Huaier extract restrains the proliferative potential of endocrine-resistant breast cancer cells through increased ATM by suppressing miR-203

Endocrine therapy is one of the main treatments for breast cancer patients in the early stages. Tamoxifen and fulvestrant are the major drugs of endocrine therapy for breast cancer patients. However, acquired drug resistance often caused treatment failure and relapse for patients, which is a major clinical problem. We investigated whether Huaier extract had effects on endocrine-resistant breast cancer cells. In our study, we aimed to demonstrate the inhibitory effects of Huaier extract on tamoxifen-resistant cells (M7-TR) and fulvestrant-resistant cells (M7-FR). Using MTT and clone formation assays, we found that Huaier extract could inhibit the proliferation in M7-TR and M7-FR cells. Flow cytometry and western blotting illustrated that Huaier extract could induce G0/G1 arrest in both endocrine-resistant breast cancer cells. Mechanistically, we present that Huaier extract significantly increased ataxia telangiectasia mutation (ATM) via down-regulation of miR-203. Huaier extract also had the inhibitory effects on tumour growth in vivo in a xenograft mouse model. These results demonstrated that Huaier extract could inhibit the proliferation of M7-TR and M7-FR cells by increasing ATM via suppression of miR-203.

extract demonstrated proliferation-inhibiting effects related to the cell cycle and DNA damage. We further investigated the roles of miR-203 and ATM in endocrine-resistant breast cancer cells after Huaier extract treatment.

Results
Huaier extract inhibited proliferation of endocrine-resistant breast cancer cells. Clone formation assay was performed to verify the proliferative ability between endocrine-resistant breast cancer cells (M7-TR and M7-FR) and endocrine-sensitive breast cancer cells (MCF-7). As shown in Fig. 1A, the growth of MCF-7 cells was significantly slower than that of M7-TR and M7-FR cells. The anti-proliferative effects of Huaier extract Representative images and the colony formation rates of M7-TR and M7-FR cell colonies after treatment with Huaier extract for 24 h. *P < 0.05 or # P < 0.01, compared with the controls. The data are presented as the mean ± SD of three separate experiments. on both resistant breast cancer cells were investigated by MTT assay and clone formation assays. As shown in Fig. 1B, Huaier extract inhibited the cell viability of both M7-TR and M7-FR cells. In both the M7-TR and M7-FR cells, a sharp decrease in cell viability was present at 8 mg/mL, independent of the treatment time. The M7-TR and M7-FR cells indicated cytotoxic effects more evidently at 48 and 72 h. As shown in Fig. 1C, a significant inhibitory effect was observed with different concentrations of Huaier extract. Huaier extract could significantly inhibit clone formation in both M7-TR and M7-FR cells.
Huaier extract induced G0/G1 cell cycle arrest in endocrine-resistant breast cancer cells. The effects of Huaier extract on cell cycle progression in M7-TR and M7-FR cells were investigated by flow cytometry. Cells were exposed to different concentrations (0, 4 or 8 mg/mL) of Huaier extract for 24 h. After examination with a flow cytometer, the results demonstrated that Huaier extract resulted in a significant accumulation of cells in the G0/G1 phase. In M7-TR cells, the percentage of cells in the G0/G1 phase increased from 36.26 to 54.3% upon Huaier extract treatment ( Fig. 2A). As shown in Fig. 2B, cells treated with Huaier extract had increasing percentages of cells in the G0/G1 phase (from 53.29 to 66.39%). To investigate the mechanism of G0/G1 arrest, we detected the protein levels of cell cycle-regulating proteins by western blotting. We detected two important The data represent the results of three independent experiments; *P < 0.05 or **P < 0.01. Changes in cell cycle progression could be due to cyclin D1 and CDK4 in the M7-TR (E) and M7-FR (F) cells. The data are presented as the mean ± SD of three separate experiments.
cell cycle-regulating proteins, cyclin D1 and CDK4, which were obviously decreased in both cell lines following treatment with Huaier extract, which might explain the G0/G1 cell cycle arrest for both cell lines ( Fig. 2C and D).

