Cell culture and reagents
All cell lines in this study were obtained from the American Type Culture Collection (ATCC, USA). MDA-MB-231, MDA-MB-468 and MDA-MB-453 were grown in L15 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in air. The BT549, T47D, MCF7, MCF-10A, HEK293 and HEK293T cells were incubated at 37 °C with 5% CO2. BT549 and T47D were grown in RPMI1640 (Gibco, USA) with 10% fetal bovine serum. MCF7, HEK293 and HEK293T were grown in DMEM (Hyclone, USA) supplemented with 10% fetal bovine serum. MCF-10A was cultured with MEBM medium (Lonza, Switzerland) supplemented with 10% fetal bovine serum. All cell lines were authenticated by STR and tested for mycoplasma contamination.
Single-cell suspension was prepared and MDA-MB-231 and T47D cells in the medium were seeded in ultra-low attachment dishes to form tumorspheres. Tumorspheres were maintained in DMEM/F12 (Gibco), supplemented with B27 serum replacement (Thermo Fisher Scientific, USA, 17504044), 20 ng/ml basic FGF (sino biological, China, 10014-HNAE), 20 ng/ml recombinant human EGF (sino biological, China, 10605-HNAY), heparin (MedChemExpress, China, HY-17567A) for up to 7 days.
Mass spectrometry (MS) analysis
MDA-MB-231 tumorsphere and MDA-MB-231 adherent cells were lysed in SDT lysis buffer. Mass spectrometry was performed in Shanghai Institute of Materia Medica, Chinese Academy of Sciences following the standard protocol. Protein quantification was based on TMT-label quantitative analysis.
RNA extraction and real-time qPCR (RT-qPCR)
Total RNA was extracted by TRIzol reagent (Invitrogen) from cells and reversed transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa, Japan). qPCR was performed to detect mRNA expression of genes by TB Green PCR Kit (TaKaRa) and primers are shown in table S2.
Western blotting and co-immunoprecipitation (co-IP)
For western blotting, anti-STAT3(9139s), anti-p-STAT3 Y705(9145s), anti-GAPDH (97166) were obtained from Cell Signaling Technology. Anti-HMGN5 (PA5-50468) was purchased from Invitrogen. Anti-FLAG (F3156) was purchased from Sigma Aldrich.
For co-IP, the clarified supernatants were first incubated with anti-FLAG (Sigma Aldrich, F3156) or anti-HMGN5 antibody overnight at 4 °C, and then incubated with Dynabeads protein G beads (Invitrogen, 10004D) for 2h at 4 °C, and the precipitates were washed five times with RIPA and analyzed by western blotting.
Cell proliferation and colony formation assay
For the cell proliferation assays, 5,000 cells were seeded into 96-well plates and cell viability was assessed by the Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) at 72h after transfection.
For the colony formation assay, 1,000 cells were seeded into 6-well plates for 10 days. Then, the cells were fixed with 4% paraformaldehyde, washed with PBS, stained with 0.05% crystal violet and washed with water. Finally, the dye was eluted with 1mL of 33% acetic acid, and the optical density (OD) was detected at 595 nm with the microplate reader.
All animal experiments were carried out according to the Institutional Ethical Guidelines on animal care and were approved by the Institutional Animal Care and Use Committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (2020-03-RJ-212, 2021-05-RJ-240). All mice were housed in a temperature-controlled pathogen-free environment on a 12h light/dark cycle, and had ad libitum access to food and water. In all trials, 4–6-week-old female BALB/c nude mice were purchased from JH-LabAnimal in China.
To explore the effect of HMGN5 on breast tumor growing, each mouse was injected subcutaneously with 2 × 106 MDA-MB-231 cells that stably expressed sh-control or sh-HMGN5. Tumor volume was measured every 3 days after injection. Four weeks later, mice were sacrificed, and then the xenografts were collected for weight mensuration and gene expression analysis.
