Background
As a member of the nuclear receptor family, the androgen receptor (AR) plays an important role in breast cancer, and has been identified as a biomarker for a specific molecular subtype of breast cancer. The AR gene expression profile can be used for further classifying receptor-negative tumors as molecular apocrine breast cancer (MABC) [
1]. MDA-MB-453 breast cancer cells have been classified as molecular apocrine by gene profiling studies [
2]. Our previous study has stated that patients with MABC develop distant metastases earlier and have poor prognosis [
3]. As MABC is characterized by increased androgen signaling, the malignant potential of MABC may partly be because of the AR. A study has shown that when the AR in MDA-MB-453 breast cancer cells is knocked down, the cell colony formation rate is significantly decreased, which verifies the fact that the AR regulates the biological behavior of MABC [
4]. Both ligand binding and translocation from the cytoplasm to the nucleus play important roles in the function of the AR [
5]. However, the specific mechanism of cytoplasmic and nuclear translocation of the AR has not been clarified in MABC.
In the absence of ligand, the AR remains in a non-active state, which forms a protein complex with heat shock proteins (HSPs) and other co-chaperones in prostate cancer, while the AR becomes active when ligand binds, and it translocates to the nucleus as a transcriptional regulator [
6]. Based on this result, HSPs play crucial roles in the process of AR activation. As a member of the molecular chaperones, HSP27 forms a chaperoning oligomer which can regulate multiple cellular survival and signaling pathways [
6]. However, whether HSP27 combines with the AR during its translocation from the cytoplasm to the nucleus in MABC cells remains unclear. Furthermore, in the process of AR translocation to the nucleus, the rapid phosphorylation of HSP27 is regulated by ligand binding [
6,
7]. There are three serine residues, serine 15, 78, and 82, reported as the sites of human HSP27 phosphorylation [
8,
9]. However, which one of these residues plays an indispensable role for AR cytoplasmic and nuclear translocation still remains unknown.
To detect the specific mechanism of AR cytoplasmic and nuclear translocation in MABC cells, androgen and siRNAs specific for HSP27 were used to analyze the location of the AR and HSP27 in vitro, and their effects on the tumorigenic capacity of MABC cells in vivo. The results of this study could determine the mechanism of the AR in regulating the malignant potential of MABC. Additionally, it aims to explore potential therapeutic targets for patients with MABC.
Methods
Cell lines and culture
Because the MDA-MB-453 cell line is classified as a model of MABC [
2], and MCF7 cells as nonMABC cell line [
10], we obtained them from American Type Culture Collection (ATCC, USA) for this study. The MDA-MB-453 cells were cultured in L15 medium (Gibco, USA), containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Life Technologies, USA). MCF7 cells were cultured in DMEM medium (Gibco, USA) which contained 10% FBS and 1% penicillin/streptomycin. Both cell lines were incubated at 37 °C in 5% CO
2.
Plasmids and transfection
The HSP27 siRNAs and control plasmids were constructed by Genechem (China). Three target sequences for the HSP27 siRNAs were studied, which included siRNA#4892-1: 5′-CTGTGAGGACTGTGGATAA-3′, siRNA#4893-1: 5′-CCCAGCAAATCCCTCTCTA-3′ and siRNA#4894-2: 5′-GGCAAGTTCCAGGCATTT-3′.
The deletion of HSP27 phosphorylation sites (Ser15, Ser78 and Ser82; CS-I0586-Lv201-01, CS-I0586-Lv201-02, and CS-I0586-Lv201-03) were carried out by GeneCopoeia (China, Additional file
1). The plasmids were amplified in
E. coli, and then extracted by using the Endotoxin-free Plasmid Size Kit (TIANGEN, China).
Cell transfections were performed as follows: firstly, cells were seeded in 6-well plates at a density of 1.0 × 104 cells per well overnight. Subsequently, 2 μg of the constructed plasmids were added to MEM (Gibco, USA), respectively, and incubated for 5 min at 37 °C. Further, the FuGENE Transfection Reagent (Promega, USA) was added and mixed. After incubating for 15 min, the complex solution was added to the cells, and replaced with complete medium 8 h later. The reactions were incubated for 48 h.
