Background
Breast cancer is one of the most common malignant tumors in women and the second major cause of cancer-related death in women [
1]. The estrogen receptor (ER) is a member of the nuclear hormone receptor family, which plays an important role in cell proliferation, differentiation, and tumor formation. Approximately 60%–70% of breast tumors are ER-positive at diagnosis, and anti-estrogen therapies, such as tamoxifen, are very important in premenopausal women breast cancer management [
2]. However, nearly 50% of breast cancer patients develop resistance to endocrine therapy, leading to tumor progression and reduced patient survival [
2]. Therefore, research on the mechanism of tamoxifen resistance is important to improve the prognosis of breast cancer patients.
Human epidermal growth factor receptor 2 (HER2)/neu overexpression or amplification is found in approximately 15%–30% of breast cancers, and increased expression of this receptor correlates with poor clinical outcome and resistance to endocrine therapy [
2,
3]. The crosstalk between ER and HER2 pathways plays an important role in both intrinsic and acquired resistance to endocrine therapy [
4], and thus current research in drug resistance has mainly focused on these pathways.
Evidence indicates that the non-genomic actions of estrogen are mediated by membrane-associated ERα, which resides in or near the cell membrane and interacts with several growth factor signaling pathways [
5]. A previous study showed that extranuclear ER colocalizes with the HER2 receptor in membrane signaling domains that modulate downstream nuclear events leading to the growth of breast cancer cells [
3].
Lipid rafts provide a functional platform for the interaction of protein molecules through dynamic aggregation [
6]. Lipid rafts are essential for the plasma membrane localization of ER and play a critical role in its membrane-initiated effects [
7,
8]. In HER2-overexpressing breast cancer cells, HER2 receptors are present on the cell surface as monomers, homodimers, and heterodimers [
9]. Signal activation and transduction require the localization of HER2 to lipid rafts, and lipid raft aggregation is necessary for HER2 activation and function. Combination of lipid rafts inhibition, tamoxifen was more effective in inhibiting the proliferation of melanoma cells [
10]. Cholesterol-rich lipid rafts were highly amplified in TAM resistant cell lines, disrupted lipid rafts acted cooperatively with TAM to reduce prosurvival mediators [
11]. However, whether HER2 interacts with ER in lipid rafts and whether this interaction is involved in tamoxifen resistance remains unclear.
Research has shown that the molecular mechanisms of estrogen involve the ability of the 17beta-estradiol (E2)-ER complex to induce gene transcription through specific coregulators (i.e. coactivators or corepressors) [
12,
13] and to evoke membrane-initiated activation of specific rapid phosphorylation cascades, such as Src/ERK/MAPK [
13,
14]. A previous study showed that E2-induced Src activation requires the formation of a protein complex that contains at least ERα and c-Src [
15]. In previous reports, membrane-associated ERα was shown to interact with HER2 in the presence of E2 [
16], and HER2 interacts with the non-receptor tyrosine kinase c-Src [
17]. No studies have examined the complex formation between HER2, ERα and c-Src.
Previous studies show that the ubiquitin ligases c-Cbl and Cbl-b are important regulators of lipid rafts [
18]. Cbl-b inhibits the aggregation of T cell receptors by preventing lipid raft aggregation, and we previously showed that Cbl-b inhibits aggregation of lipid rafts [
18]. Recent work from our group also showed that c-Cbl and Cbl-b overexpression inhibits lipid raft aggregation in gastric cancer cells [
19]
.
In this study, we hypothesized that the ER-c-Src-HER2 complex formation is involved in HER2-mediated tamoxifen resistance and that lipid rafts are important sites for the formation of these complexes. We also investigated the potential role of c-Cbl in modulating the ER-c-Src-HER2 complex formation and function through its established effects on lipid raft formation.
Methods
Reagents and antibodies
Anti-HER2 was purchased from NeoMarker (Waltham, MA, USA), anti-ER, anti-PR, anti-c-Src, anti-p-HER2 (Tyr1248), anti-p-c-Src (Tyr416), and anti-p-ER (Ser118) were purchased from CST (Danvers, MA, USA). Anti-actin, anti-c-Cbl, anti-Caveolin1 and anti-HER2 labeled with phycoerythrin were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Tamoxifen, 17-β estradiol, Nystatin, PP2were purchased from Sigma (St. Louis, MO, USA). The c-Cbl-3 × flag-CMV9 plasmid was constructed by Taihe Biotechnology (Beijing, China). The HER2-pEGFP-N1 plasmid was constructed by GENEWIZ (Suzhou, China). The PSVL-cbl-70Z plasmid was kindly gift from Professor Kiyonao Sada (Kobe University Graduate School of Medicine). The lentiviral system was purchased from Genechem (Shanghai, China).
