MCF-10A, MDA-MB-436, MDA-MB-231, MDA-MB-468, MDA-MB-453 and MCF-7 cells were obtained from Xiamen Immocell Biotechnology Co., Ltd., and were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured at 37°C and 5% CO₂ in an incubator.

A total of 10 pairs of breast cancer tissue samples and adjacent normal tissue samples (1 cm away from the edge of the tumor and free of tumor cells) were obtained from Sanming First Hospital Affiliated to Fujian Medical University (Sanming, China). The age range of the 10 female patients was 43–62 years (mean age, 53±6.5 years). All of the patients did not receive any other treatment, such as chemotherapy, target therapy or other preoperative treatment, except for surgical operation.

Plasmid encoding His-ubiquitin (His-Ub) was obtained from Xiamen Anti-HeLa Biological Technology Trade Co., Ltd. The plasmids encoding hemagglutinin (HA)-OTUD7B and Flag-FOXM1 were constructed by subcloning OTUD7B and FOXM1 cDNA into pCDNA3.1-HA and pCDNA3.1-Flag vectors (Xiamen Anti-HeLa Biological Technology Trade Co., Ltd.), respectively. FOXM1 cDNA was also inserted into plv-EF1a-MCS-c-Flag-bsd vector (Xiamen Anti-HeLa Biological Technology Trade Co., Ltd), and the resulting plasmid was named plv-Flag-FOXM1, which was used to produce stable OTUD7B-knockdown and FOXM1-overexpression cells. Plasmids encoding a short hairpin (sh)RNA targeting OTUD7B (shOTUD7B) and a negative control shRNA (shNC) were constructed using the pLKO.1 vector (Xiamen Anti-HeLa Biological Technology Trade Co., Ltd). The sequences for plasmid construction are listed in Table I.

To determine the knockdown or overexpression efficiency of plasmids in different breast cancer cells, MDA-MB-468, MDA-MB-453 and MCF7 cells (2×10⁶ cells/well) were cultured in six-well plates and 5 µg shNC, shOTUD7B, pCDNA3.1 vector, HA-OTUD7B or Flag-FOXM1 were transfected into cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 24 h, cells were collected for western blotting. The results of which showed that plasmids encoding Flag-FOXM1 or HA-OTUD7B markedly increased the expression of FOXM1 or OTUD7B in cells, whereas shOTUD7B markedly decreased the expression of OTUD7B in cells. Notably, shOTUD7B-1 had the most significant effect and was thus used in subsequent experiments (Fig. S1).

Stable OTUD7B-knockdown MDA-MB-468, MDA-MB-453 and MCF7 cells; and stable OTUD7B-knockdown and FOXM1-overexpressing MDA-MB-468, MDA-MB-453 and MCF7 cells were constructed as follows: 9 µg shNC, shOTUD7B or plv-Flag-FOXM1, 3 µg pMD2G (Xiamen Anti-HeLa Biological Technology Trade Co., Ltd) and 6 µg psPAX2 (Xiamen Anti-HeLa Biological Technology Trade Co., Ltd) were co-transfected into 293T cells (Xiamen Immocell Biotechnology Co., Ltd.) in a 10-cm dish using Lipofectamine 2000. After 48 h cultivation at 37°C, the supernatant containing the lentivirus encoding shOTUD7B or FOXM1 was collected. The lentivirus was enriched and the titer was determined. To construct the stable OTUD7B-knockdown cell lines, the lentivirus encoding shOTUD7B was transduced into MDA-MB-468, MDA-MB-453 and MCF7 cells at a multiplicity of infection of 10 in the presence of 8 µg/ml polybrene. After 48 h, the medium was replaced with fresh medium containing 1 µg/ml puromycin (Gibco; Thermo Fisher Scientific, Inc.). At 10 days post-infection, cells were collected for gene expression analysis. To construct the stable OTUD7B-knockdown and FOXM1-overexpressing cell lines, the lentivirus encoding FOXM1 was transduced into stable OTUD7B-knockdown MDA-MB-468, MDA-MB-453 and MCF7 cells at a multiplicity of infection of 10 in the presence of 8 µg/ml polybrene. After 48 h, the medium was replaced with fresh medium containing 5 µg/ml blasticidin (Gibco; Thermo Fisher Scientific, Inc.). At 10 days post-infection, cells were collected for gene expression analysis.

MDA-MB-468, MDA-MB-453 and MCF7 cells; stable OTUD7B-knockdown MDA-MB-468, MDA-MB-453 and MCF7 cells; stable OTUD7B-knockdown and FOXM1-overexpressing MDA-MB-468, MDA-MB-453 and MCF7 cells were seeded into 96-well plates (1×10⁴ cells/well) and cultured overnight. After 0, 24, 48 or 72 h, 10 µl Cell Counting Kit 8 (CCK8) solution (Beyotime Institute of Biotechnology) was added and cells were incubated for 4 h at 37°C. The optical density was determined using an absorbance reader (SpectraMax^®; Molecular Devices, LLC) at 450 nm.

Clusters of cells containing >50 cells were considered colonies. The same cells used in the cell proliferation assay were seeded in 6-well plates at a density of 1×10³ cells/well. After 14 days of culture, cell colonies were fixed with 4% paraformaldehyde overnight at 4°C. The fixed colonies were then stained with 0.1% crystal violet for 10 min at 26°C. A light microscope was used to count the colony numbers.

