Contributed equally
Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Our previous studies find that GPER and its down-stream signaling play a pivotal role in the development of tamoxifen (TAM) resistance. cDNA array analysis indicated a set of genes associated with cell apoptosis are aberrant in GPER activated and TAM-resistant MCF-7R cells compared with TAM-sensitive MCF-7 cells. Among these genes, Bim (also named BCL2-L11), a member of the BH3-only pro-apoptotic protein family is significantly decreased, and TRIM RING finger protein TRIM2 (a ubiquitin ligase) is highly expressed in MCF-7R. To understand the mechanism of TAM-resistance in GPER activated ER+ breast cancer, the function of TRIM2 and Bim inducing cell apoptosis was studied. By using immunohistochemical and western blot analysis, there is an adverse correlation between TRIM2 and Bim in TAM-resistant breast tumor tissues and MCF-7R cells. Knockdown Bim in TAM-sensitive MCF-7 cells or overexpression of Bim in TAM-resistant MCF-7 cells significantly changed its sensibility to TAM through altering the levels of cleaved PARP and caspase-3. Activation of GPER and its downstream signaling MAPK/ERK, not PI3K/AKT, led to enhanced TRIM2 protein levels and affected the binding between TRIM2 and Bim which resulted in a reduced Bim in TAM-resistant breast cancer cells. Thus, the present study provides a novel insight to TAM-resistance in ER-positive breast cancer cells.
Tamoxifen, a selective estrogen receptor (ER) modulator, is the most common endocrine therapy worldwide to women with ER-positive metastatic breast cancer or as adjuvant therapy for early stages of the disease (
The tripartite motif (TRIM) proteins, one of the subfamilies of the RING type E3 ubiquitin ligases), are involved in a broad range of biological processes and their alterations are associated with disease incidence and progression relevant to the development of common human cancers (
G protein-coupled estrogen receptor (GPER), a novel estrogen receptor, has been reported to be activated by TAM and the pure anti-estrogen fulvestrant and play an important role in the acquired TAM-resistance (
In the present study, we elucidated that the degradation of Bim in MCF-7R plays a key role in GPER-mediated tamoxifen resistance in ER. Knockdown Bim in TAM-sensitive MCF-7 cells or overexpression of Bim in TAM-resistant MCF-7 cells significantly changed its sensibility to TAM. The activation of GPER acts as a direct mediator of the transcription of TRIM2 and the binding between TRIM2 and Bim, which regulates the expression of Bim in TAM-resistant breast cancer cells. These findings may provide a novel insight to understand the mechanism of GPER in acquired TAM-resistance in ER+ breast cancer.
The primary antibodies against Bim, ERK and phosphorylated ERK1/2 (pT202/Y204) were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA). The primary antibody of goat anti-Trim2 was obtained from Sigma-Aldrich (Steinheim, Germany). Antibodies against AKT and phosphorylated AKT (pS473) were from Cell Signaling Technology (New England Biolabs, Hertfordshire, UK). The antibody against GPER was purchased from Abcam (Cambridge, MA, USA). β-actin antibody, goat antimouse IgG-HRP, and goat antirabbit IgG-HRP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The reagents of GPER-specific agonists G1 and specific inhibitor G15 were purchased from Tocris Bioscience (St. Louis, MO, USA) and 4-hydroxytamoxifen (TAM), (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma-Aldrich. Lipofectamine™ 2000 was purchased from Life Technologies (Carlsbad, CA, USA).
Human breast cancer cells (MCF-7) were routinely grown in RPMI-1640 (Gibco, Mulgrave VIC, Australia) containing 5% fetal bovine serum (FBS; Gibco), 10
The expression vector encoding Bim was constructed by inserting human Bim cDNA into pcDNA3.0 vector (Promega, Madison, WI, USA). The pcDNA-Bim and its control vector were transfected into TAM-resistant MCF-7R cell lines using Lipofectamine 2000 (Life Technologies). The siRNAs used in the present study were obtained from Shanghai GenePharma, Co., Ltd. (Shanghai, China). Bim (Bcl2-L11)-specific siRNA or control siRNA (100 nM) were transfected into MCF-7; Trim2-specific siRNA and control siRNA transfected into MCF-7R using Lipofectamine 2000 according to the manufacturer's instructions. The target sequences for Bim (Bcl2-L11) siRNA are 5′-GACAGAGCCACAAGGUAAUTT-3′ and 5′-AUUA CCUUGUGGCUCUGUCTT-3′. The control siRNA sequences are 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). The efficiency of gene knockdown was determined by qRT-PCR and western blot analysis.
Cell growth was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay. The cells were plated at 5×103 cells/well in 96-well microtiter plates. After incubation and treated with designed concentration of TAM for the specified time, MTT (5 mg/ml) was added to each well and incubated for 4 h. The absorbance was recorded on a digital spectrophotometer at a wavelength of 570 nm. The experiment was repeated three times.
