1. Therapeutic agents
17β-Estradiol (E2), Tamoxifen, Fulvestrant and Ezrin-inhibitor (NSC668394) were purchased from MCE (MedChem Express).
2. Cell culture
The human breast cancer cell lines MCF7 (NCI-DTP Cat# MCF7, RRID: CVCL_0031) and T47D (BCRC Cat# 60250, RRID:CVCL_0553) were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). MCF7 were cultured in MEM (Gibco, Thermo Fisher Scientific, USA), supplemented with 10% fetal calf serum, 1×MEM non-essential amino acid (NEAA), 0.01 mg/ml bovine insulin, 100 U/ml penicillin and 100mg/ml streptomycin. T47D were cultured in RPMI‑1640 medium, supplemented with 10% fetal calf serum, 0.0075 mg/ml bovine insulin ,100 U/ml penicillin and 100mg/ml streptomycin. All cells were grown to 85% confluence for the experiments. All the cell lines were authenticated using SNP profiling. All the cell lines were tested negative for mycoplasma. All cell lines were cultured in a humidified incubator with 5% CO2 at 37℃.
3. Generation of resistant cell lines
MCF7 and T47D cells were maintained in phenol-red-free MEM (Gibco, Thermo Fisher Scientific, USA) and RPMI‑1640 medium (Gibco, Thermo Fisher Scientific, USA) respectively for 3 days, and the media was supplemented with 5% charcoal/dextran-treated FBS. The resistant cell lines were generated by long-term treatment with 1 µM tamoxifen or fulvestrant as previous reports [34, 35].
4. Cell proliferation
The cell survival of cancer cell lines (sensitive or their resistant counterparts) was determined by CCK-8 assay (Promega, America). Equal numbers of cells (5000 cells/well) were seeded into 96-well plates for the proliferation experiment. After increasing doses of inhibitor exposure for 3 days, the CCK-8 reagent (20ul) was added to each well and incubated at 37°C for 2 h. The optical density at 490 nm was measured using an automatic microplate reader (Epoch; BioTek, USA).
5. 5-Ethynyl-20-deoxyuridine (EdU) assay
Cell proliferation was analyzed using EdU assays with a Cell‑Light EdU DNA Cell Proliferation kit (Beyotime Institute of Biotechnology,China). Naive and endocrine-resistant breast cancer cells (1 × 104) were seeded in 96-well plates separately .After incubation for 48 h, 10 mM EdU was added and incubated at 37˚C for another 2 h. Cells were fixed with 4% paraformaldehyde and stained with azide (30 min, for proliferating cells) and Hoechst 33342 (10 min, for all cells) at room temperature. Images were captured by fluorescence microscope (Nikon). The percentage of proliferating cells was calculated using ImageJ V1.50 software (ImageJ, RRID:SCR_003070, National Institutes of Health).
6. Western blot and Antibodies
Cell lysis buffer (RIPA) (Beyotime, China) was used for protein extraction. Then, total protein concentration was determined by a bicinchoninic acid protein assay kit (Sigma, USA). Total cell lysates were collected, and equal quantities of protein were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene difluoride (PVDF) membrane. PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% skimmed milk powder for 1 h, and incubated with Has2 antibody (Abcam, ab131364) at 4°C overnight. Then, the membranes were washed with TBST buffer for three times (10 min each time) and incubated with horseradish peroxidase (HRP)-conjugated polyclonal secondary antibody for 1 h. The membranes were developed using the enhanced plus chemiluminescence assay (Thermo, USA) according to the manufacturer’s instructions. Images were analyzed using Image Pro-Plus 6.0. Primary antibodies were pAKT308 (D25E6) XP Rabbit antibody (Cell Signal, 13038T), pAKT473 (D9E) XP Rabbit antibody (Cell Signal, 4060T), pERK (D13.14.4E) XP Rabbit antibody (Cell Signal, 4370P), ERK (C67E7) Rabbit antibody (Cell Signal, 4691P), Ezrin (Cell Signal, 3145s), ERα (Abcam, ab32063, RRID:AB_732249), pERα (s118) (16J4) Mouse antibody (Cell Signal, 2511S).