Huaier extract up-regulated ATM in endocrine-resistant breast cancer cells.
To clarify the molecular mechanism of the inhibition of proliferation of M7-TR and M7-FR cells, we detected ATM in M7-TR and M7-FR using qRT-PCR and western blot assays. Cells were incubated with Huaier extract (8 mg/mL) for 48 h. Compared to the controls, Huaier extract could significantly increase the mRNA levels of ATM in M7-TR and M7-FR cells (Fig. 3A). γ-H2AX was phosphorylated by ataxia-telangiectasia mutation (ATM) 19 . Replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex, was downstream target of ATM 20 . As shown in Fig. 3B and C, the protein levels of ATM and p-RPA increased in both M7-TR and M7-FR cells by treatment with different concentrations of Huaier extract (4 or 8 mg/mL). γ-H2AX was increased significantly in M7-TR cells.
ATM was regulated by miR-203 in endocrine-resistant breast cancer cells. To explore the regulation of ATM, we detected miR-203 expression based on data mining and miRNA prediction. We used the  transfection for 48 h, MTT and clone formation assays were used to measure cell viability. As shown in Fig. 4C, overexpression of miR-203 led to decreased anti-proliferative activity of Huaier extract. Clone formation assay revealed that overexpression of miR-203 increased the clone formation ability (4 mg/mL and 8 mg/mL) of both cancer cell lines (Fig. 4D).

Huaier extract increased ATM by suppressing miR-203 in endocrine-resistant breast cancer cells.
To explore the further mechanism between ATM and miR-203 in M7-TR and M7-FR cells, we transfected miR-203 mimics to over-express miR-203 in both cell lines. Subsequently, cells were treated with Huaier extract or not for 48 h. As shown in Fig. 5A, the mRNA level of ATM was detected by qRT-PCR. Overexpression of miR-203 significantly reversed the expression of ATM in both cell lines. Compared to controls, ATM was increased after being combined with Huaier extract. To further confirm this finding, western blot analysis confirmed that miR-203 overexpression increased the level of ATM after combination with Huaier extract (Fig. 5B and C).
Huaier extract inhibited the growth of subcutaneous tumours. To demine the curative effect of Huaier extract in vivo, M7-TR and M7-FR cells were subcutaneously injected into the right flanks of BALB/c nu/ nu mice. As shown in Fig. 6A, xenograft tumour growth was reduced after Huaier extract treatment, compared to in the control group. To further investigate the mechanism underlying the inhibition of tumour growth by Huaier extract in vivo, we detected the miR-203 (Fig. 6B) and ATM (Fig. 6C) mRNA levels using qRT-PCR. Furthermore, we measured ATM and γ-H2AX protein levels using immunohistochemical staining and western blot analysis. Huaier extract increased ATM expression levels (Fig. 6D). Compared to controls, ATM, γ-H2AX and p-RPA were  increased by treatment with Huaier extract (Fig. 6E and F). These results demonstrated that Huaier extract had inhibitory effects on endocrine resistance in vivo.

