Pan-cancer analysis was conducted using TIMER2.0 database (http://timer.cistrome.org//). The differential level of AZU1 between normal tissues and BCs were analyzed using TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). One-way survival analysis was performed based on Kaplan-Meier plotter (https://kmplot.com/analysis/). Overall survival, first progression survival and post-progression survival were used as indicators for survival analysis.

MDA-MB-231 (TNBC), MCF-7 (luminal A) and BT-549 (TNBC) cell lines were cultured in high-sugar DMEM or RPMI-1640 containing 10% fetal bovine serum (FBS; all from Zhejiang Ginocell Biological Technology Co., Ltd.; www.genomcell.com) plus 1% streptomycin/penicillin at 37°C in an environment of 5% CO₂. Subsequently, the cells were pre-treated with 50 µM pyroptosis inhibitor (VX765; MedChemExpress) which was prepared in DMSO and was further diluted in cell culture medium for 2 h, followed by the exposure to various concentrations (0, 250, 500 and 1,000 ng/ml) of AZU1 (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h at 37°C in an environment of 5% CO₂. Then, 10 µl of Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) reagent was added to each well and incubated at 37°C for 2 h to detect the viability of BC cell lines.

The three samples of TNBC paired with adjacent normal mammary tissues were obtained from surgical specimens of patients with BC (aged, 67, 47 and 71 years) at Hunan Provincial People's Hospital (Changsha, China) from September 2021 to August 2022. The inclusion criteria were as follows: i) Patients with pathologically confirmed TNBC (19); ii) patients >18 years old; iii) patients with no indication for surgery; and iv) patients with no acute or chronic inflammation, hematological and autoimmune diseases. The exclusion criteria were as follows: i) Patients with other malignant tumors; and ii) patients with severe heart, brain or kidney diseases. Samples were frozen in liquid nitrogen immediately after surgical removal and maintained at −80°C until protein extraction. All studies were approved (approval no. 2021-036) by the Institutional Ethics Committee of Hunan Provincial People's Hospital (Changsha, China), and written informed consent was obtained from all the patients.

Proteins were extracted and harvested from MDA-MB-231, MCF-7 and BT-549 cell lines with radio immunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). The isolated proteins were centrifuged at 10,000 × g for 10 min at 4°C. A BCA assay was then used to measure protein concentration and densitometry was detected using microplate reader (Biotek ELX808). Total protein (20 µg per lane) was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF). The PVDF membrane was soaked in 5% skim milk which was dissolved in Tris-buffered saline with Tween-20 (TBST; containing 2% Tween-20) for 1 h at room temperature. Subsequently, the soaked PVDF membrane was incubated overnight at 4°C with a rabbit polyclonal antibody against β-actin (product no. 4970) or GAPDH (product no. 92310; both from Cell Signaling Technology, Inc.), GSDMD (cat. no. ab219800; Abcam), p65 NF-κB (cat. no. 310099) and p-p65 NF-κB (cat. no. 310013; both from Chengdu Zhengneng Biotechnology, Inc.; www.zen-bio.cn), caspase-1 (cat. no. ab179515; Abcam), apoptosis-associated speck-like protein containing a CARD (ASC; cat. no. DF6304; Affinity Biosciences), NLRP3 (cat. no. A5652; Abclonal Biotech Co., Ltd.), cleaved caspase-3 (product no. 9664), Bax (product no. 2772) and Bcl-2 (4223; all from Cell Signaling Technology, Inc.) at a dilution of 1:1,000 (for GSDMD, p65, p-p65, ASC, NLRP3, cleaved caspase-3, Bax and Bcl-2) or 1:2,000 (for GAPDH and β-actin). Subsequently, the membrane was washed with TBST three times and incubated with a secondary HRP goat anti-rabbit antibody (cat. no. AS014; Abclonal Biotech Co., Ltd.) for 1 h at room temperature at a dilution of 1:5,000. Similarly, the PVDF membrane was washed three times again using TBST. Finally, specific bands were visualized using a chemiluminescent substrate (cat. no. P0018M; Beyotime Institute of Biotechnology) and detected using the Omega Lum C Gel Imaging System. The relative expression of proteins was analyzed using ImageJ 1.48v (National Institutes of Health).

For the colony formation assay, 3×10² MDA-MB-231 or BT-549 cells were plated into 60-mm culture plates, and cultured in DMEM or RPMI-1640 culture medium supplemented with 10% FBS. The cells were then treated with various concentrations of AZU1 (0, 250, 500 and 1,000 ng/ml). After 14 days of incubation at 37°C in a 5% CO₂ environment, cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 10 min at room temperature. Finally, the number of colonies that contained >50 cells were counted manually. All experiments were performed at least three times.

A total of 5×10³ MDA-MB-231 or BT-549 cells were seeded into 24-well plates and cultured in DMEM or RPMI-1640 complete culture medium supplemented with 10% FBS, followed by treatment with 0, 250, 500, 100 ng/ml of AZU1 for 24 h at 37°C in a 5% CO₂ environment. An EdU proliferation assay was then used to examine the proliferation capability of MDA-MB-231 or BT-549 cells, according to the manufacturer's protocol. Firstly, the cells were incubated with 10 µM EdU (product no. CX002; Shanghai Epizyme Biotech Co., Ltd.) for 2 h at 37°C in a 5% CO₂ environment. Subsequently, the medium was removed and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with Triton X-100 for 5 min (cat. no. T8200; Beijing Solarbio Science & Technology Co., Ltd.) and 500 µl Click Additive Solution (included in the EdU kit) was added at room temperature. A total of 500 µl DAPI solution (cat. no. D1306; Thermo Fisher Scientific, Inc.) was added after 30 min and the fluorescence intensity was measured using a fluorescence microscope at a magnification of 40.