For in vivo metastasis analysis, 25 mice were randomly allocated to sh-control and sh-HMGN5 group. 2 × 106 MDA-MB-231 cells stably expressing luciferase and sh-control or sh-HMGN5 were injected into the tail vein of mice. Bioluminescence imaging was performed using the IVIS Lumina II (Caliper LS) every week. For luminescence monitoring, the mice were anesthetized using 2% isoflurane, followed by D-luciferin (J&K, China) injection according to the manufacturer’s protocol. Eight weeks after cell injection, the lungs of mice were collected for counting the pulmonary metastatic nodules. Mice bearing over 20 % weight loss and poor condition were euthanized earlier according to “AVMA Guidelines for the Euthanasia of Animals: 2020 Edition”. The lungs of all mice were fixed in formalin and processed with paraffin embedding and Hematoxylin and Eosin (H&E) staining.
For [email protected] treatment experiment, each mouse was injected subcutaneously with 2 × 106 MDA-MB-231 cells. When tumors reached a volume of about 50 mm3, the animals were randomized into treatment and control groups of 7 mice per group. HMGN5 siRNA loaded into polymersomes was injected intravenously at 4.5mg/kg twice a week. The tumors were measured every 3 days for 3 weeks, and the tumor volume (mm3) was calculated using the following formula: V=1/2× length× width2. After 3 weeks, the mice were sacrificed, and the tumor tissues were obtained for weight mensuration and gene expression analysis.
Migration and invasion assays
The cell invasion and migration assays were performed using the Transwell Permeable Supports with 8μm pore (Corning, USA, 3422). Briefly, 1.5 × 105 cells/cm2 were seeded into apical chambers with the basement membrane pretreated with (for cell invasion assay) or without (for cell migration assay) 20μg Matrigel (Corning, 354248). After 24 h, the transwell was fixed with 4% PFA for 15 min and stained with hematoxylin for 15 min. The cells on the upper side of basement membrane were gently removed by cotton swabs, while the cells on the lower side of basement membrane were imaged. ImageJ was then used to calculate the number of transferred cells.
Human BC TMA
Tissue microarray (TMA) containing 161 tissue spots from 152 breast cancer patients was obtained from Shanghai Outdo Biotech Co.,Ltd. The TMA was stained with antibodies against p-STAT3 Y705 (Abcam, ab76315) and HMGN5 (Invitrogen, PA5-50468). Positive signal was graded by two automatized histology quantification program (Definiens Tissue Studio® 4.0 or ZEN).
Immunohistochemistry staining (IHC)
Five-micrometer-thick paraffin sections of human BC patients were obtained from the Second Affiliated Hospital of Zhejiang University. Antigen repair was performed for 10 min in the citric acid buffer (PH 6.0). Then, the treated sections were incubated with primary antibodies against HMGN5 or p-STAT3 at 4 °C overnight. HRP conjugated Goat anti-Rabbit lgG (Active Motif, USA, 15015) and DAB Kit (Zhongshanjinqiao, China, ZLI9018) was then used for immunochemical reaction. IHC score was assessed by pathologists according to the percentage of stained cells (0–4) and staining intensity (0–3).
RNA-Seq and analysis
Briefly, mRNA was enriched with Oligo-dT magnetic beads followed by reverse transcription, fragmentation and library construction. Sequencing was conducted on an BGISEQ at BGI-Wuhan (China). For RNA-Seq analysis, raw reads were trimmed and filtered first. Then filtered reads were aligned to the GRCh38 human reference transcriptome using salmon (v1.4.0) for count matrix generation . Differentially expressed genes were identified using R package DESeq2 (v1.32.0) . The genes with adjusted p-value <0.05 and fold change >1.5 were considered as differentially expressed genes. GSEA was calculated by R package fgsea (v1.18.0)  and gene sets of hallmark gene sets and curated gene sets were obtained from MSigDB (https://www.gsea-msigdb.org/gsea/msigdb ). Batch correction was performed by ComBat_seq function in R package sva (v3.40.0) when needed. The RNA-Seq data generated in this study are deposited in GEO database (accession number GSE193507).
