4-Hydroxytamoxifen (TAM) was purchased from Sigma-Aldrich; Merck KGaA. Doxorubicin, paclitaxel and cisplatin were purchased from Beijing Solarbio Science & Technology Co., Ltd. Antibodies against Erα (cat. no. 8644S), human epidermal growth factor receptor 2 (HER2) (cat. no. 2242S), GAPDH (cat. no. 5174S) and AKT (cat. no. 9272S) were purchased from Cell Signaling Technology, Inc. Antibodies against c-MYC (cat. no. ab32072), cyclin D1 (cat. no. ab226977, phospho (p)-AKT (Ser473) (cat. no. ab81283) and E-cadherin (cat. no. ab133597) were purchased from Abcam. Antibodies against p21 (cat. no. 27296-1-AP) and β-catenin (cat. no. 51067-2-AP) were purchased from ProteinTech Group, Inc., and an antibody against vimentin (cat. no. BS1491) was purchased from Bioworld Technology, Inc.

The human breast cancer cell lines, MCF-7 and T47D, were purchased from the American Type Culture Collection. The TAM-resistant cell lines, MCF-7R and T47DR, were generated by exposing parental MCF-7 and T47D cells to progressively increasing concentrations of TAM (up to 5 µM) over a duration of ~8 months as previously described (24,25). All cell lines were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology) at 37°C in an humidified incubator with 5% CO₂.

Cell viability was measured using a Cell Counting kit-8 assay (CCK-8; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, 5×10³ cells/well were seeded in a 96-well plate in 100 µl medium. After 24 h, the cells were treated with the different drugs (TAM: 5, 10, 11, 12, 13, 14, 15, 16, 18, 20 and 25 µM; doxorubicin: 0.25, 0.5, 1, 2, 4, 8 and 16 µM; paclitaxel: 0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 nM; cisplatin: 2, 4, 8, 16, 32, 64, 128 and 256 µM) (5 wells for each drug) for 24-72 h. Subsequently, 10 µl CCK-8 solution was added to the medium and the cells were incubated for a further 2 h at 37°C. Optical density (OD) values were measured using a digital spectrophotometer (Bio-Rad Laboratories, Inc.) at a wavelength of 450 nm, and cell viability rates were expressed as a percentage relative to the corresponding control cells. The IC₅₀ value was determined from the concentration-effect curve. After the data were log-transformed, the specific value was obtained through the curve parameter equation, and the viability of the control cells was considered as 100% (26).

The MCF7, T47D, MCF7R or T47DR cells were seeded in the upper chamber of a Transwell insert (3×10⁵ cells/well; EMD Millipore) coated with Matrigel (1:7.5) in 200 µl of serum-free medium. Supplemented RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added to the lower chamber. The cell lines were incubated for 24-48 h at 37°C, and the cells were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.) for 15 min at room temperature and stained with 0.5% crystal violet (Wuhan Boster Biological Technology, Ltd.) for 5 min at room temperature. the number of migrating cells was determined in 5 random fields at an inverted light microscope (magnification, ×200; TE2000-U; Nikon Corp.).

The proportion of cells in the S phase of the cell cycle was analyzed by flow cytometry using a standard protocol. Briefly, the cells were seeded in 6-well plates (2×10⁵ cells/well) and cultured for 24 h. The cells were then harvested and washed twice with cold PBS, fixed with 70% ethanol at 4°C for 1 h, incubated with Rnase for 30 min at 37°C and stained with propidium iodide for 30 min at room temperature (cat. no. P4170; Sigma Aldrich; Merck KGaA). Cell cycle distribution was analyzed by flow cytometry using a FACSVantage SE instrument (BD Biosciences).

Total cellular RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Reverse transcription and quantitative PCR were performed using the PrimeScript RT Master mix kit (Takara Bio, Inc.) and SYBR Pre-mix Ex Taq™ II (Takara Bio, Inc.) according to the manufacturer's protocols. GAPDH was used as the reference gene, and the thermocycling conditions were as follows: 2 min at 95°C, followed by 39 cycles at 95°C for 30 sec, 30 sec at 56.5°C and 20 sec at 72°C. Relative gene expression was normalized to GAPDH and calculated using the 2^−∆∆Cq method (27). The sequences of primers used for amplifying GAPDH, c-MYC, multidrug resistance-related protein (MRP), multidrug resistance protein (MDR) and breast cancer resistance protein (BCPR) are presented in Table SI.

