Bioinformatics analysis

In this study, The Cancer Genome Atlas Research Network (TCGA,, UALCAN ( and METABRIC database were explored. We used the Kaplan–Meier plotter (KM plotter, database and the survival R packages were employed to analyze clinical outcomes. Linkedomics [24], cBioportal ( and Gene Expression Profiling Interactive Analysis (GEPIA, databases were applied, alongside R4.0.5.

Tissue specimens and patients

Totally 10 pairs of fresh breast tissues (paired BRCA tumor samples and matched adjacent normal tissue samples) were obtained from randomly selected patients for western blot (WB). We collected 28 normal samples and 362 BRCA samples from patients who were treated by surgical removal in 2007 for tissue microarray, with follow-up until July 2021.The overall survival (OS) and disease-free survival (DFS) were calculated as time from surgery until the occurrence of death and relapse, respectively. In addition, pathological sections from 50 BRCA patients were used for correlation analysis. All clinical samples were collected at Harbin Medical University Cancer Hospital and verified by histological and pathological examinations. This study was approved by the Ethics Committee of Harbin Medical University. All participating patients had provided written informed consent.

Immunohistochemistry (IHC)

The IHC assay was performed as previously described [25]. Primary antibodies were listed in an Additional file 1: Table S1. The staining results were scored according to the following criteria: percentage of immunoreactive cells: 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%); and staining intensity: 0 (negative), 1 (weak), 2 (moderate), or 3 (intense). The final score for TPI1 expression was the multiplication of percentage and intensity. For statistical analysis, a final staining score of ≤ 7 was defined as low expression, whereas a score > 7 as high expression.

Cell culture, transfection and treatment

Human BRCA cell line and MCF-10A were secured from the Cancer Research Institute of Heilongjiang Province. 10% fetal bovine serum (ExCell Bio, Australia) and 1% penicillin/streptomycin (Solarbio, China) were added to all media. MCF7, T47D, UACC-812, and HCC70 cells were cultured in DMEM (Gibco, Life Technologies, California, USA), whereas MDA-MB-453 and SKBR-3 cells in RPMI 1640 (Gibco, California, USA), and MCF-10A cells in complete medium purchased from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China [CAS: ZQ1311]). Cell lines were incubated in a humidified incubator at 37 °C with 5% CO2. MDA-MB-231 and MDA-MB-468 cells cultured in Leibovitz’s L-15 (PM151010, Procell, China) were placed in an incubator without CO2.

TPI1 knockdown (sh1, sh2, sh3) and vector (NC) by lentiviral system (Hanbio, Shanghai, China) with a puromycin selection marker were stably transfected into T47D cells. Furthermore, overexpressed TPI1 controlled with lentiviral system (Hanbio, Shanghai, China) was stably transfected into MDA-MB-231 cells according to the manufacturer’s instructions. Sequences of the interference TPI1 were listed in an Additional file 1: Table S2. Stable cells with lentiviral system were yielded after treatment with 1 μg/ml puromycin for 2 weeks. CDCA5 plasmid (CDCA5) and P62 plasmid (P62) were transfected into BRCA cells using jetPRIME® (Polyplus-transfection S.A, Illkirch, France) according to the manufacturer’s instructions, with empty vector plasmid as a negative control. All plasmids were purchased from Sino Biological (Beijing, China). Transfection efficiency was determined by qRT-PCR and WB, respectively. For cycloheximide (CHX) chase assay, cells were incubated with 200 μg/ml CHX (HY-12320, MCE, USA) for indicated durations (0, 2, 4, 6, 8, 10, and 12 h, respectively). LY294002 was used in cell-based assay (50 μM, 24 h) and mice models (75 mg/kg).

Western blot (WB) and qRT-PCR

WB was conducted as previously described [25]. Briefly, cell lysates were obtained using RIPA lysis buffer (Beyotime, Shanghai, China). Concentrations of protein were confirmed by a BCA Protein Assay Kit (Thermo Scientific). Protein was separated using 10% or 12.5% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% milk, the membranes were incubated with primary antibody overnight at 4 °C. The next day, it was incubated for 1 h at room temperature with corresponding secondary antibodies and then was visualizing by an ECL Plus kit (Xin Saimei, China). The primary antibodies were listed in Additional file 1: Table S3.

Total RNA was extracted using TRNzol reagent (TIANGEN Biotech, Beijing, China). Complementary DNA was synthesized using a high-capacity cDNA reverse transcription kit. qRT-PCR was conducted with the Applied Biosystems 7500 Real-Time PCR System using a SYBR Green Real-Time PCR Master Mix kit (Takara, Dalian, China). Primers for qRT-PCR were listed in Additional file 1: Table S4.

Cell proliferation assays

For Cell Counting Kit-8 (CCK-8) assay, 5 × 103 cells/well were seeded in 96-well plates (Jet Biofil, Guangzhou) and cultured at 37 °C. 10 μl of CCK-8 (SEVEN BIOTECH, China) was mixed with 90 μl of medium. The mixture was added to each well at time points of 1, 2, 3, 4, and 5 days, respectively, after seeding, incubated for 2 h, and measured for absorbance at 450 nm.

For colony formation assay, cells (8 × 102/well) were seeded in 6-well plates, culture medium was changed every 2–3 days. Two weeks later, living cells were fixed, stained, and counted.