Cut&Tag assay and data processing
50,000 cells were harvested, and the CUT&Tag assay was performed using a commercial kit (Vazyme, China, TD901-TD902) according to the manufacturer’s protocol. Briefly, cells were collected and washed followed by incubation with Concanavalin A-coated magnetic beads. Next, cells were incubated with anti-Acetyl-Histone3(K27) (Cell Signaling Technology) primary antibody for 2h, and then 1h with secondary antibody at room temperature. Hyperactive pG-Tn5 transposonase was then added to perform tagmentation. The reaction was terminated, and DNA fragments were extracted by PCI, followed by PCR amplification using indexed P5 and P7 primers. Purified libraries were then sent for paired-end sequencing on NovaSeq 6000 (Illumina).
The STAT3 ChIP-Seq data were obtained from GEO database (GSM4608989). ChIP-Seq reads were aligned to human reference genome GRCh38 using Bowtie2 . Then the STAT3 binding sites were identified by peak-calling using MACS2 (v18.104.22.168)  with q-value 0.05. The H3K27ac Cut&Tag sequencing reads were filtered and trimmed before alignment. H3K27ac enriched peaks were then called using MACS2 (v22.214.171.124) with broadpeak option on. H3K27ac peak density were calculated by annotatePeaks.pl function in HOMER2 . The STAT3 ChIP-Seq and H3K27ac Cut&Tag tracks were visualized in IGV browser . The Cut&Tag data generated in this study are deposited in GEO database (accession number GSE193507).
Immunofluorescence (IF) staining
Cells treated with IL6 (sinobiological 10395-HNAE) for 15min were fixed in 4% paraformaldehyde for 5 min and then fixed in methanol stored at -20 °C for 10min. Next, cells were permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 1% BSA in PBS for 1 h, the cells were then incubated with primary antibodies overnight at 4°C and then the secondary antibody conjugated with Alexa Fluor 488 (green) (Thermo Fisher Scientific, 35502) or Alexa Flour 594 (red) (Invitrogen, A24207). Nuclei were stained with DAPI (Beyotime, China). A confocal microscope (Leica-SP8) was used for image capture.
The FLAG-STAT3 and FLAG-HMGN5 were purchased from OBiO (China). The FLAG-STAT3C plasmids were constructed as described previously. The double strand DNA coding HMGN5 shRNA and STAT3 shRNA were synthesized by Sangon Biotech (Shanghai, China) and cloned into the lentiviral vector pLKO.1-Puro (Addgene, 8453).
30nM synthesized siRNAs were transfected with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. The oligonucleotide sequences targeting STAT3 and HMGN5 mRNA are shown in table S3.
The luciferase reporter assay
The promoter region and mutant of HMGN5 was cloned into pGL4.10 luciferase reporter vectors. HEK293 cells were first transfected with siRNA (30nM) for 24h, and then transfected with the reporter constructs (0.5μg) in 24-well plates. SV40promoter-Renilla plasmids were co-transfected as internal control. Forty-eight hours after transfection, cell extracts were prepared, and the luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega, USA). The primers used for the cloning HMGN5 promoter and its mutant are listed below.
Primers for HMGN5 promoter: 5′-GTAAACGTGCTGCTCTACGACATGA-3′ and 5′-AGCTCCAACTGAAGGTCCCTGA-3′. Primers for HMGN5 promoter mutant: 5′-GGAAAGTCTAAGAGGGCGAGGAGAAGA-3′ and 5′-GGAATCGTCAAAAATCAATGTTTCAACAGATCT-3′
Chromatin immunoprecipitation (ChIP) assay
MDA-MB-231 cells were cross-linked by 1% formaldehyde for 10 min at room temperature. ChIP assay was performed using the ChIP Assay Kit (Cell Signaling Technology, 9003) according to the manufacturer’s protocol. Anti-STAT3 (9139s) antibody was purchased from Cell Signaling Technology. Primers at predicted STAT3 binding sites were used for qPCR assays.
All in vitro experiments were performed three times independently. Quantitative data from in vitro experiments were shown as mean ±SD; while the data from in vivo experiments were shown as mean ±SEM. One-Way ANOVA test and Student's two-tailed t-test was used for statistical analysis with multi-group and two-group experiments, respectively. Two-way ANOVA test was used for analyzing the statistical change of tumor volume in tumor xenograft experiments. Kaplan-Meier (KM) survival analysis was performed using survival package in R and the p-value was assessed by log-rank test. p < 0.05 was considered significant statistically.