Cell lines and cell culture

The human breast cancer MCF-7, T47D, MDA-MB-175, and HEK-293T cells were purchased from American Type Culture Collection (ATCC). Cell line authentication was performed via short tandem repeat (STR) using the Promega Power Plex 21 system. These cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, D6429, Sigma-Aldrich), supplemented with 10% Fetal Bovine Serum (FBS, 10270-106, Gibco), 1% penicillin–streptomycin–gentamicin Solution (C0223, Beyotime), 4.5 g/L glucose, 4 mM l-glutamine, and incubated at 37 °C with 5% CO2. For E2 assays, cells were cultured in charcoal-stripped FBS (Gibco, 12676-029) treated with phenol red-free DMEM (Gibco, 21063029) and then supplemented with 17b-estradiol (E2; Sigma-Aldrich) dissolved in ethanol.

To generate lentiviruses for PSMD14 depletion, short hairpin RNA (shRNA) lentiviral particles against PSMD14 were transduced into HEK-293T cells, following the manufacturer’s protocol. The transfection was conducted by co-transfecting the cells with pMD2G and psPAX2 envelop plasmids. The lentivirus was obtained after 2 days of transfection. T47D (ERα positive breast cancer cells) were incubated with 2 mL antibiotic-free medium containing 200 μL lentivirus. To generate stable cell lines, infected cells were selected using 2 μg/mL puromycin (Merck Millipore).

Plasmids and siRNA

The Myc-PSMD14 plasmid, EGFP-PSMD14 plasmid, HA-ERα and Flag-ERα plasmid were acquired from Origene Company (https://www.origene.com). HA-Ub, HA-K48, HA-K48R, HA-K63 and HA-K63R plasmids were used in previous study. The Lipofectamine 2000 (1662298, Invitrogen) was used for the transfection of plasmids. We used the small interfering RNAs to knockdown the specific gene. The PSMD14 siRNA sequences were siRNA#1: GGC AUU AAU UCA UGG ACU ATT; siRNA#2: GAU GGU UGU UGG UUG GUA UTT; Negative control: UUC UCC GAA CGU GUC ACG UTT. The RNA iMAX reagent (13778150, invitrogen) was used for the transfection of siRNA. The PSMD14 shRNA sequences used were showed as following: GCA GCA GAA CAA GTC TAT AT; Negative control: UUC UCC GAA CGU GUC ACG U.

DUB siRNA library screening

The siRNA library consisting of 76 human DUBs was purchased from Human Deubiquitinating Enzyme (ON-TARGET plus) from Dharmacon siRNA Library, GU-104705). ERα positive breast cancer cells were transfected with different siDUBs in MCF-7. After transfection for 48 h, RNA was extracted, and RNA was reverse transcribed into cDNA. The levels of the ERα classical downstream gene, TFF1, were measured to screen for significant regulation of the ERα signaling pathway by the results of this study focused on the following Using the preliminary results, this study focused on the deubiquitinating enzyme PSMD14.

Real-time quantitative PCR (qRT-PCR)

We used Rneasy Puls Mini Kit (Qiagen, China; Cat: 4992235) to extract total RNA and reverse-transcribed into cDNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo, Lithuania), and then amplified by PCR using specific primers and SYBR Green (A25742, Thermo Fisher) on a 7500 real-time fluorescence quantitative PCR system (Applied Biosystems, Singapore) with the expression of 36B4 as the internal reference. Primer sequences are shown in Supplementary Table S1.

Western blotting

Cells were lysed with RIPA buffer (Beyotime). Protein samples were loaded on to and separated using SDS/PAGE, transferred on to PVDF membranes blocked and probed with the primary antibodies. After blocking the membranes for 1 h at room temperature in 5% skim milk powder dissolved in Tris-buffered saline containing 5% Tween-20 (TBST), membranes were incubated overnight at 4 °C with the corresponding antibodies. Then Membranes were washed by PBST for three times and probed with secondary antibodies. After washing, the blots visualized using the ECL system (Bio-rad ChemiDoc).

Luciferase assay

The cells were transfected with 50 nM PSMD14 siRNA or 1 µg Myc-PSMD14 plasmids, the luciferase reporter and the renilla plasmid. About 24 h later, Cells were harvested, and Luciferase activity was measured. The luciferase activity of ERα were monitored using the Dual Luciferase Reporter kit (Promega).