An Edu assay, a total of 5 × 103 cells/each well were seeded in 96-well plates for 2 days, added with Edu at a concentration of 50 μM, and cultured 2 h at 37 °C before fixation. This assay was carried out following the manufacturer’s protocol of Click-IT Edu Imaging Kits (Yuheng, Suzhou, China). Images were taken under an inverted fluorescence microscope.

Migration, invasion and wound healing assays

For migration assays, cells (3 × 104–7 × 104) were inserted in 200 μl of serum-free medium, and 600 μl medium with 10% FBS was added to the bottom chambers. After 24 h, cells were fixed in 4% paraformaldehyde and stained with crystal violet solution for 2 h. The number of cells per five randomly selected fields was counted under an inverted microscope (Leica, Germany). Then, cells were photographed and counted in five randomly selected fields.

For invasion assays, the upper basement membrane of the chamber was precoated with 30 μg Matrigel (356243, BD Biosciences) and cultured for 48 h at 37 °C. The procedure was the same as migration assay.

For wound healing assays, wounds were scratched on a monolayer cell by 10 μl pipette tips until 95% of the cells were covered in a 6-well plate. Then, wound healing images were taken at appropriate time points (24 h/48 h). The migration distance between the dotted lines was measured and normalized according to that of control cells.

Glucose uptake and lactate production measurement

Cells were cultured in 96-well plates for 48 h to examine glucose consumption rate and lactate production. The supernatants were collected for detection, measured for absorbance at 490 nm, and calculated according to the manufacturer’s instructions. The Glucose Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and Lactate Assay Kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute) were applied for respective experiments.

In vivo assay

Female BALB/c nude mice (4–5 weeks old) were obtained from the Beijing Vital River Laboratory and housed under standard specific-pathogen-free conditions of the Animal Center of the Second Affiliated Hospital of Harbin Medical University.

The mice were randomly divided into three groups (n = 5/group). Two groups of mice were subcutaneously injected with MDA-MB-231 cells overexpressing TPI1-luciferase (5 × 106 cells in 200 µl phosphate buffered saline (PBS)/Matrigel [3:1]), while the remaining group was injected with MDA-MB-231-luciferase vector control cells. After primary tumor formation, five mice from TPI1 overexpressing group were randomly selected to receive LY294002 (75 mg/kg) intragastric administration 3 times a week for 3 weeks. T47D vector cells or shTPI1 cells (5 × 106 cells in 200 µl PBS/Matrigel [3:1]) were injected subcutaneously (n = 6/group). The tumor volume was monitored using vernier calipers every 4–5 days for a month, calculated by the formula (width2 × length)/2 (mm3). The mice were sacrificed after 35 days and tumor weight was measured. Tumor tissues were used for WB, H&E and IHC assays.

Immunoprecipitation (IP), silver staining and mass spectrometry assay

The protein A/G magnetic beads (MCE, HY-K0202) were incubated with antibodies at 4 °C for 1 h, followed by incubation with cell lysates based on the instructions. Subsequently, the beads were collected and subjected to immunoprecipitation (IP). A silver staining assay was performed according to the protocol provided by Beyotime Technology (P0017S, Beyotime, Shanghai, China). The antibody, magnetic beads and antigen were combined and boiled at 95 °C, and then the mass spectrometry results were obtained by Wuhan GeneCreate Biological Engineering. The antibodies used for the co-IP were TPI1 (Proteintech, 10713-1-AP), CDCA5 (Santa Cruz, sc-365319), and sequestosome-1/P62 (SQSTM1/P62) (Proteintech, 66184-1-Ig).

Immunofluorescence (IF) assays

The cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 for 10 min, and blocked by 10% normal goat serum for 30 min at room temperature. Cells were washed 3 times with PBS. Primary antibody was administered overnight at 4 °C. The next day, the cells were incubated with secondary antibody for 1 h and stained with DAPI (Beyotime, Shanghai, China) for 5 min at room temperature in a dark room. During each step, cells were washed 3 times with PBST. Then, pictures were taken under a confocal microscope (ZEISS LSM 800). The antibodies used were CDCA5 (Proteintech, 1:80 dilution, 67418-1-Ig), TPI1 (Proteintech, 1:50 dilution, 10713-1-AP), DyLight 649 Goat Anti-Rabbit IgG (A23620, Abbkine) or DyLight 488, and Goat Anti-Mouse IgG (A23210, Abbkine).

Ubiquitination assay

MDA-MB-231 cells transfected with Flag-Ub plasmid were pretreated with MG132 (10 μM) (MCE, New Jersey, USA) for 6 h and lysed. Then, co-IP was performed and analyzed by WB using an anti-ubiquitin antibody (1:100 dilution; ab134953, Abcam, Cambridge, MA, USA).

Statistical analysis

We performed statistical analyses using SPSS 22.0 software and GraphPad Prism 8.0 software, collecting data from no fewer than three independent experiments. The statistical results were expressed as mean ± standard deviation. The differences between two groups were analyzed using the student’s t-test and the chi-square test. Survival was calculated using the KM plotter method and the log-rank test. Spearman’s rank correlation coefficient (r) was used to quantify correlation analysis. A p value of < 0.05 (two-tailed) was considered statistically significant.

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