Cells and cell culture
The breast cancer BT474 and T47D cell lines were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). BT474 (catalogue number TCHu143) is an ER-positive and HER2-overexpressing cell line. BT474 cells were cultured in RPMI 1640 medium (Hyclone, GE, USA) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). T47D (catalogue number TCHu87) is an ER-positive and low HER2-expressing cell line. T47D cells were maintained in DMEM (Hyclon, GE, USA) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). Cells were cultured at 37 °C in 5% CO2. The cells were routinely subcultured every 3–5 days, and cells used for experiments were from the logarithmic growth phase. All cell lines were mycoplasma negative by PCR reaction.
Cell viability assay
Cell proliferation was measured with the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cells (5 × 103 cells/well) were seeded in 96-well plates for 24 h and then treated with E2 (10 nmol/L) and/or tamoxifen (1 μmol/L). Absorbance was measured at 570 nM at 48 and 72 h. The data represents the mean ± SD of at least nine wells from three independent experiments.
Cells were plated in 6-well plates (800 cells/well), and after 24 h the cells were treated with E2 (10 nmol/L) and/or tamoxifen (1 μmol/L). Treated cells were then cultured for another 14 days. After removing the medium from the wells, colonies were stained with Giemsa and images were captured.
Western blot analysis
Western blot analysis was performed as described in our previous studies [
20]. Cells were solubilized in 1% Triton lysis buffer and quantified with the Kaumas blue method. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. The PVDF membranes were blocked by 5% skim milk powder in TBST buffer for 2 h at room temperature. The membranes were then incubated with primary antibodies overnight (HER2 1:1000, p-HER 1:500, ER 1:1000, p-ER 1:500, c-Src 1:1000, p-c-Src 1:250, c-Cbl 1:1000, Caveolin1 1:250, Actin 1:1000) at 4 °C, followed by incubation with secondary antibodies(CST, 1:2000) for 30 min at room temperature. The proteins were detected with enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, USA) and visualized with the Electrophoresis Gel Imaging Analysis System (Hercules, California, USA).
Immunoprecipitation
Immunoprecipitates was performed as described in our previous studies [
21]. Briefly, the collected cell lysates were incubated with anti-c-Src antibody or immunoglobulin-G (CST), and precleared protein G-agarose beads for 6 h at room temperature. Immunoprecipitates were washed four times in mild lysis buffer and then subjected to western blot analysis using anti-HER2(1:1000), anti-ER(1:1000), and anti-c-Src antibodies(1:1000).
Inhibition of lipid rafts
Lipid rafts are dynamic assemblies of proteins and lipids that harbour many receptors and regulatory molecules and so act as a platform for signal transduction, nystatin inhibits lipid rafts by affecting lipid metabolism [
22]. To inhibit the function of lipid rafts, cells were treated with nystatin (5 μg/mL) for 2 h before the experiments.
HER2 siRNA
Three siRNAs against HER2 and a scrambled control were purchased from VIEWSOLID BIOTECH (Beijing, China) The si-HER2 target sequences were as follows:5′-GUUGGAUGAUUGACUCUGATT-3′,5′-GGAGACCCGCUGAACAAUATT-3′, and 5′-GCUCAUC GCUCACAACCAATT-3′. SiRNA transfection was performed using Lipofectamin 2000 (Invitrogen, Carlsbad, CA, USA), according to the protocol previously [
21].
Cell transfection and lentiviral infection
Plasmid transfection was performed using Lipofectamine 2000, according to the protocol previously [
21]. For in vivo experiments, c-Cbl was overexpressed with a lentiviral system. Lentiviral production and infection were performed following the standard procedure recommended by the company (Shanghai Genechem Co., Ltd.). At 72 h, the virus-infected cells were used for experimental analysis.
In vivo xenograft animal model
Pathogen-free female BALB/c nude mice (4 weeks of age) were purchased from WeiTongLiHua (Beijing, China). The animals were housed in accordance with institutional ethical guidelines of animal care. Mice were housed in specific pathogen-free conditions, three per cage, and maintained at constant temperature (22 °C) and humidity. At 5 weeks of age, the mice were randomized into two groups of six mice each. To establish xenograft tumors, a breast cancer cell suspension (1 × 10
7 cells in 0.2 mL of PBS was injected subcutaneously in the right flank of each nude mouse. One group was subcutaneously inoculated with BT474 cells infected with lentiviral vector control and the second group was inoculated with BT474 cells infected with c-Cbl lentivirus. Tumor growth was measured with fine calipers twice each week and the tumor volume was calculated by the formula shown below:
$$ V=\left(L\times {W}^2\right)/ 2 $$
We observed 100% tumor incidence for both groups. When the volume of xenografts reached 50–100 mm3, each group was divided into two treatment subgroups: vehicle control or tamoxifen treatment (three mice per group). The control groups received 200 μL saline daily from days 1–7. The tamoxifen gavage group received tamoxifen (20 mg/kg) daily from days 1–7. Tumor volume and body weight were measured twice per week. Treatments continued for 7 days, and then mice were observed for 1 week. At the end of the experiment, all mice were humanely euthanized and necropsied. Tumor tissues were harvested, rinsed in saline, weighed, and immediately formalin-fixed.