Discussion
Endocrine therapy has been the first-line treatment for hormone-positive breast cancer patients over the past 30 years 21,22 . However, acquired endocrine resistance is a serious problem in clinical therapy. Acquired endocrine resistance often causes failure of endocrine therapy and relapse during clinical therapy 22 . Increasing research has investigated the underlying mechanism of endocrine resistance to search for a new therapeutic target or biomarker, including regulators of the ER pathway 23 and modulations in the cell cycle and apoptotic machinery 24 . In this study, we revealed that Huaier extract, which has been widely used for antitumour effects in several cancers, inhibited proliferation in endocrine-resistant breast cancer cells.
Accumulating evidence has underscored that TCM is a rich source for finding new drugs, and increasingly, TCM has been found to have anti-tumour effects on cancer 25,26 . Furthermore, TCM has revealed various effects on drug or radiation resistance [27][28][29] . Huaier extract is classified as an official fungus, and it has been widely used as a complementary agent for cancer therapy in recent years 30 . In a previous study, our group revealed that Huaier extract showed strong anti-proliferation effects on breast cancer 31 and synergized with endocrine drugs and radiosensitization via various pathways 10,32 . As shown in MTT and clone formation assays, we found that Huaier extract had strong inhibitory effects of the proliferation of M7-TR and M7-FR cells. Our founding revealed that Huaier extract induced G0/G1 cell cycle arrest. Cell cycle arrest is often related to cell cycle-regulating proteins. Cyclin D1 is an important checkpoint in cell cycle progression in G1 to S transition by binding to CDK4, which is required for transition from the G1 to S phase 33 . In this study, Cyclin D1 and CDK4 were decreased by treatment with Huaier extract.
Currently, DNA damage was negatively regulated for tumourigenesis and drug resistance in cancer 34,35 . Ataxia telangiectasia mutation (ATM) is a tumour-suppressor gene encoding a serine/threonine kinase, and it plays a key role in DNA double-strand breaks (DSBs) and activation of cell cycle checkpoints 17,36 . As measured by western blot, Huaier extract could significantly increase ATM and p-RPA in M7-TR and M7-FR cells. In previous studies, ATM was regulated by several miRNAs, such as miR-203, in various cancers 18,37 . Our study demonstrated that Huaier extract up-regulated ATM by inhibiting miR-203 in M7-TR cells. Overexpression of miR-203 could not significantly inhibit ATM expression in M7-FR cells (Fig. 5B). Therefore, there are likely other functional mediators of Huaier extract's effects on M7-FR cells. In addition, xenograft tumourigenicity assay demonstrated that Huaier extract could inhibit the tumourigenesis of endocrine-resistant cells in vivo.
In conclusion, our results demonstrated that Huaier extract has effects on cell cycle arrest by regulating cell cycle-related proteins, and it induced DNA damage through the miR-203/ATM pathway in vitro and in vivo. These data suggested that Huaier extract should be investigated as a potential supplementary drug for breast cancer patients with endocrine resistance. Additionally, the effects of Huaier extract on endocrine-resistant breast cancer cells in other molecular mechanisms, such as apoptosis, autophagy, tumour-associated macrophages, should be investigated in further studies. However, further clinical correlation studies should also assess the toxicity and efficacy of Huaier extract treatment, and our current study could provide the proof suggesting that Huaier extract is promising for further clinical investigation in breast cancer patients with endocrine resistance.

Materials and Methods
Cell lines and reagents. Tamoxifen Cell proliferation assay. Colony formation assay was used to assess cell proliferation. The cells were seeded at a density of 1.5 × 10 3 cells in a 6-cm Petri dish. The cells were cultured for another 14 days. The medium was refreshed every three days. Thereafter, the cells were washed with phosphate-buffered saline (PBS) and were fixed with paraformaldehyde and stained with 0.5% crystal violet. Images of clones were obtained using an Olympus digital camera (Olympus, Tokyo, Japan).
Cell viability assay. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. M7-TR and M7-FR (3 × 10 3 cells/well) cells were seeded into 96-well culture plates and were incubated in 5% CO 2 at 37 °C. After incubation overnight, the cells were exposed to vehicle or Huaier extract and were incubated for 24, 48, and 72 h. Then, 20 μL of MTT (5 mg/mL) were added to each well, and the cells were incubated for another 4 h at 37 °C. After removal of MTT, 100 μL of DMSO were added to each well, and the plate was gently shaken for 10 min at room temperature. The absorbance was measured by a Microplate Reader (Bio-Rad, Hercules, CA, USA) at 490 nm. All of the experiments were repeated at least three times.
Colony forming assay. M7-TR and M7-FR cells were incubated for 24 h. Subsequently, the cells were treated with different concentrations of Huaier extract for 24 h. M7-TR and M7-FR cells in single-cell suspension were seeded in a 6-cm Petri dish (1500 cells per dish) of completed medium. The cells were cultured for another 14 days. The medium was refreshed every three days. Thereafter, the cells were washed with phosphate-buffered saline (PBS) and were fixed with paraformaldehyde and stained with 0.5% crystal violet. Only colonies containing >50 cells were counted. Images of clones were obtained using an Olympus digital camera (Olympus, Tokyo, Japan).
Cell-cycle analysis. Cells at a density of 3 × 10 5 cells/well were seeded into a 6-cm Petri dish and incubated with completed medium at 37 °C for 24 h. Subsequently, the cells were treated with Huaier extract for 48 h, and the total cells were collected. The cells were fixed with 75% cold ethanol (1 mL of PBS and 3 mL of absolute ethanol) at −20 °C overnight. Then, the DNA of cells was stained with 200 of μL RNase A (1 mg/mL) and 500 μL of propidium iodide (PI, 100 μg/mL) (Liankebio, Zhejiang, China) for 30 min at room temperature in the dark, and they were analysed using a FACScan flow cytometer. The data were analysed using ModFitLT software, version 2.0 (Becton Dickinson, Franklin Lakes, NJ, USA).