Cells were lysed using RIPA lysis buffer with phenyl-methanesulfonyl fluoride (Wuhan Boster Biological Technology, Ltd.), and the protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). A total of 50 µg protein was loaded on a 10% SDS gel and resolved using SDS-PAGE. The resolved proteins were transferred to PVDF membranes. Non-specific binding sites were blocked by incubating the membranes with 5% non-fat milk, after which the membranes were incubated overnight at 4°C with the primary antibodies: ERα (1:1,000); HER2 (1:1,000); AKT (1:1,000); p-AKT (1:1,000); c-MYC (1:1,000); cyclin D1 (1:1,000); vimentin (1:1,000); E-cadherin (1:1,000); p21 (1:500); β-catenin (1:500); GAPDH (1:1,000) Membranes were subsequently incubated with a horseradish peroxidase-conjugated anti-rabbit/mouse immunoglobulin G secondary antibody (1:1,000; cat. no. BA1075 and BA1051; Wuhan Boster Biological Technology, Ltd.). Signals were visualized using enhanced chemiluminescence reagent (EMD Millipore). Densitometry analysis was performed using ImageJ and normalized to the respective expression of GAPDH.

siRNAs targeting c-MYC mRNA and a control siRNA were purchased from Shanghai GenePharma Co., Ltd. siRNAs were transfected into the cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The efficiency of RNA-interference was assessed using RT-qPCR 24-36 h following transfection. Cell lysates were collected 48-72 h following transfection and used for western blot analysis. The sequences of the siRNAs targeting c-MYC mRNA are presented in Table SII.

A total of 178 consecutive patients with recurrent and/or metastatic ER⁺ breast cancer, treated at the Breast Cancer Center of Chongqing at The First Affiliated Hospital of Chongqing Medical University between July, 2012 and July, 2017, were recruited for the present study. The outcomes of endocrine therapy were investigated for all patients, and 122 patients with TAM resistance were identified (Table I). At the time of diagnosis of recurrent and/or metastatic disease, 125 patients (70.2%) had received various chemotherapeutic regimens, whereas the remaining 53 patients continued to receive endocrine therapy. The Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1) (28) were utilized to assess chemotherapy responses as follows: Partial response (PR), a reduction of all target lesion diameters of ≥30%; progressive disease (PD), growth of all target lesion diameters by ≥20%; stable disease (SD), neither a sufficient reduction to be classified as PR, nor sufficient growth to be classified as PD (29). A total of 28 paired, archived paraffin-embedded breast cancer specimens (primary and metastatic breast cancer tissues) from the aforementioned TAM-resistant patients were obtained from the Clinical Diagnostic Pathology Center of Chongqing Medical University (Chongqing, China). Detailed information pertaining to the specimens is presented in Table SIII. The present study was approved by the Ethics Committee of Chongqing Medical University and written informed consent was obtained from all patients.

IHC staining was performed as previously described (19). For histochemical analysis, Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) was used to assess the percentage of positive cells. The detailed steps were as follows: Deparaffinized specimens were then sectioned (4-µm-thick slices). Following antigen repair and blocking for non-specific binding, the slices were incubated with specific primary antibodies against c-MYC (1:200; cat. no. ab32072, Abcam) overnight at 4°C. Subsequently, the sections were treated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:200; cat. no. TA140003; OriGene Technologies, Inc.) for 30 min at room temperature. After staining with diaminobenzidine (OriGene Technologies, Inc.) and hematoxylin for 5 sec at room temperature, images were captured using a Nikon Eclipse 80i microscope (magnification, ×200; Nikon Corp.). The mean OD in 5 randomly selected areas (MOD=IOD/area) was used to evaluate the levels of protein expression. The c-MYC staining intensities in tumor tissues were scored as follows: 0, no staining; 1, weak staining; 2, intermediate staining; 3, strong staining; and 4, very strong staining. Finally, the IHC data were quantified by multiplying the staining intensity by the proportion of positive cells as previously described (30).

The microarray analysis data of MCF-7 cells and TAM-resistant cell lines were downloaded from GEO (accession no. GSE26459). c-MYC expression in breast cancer tissues, para-cancerous tissues and in different molecular subtypes of breast cancer were obtained from TCGA, and survival curves based on c-MYC expression in breast cancers were obtained from Kaplan-Meier plotter: c-MYC expression data in tumors and follow-up information for patients with ER-positive breast cancer treated with adjuvant TAM were also downloaded from GEO (accession nos. GSE6532, n=87; and GSE9195, n=77), and the log-rank test was then used to assess the association between c-MYC expression and survival in breast cancer patients.