CCK8 assay

Twenty-four hours after silencing PSMD14, both the silenced group and control group were plated onto 96-well plates with 4000 cells per well at 24, 48, and 72 h. Cell counts were determined at these time points using the CCK8 absorbance assay.

EdU assay

Cell proliferation was determined by EdU (5-ethynyl-20-deoxyuridine) assay using the EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China). MCF-7, T47D and MDA-MB-175 cells were spread into 96-well plates after corresponding treatment. After 24 h, cells were added with 50 mM EdU and continued to incubate for 2 h. Then fix the cells with 4% paraformaldehyde and stain with proliferating cells using Apollo dye solution. Nucleic acid was stained with Hoechst 33342. The cell proliferation rate was calculated using the imageJ.

Flow cytometric analyses

MCF-7, T47D and MDA-MB-175 cells were treated with Thiolutin for cell cycle analysis. For siPSMD14 knockdown experiments, MCF-7 cells were transfected with siPSMD14 or siControl. Similarly, for MCF-7 Y537S cells, they were treated with siPSMD14 or Tamoxifen. After 24 h, the treated cells were digested and resuspended into a single cell suspension, followed by washing in PBS. Ethanol was used to fix the cells, which were subsequently stained with propidium iodide. Fluorescence intensity was measured using BD LSR flow cytometry.

Co-Immunoprecipitation assay

Cell proteins were collected with Western and IP lysates, adding proteasome inhibitor (ST506 P0013, Beyotime). 12,000 × g at 4 °C after centrifugation for 30 min, the collected supernatant was incubated with the required antibody or control IgG and protein A/G agarose (P2051/P2053, Beyotime) at 4 °C overnight. The next day at 4 °C, 3000 × g after centrifugation for 10 min, rinse with lysis buffer (P0013F, Beyotime) for three times and discard the supernatant. Then add 2× SDS-PAGE buffer, 99 °C boiling 10 min, and going on immunblotting.

Protein stability assay

Cycloheximide (CHX) chase assay was used to determine the half-life of endogenous or ectopically expressing ERα. Cells were cultured in 12-well plates, with about 105 cells per well, and were transfected with 50 nM siControl/siPSMD14 or 1 µg vector/EGFP-PSMD14 WT/EGFP-PSMD14 Mutants. After 48 h, the cells were treated with 100 µM cycloheximide (C7698, Sigma) for the indicated time points. Subsequently, equal amounts of boiled lysates were analyzed by immunoblotting western blotting to detect ERα degradation.

Poly-ubiquitination detection assay

K48-linked poly-ubiquitination as an example. To directly detect K48-linked poly-ubiquitination of ERα from cell extracts, the cells were co-transfected with 2 µg of Myc-PSMD14 or Myc tagged with 0.5 µg of K48 Ubi plasmids and 0.5 µg of Flag ERα for 24 h. After that, the cells were treated with 10 μM MG132 for 6 h. Protein extraction was performed and then subjected to preclearance with 30 µL of protein A (P2051, Beyotime) for 2 h. Next, the extract was incubated overnight with anti-ERα antibody or anti-FLAG antibody, followed by incubation with protein A/G beads for 1 h at 4 °C. Finally, the western blotting technique was employed to detect total polyubiquitinated ERα or K48-polyubiquitinated ERα using anti-HA antibody.

Immunofluorescence assay

MCF-7 cells were fixed with 4% paraformaldehyde (p0099, Beyotime) for 10 min, permeabilized with PBS containing 0.2% Triton X-100 (T8200, Solarbio) for 10 min at room temperature. And then blocked by PBS plus 5% BSA (ST025, Beyotime) for 1 h. Rabbit anti-PSMD14 polyclonal antibody (HPA002114, SIGMA) and mouse anti-ERα Monoclonal antibodies (SC-56833, Santa Cruz) were used overnight at 4 °C, followed by Alexa flow 647 (Invitrogen) anti rabbit antibody and FITC coupled anti mouse antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature in dark. After three times washing, acquiring a final concentration of 0.1 μg/ml DAPI (Sigma) to stain nucleus. The images were captured by (acquired with) confocal laser scanning microscope. The collected images are further processed and analyzed by ImageJ.

Clinical breast tumor samples

One hundred and sixteen breast cancer specimens were collected from the pathology department of Qilu Hospital, Shandong University. The PSMD14 status, ER alpha status, PR status, and HER2 status of all breast tumor samples were examined by pathologists. Pathological specialists also examined the pathological grading and lymph node metastasis status of each sample. The study of clinical samples, which had received written informed consent from all the patients, was reviewed and approved by the Ethical Board at Shandong University.