Microrna and Transfection
MicroRNA mimics and the corresponding negative controls (NCs) were obtained from Guangzhou RiboBio (Guangzhou, China). To overexpress miR-203, we transfected mimics of miR-203 with Lipofectamine 2000 (Invitrogen) into M7-TR and M7-FR cells, according to the manufacturer's protocol. Then, the cells were harvested after 48 h for mRNA and MTT and for transwell and protein analysis.
Quantitative RT-PCR analysis. RNA was extracted using TRIzol (Takara, Dalian, China) reagents. Total RNA was used for RT reactions and quantitative (q)RT-PCR, according to the manufacturer's protocol (Takara). The expression of U6 was used as an endogenous control for the analysis of miRNA expression, and GAPDH was used as an endogenous control for the analysis of other mRNA expression levels. The experiments were repeated in triplicate at a minimum.
Western blot analysis. Cells were lysed with radio immunoprecipitation assay (RIPA) and PMSF (Biocolors, Shanghai, China) and were quantified using a BCA protein concentration assay kit (Merck, Darmstadt, Germany). Fifty micrograms of proteins were separated by 10% SDS-PAGE and were electro-blotted onto a PVDF membrane using a semidry blotting apparatus (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The next day, the membranes were labelled with secondary antibody, and signals were detected using a Luminescent Image analyser (GE Healthcare Bio-Sciences, Uppsala, Sweden). β-actin was used as the endogenous control.
Xenograft tumourigenicity assay. Cells (5 × 10 6 in 0.2 mL PBS) were injected subcutaneously into 4-week-old BALB/c nu/nu female mice (Taconic). After 2 days, the mice were randomly assigned to vehicle control (tri-distilled water) or Huaier extract alone (5 animals in each group). The Huaier group was administered 100 μL of solution containing 50 mg. Drugs were administered by gavage once every three days. Tumour growth was measured every 5 days, and tumour volume was calculated using the following equation: = × volume (width length)/2 2 . After 40 days, the mice were sacrificed, and the xenografts were removed for immunohistochemical staining and western blot assays.
All of the animal care and experiments were conducted in accordance with the Guideline for Animal Experiments of Qilu Hospital and were approved by the Ethics Committee of Qilu Hospital for Animal Research (approval number KYLL-2016-221).
Immunohistochemistry. The tumours in 4-week-old BALB/c nu/nu female mice previously injected with M7-TR and M7-FR cells were submitted to analysis. After excision, the tumour tissues were stored in 10% neutral-buffered formalin. After 24 h, the samples were paraffin-embedded and sliced into 4 μm sections. Immunohistochemistry was performed according to the manufacturer's protocol. Tissue sections were then incubated with streptavidin-HRP complex, followed by haematoxylin. For negative controls, the antibody solution was replaced with PBS. Statistical analysis. The results were analysed using SPSS software (SPSS, Chicago, IL, USA). Student's two-tailed t-test and one-way ANOVA were performed to determine significance. Each experiment was performed three times, and differences were considered significant when p-values < 0.05.