A total of 77 breast cancer specimens and their matched primary tumor tissues (PTs) were obtained from patients with breast tumors resected at the First Affiliated Hospital of Chongqing Medical University under permission by the ethics committee of Chongqing Medical University (Chongqing, China). All of the patient details and exclusion criteria have been previously described (
GPER scores were assigned as follows: the percentage of positive cells was categorized as 0 (negative staining in all cells), 1 (<1% cells stained), 2 (1–10% of cells stained), 3 (11–40% cells stained), 4 (41–70% cells stained) or 5 (71–100% cells stained), and staining intensity was categorized as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). Percentage and intensity scores were added to give total immunohistochemical scores, ranging from 0 to 8. Specimens that scored ≥2 were defined as GPER+.
The Bim expression was scored based on intensity (0–3) and extent (0, <10%, 1, 10–25%, 2, 26–50% and 3, >50%). The individual categories were multiplied to give a total immunohistochemical score ranging between 0 and 9. Samples that scored ≥3 were defined as positive immunohistochemical results.
Total RNA was extracted using TRizol® reagent (Takara Biotechnology, Dalian, China) from MCF-7 and MCF-7R cells and reverse transcription was performed using the PrimeScript RT reagent kit (Takara Biotechnology) following the manufacturer's instruction. Quantitative real-time PCR was performed with SYBR Premix Ex Taq™ II (Takara Biotechnology). The specific primers for Bim are: 5′-CCTTTCTTGGCCCTT GTTCC-3′ (sense) and 5′-TTGTGGCTCTGTCTGTAGGG-3′ (antisense); the specific primers for Trim2: 5′-CACCAAGGA CAAAGACGGTG-3′ (sense) and 5′-ATCAGCGGATCGGAT CACTT-3′ (antisense). β-actin was used as internal control for normalizing different samples. The primer sequences for α-actin are: 5′-TGACGTGGACATCCGCAAAG-3′ (sense) and 5′-CTGGAAGGTGGACAGCGAGG-3′ (antisense). All experiments were performed at least three times.
The total protein was acquired using RIPA protein extraction buffer with protease inhibitor (Beyotime Institute of Biotechnology, Haimen, China). Cell lysates were electrophoresed with 10 or 12% SDS-PAGE, and the specific primary antibody targeting to each indicated protein was incubated with the membranes at 4°C overnight. The membranes were then incubated with the appropriate secondary antibody conjugated to horseradish peroxidase for 1 h and visualized by using the enhanced chemiluminescence imaging system (Amersham Pharmacia Biotech, Tokyo, Japan). The gray level of each band was quantified using the Quantity One 4.62 software (Bio-Rad Laboratories, Hercules, CA, USA). The results were expressed as fold change relative to the loading control (β-actin).
Cells were seeded into 6-wells plates and grown to 60% confluence. After cultured in FBS- and phenol-free medium for 24 h, the cells were then treated with or without TAM for another 24 h. At the end of the treatment, cells were washed with phosphate-buffered saline (PBS) twice and stained with 5 ml Annexin V-FITC and 5 ml propidium iodide following the manufacturer's instructions (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China). Apoptotic cells were determined using a BD FACScan flow cytometer (BD Biosciences, Mansfield, MA, USA).
Coimmunoprecipitation was performed as previously described (
Statistical analysis was performed using the SPSS standard version 19.0 software (SPSS, Inc., Chicago, IL, USA). Data are represented as mean ± standard deviation from at least three independent determinations. The independent Student's t-test was calculated to compare the results between the two groups. P<0.05 was considered to be statistically significant.
A total of 114 breast cancer tissues were eligible for analysis according to our previous inclusion criteria; of these, 106 recurrent breast tumor samples (66 local and 40 distant metastases) were GPER positive identified by IHC staining. Among the 106 GPER+ specimens, GPER expression was increased in 73.58% (78/106), decreased in 5.66% (6/106) and unchanged in 20.76% (22/106) compared with the matched primary tumor tissues (PTs). All these GPER+ recurred tumor tissues (RTs) and the paired PTs were used to determine the expression of Bim (Bcl2-L11), an aberrant downregulated gene identified by cDNA-array in TAM-resistant breast cancer cells. Bim and GPER were shown to be located in the cell cytoplasm (
As shown in our reports, the mean IHC score for GPER is significantly increased in RTs (6.23±0.91) compared with that in PTs (3.46±1.07) (P=0.001). To understand whether Bim is involved in GPER-mediated TAM-resistance, Bim expression was scored in PTs and the paired RTs. Bim expression was decreased in 83.02%, 44/53), increased in 3.78% (4/106) and unchanged in 13.21% (14/106) of these 53 GPER+ tumors which relapsed during TAM treatment (
It has been reported that Bim plays an important role in cell apoptosis (
To further confirm the role of Bim on apoptosis in response to TAM in MCF-7, specific siRNAs against Bim (
To understand why Bim was decreased in MCF-7R, bioinformatics was applied. It was found that a tripartite motif protein 2 (TRIM2) upregulated in MCF-7R cells (