The apoptosis of MDA-MB-231 or BT-549 cells were analyzed using flow cytometry. Firstly, the cells were trypsinized with 0.25% trypsin (Beijing Solarbio Science & Technology Co., Ltd.) and washed with 1X PBS twice. Subsequently, the cells were resuspended in PBS and 5 µl Annexin V-fluorescein isothiocyanate and propidium iodide [cat. no. AT101; Hangzhou Multisciences (Lianke) Biotech Co., Ltd.] were added to the sample. After 15 min of incubation at room temperature, the apoptosis of the cells was detected using a flow cytometer (CytoFLEX; Beckman Coulter, Inc.). The acquired data was analyzed using FlowJo v10.8.1 (BD Biosciences). All experiments were performed at least three times.

All data are presented as the mean ± standard deviation and were statistically analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc.; Dotmatics). The Student's unpaired t-test was used to determine statistical significance between two groups. Statistical analyses for more than two groups were performed using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To clarify the role of AZU1 in tumor growth, pan-cancer analysis was conducted in tumors and adjacent normal tissues using TIMER2.0. The results revealed that AZU1 mRNA expression was strongly downregulated in BC, especially in TNBC (Fig. 1A-C). In order to further validate its expression, the cancerous tissues (Ca) and para-cancerous tissues (P-Ca) of patients with TNBC were isolated for western blot analysis. The results validated the bioinformatics analysis data (Fig. 1D and E). To analyze the association between AZU1 expression level and prognosis of patients with TNBC, survival analysis was performed in the present study. In Fig. 1F and G, the abscissa axis represents the survival time and the vertical axis indicates the survival rate of patients with BC or TNBC. In addition, the red line indicates the BC or TNBC patients with a high expression level of AZU1, while the black line indicates the patients with BC or TNBC with a low expression level of AZU1 (Fig. 1F and G). The results of the survival analysis revealed that patients with TNBC and a low expression level of AZU1 exhibited a low survival rate (Fig. 1G). These data suggested that AZU1 exerted a vital role in the progression of TNBC.

To further explore the role that AZU1 exerted in the pathogenesis of TNBC, various concentrations of AZU1 (0, 7.8, 15.6, 31.2, 62.5, 125, 250, 500 and 1,000 ng/ml) were utilized to stimulate the cell lines of TNBC (MDA-MB-231 and BT-549 cells). CCK-8 assays demonstrated that a high concentration of AZU1 (above 250 ng/ml) was able to inhibit the proliferation of MDA-MB-231 and BT-549 cells (Fig. 2A). Therefore, 250, 500 and 1,000 ng/ml AZU1 was used in the subsequent experiments. The results of the EdU assays also showed that a higher concentration of AZU1 led to a weak proliferation capability of the cells (Fig. 2B). The colony formation assay results were highly similar with the EdU data (Fig. 2C).

For further exploration of the underlying mechanism of AZU1, exerted in TNBC progression, MDA-MB-231 cells and BT-549 cells were treated with various concentrations of AZU1 and flow cytometry was conducted to detect the apoptosis levels. As revealed in Fig. 3A, a high concentration of AZU1 led to an increased percentage of apoptosis (Fig. 3A). Subsequently, the expression levels of apoptosis associated proteins, including anti-apoptotic and pro-apoptotic-related proteins, were detected using western blot analysis. As demonstrated in Fig. 3B, the anti-apoptotic-related protein, Bcl-2, was decreased following AZU1 treatment, whereas the pro-apoptotic proteins, cleaved caspase-3 and Bax, were increased in MDA-MB-231 cells and BT-549 cells. Collectively, the data of the present study showed that high concentrations of AZU1 promoted the apoptosis of TNBC cell lines.

Pyroptosis, a form of programmed cell death, has been revealed to exert crucial roles in modulating the aberrant proliferation of tumor cells (20). As GSDMD is its specific biomarker, the protein level of GSDMD in TNBC cell lines was investigated. The results revealed that the effects of AZU1 on the levels of GSDMD, both the full-length GSDMD (GSDMD-FL) and the N-terminal cleavage product of GSDMD (GSDMD-N), were dose- and time-dependent, especially after 24 h of the treatment (Fig. 4A and B).

As the NF-κB/NLRP3 axis is an effective and functional regulator of pyroptosis (21), the role that AZU1 exerted on the NF-κB/NLRP3 axis was subsequently assessed. Firstly, TNBC cell lines were treated with various concentrations of AZU1 and western blot analysis was utilized to detect the expression of p-65 NF-κB, NLRP3, caspase-1 and ASC. As depicted in Fig. 5, p-65 NF-κB, NLRP3, caspase-1 and ASC protein levels revealed a significant increase under the treatment of AZU1 after 24 h. These findings indicated that AZU1 promoted the pyroptosis of TNBC cell lines through the modulation of the NF-κB/NLRP3 axis.

As it was demonstrated that AZU1 promoted the pyroptosis of TNBC cell lines through the modulation of the NF-κB/NLRP3 axis, next, it was further explored whether AZU1 functioned to inhibit the proliferation of TNBC cell lines through pyroptosis. Therefore, TNBC cell lines were treated with a pyroptosis inhibitor and CCK-8 along with colony formation assay were used to assess the proliferation of the cells. The results revealed that the proliferation of TNBC cell lines was significantly inhibited when treated with various concentrations of AZU1, which was partially reversed when the cells were co-incubated with the pyroptosis inhibitor and AZU1 (Fig. 6). These findings suggested that AZU1 inhibited TNBC proliferation through the modulation of pyroptosis.