Publicly available clinical data analysis

PSMD14 tumor RNA-seq data in breast cancer can be obtained from the Genomic Data Commons (GDC) data portal website (https://portal.gdc.cancer.gov/). The acquired data were analyzed and calculated by Prism 8.0 (GraphPad). Analysis of PSMD14 correlation with ERα target genes (MCM6, TFF1, and GREB1) was carried out by the Cancer Genome Atlas (TCGA), database using 879 breast cancer samples. PSMD14 expression analysis of ER-positive, HER2-positive, triple-negative breast cancer tissues, and normal tissues, as well as the expression of individual breast cancer stages, was conducted by the TCGA database (https://www.genome.gov/Funded-Programs-Projects/Cancer-Genome-Atlas). Analysis of the association of PSMD14 expression with clinical prognosis was implemented using the KMPLOT database (https://kmplot.com).

Computational analysis of RNA sequencing data

Gene set enrichment analysis (GSEA) was performed using the GSEA program provided by the Broad Institute (http://www.broadinstitute.org/gsea/index.jsp). GSEA was used to assess the relative enrichment of ERα positive regulated genes in two different groups, siControl and siPSMD14. The enrichment analysis utilized Hallmark gene sets and KEGG pathways by Metascape (https://metascape.org), which allowed for exploration of pathways associated with differentially expressed genes (DEGs). Additionally, the OmicStudio tools (https://www.omicstudio.cn/tool) were used to provide a volcano plot of the DEGs, with a threshold of P < 0.05 and fold change > 1.5.

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was conducted to analyze MCF-7 and T47D cells. The cells were fixed for 30 min to enable cross-linking. Following fixation, a mixture of 0.1375 mol/L glycine was added to the cells for neutralization. The cells were then washed with cold PBS/1 mmol/L PMSF and subsequently scraped into PBS/1 mmol/L PMSF, followed by centrifugation. After centrifugation, the cell pellet was treated with SDS lysis buffer. Next, the cells were sonicated for 10 min (30 s on/off) to fragment the chromatin. The ChIP assay kit (Millipore, 17-295) was utilized for the subsequent steps of the assay. In the ChIP experiments, the anti-ERα rabbit antibody (D8H8, #8644) was employed. Quantitative PCR analysis was performed with a DNA extraction kit (Qiagen, Cat. No. 28106). Primer sequences for ChIP-qPCR are displayed here: PSMD14 F: GGG GGC ACG CTA GAA TAA ACT, R: CAA CGC AGC CCT GTT TTG AA; TFF1 F: GGG CTT CAT GAG CTC CTT C, R: TTC ATA GTG AGA GAT GGC CGG; The ChIP-seq analysis of ER binding to the PSMD14 promoter region shown in Fig. 7A utilized data from GEO with accession numbers GSE128208.

Xenograft mouse models

In vivo tumorigenic experiment, we used the 5-week-old female BALB/c nude mice which were purchased from Shanghai Model Organisms Center, inc. Slow-release 17 beta-estradiol pellets (0.72 mg/90 days, Innovative Research of America) were implanted into the mice. After 24 h, 4 × 106 cancer cells were injected into the mammary fat pad of each mouse, using 150 μL of PBS. Tumor formation in the mice was then monitored for approximately 6 weeks. The tumor volume was calculated using the formula: tumor volume = length × width2/2. In Thiolutin treatment experiments, mice were randomly assigned to experimental groups containing 6 mice each. Thiolutin was injected intraperitoneally at a dosage of 1 mg/kg per day for one month, while the control group received solvent injections. All experimental procedures involving mice were conducted in accordance with the guidelines approved by the Xin Xiang Medical University Animal Care Commission.

Patient-derived explant (PDEx) assay

The excised tissues for research purposes, approved by the ethical committee of Qilu Hospital, Shandong University, were processed according to the ex vivo culture protocol. In general, breast cancer tissues were cultured on gelatin sponges with the cell culture medium containing 10% FBS. The tissues were either treated with vehicle or Thiolutin for 48 h. Subsequently, the tissues were fixed in 10% formaldehyde at 4 °C overnight. To confirm the quality, the tissues were stained with hematoxylin and eosin. After that, the immunohistochemistry was performed to examine the indicated markers.

Statistics

Student’s t-test, Pearson correlation coefficient, and Cox regression analysis were Publicly available data. Data are expressed as the mean ± SEM. Differences were considered to be statistically significant when P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.