Quantitative real-time PCR (qPCR)
The RNAs used in this study were extracted using Trizol reagent (Takara, Japan). Reverse transcription was carried out using the SuperScript RT kit (Takara, Japan). qPCRs were performed according to the manufacturer’s protocol using the SYBR Green PCR kit (Toyobo, Japan). The transcript level of GAPDH was adopted as an internal control, and the primers used were as follows: GAPDH: 5′-GGAAGGTGAAGGTCGGAGTC-3′ and 5′-GTCTTCTGGGTGGCAGTGAT-3′; AR: 5′-GGAATTCCTGTGCATGAAA-3′ and 5′-CGAAGTTCATCAAAGAATT-3′; HSP27: 5′- GCGTGTCCCTGGATGTCAAC-3′ and 5′-TGTATTTCCGCGTGAAGCAC-3′. Each sample was assayed in triplicate.
Western blot analysis
Total proteins were extracted with RIPA buffer (Thermo Scientific, USA) and 1 mM PMSF. Cytoplasmic and nuclear subcellular fractionation was performed according to the manufacturer’s instructions using the Nuclear and Cytoplasmic Isolation Kit (KeyGEN, China). All proteins were separated on 10% SDS-PAGE (Invitrogen, USA) gels, transferred onto PVDF membranes (Millipore, USA), and then blocked using 5% skim milk. The proteins were detected by incubating the following primary antibodies: anti-AR (AR441; Abcam, USA), anti-estrogen receptor (ER; D8H8; CST, USA), anti-progesterone receptor (PR; 6A1; CST, USA), anti-HSP27 (G3.1, Abcam, USA), anti-HSP27 (phospho S15) (EP2293Y, Abcam, USA), anti-HSP27 (phospho S78) (Y175, Abcam, USA), anti-HSP27 (phospho S82) (EPR7278, Abcam, USA), anti-β-actin (8H10D10, CST, USA), and anti-Histone H3 (D18C8, CST, USA) overnight; and incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG, followed by detection using the ECL detection kit (Solarbio, China). Each sample was analyzed in triplicate.
Co-immunoprecipitation(Co-IP) and western blot
Co-IP of the AR and HSP27 was carried out according to the manufacturer’s protocol using the Pierce Co-IP kit (Thermo Scientific, USA). Briefly, AR or HSP27 antibody (10 μL) was first incubated with the AminoLink Plus coupling resin. The antibody-coupled resin was incubated with protein lysates overnight. Subsequently, the resin was washed, followed by elution of the protein complexes, which bound to the AR or HSP27 antibody. Subsequently, the samples were detected by western blot using the HSP27 or AR antibody as described previously. Each sample was assayed in triplicate.
Cell counter kit-8 (CCK8) cell proliferation and clonogenic assays
Cells (DHT treatment or HSP27 knock-down) were suspended and seeded in 96-well plates at 5000 cells per well and incubated for 24 h. Further, 10 μL of CCK8 (KeyGEN, China) solution was added into each well on day 1, 2, 3, 4 and 5. After 1–4 h, the absorbance of each well was measured at 450 nm using a microplate reader. Clonogenic assays were carried out using 6-well plates with 1000 cells per well. After 15 days, cells were collected and stained with crystal violet, and then the number of cell colonies was counted. Each sample was assayed in triplicate.
Immunofluorescence (IF) assay
Cells were seeded in 24-well plates. After 24 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with bovine serum albumin, incubated with the primary antibodies and fluorescein-labeled secondary antibody, and then DAPI (Thermo Scientific, USA) was used to stain the nucleus. Images were visualized and analyzed using a fluorescence microscope. Each sample was analyzed in triplicate.
In vivo experiments
Eighteen female BALB/c-nude mice (4–6 weeks old, 18–20 g) were purchased and randomly divided into three groups: MDA-MB-453 cells with DHT treatment,
HSP27 knock-down, and the control group. Cells (2 × 10
6) were inoculated subcutaneously in the groin. Care and procedures of the mice were provided by the Institution of Animal Use and Care Committee of Tianjin Medical University Cancer Institute and Hospital. All mice were sacrificed until 55 days. Tumor volumes were calculated as previously reported [
11].
All the tumors, livers, and lungs were paraffin-embedded and stained with hematoxylin-eosin (HE). HSP27 and Ki67 expression of mouse tumors were analyzed by immunohistochemistry as previously reported [
3]. In order to detect apoptotic cells, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out according to the manufacturer’s instructions, and the kit was obtained from KeyGEN (China).
Statistical analysis
Statistical analyses were performed using SPSS 19.0 software. The data were recorded as means ± standard deviation from at least three independent experiments, and analyzed by one-way ANOVAs and T-tests. P < 0.05 was considered as statistically significant.
