Patient and Tissue Sample Collection
This study included 108 patients with breast cancer who underwent neoadjuvant therapy with paclitaxel at Tianjin Medical University Cancer Institute and Hospital from 2021 to 2023, with approval from the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. Tissue samples for analysis were collected during surgeries performed for clinical indications and were subjected to immunohistochemical staining after sectioning.
Immunohistochemical Analysis
IHC was conducted on 4 µm paraffin-embedded tissue sections from patients with breast cancer and mouse tumors. Sections were deparaffinized, rehydrated, and heated in EDTA (pH 9.0). After blocking with 3% H2O2 followed by PBS washes, sections were incubated with primary antibodies overnight at 4°C. The next day, after PBS washes, the sections were incubated with HRP-labeled secondary antibody for 50 min. DAB chromogenic reagent (ZSGB-Bio, Beijing, China) was applied according to manufacturer's instructions, followed by counterstaining with hematoxylin, xylene rinsing, and resin mounting. Images were captured with a Orthostatic fluorescence microscope and imaging system (OLYMPUS BX61, Japan).
Cell Culture and Treatments
Human breast cancer cell lines MDA-MB-231 and MCF-7 were acquired from American Type Culture Collection. These cells were cultured in DMEM, supplemented with 10% FBS (Gibco, Waltham, MA, USA), and 1% penicillin-streptomycin (100 U/mL penicillin and 100 mg/mL streptomycin), in a 5% CO2, humidified atmosphere at 37°C. Cells were treated with 10 µM MG132 (Selleck, TX, USA) or 50 µM chloroquine (CQ, Sigma) for 8 h for proteasome and lysosome inhibition, respectively. For transwell assays, cells were treated with 40 nM paclitaxel (GLPBIO, Montclair, USA) or 10 mM IWR-1 (Selleck, TX, USA). For protein synthesis inhibition, cells were treated with 100 µg/mL cycloheximide (CHX, Sigma) for various durations (0, 2, 4, 6, 8 h). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect cells with plasmid or small interfering RNA (siRNA; Hanbio, Shanghai, China) according to the instructions of the manufacturer. The siRNA sequences used in this study are listed in Supplementary Table 1.
RNA Isolation and RT-qPCR
Total RNA was extracted using TRIzol Reagent (Invitrogen, San Diego, CA, USA). For cDNA synthesis, 5 µg of RNA was reverse transcribed using M-MLV reverse transcriptase (ThermoFisher). RT-qPCR was conducted using SYBR Green I on the ViiA™ 7 Real-Time PCR Detection System (BioRad, Hercules, USA), following a specific thermal profile. Gene expression was calculated using the 2 − ΔΔCt method relative to β-actin. All experiments were performed independently in triplicate. PCR primers are listed in Supplemental Table 2.
Western Blot Assay
The cells were washed with cold PBS, lyzed, and proteins were extracted using RIPA buffer (Solarbio, Beijing, China) with added PMSF. Protein concentrations were determined using a BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). Equal quantities of protein (50 µg) were separated by SDS-PAGE and transferred to PVDF membranes (Roche, Basel, Switzerland). Membranes were blocked with 5% non-fat milk in TBST for 1 h, then incubated with primary antibodies overnight at 4°C. This was followed by incubation with HRP-linked secondary antibodies and detection using Amersham ECL Plus Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). Quantification was performed using ImageJ where necessary. Antibodies are listed in Supplemental Table 3.
Cell Apoptosis Assay
Cell apoptosis was detected using a FITC Annexin V Apoptosis Detection Kit I (Simubiotech, Tianjin, China), following the manufacturer's protocol. Cells were first washed with cold PBS, then resuspended in binding buffer, and stained with Annexin V-APC and Propidium Iodide. Cell death was quantified using a FACSVerse flow cytometer (BD Bioscience, San Diego, CA, USA) and analyzed with FlowJo software. Gating strategies were applied to exclude debris and doublets. Cells were categorized as viable, early apoptotic, late apoptotic, or necrotic based on Annexin V-APC and PI staining.
Immunoprecipitation and Co-immunoprecipitation Assays
For immunoprecipitation, cells were washed with PBS, lyzed in RIPA buffer with inhibitor cocktail (Thermo, USA), and processed using protein A/G agarose (Thermo) following the manufacturer's instructions. Lysates containing 1 mg of total protein were incubated overnight at 4°C with magnetic beads conjugated to Flag, FATS, or control IgG antibodies. After washing, proteins were eluted and analyzed by western blotting. Immunoprecipitation of FATS and MYH9 was performed using a kit (Sangon Biotech, China), with 4 µg of anti-FATS or anti-IgG antibodies, and anti-MYH9 antibodies for detection. For co-immunoprecipitation, after FATS and MYH9 were co-transfected into cells, the cells were processed as mentioned above before detection. At the end of gel electrophoresis, the results were visualized by western blotting and silver staining using a Fast Silver Stain Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Antibodies are listed in Supplemental Table 3.