7. Quantitative real-time PCR
Real-time PCR was performed to verify the expression of HAS2 genes to the response of MCF7 cells and T47D cells with the treatment of ER inhibitors. According to the manufacturer's instructions, total RNA was extracted from cultured cells using RNAiso Plus (Takara, Japan). The RNA concentration was measured using NanoDrop system (Thermo Fisher Scientific, USA). Then, 1 µg mRNA was reverse transcribed using the PrimeScript™ RT Reagent kit with gDNA Eraser (Takara, Japan). qPCR assays were performed with SYBR‑Green mix (Takara, Japan) according to the manufacturer's protocol. The relative RNA expression was analyzed by the change-in-threshold (2−∆∆CT) method of the specific gene over the housekeeping genes, GAPDH. Sequences of primers used are as follow:
Has2: Forward: 5′-TTATGGGCAGCCAATGTA‐3′
Reverse: 5′-ACTTGCTCCAACGGGTCT‐3′
Ezrin: Forward: 5′-ACCAATCAATGTCCGAGTTACC‐3′
Reverse: 5′-GCCGATAGTCTTTACCACCTGA‐3′
FOS: Forward: 5′-GACTGATACACTCCAAGCGG‐3′
Reverse: 5′-CATCAGGGATCTTGCAGGC‐3′
PBX1: Forward: 5′-CAGTGAGGAAGCCAAAGAGG‐3′
Reverse: 5′-CAGCTGTTTTGGCAGCATAA‐3′
c-MYC: Forward: 5′-CCTCCACTCGGAAGGACTATC‐3′
Reverse: 5′-TGTTCGCCTCTTGACATTCTC‐3′
GAPDH: Forward: 5′-AACGGATTTGGTCGTATTGGG‐3′
Reverse: 5′-TCGCTCCTGGAAGA TGGTGAT‐3′
8. CRISPR-Cas9 knockout of Has2 in MCF7 cells
MCF7 was subjected to CRISPR/Cas9-mediated knockout of Has2 by plasmid transduction using particles from Santa Cruz Biotechnology (sc-401032). The guide RNA vector was cloned into a pCas-Guide vector which expresses Cas9 behind CBh and U6 promoters. To target the Has2, guide RNA vector including target sequence (CTCGCAACACGTAACGCAAT; TCCAGTGATAATCGCTTCGT; ACCTACGAAGCGATTATCAC) was prepared by following the depositor's instruction. This vector was co-transfected with the donor template including homologous arms and a functional GFP-puromycin cassette using UltraCruz® Transfection Reagent (sc-395739) as the delivery reagent. MCF7 cells were passaged at 48 h post-transfection for 3 passages. Cells were treated with 2µg/mL puromycin daily until pooled population of all puromycin resistant cells was expanded. The pooled population of CAS9/Control cells and Has2 KO cells were then used for functional in vitro.
9. Cell transfection
In this study, namely Lipofectamine 3000, a cationic lipid-based transfection reagent was used (Thermo Fisher Scientific, USA). The confluency of 5×104 tamoxifen- or fulvestrant-resistant cells (TamR or FulR) was reached in 24-well cell culture plates. There were also non-transfected cells for each cell line as a negative control. Lipofectamine 3000 was initiated by adding separately 0.5 µg of HAS2 plasmid vector (RRID:Addgene_106837)and 1.5 µl of Lipofectamine 3000 to 25 µl of serum-free medium, Opti-MEM (Gibco, Thermo Fisher Scientific, USA). Finally, 1 µl of P3000 reagent (provided in Lipofectamine 3000 kit) was added to each mixture and mixed gently by pipetting. The plates were incubated at 37°C and 5% CO2 for 72h in the case of removing the inhibitor.
10. Human breast cancer samples
This study was approved by the ethics committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Written informed consent was obtained from all individual participants in accordance with the Declaration of Helsinki of the World) Medical Association. We analyzed tissue specimens (n=18) that were obtained from Shanghai Jiao Tong University Affiliated Sixth People’s Hospital between November 1, 2020, and July 31, 2021. TNM staging system was used to stage tumors.
11. Immunohistochemistry
Tissue samples from breast cancer patients were fixed in 4% buffered neutral formalin, embedded in paraffin, and were mounted on glass slides. After dewaxing, dehydration, antigen retrieval, and inhibition of endogenous peroxidase activity and nonspecific binding. Then, mouse anti-human has2 (1: 100; Abcam) antibodies, rabbit anti-human ERα (1: 1000; Abcam) antibodies or rabbit anti-human Ezrin (1: 100; CST) antibodies were added, and the slides were incubated at 4°C overnight. After washing with PBS, the slides were incubated with or without biotinylated secondary antibodies for 1 h, followed by streptavidin-ABC at room temperature. Then, the slides were developed with a 2,4-diaminobutyric acid Substrate Kit and counterstained with hematoxylin. The intensities of Has2, ERα, and Ezrin were quantitatively analyzed using IMAGE-PRO PLUS 6.0 software (ImageJ, RRID:SCR_003070) (Media Cybernetics, Bethesda, MD, USA).
