Bioinformatic analysis

In the GSE147271 datasets (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147271), the BC tissue samples were obtained from 62 patients before tamoxifen treatment and 40 patients after tamoxifen treatment. The 40 patients received an oral loading dose of 40 mg of tamoxifen twice daily for the first seven days, followed by a daily dose of 20 mg until surgery. GSE241654 datasets (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241654) include mRNA profiles of human breast cancer cell lines, including parental MCF7 cells and MCF7-derived tamoxifen-resistant cell lines MCF7-TR1 and MCF7-TR2. The gene expression in these datasets was shown as heatmaps and volcano plots. Venn diagrams were used to find overlapped genes from the two datasets. The overlapped genes were further used for GO-BP analysis. Bioinformatics analysis using Jaspar (https://jaspar.elixir.no/) was conducted to find genes that are regulated by Nanog a potential transcription factor.

Collection of BC tissue samples

BC and adjacent normal tissues were obtained from twenty female patients who underwent breast mass resection at the Fourth Affiliated Hospital of Harbin Medical University (Harbin, China) from 2016 to 2017; patients were excluded if they lacked histologically confirmed estrogen receptor (ER)-positive invasive ductal carcinoma, had a history of radiotherapy, neoadjuvant therapy, or endocrine therapy prior to surgery, showed evidence of distant metastasis at diagnosis, or did not receive NCCN guideline-adherent standard postoperative treatment (consisting of radiotherapy or chemotherapy followed by tamoxifen-based endocrine therapy). Furthermore, this study collected ER-positive BC tissue samples from ten patients who showed resistance to Tamoxifen after long-term Tamoxifen-based therapy; these patients were also required to meet the same exclusion criteria defined above. The detail clinical information of the patients was shown in Table 1. The collected tissue samples were used for PCR, western blot, and immunohistochemistry assays. In addition, the tissues samples were sent to Ptm-Biolab Inc. Company (Hangzhou, China) for Lactylation-quantitative proteomics analysis. The research protocol was approved by the Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University (Approval code: 2024-77), and all patients involved provided written informed consent.

Table 1 The detail clinical information of the patientsCell culture and treatments

T47D and MCF-7, two ER-positive BC cell lines, were purchased from the American Type Culture Collection (Manassas, VA, US). T47D-TR and MCF-7-TR, which show strong resistance to Tamoxifen, were obtained by incubating MCF-7 and T47D cells with gradually increased Tamoxifen (from 1 μM to 10 μM) for 6 months (Chinese Academy of Sciences, Shanghai, China). T47D and T47D-TR cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Thermo Fisher Scientific, Inc. Shanghai, China), while MCF-7 and MCF-7-TR cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen). The culture medium was supplemented with 10% fetal bovine serum (FBS; Hyclone, GE Healthcare Life Sciences), 100 nM oestradiol (E2) (Sigma-Aldrich), and 1% penicillin-streptomycin (Sigma-Aldrich). Cell samples were sent to Oebiotech Co., Ltd., (Shanghai, China) for metabolomics analysis. Metabolites were extracted from cells using organic solvents (methanol/acetonitrile/water mixtures), followed by centrifugation and filtration to remove proteins and particulates. LC-MS was adopted with reversed-phase (RP) chromatography for hydrophobic metabolites and hydrophilic interaction chromatography (HILIC) for polar compounds. Data acquisition modes include full-scan (untargeted metabolomics). Raw data are processed using XCMS software for peak alignment, annotation (via HMDB databases), and statistical analysis.

Sodium L-lactate (LLA, #L7022) and sodium oxamate (OXA, #565-73-1) were obtained from Millipore Sigma (USA) to promote or inhibit lactylation in cells, respectively. Cycloheximide (CHX, #66-81-9, Millipore Sigma) was used to inhibit protein synthesis. MG132 (HY-13259, MedChemExpress, USA) was used to prevent ubiquitin-mediated protein degradation in the proteasome.

Endogenous ZMIZ1 and Nanog were knocked down by shRNAs (GenePharma Co., Ltd., Shanghai, China). The shRNAs (shown in Table 2) and scrambled shRNAs (shRNA-NC) were transfected into the cells using the Lipofectamine 2000 kit (Invitrogen). The coding regions of ZMIZ1 and Nanog were cloned into the enhanced green fluorescent protein plasmid-C1 vector (GenePharma) to construct overexpression vectors. The knockdown and overexpression were confirmed by PCR and western blot assays. This study constructed ZMIZ1 wild-type vectors (HA-labelled WT) and mutant vectors with mutations at each or both K537 and K843 [HA-MT(K537R), HA-MT(K843R), and HA-MT(K537R/K843R)]. These vectors were transfected into T47D-TR and MCF-7-TR cells.