Immunofluorescence Assay
Treated cells in 24-well plates were washed twice with pre-cooled PBS and fixed with 500 µl 4% paraformaldehyde per well for 20 min. After washing three times with pre-cooled PBS, the cells were permeabilised with 300 µl of 0.2% triton-X100 per well with shaking for 15 min. After blocking with 5% BSA (Biosharp, China), the cells were incubated with specific primary antibodies overnight at 4°C, washed, and then incubated with secondary antibodies for one hour on a shaker, protected from light.Antibodies are listed in Supplemental Table 3. Finally, the coverslips were sealed with 3–5 µl of DAPI (Beyotime, Shanghai, China), and then observed with a Confocal laser scanning microscope (Leica TCS SP5, Germany).
Molecular Docking Assay
The PDB ID of MYH9 is 4ETO and the uniprot ID of FATS is Q96M02. The process of molecular recognition was simulated on AutoDock 4.2 on a computer to find the optimal binding conformation of the protein and its ligand and to ensure that the overall binding free energy of the complex was minimized.
Plasmids and cloning
Flag-FATS plasmid was constructed by in-frame insertion of full-length FATS cDNA into the p3xFlag-myc-CMV-26 vector (Sigma, Buchs, Switzerland). Flag-MYH9 plasmid was constructed by in-frame insertion of full-length MYH9 cDNA into the p3xFlag pLVX-PURO vector (Hanbio, Shanghai, China).
Cell Viability and Clonogenic Assays
For cell viability, MDA-MB-231 or MCF-7 cells were seeded in 96-well plates(1000 cells/well). CCK-8 reagent (US Everbright, Suzhou, China) was added at a series of time points (0, 24, 48, 72 h) followed by incubation for 2 h at 37°C. Absorbance at 450 nm was measured using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). For clonogenic assays, cells were seeded in 6-well plates(1000 cells/well) and allowed to form colonies over 7–10 days. Colonies were fixed, stained, and counted.
Transwell Assay
The invasive and migratory capacity of MDA-MB-231 and MCF-7 cells was assessed using a Transwell chamber (COSTAR, Suzhou, China). Cells were treated with various agents and seeded onto Matrigel-coated membranes. After 24 h, migrated cells were fixed, stained, and counted. This experiment was replicated three times.
In vivo ubiquitination assay
Ubiquitin (Ub) was transfected into MCF-7 and MDA-MB-231 cells expressing vector or FATS. Cell lysates were prepared 24 h post-transfection and incubated with Ni-NTA beads (Qiagen, Venlo, The Netherlands) in binding buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, 5 mM EDTA, 2 mM imidazole) containing protease and phosphatase inhibitor cocktail (Thermo, USA). The beads were collected and washed with the binding buffer a total of four times, followed by analysis by western blotting.
Tumor-Xenografted Mice Model
The study protocol for C57BL/J mice was approved by the Animal Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. Mice care and experimental procedures complied with national and international guidelines. Female C57BL/J mice (4 weeks old) were purchased from GemPharmatech LLC. MDA-MB-231 cells were suspended in PBS, mixed with Matrigel (REF354234, Corning, USA), and injected subcutaneously into mice. Post-tumor formation, mice were treated with 5mg/kg paclitaxel (GlpBio, Montclair, USA), 5mg/kg IWR-1 (Selleck, TX, USA), or both agents combined. Body weight and tumor volume were monitored, and tumor tissues were collected for analysis and histological examination.
Haematoxylin and Eosin (H&E) Staining
H&E staining was performed on paraffin-embedded tumor tissues from mice xenografted with MDA-MB-231. After deparaffinization and rehydration, sections were stained with hematoxylin, rinsed, treated with acidic ethanol, and stained with eosin. After dehydration, images were captured with a Orthostatic fluorescence microscope and imaging system (OLYMPUS BX61, Japan).
Statistical Analysis
Data normalization was performed relative to control values and expressed as mean ± SD. The Chi-squared, Fisher's exact, and two-tailed Student's t tests were utilized to compare date groups as appropriate. GraphPad Prism 9.0 software facilitated both statistical analysis and graphical representation. Independent prognostic factors were identified using a multivariate Cox regression model. Statistical significance was established at p < 0.05: the specific values are reported or indicated in Figure legends as *, p < 0.05; **, p < 0.01; ***, p < 0.001.