERE sites. ChIP assay and Re-ChIP provided enough evidenceses to confirm the self regulation of
ERRβ through ERα in the presence of estrogen. Furthermore, luciferase assay confirmed the regulation of
ERRβ by ERα. Surprisingly,
ERRβ alone has no effect on promoter activity. These findings demonstrate that ERα can regulate the transcriptional activity of
ERRβ.
In normal cells the cell division is tightly regulated and a fine balance amongst the cell cycle modulators does exist [
61]. The impairment of this fine balance is one of the major causes of cancer. p21
cip is an inhibitor of cyclin dependent kinase belongs to cip and kip family [
62], primarily inhibits CDK2 by which it can inhibit cell cycle progression [
63,
64]. p21
cip arrests G1-G2 transition in cell cycle through binding to PCNA in P53 deficient cells [
65]. p18 belongs to INK4 family and can inhibit cyclin dependent kinases potentially. Reduced levels of p18 were detected in hepatocellular carcinoma [
66]. In this study, we have established the correlation between the expression of ERRβ and various cell cycle markers such as p21
cip, p18 and cyclin D1 in breast cancer cells. The elevated levels of p21
cip, p18 and decreased expression of cyclin D1 in ectopically expressed ERRβ breast cancer cell lines were observed. Cell cycle analysis (FACS) provided enough evidence of cell cycle regulatory role of ERRβ in MCF7 cells. p21
cip protein levels were directly correlated with the expression of ERRβ in prostate cancer cells and it has been proved that p21
cip is a direct target for ERRβ [
36]. Interestingly p21
cip was demonstrated as a direct target for both ERRα and ERRγ and their protein levels were negatively correlated with each other [
67,
68]. Thus, not only for ERRβ, p21
cip is a direct target for all ERRs. Prostate and breast cancer cells showed inhibition of ERRα using XCT790 (inverse agonist) leads to reduction in cell proliferation [
67]. However, ERRβ and ERRγ were served as tumor suppressors in prostate cancer cells [
36,
68]. Recent studies also demonstrated the tumor suppressor role of ERRβ through BCAS2 in breast cancer cells [
37]. Our results were in agreement with the previous studies and this cell cycle regulatory and tumor suppressor roles of ERRβ in breast cancer cells suggest that ERRβ can be considered as a potential therapeutic target for the treatment of breast cancer.
One might surprise with the tumor suppressive role of an estrogen induced gene. It is well established that estrogen promotes cell proliferation in ER + ve breast cancer cells but also induces the expression of p53 and BRCA1. Interestingly, not only p53 but also BRCA1 gene is associated with inhibition of cell growth, DNA repair and apoptosis [
69‐
73]. P53 and BRCA1 both physically interact with ERα and inhibit ERα-mediated transactivation [
74,
75]. Recent studies also showed that estrogen up-regulate the expression of RERG a novel tumor suppressive gene which is highly expressed in ER + ve breast cancers [
76]. In our study we showed that ERRβ is an estrogen responsive gene and it exhibits tumor suppressor role in breast cancer cells. Recent studies showed that ERRβ interacts with ERα in the presence of estrogen and ERRβ decrease the intranuclear mobility through which it can inhibit the transcriptional activity of ERα [
48]. This phenomenon might be playing an important role in the inhibition of estrogen responsive target genes. Hormonal activation of tumor suppressive genes such as p53, BRCA1, RERG and ERRβ do play a vital role in the regulatory pathways that inhibit the estrogen induced cell growth and differentiation.
Acknowledgements
BMK, SC and SS thank the Department of Biotechnology, Government of India for research fellowships. DRM thank Indian Council of Medical Research, Government of India for research fellowship. SKN thank the Council of Scientific and Industrial Research, Government of India for research fellowship. We thank Dr. S. Senapati for extending his help regarding TMA related work. We thank Dr. Philippa Saunders, Director, MRC Centre for Reproductive Health, The Queen’s Medical Research Institute, Edinburgh, for providing ERRβ-YFP and ERα-CFP constructs. We would like to thank Mr. Ravi Chandra Tagirasa and Mr. Sashi bhusana sahoo for extending their help in performing FACS and technical support respectively.