Table 2 Informaton of shRNAsQuantitative PCR (qPCR)

Total RNA was extracted from both cells and tissues using TRIzol reagent (KeyGEN BioTECH, KGA1201, China) and then reverse-transcribed to cDNA using a First-strand cDNA synthesis kit from KeyGen BioTECH (KGA1316). qPCR was performed using Realtime PCR Master Mix (SYBR Green) from KeyGen BioTECH (KGA1339). The primers used for qPCR are shown in Table 3.

Table 3 The primer informationImmunohistochemistry (IHC)

The sections were deparaffinized in xylene and rehydrated in a graded ethanol series (80–100%). Non-specific peroxidase reactions were blocked using hydrogen peroxide. The sections were incubated overnight at 4 °C with primary antibodies against ZMIZ1 (Abcam, ab113294, 1:200) and Nanog (Abcam, ab214549, 1:200). The sections were then incubated with a biotin-conjugated goat anti-rabbit polyclonal antibody (1:1000, Abcam, ab6720) at 37 °C for 30 minutes. Visualization was achieved using diaminobenzidine and haematoxylin (ZSGB-BIO, ZLI-9019) for 10 minutes.

Western blotting

Total proteins were extracted from cells and tissues using RIPA buffer and quantified using a BCA Protein Assay Reagent (Bestbio, #BB-3401, China). Fifteen micrograms of protein were loaded onto a 10% SDS-PAGE gel for electrophoresis. The proteins were transferred to a nylon membrane and labelled with primary antibodies including anti-ZMIZ1 (Abcam, ab113294, 1:200), anti-Nanog (Abcam, ab214549, 1:200), anti-NPC2 (Abcam, EPR19993, 1:200), anti-HA tag (Ptgcn, 51064-2-AP, 1:2000), anti-lactyl-lysine (PTM, PTM-1401, 1:300), and β-actin (Ptgcn, 66009-1-Ig, 1:2000). The next day, the membrane was washed three times with PBS supplemented with 0.1% Tween20 (PBST), followed by a 1-hour incubation with appropriate secondary antibodies at room temperature. HRP substrate (BestBio, #BB-3501, China) was added for chemiluminescent detection.

Immunoprecipitation

Cells were lysed using 1 mL of a buffer containing 1% NP-40, 0.1% SDS, 50 mM DTT, 2 μg/mL Aprotinin, 2 μg/mL Leupeptin, and 1 mM PMSF. The mixture was supplemented with Protein A+G Agarose beads linked with primary antibodies, including anti-ZMIZ1 (Abcam), anti-Nanog (Abcam), and anti-HA tag (Ptgcn). After centrifugation at 2500 rpm for 5 minutes, the precipitate was heated in a boiling water bath for SDS-PAGE electrophoresis and western blot analysis.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA), and then briefly rinsed with PBST. They were then incubated overnight with primary antibodies against ZMIZ1 and Nanog, followed by secondary antibodies (1:1000, Thermo Fisher, USA) in the dark for 1 hour. Fluorescence was observed using a Nikon confocal microscope (Japan).

Proximity ligation assay (PLA)

Cells were fixed in 4% PFA, permeabilized in PBS-Triton 0.5%, and incubated for 60 min at 37 °C with Duolink Blocking Solution for PLA (Sigma-Aldrich, USA). Cells were then incubated overnight at 4 °C with the following primary antibodies: anti-ZMIZ1 (Abcam) and anti-Nanog (Abcam). Afterward, cells were incubated with Duolink PLA probes conjugated to oligonucleotides for 1 hour at 37°C. Ligation and amplification were performed following the manufacturer’s instructions. Oligonucleotides hybridize to generate a closed circle when proteins are in close proximity (30 nm) to each other. Slides were then mounted using Duolink in Situ Mounting with DAPI. Single primary antibody staining was performed as a background control.

Dual luciferase reporter assay

The OCT4 and NPC2 gene promoters were cloned into a pGL4 vector (Addgene, Inc., Cambridge, MA, USA) upstream of the firefly luciferase coding region within restriction sites XhoI and NotI (Takara Bio, Inc., Otsu, Japan). The putative target region of Nanog on the NPC2 gene promoter was mutated and cloned into the pGL4 vector to construct NPC2 mutant-type (MT) reporters. The vectors were transfected into cells using Lipofectamine 2000 (Invitrogen). Cells were harvested at 48 hours, and the activity of firefly luciferase was normalized to that of Renilla luciferase.

Cell viability and proliferation assay

Cell viability was assessed using a CCK-8 kit (Beyotime, C0038, Shanghai, China). Briefly, 10 μL of CCK-8 reagent was added to cells in each well, followed by incubation at 37 °C for 1 hour. The colour change of the CCK-8 reagent was quantified at 450 nm in a plate reader to evaluate cell viability. Cell proliferation was assessed using 5-ethynyl-2′-deoxyuridine (EdU) staining with the BeyoClick™ EdU Cell Proliferation Kit (Beyotime), according to the manufacturer's instructions.