Immunofluorescence
Tumor samples were formalin-fixed, paraffin-embedded and then cut into 4 μm sections. All sections were de-paraffinized in xylene and hydrated through a graduated alcohol series and then immunofluorescence was performed based on standard procedures [
21]. The sections were permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin for 1 h and then incubated with phycoerythrin-labeled anti-HER2 (Sc-33684PE), anti-c-Src (SC8056), and anti-ER (SC543) antibodies (all purchased from Santa Cruz) overnight at 4 °C. The next day, Alexa Fluor 405-conjugated goat anti-mouse IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) antibodies were added in blocking solution for 1 h at room temperature in the dark. DAPI (4′6′-diamidino-2 phenylindole) was used to stain nuclei for 1 min. After mounting with the Slow Fade Antifade Kit (Molecular Probes, Eugene, OR, USA), the sections were visualized by fluorescence microscopy (BX61, Olympus, Japan).
Statistical analysis
All experiments were performed at least in triplicate and repeated in three independent studies. The data are expressed as the mean ± SD. Differences between groups were compared using the Student’s t-test. SPSS Statistics 17.0.1 (SPSS, Chicago, IL, USA) was used for statistical analysis. P < 0.05 was considered significant.
Discussion
Innate and acquired tamoxifen resistance is an important problem in ER+ breast cancer. HER2 is overexpressed in approximately 15%–30% of human breast cancers and plays a role in tamoxifen resistance [
24]. In the previous study, the mechanisms of tamoxifen resistance have been explord, the reports included that pharmacologic mechanisms, loss or modification in estrogen receptor expression, alterations in co-regulatory proteins, autophagy, microRNA, and tumor microenvironment [
25‐
27]. However, the underlying mechanism remains unclear.
BT474 is an ER- positive invasive human breast ductal carcinoma cell line with very high HER2 expression, which is very sensitive to Herceptin but resistant to tamoxifen [
28]. We confirmed HER2-overexpressing breast cancer BT474 cells are relatively resistant to tamoxifen. The ER-c-Src-HER2 complex was detected in BT474 cells; this complex participated in tamoxifen resistance, inhibiting the formation of this complex reversed tamoxifen resistance. In Fig.
4, E2 along does not have much effect on cell proliferation after knockdown HER2, but this does not mean that the effect of tamoxifen is independent of ER in BT474 cells. Many previous studies have confirmed the reactivity of BT474 cells to estrogen [
29‐
31]. In our study, the MTT assays were conducted in BT474 cells transfected with siRNA targeting HER2 24 h, and treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for another 48 h. BT474 is a cell that doubles for a long time. It takes about 96 h to multiply. So in this experiment, the effect of estrogen on the proliferation of BT474 cells is not yet fully reflected.
Lipid rafts are required for HER2 activation and signal transduction [
32]. However, whether lipid rafts play a role in the formation of an ER-c-Src-HER2 complex has not been reported. In this study, we showed that lipid rafts are essential for the formation of the ER-c-Src-HER2 complex, as inhibition of lipid raft formation by nystatin reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. Our findings indicated that formation of the ER-c-Src-HER2 complex may play a key role in the crosstalk between ER and HER2 signaling pathways, and our results provide new insight into the mechanism of tamoxifen resistance.
Studies have shown that c-Cbl selectively regulates receptor translocation into lipid rafts [
33], and c-Cbl is involved in the regulation of lipid rafts in several cell types [
34,
35]. Overexpression of c-Cbl depleted Lck from lipid rafts in Jurkat cells [
36]. Our previous study showed that the Cbl ubiquitin ligase family inhibits the function of lipid rafts [
9,
16]. In vitro and in vivo experiments confirmed that overexpression of c-Cbl reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance in BT474 cells. We also found that the ubiquitin ligase activity of c-Cbl may play a key role in the reversal of tamoxifen resistance. One hand, c-Cbl may reduce the formation of ER-c-Src-HER2 complex through inhibition of lipid rafts aggregation; the other hand, c-Cbl may promote ER-c-Src-HER2 complex ubiquitination through its ubiquitin ligase activity. Therefore, c-Cbl may be used as a therapeutic target in breast cancer patients resistant to tamoxifen, and the regulation of c-Cbl expression should be study furthermore.
Acknowledgements
The authors thank Jia Liu (Animal Center of China Medical University) for kindly providing technical support. We thank Gabrielle White Wolf, PhD, from Edanz Group (
https://www.edanzediting.com) for editing a draft of this manuscript.