Discussion
Breast cancer is a heterogeneous tumor that can be reflected in different aspects, such as the classical histopathological features and the more modern molecular classification. MABC is defined as ER-negative, PR-negative, and AR-positive [
12]; furthermore, with a poor prognosis by a series of clinical samples [
3]. In this study, we further explored the mechanisms by which the AR regulates the malignancy of MABC
. The results showed that HSP27 phosphorylation induced by androgen played a vital role in the process of AR translocation from the cytoplasm to the nucleus, which could affect the proliferation of tumor cells and tumorigenic capacity.
As a model of MABC, the proliferation of MDA-MB-453 cells is regulated by androgen in an AR-dependent manner [
13‐
15]. In MABC cells, HSP27 was highly expressed and played a pivotal role in cell proliferation. In accordance with previous studies [
2,
14,
16], we found that androgen could stimulate the proliferation of MDA-MB-453 cells. In contrast to the promotion effect on MABC cells, androgen has a predominantly inhibitory proliferative effect on MCF7 cells [
17], a model of luminal breast cancer [
10]. However, the effects of androgen on MDA-MB-453 cells could be partly rescued by down-regulation of HSP27, suggesting that knocking-down
HSP27 could reduce the androgen promotion effect on MDA-MB-453 cells. Additionally, HSP27 can decrease the toxic effects of oxidized proteins and reduce reactive oxygen species, which results in inhibiting cancer cell apoptosis [
18‐
20]. These results suggest that androgen and HSP27 may interact with each other and co-regulate the proliferative ability of MABC cells.
HSP27 is a member of the HSPs family and has been stated to regulate the functions of the AR, such as AR cytoplasmic/nuclear translocation and AR transactivation [
6,
21]. Studies confirm that gain and loss of function of HSP27 are strongly related to the expression of the AR in myogenic cells and prostate cancer cells [
22,
23]. We also found that
HSP27 knock-down significantly decreased the level of AR protein, but no difference could be found in the level of AR mRNA, which suggests that HSP27 might only regulate the protein translation of AR in MABC cells. However, Stope et al. [
23] stated that the decrease in the AR protein level is paralleled with a down-regulation in AR mRNA levels. This difference might be owing to the different cancer cells used for the research; however, the related mechanism should be further analyzed.
In malignant tumor cells, the expression of HSP27 is obviously high [
24,
25]. The main mechanism by which the AR and HSP27 regulate the malignant behavior of MABC may rely on the the androgen-triggered phosphorylation of HSP27 that accompanies the AR tanslocation from the cytoplasm to the nucleus where the AR interacts with androgen response elements to promote its genomic activity. HSP27 is directly phosphorylated at three serine residues via the p38 mitogen-activated protein kinase (MAPK) pathway, which affects its subcellular distribution [
26‐
28]. The phosphorylation sites at serine 78 and 82 are identified as the predominant residues of HSP27, and serine 78 can be substituted by asparagine in some animals, but serine 82 is conserved throughout the animals [
9]. In MABC cells, deleting residue serine 82 of the HSP27 phosphorylation sites induced a significant decrease in the expression levels of AR in the nucleus compared to the control group, which indicated that HSP27 phosphorylation at serine 82 might be the critical site for AR cytoplasmic and nuclear translocation.
Several studies have confirmed that the positive expression of AR is significantly associated with poor survival, and increased mortality in AR-positive triple negative breast cancer (TNBC) [
3,
29‐
31]. HSP27 is reported to be associated with a decreased survival in breast cancer [
32]. In accordance with clinical research, we found that DHT treatment increased xenograft tumor volume and distant metastasis, while HSP27 knock-down inhibited tumor growth greatly, which indicated that the AR and HSP27 might interact with each other and co-influence the development of MABC. Studies have stated that AR antagonists are able to induce breast cancer cell apoptosis and decrease tumor proliferation significantly in TNBC cell lines [
33,
34]. In addition, the HSP second generation antisense oligonucleotide targeting HSP27 can increase drug efficacy in pancreatic and prostate cancer xenograft models [
35‐
37]. Based on these results, although no valid endocrine therapy is suggested for MABC, the combination of AR antagonists and HSP27 inhibitors could be a potential therapeutic regimen. Of course, more xenograft models and clinical trials should be carried out to confirm this hypothesis.
Conclusions
In conclusion, this study confirmed that activation of AR and HSP27 by androgen could increase the proliferative ability of MABC cells and the growth of xenograft tumors. HSP27 phosphorylation on residue serine 82, induced by androgen, played a critical role in the process of AR translocation from the cytoplasm to the nucleus. The AR and HSP27 could form a protein complex, which was the main factor in AR regulating the malignancy behavior of tumor cells, and could present a new therapeutic regimen in clinical therapies of MABC.