12. TCGA Data Analysis
As required by experiment, the mRNA expression of HAS2 and ER were analyzed in 543 cases of primary luminal-like breast tumors. Analyses of TCGA data are performed on primary luminal-like breast cancers samples (n=543) with RNA-sequencing data. A total analysis of 543 luminal-like samples with RNA-sequencing data was obtained. The same method is used for other TCGA data.
13. Generation of stably ER-expressing cell lines through lentiviral transduction
Full-length human ESR1 plasmid (NM_001291230) was purchased from Shanghai Jikai Biotechnology. ESR1 lentivirus was overexpressed in MCF7 cells. Stable ER-expressing cells were chosen post transfection using puromycin. The stably Has2-expressing cell lines were prepared through lentiviral transduction as previously described [28]. The pCMVIE-IRES-HAS2 vector encoding Has2 were purchased from Hanbio (Shanghai, China). Stable Has2-expressing cells were chosen using puromycin. Has2-shRNA-targeting lentiviral particles were obtained from Santa Cruz Biotechnology (sc-45328-V). Stablely transfected cells were also purified by using puromycin (5µg/ml).
14. Immunofluorescence
The expression of ER and Ezrin was determined by immunofluorescence assay. Cells were fixed in 4% paraformaldehyde, permeabilized with Triton X-100/phosphate-buffered saline (PBS), and blocked in 1% BSA. Cells were incubated with primary antibodies overnight at 4°C in incubation fluid (Dako, Denmark). On the second day, cells were washed in PBST for 3 times (10 min each time) and then incubated with secondary antibodies conjugated with Alexa Fluor 647 (Abcam, ab150083) for 1 h at room temperature. Then cells were incubated with Phalloidin-iFluor 488 reagent(abcam,ab176573)for 90 minutes at room temperature. Cells were washed in PBST three times (10 min each time). Images were analyzed under confocal microscopy (Nikon A1, Tokyo, Japan). Primary antibodies against ERα and Ezrin were used.
15. Small-interfering RNA (SiRNA) for ezrin
The expression of ezrin was downregulated with siRNAs. SiRNA transfection was performed using riboFECTTMCP (RiboBio, Shanghai, China) according to the manufacturer’s protocol using 100nM of ezrin siRNA (pool of three: 5ʹ- GGATTACTGCGTCGATTCA-3ʹ, 5ʹ- CCATGGCTTTCCCGGATAA-3ʹ, 5ʹ- GTCGAGGCATGGAGTTCAA-3ʹ, RiboBio).
16. Co-immunoprecipitation
For the immunoprecipitation experiments, cells were first lysed in cell lysis buffer (RIPA) (Beyotime, China). Just before use, protease inhibitor, phosphatase inhibitor and PMSF were added to complete the buffer according to the manufacturer’s protocol. Insoluble fractions were removed by centrifugation (12000rpm, 10min, 4°C). The total protein concentration was determined by a bicinchoninic acid protein assay kit (Sigma, USA) before being pre-cleared by incubation with 25µL Protein A/G Plus agarose beads (Yuekebio, Shanghai, China) and 3µg normal IgG antibody whose source is the same as primary antibody for 2-3 h at 4°C with gentle inversion. Then removed the Protein A/G Plus agarose beads by centrifugation (300g 3min) and subsequently transferred supernatant to new tubes. 3µg antibody of Ezrin or ERα protein was added to the tube. Then fresh Protein A/G Plus beads were added to the antibody mix and inverted gently for overnight at 4°C. The beads were pelleted by centrifugation at 3000rpm for 3 min, the supernatant was removed and 200µL iced PBS was added. This was repeated for a total of five washes. Kept 30µL iced PBS after last wash. Following the washing step, beads were resuspended in 5×SDS loading buffer and incubated in a boiling water bath for 7 min before being separated by 12% SDS–PAGE. The following work was the same as western blot assay.
Statistical analysis
Statistical analysis was performed by GraphPad Software. The significance of differences among groups was determined by one-way ANOVA, t test, or Fisher exact test accordingly. Statistically significance was considered as p< 0.05.