Transwell assay

Cell invasion was evaluated using Transwell 24-well plates (5 μm pore, Corning). A total of 5×104 cells suspended in serum-free medium were placed into the upper chamber. The invaded cells were stained with haematoxylin and eosin and quantified using a Nikon confocal microscope (Japan).

Colony-formation assay

Cells in the logarithmic growth phase were seeded into 3 cm dishes at a density of 500 cells per dish. The dishes were maintained at 37 °C in an environment of 5% CO2 and humidity for 2 weeks. The colony-formation rate was determined by dividing the number of colonies by the initial number of cells seeded.

Measurement of oxygen consumption rate

The oxygen consumption rate (OCR) was measured using an Oxygen Consumption Rate Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Briefly, cells were treated with MitoXpress-Xtra for the indicated times, with Mineral Oil layered over each well. The MitoXpress-Xtra fluorescence was then detected with an Infinite F500 microplate reader (TECAN, Männedorf, Switzerland).

Determination of glucose concentration in culture medium

Glucose concentration was measured using the glucose oxidase/peroxidase method, in which glucose oxidase converts glucose and O2 into gluconic acid and H2O2. The H2O2 further reacts with 1,5-dimethyl-2-phenyl-4-aminopyrazoline and phenols, generating coloured compounds that have a maximum absorption at 570 nm.

Measurements of lactate acid level and lactate dehydrogenase (LDH) activity

Intracellular L-lactate acid was measured using a Lactate Detection Kit (Biovision, #K607-100) according to the manufacturer’s instructions. Briefly, cells were homogenized and centrifuged at 13,000×g for 10 minutes. The supernatant underwent deproteinization to remove lactate dehydrogenase. A colorimetric detection was then performed using a plate reader at 570 nm. LDH enzyme activity was measured using a detection kit from Beyotime (C0016, Shanghai, China). Cells were treated with LDH detection working solution and incubated at room temperature (~25°C) in the dark for 30 minutes. Absorbance was measured at 490 nm.

Measurement of complex I activity

Mitochondrial respiratory chain complex I activity was examined using a detection kit from Solarbio (BC0510, Beijing, China). The collected cells were mixed with the kit reagents, and absorbance was measured at 340 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyses the consumption of 1 nmol NADH per minute per milligram of protein.

Extracellular medium acidification rate (ECAR) measurements

ECAR was quantified using the Seahorse XF Glycolysis Stress Test Kit (Agilent, 103020-100). Cells were seeded into Seahorse XF 96-well culture plates at 2 × 104 cells per well. Before measurements, the medium was replaced with 500 μL of DMEM containing 10 mmol/L D-glucose and 2 mmol/L L-glutamine. After acquiring steady-state extracellular acidification rates, the ATP synthase inhibitor oligomycin (5 μM) was added to observe changes following the blockage of mitochondrial respiration. Finally, 2-deoxyglucose (100 mM) was added to suppress glycolysis.

Cholesterol measurement

Cellular free cholesterol concentration was measured using a Cholesterol Assay Kit (ab65390, Abcam) according to the manufacturer’s instructions. Briefly, cells were lysed and incubated with cholesterol oxidase. The fluorescence (Ex/Em = 538/587 nm) was detected with an Infinite F500 microplate reader (TECAN). Cholesteryl ester was tested by first converting it to free cholesterol using cholesterol esterase. The total cholesterol was the sum of free cholesterol plus cholesteryl ester.

In vivo orthotopic tumour formation assay

T47D and T47D-TR cells were selected for animal studies. A total of 18 female BALB/c-nu nude mice (aged 4–6 weeks) were used, with 3 mice in each group. Mice were randomly assigned to each group by researchers blinded to the treatment groups. Non-treated cells (1 × 107), T47D cells with stable overexpression of ZMIZ1, and T47D-TR cells with stable knockdown of ZMIZ1 were subcutaneously injected into the flanks of 6-week-old female BALB/c mice. All mice were also subcutaneously injected with estradiol benzoate (1 mg/kg body weight) once per week to increase the growth rate of xenograft tumors. In Tamoxifen treatment groups, mice were orally administered 5 mg/kg Tamoxifen daily. All procedures for transplanting tumour cells in mice were conducted in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee of the Fourth Affiliated Hospital of Harbin Medical University. These nude mice were obtained from and maintained at the Laboratory Animal Centre of Harbin Medical University Cancer Hospital (China). All mice were housed in a specific pathogen-free (SPF) environment with a 12-hour light/12-hour dark cycle at 21–24°C and 40–60% humidity. Tumour dimensions were measured using the formula: Volume = (length × width2) × 0.5.

Statistical analysis

Data are presented as the mean ± SEM from three independent experiments. Statistical analysis was conducted using an unpaired Student’s t-test for comparisons of two groups, or a one-way ANOVA for comparisons of more than two groups. Prism GraphPad software was used for these analyses. A p-value below 0.05 was considered statistically significant.