SPSS version 22.0 (SPSS Inc.) was used for the data analysis. Data are presented as the means ± standard deviation of 3 repeats. A one-way ANOVA followed by Tukey's post hoc test was used to compare differences among multiple groups. A χ² test was used to evaluate clinical response to chemotherapy in patients with recurrent and/or metastatic breast cancer. A value of P<0.05 was considered to indicate a statistically significant difference.

In agreement with a previous study (19) by the authors, the morphology of the MCF-7R and T47DR cells exhibited a flatter and more polygonal shape compared with that of the parental control cells, and the cells were more mesenchymal phenotypically (Fig. 1A). To validate TAM resistance in these established cells, MCF-7R and T47DR cells, and their parental cells were treated with various concentrations of TAM and the cell survival rates were measured compared with a negative control. As shown in Fig. 1B, survival was higher in the MCF-7R and T47DR cells compared with the respective parental cells treated with the same concentration of TAM. The IC₅₀ values of the 4 cell lines to TAM were 12.53±0.95 µM for the MCF-7, 26.93±1.76 µM for the MCF-7R, 9.29±1.23 µM for the T47D and 17.27±1.16 µM for the T47D-7R cells (data not shown). In addition, the protein expression levels of ERα and HER2 were significantly higher in the TAM-resistant cells (Fig. 1C and D), consistent with previous preclinical models and clinical observations (24,31-33).

To evaluate the proliferation of the TAM-resistant cells, cell cycle progression was analyzed by flow cytometry, and the results revealed that the percentage of cells in the G0/G1 phase was decreased and the percentage of cells in the S phase was increased significantly in the TAM-resistant cells (Fig. 2A and B), suggesting that proliferation of the TAM-resistant cells was increased. Consistently, the expression of the cell cycle checkpoint-associated protein, p21^waf1, decreased significantly in the TAM-resistant cells, whereas the expression of cyclin D1 was increased (Fig. 2C and D). Furthermore, the TAM-resistant cells were significantly more invasive compared with the parental cells (Figs. 2E and F, and S1A and B). E-cadherin protein expression was decreased and vimentin expression was increased in the TAM-resistant cells compared with the respective parental cells (Figs. 2G and H, and S1C and D), suggesting the cells has undergone epithelial-mesenchymal transition, consistent with the morphology of resistant cells.

To explore the potential mechanisms through which TAM resistance is acquired, microarray data from TAM-resistant cells were downloaded from GEO (accession no. GSE26459) and used to identify TAM resistance-associated genes. A heatmap depicting the expression of proliferation-related genes in the TAM-resistant cells relative to the parental controls is presented in Fig. 3A. c-MYC expression was found to be abnormally high in the TAM-resistant cells (P<0.001, Fig. 3B). c-MYC is one of the primary target genes of the ER signaling pathway and possesses similar functions to ER (34-36). The expression of c-MYC was observed in the TAM-resistant cells used in the present study (Fig. 3C and D). Consistently, the activation of PI3K/AKT and β-catenin signaling in TAM-resistant cells was also confirmed in the cells used (Fig. 3C and D). To further examine the role of c-MYC in TAM resistance, the siRNA-mediated knockdown of c-MYC in the MCF-7R cells (Fig. 3E-G) was shown to enhance the sensitivity of MCF-7R cells to TAM (Fig. 3H). The TAM IC₅₀ values of the MCF-7R cells prior to and following the knockdown of c-MYC were 20.26±0.76 µM for the MCF-7R, 18.88±0.69 µM for the MCF-7R/NC,13.95±1.82 µM for the MCF-7R/siRNA1 and 13.71±1.63 µM for the MCF-7R/siRNA2 cells (data not shown). Taken together, these data demonstrate that c-MYC plays a critical role in TAM resistance in vitro.

To further confirm the association between c-MYC and clinical TAM-resistance in patients with breast cancer, 28 pairs of primary and recurrent/metastatic tumor specimens from the same patients who underwent adjuvant TAM treatment were collected. As shown in Fig. 4A, c-MYC expression in the primary tumors was weaker, but was increased significantly in the metastatic lesions following the development of TAM in the same patients (all P<0.001; Fig. 4B and C), consistent with the results observed in TAM-resistant cell lines.

To assess c-MYC expression in breast cancer, TCGA was used for bioinformatics analysis. As shown in Fig. S2A and B, c-MYC expression was relatively low in breast cancer tissues compared with normal tissues (P<0.05); however, c-MYC was highly expressed in basal subtypes (endocrine therapy-resistant), which indicated an association with the therapeutic efficacy of TAM. Furthermore, Kaplan-Meier survival analysis revealed that a higher c-MYC expression was associated with a shorter distant metastasis-free survival in patients with breast cancer (P=0.011; Fig. S2C), and its expression was also associated with a poor relapse-free survival in patients with breast cancer that received endocrine therapy (P=0.032; Fig. 4D). In addition, a high c-MYC expression predicted an improved post-progression survival of patients with ER⁺ breast cancer (P=0.023), but not of the patients with ER-negative breast cancer (P=0.27; Fig. S2D and E).