Previous reports have revealed an activated GPER in TAM-resistant breast cancer cells (
It has been previously demonstrated that TAM activates the MAPK/ERK and PI3K/AKT pathways through GPER/EGFR signaling axis in MCF-7R cells (
Estrogen plays a crucial role in ER+ breast cancer development and ER is the main mediator of the estrogenic effect. Tamoxifen, an estrogen receptor antagonist, has been widely used in clinical endocrine therapy (
Bim is the BH3-only protein that may function as death sensor that mediates the activation of the mitochondrial or caspase-independent apoptosis pathway. It has been demonstrated that Bim is a drug-induced apoptosis regulatory protein in breast cancer (
TRIM2 functions as a direct regulator to Bim degradation in TAM-resistant breast cancer cells. TRIM2 was increased to GPER activation in TAM-resistant MCF-7R cells. Under treatments of TAM and the GPER-specific agonist G1, TRIM2 expression was dramatically enhanced along with reduced Bim protein in MCF-7R cells, indicating a GPER-dependent expression of TRIM2 in TAM-resistant breast cancer cells. Notably, the transcription activity of Bim was not changed under these stimulators, suggesting a post-transcription regulation of Bim in MCF-7R cells. Our data show that the binding between TRIM2 and Bim under GPER activation may lead to Bim degradation in TAM-resistant cells. These findings were supported by other studies (
The present study provided evidence that GPER-EGFR-MAPK/ERK signaling, not GPER-EGFR-PI3K/Akt signaling, is the key regulator for Bim degradation in TAM-resistant breast cancer cells. Here, we verified that GPER-MAPK/ERK is necessary for Bim degradation. The binding between TRIM2 and Bim was dependent on the levels of TRIM2 protein and regulated by the GPER-MAPK/ERK signaling. Blockage of GPER-MAPK/ERK signaling using G15 or U0126 decreased TRIM2 protein levels and reduced the binding between TRIM2 and Bim in MCF-7R cells. TRIM2 has been defined as E3 ubiquitin ligase and plays a key role in ubiquitinylation degradation of proteins (
In conclusion, our findings provide new evidence to GPER in TAM-induced resistance of ER+ breast cancer. Targeting TAM/GPER/ERK signaling and TRIM2 gene may be a more effective way in overcoming TAM-resistance for ER+ breast cancer patients. However, more prospective clinical investigations need to be undertaken in the future.
The present study was supported by the National Natural Science Foundation of China (NSFC 81072149). Manran Liu is supported by the Program of National Natural Science Foundation of China (NSFC 81472476 and 31171336).
Immunohistochemical staining displays high levels of GPER and lower levels of Bim expression in breast cancer tissues. (A) The staining of GPER and Bim in carcinoma tissues. (a–d) the representative images for GPER negative (a), weak (b), moderate (c), and strong positive staining (d). (e–h) The representative images for Bim strong positive (e), moderate (f), weak (g) and negative staining (h). (B) Comparison of Bim expression in primary tumors (PTs) and their corresponding recurred tumor tissues (RTs) (n=53). (C) The correlation of Bim and GPER expression in RTs was revealed through pair-wise scatter plots. Magnification, ×200.
Bim is an apoptotic effector protein in response to tamoxifen treatment in MCF-7 cells. (A) mRNA expression of Bim in MCF-7 and MCF-7R cells determined by qRT-PCR. (B and C) The protein levels of Bim were detected in MCF-7 and MCF-7R cells (B), and cells treated with different concentration of TAM (1–10
Tamoxifen induces apoptosis in MCF-7 cells through Bim-dependent activation of PARP and caspase-3. (A) The efficiency of siRNA against Bim was determined by western blot analysis in MCF-7 cells. β-actin was used as a loading control. (B) MCF-7 cells transfected with specific siRNA targeting Bim (MCF-7/Bim siRNA) and control siRNA (MCF-7/Ctrl siRNA), MCF-7R cells transfected with Bim (MCF-7R/Bim) or vector constructor (MCF-7R/Vec) were treated with ethanol or TAM (6
Protein binding between TRIM2 and Bim leads to reduction of Bim in MCF-7R cells. (A) mRNA expression of Trim2 in MCF-7 and MCF-7R cells measured by qRT-PCR. (B) Western blotting was used to test the levels of TRIM2 in MCF-7 and MCF-7R cells. Data are shown as fold changes in OD compared with MCF-7 and normalized to β-actin. *P<0.05. (C) Protein binding between TRIM2 and Bim in MCF-7R was confirmed by immunoprecipitation analysis. (D) The protein levels of TRIM2 and Bim were determined using western blot analysis in MCF-7R cells transfected with siRNA targeting TRIM2 (MCF-7R si/TRIM2) or control siRNA (MCF-7R si/Ctrl) under treatment of TAM (6
Activated GPER governs the protein levels of Bim in MCF-7R cells. (A) Western blotting was used to detect the levels of Bim, TRIM2 and GPER in breast cancer cells. β-actin was used as a loading control. (B) Under treated with TAM (6
MAPK/ERK, the downstream signaling pathway of GPER, promotes the degradation of Bim via upregulating Trim2 expression and the binding between TRIM2 and Bim. (A) MCF-7R cells were treated with ethanol, TAM (1