To determine whether c-MYC expression may serve as a direct predictor of favorable outcomes following adjuvant TAM therapy, tumor gene expression levels and corresponding follow-up data from patients with ER⁺ breast cancer treated with TAM as an adjuvant were downloaded from GEO (accession nos. GSE6532 and GSE9195). The results revealed that an increased c-MYC expression was associated with a reduced relapse-free survival (P=0.034; Fig. 4E), suggesting that c-MYC expression was closely associated with clinical TAM resistance.

According to standard clinical chemotherapy, 3 major chemotherapeutic drugs (doxorubicin, paclitaxel and cisplatin) were evaluated to determine the alterations in chemosensitivity following the development of TAM resistance. As shown in Fig. 5A-C, the MCF-7R and T47DR cells exhibited an increased resistance to doxorubicin and paclitaxel; however, they exhibited a greater sensitivity to cisplatin, in comparison with the parental cells. The IC₅₀ values of ADR, PTX and CP to the MCF-7, MCF-7R, T47D and T47DR cells were 5.30±0.85, 8.37±1.05, 1.36±0.36, 1.94±0.26 µM for ADR, 14.10±1.63, 57.78±3.85, 16.20±2.37, 102.89±5.37 nM for PTX, and 42.37±3.35, 20.76±2.86, 84.32±4.35, 25.76±3.24 µM for CP, respectively (data not shown). RT-qPCR assays revealed that the mRNA expression levels of genes associated with multidrug resistance (MRP, MDR and BCPR) were significantly higher in the TAM-resistant cells compared with the parental cells (Fig. 5D), which may partially explain the increase in resistance to doxorubicin and paclitaxel.

Thus, the mechanisms underlying increased cisplatin sensitivity were examined. It is hypothesized that cisplatin-induced DNA lesions are most cytotoxic in the S phase of the cell cycle (37-39) and that c-MYC accelerates the G1/S phase transition (40,41). In the present study, it was hypothesized that a high c-MYC expression increased cisplatin sensitivity by increasing the proportion of cells in the S phase. To test this hypothesis, the MCF-7R cells were treated with a medium concentration of cisplatin for 24 h, and the dead cells were washed away. Western blot analysis was performed to evaluate c-MYC protein expression levels in the residual adherent cells. As shown in Fig. 6A and B, c-MYC protein expression significantly decreased in the surviving cells following cisplatin treatment, indicating that the cells with a high c-MYC expression were sensitized to cisplatin-induced cell death. Furthermore, the percentage of MCF-7R cells in the S phase decreased significantly following the knockdown of c-MYC expression (Fig. 6C and D), and c-MYC knockdown desensitized the MCF-7R cells to cisplatin (Fig. 6E). The IC₅₀ values of the MCF-7R cells to cisplatin prior to and following knockdown of c-MYC were 24.07±2.25 µM for the MCF-7R, 28.89±3.02 µM for the MCF-7R/NC and 54.23±3.62 µM for the MCF-7R/siRNA cells (data not shown). Collectively, these results suggest that a high c-MYC expression in TAM-resistant cells increased cisplatin sensitivity by regulating the cell cycle.

A total of 122 patients were shown to possess TAM-resistant breast cancer among the 178 patients with recurrent and/or metastatic ER⁺ breast cancer based on their endocrine therapy regimen (Fig. 7A). The median age of the patients at the time of recurrence/metastatic disease was 42.2 years (range, 25-65 years), and the median time from TAM therapy to recurrence and metastasis was 36.0 months (range, 5-132 months). In addition, 23 patients (18.9%) received treatment with TAM for >5 years. The baseline characteristics of the TAM-resistant patients are presented in Table I.

Following diagnosis with tumor recurrence and metastasis, 125 patients (93 with TAM resistance) received chemotherapy. The chemotherapeutic regimens and therapeutic responses are detailed in Fig. 7B. Following the analysis of the clinical remission efficiencies of patients that had undergone different chemotherapy regimens, it was determined that the TAM-resistant patients who received cisplatin-based chemotherapy exhibited improved clinical responses compared with those who received other regimens (P=0.012; Fig. 7C). Furthermore, the patients treated with TAM exhibited higher clinical remission rates when treated with cisplatin-based chemotherapy, compared with patients that had undergone other endocrine treatments (P=0.003; Fig. 7D).