The same cells used in the cell proliferation assay were seeded in 6-well plates (1×10⁴ cells/well) containing serum-free DMEM supplemented with 2% B27 NeuroMix (Absin Bioscience, Inc.), 20 ng/ml epithelial growth factor (Sigma-Aldrich; Merck KGaA), 20 ng/ml basic fibroblast growth factor (Sigma-Aldrich; Merck KGaA) and 4 µg/ml insulin. After 7 days of culture at 37°C, the sphere diameters were measured under a light microscope.

The same cells used in the cell proliferation assay were plated in 6-well plates (2×10⁶ cells/well). After 24 h, the cells were harvested and incubated with FITC-conjugated antibodies for CD44 (cat. no. 12211-MM02-F; 1:200; Sino Biological, Inc.), CD24 (cat. no. 655154; 1:200; BD Biosciences) or EpCAM (cat. no. 10694-MM06-F; 1:200; Sino Biological, Inc.) at 26°C for 30 min. The fluorescence intensity was determined using a NovoCyte flow cytometer (ACEA Biosciences; Agilent Technologies, Inc.) and NovoExpress software (1.4.1; ACEA Biosciences; Agilent Technologies, Inc.).

Total RNA was isolated from MCF-10A, MDA-MB-436, MDA-MB-231, MDA-MB-468, MDA-MB-453 and MCF-7 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RNA was reverse transcribed to cDNA using a reverse transcriptase cDNA synthesis kit (cat. no. RR036Q; Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was conducted on the obtained cDNA with a Roche Lightcycler 480 system (Roche Diagnostics) and TB Green^® premix (cat. no. RR820Q; Takara Biotechnology Co., Ltd.). The qPCR program included preheating (95°C for 30 sec), 40 cycles of heating (95°C for 5 sec), cooling and extension (60°C for 30 sec), and dissolution curve procedures (95°C for 5 sec, 60°C for 60 sec and 95°C for 1 sec). The relative expression levels of mRNA were calculated using the 2^−ΔΔCq method (19). The qPCR primers are listed in Table II with 18S used as the reference gene.

MCF-10A, MDA-MB-436, MDA-MB-231, MDA-MB-468, MDA-MB-453 and MCF-7 cells; stable OTUD7B-knockdown MDA-MB-468, MDA-MB-453 and MCF7 cells; stable OTUD7B-knockdown and FOXM1-overexpressing MDA-MB-468, MDA-MB-453 and MCF7 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Beyotime Institute of Biotechnology). Protein quantification was performed using the bicinchoninic acid method. A total of 20 µg protein/lane was separated by SDS-PAGE on 10% gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk at 25°C for 1 h, the membranes were incubated with primary antibodies against OTUD7B (cat. no. 16605-1-AP; 1:1,000), Nanog (cat. no. 14295-1-AP; 1:1,000), SOX2 (cat. no. 11064-1-AP; 1:1,000), Flag (cat. no. 20543-1-AP; 1:20,000), HA (cat. no. 51064-2-AP; 1:5,000), His (cat. no. 10001-0-AP; 1:1,000), β-actin (cat. no. 20536-1-AP; 1:1,000), β-tubulin (cat. no. 10094-1-AP; 1:2,000), FOXM1 (cat. no. 13147-1-AP; 1:2,000) or GAPDH (cat. no. 10494-1-AP; 1:5,000) (all from Proteintech Group, Inc.) at 25°C for 2 h, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. SA00001-2; 1:2,000; Proteintech Group, Inc.) at 25°C for 1 h. An enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) was used to visualize the membranes. ImageJ v1.48 (National Institutes of Health) was used for densitometry. β-actin, β-tubulin and GAPDH were used as internal reference proteins.

MDA-MB-468 cells were seeded in 6-well plates (2×10⁶ cells/well) overnight at 37°C and then transfected with 1.6 µg pCDNA3.1 vector, pCDNA3.1-HA-OTUD7B, pCDNA3.1-Flag-FOXM1 or His-Ub per well with Lipofectamine 2000 following the manufacturer's instructions for 24 h at 37°C. Subsequently, cells were harvested and lysed in immunoprecipitation buffer (150 mmol/l NaCl; 50 mmol/l Tris-HCl, Ph 7.4; 40 mmol/l β-glycerophosphate; 1 mmol/l Na₄OV₃; 10 mmol/l NaF; 2 mmol/l EDTA) supplemented with 1 mmol/l PMSF and protease inhibitor on ice for 30 min. The cells in immunoprecipitation buffer were centrifuged at 4°C at 14,000 × g for 15 min, and the supernatant was immediately transferred to a new centrifuge tube to obtain the cell lysate. protein A/G agarose beads (50 µl; Beyotime Institute of Biotechnology) were incubated with anti-HA (cat. no. 66006-2-Ig; 1 µg) or anti-Flag (cat. no. 66008-4-Ig; 1 µg) (both from Proteintech Group, Inc.) at 4°C overnight, followed by incubation with 500 µl cell lysates at 4°C for 6 h. The mixture was centrifuged at 14,000 × g at 4°C for 15 min and the supernatant was discarded. The agarose bead-antigen-antibody complex was then cleaned three times with immunoprecipitation buffer. After the supernatant was discarded, the precipitation was added to 60 µl 2X loading buffer, heated at 100°C for 5 min, and then subjected to western blotting.

MDA-MB-468 cells were seeded in 6-well plates (2×10⁶ cells/well) overnight at 37°C and then transfected with 1.6 µg pCDNA3.1 vector, pCDNA3.1-HA-OTUD7B or pCDNA3.1-Flag-FOXM1 with Lipofectamine 2000 for 24 h at 37°C, according to the manufacturer's instructions. Subsequently, the cells were cultured in medium containing 50 µg/ml cyclohexane (MilliporeSigma) for 0, 2, 4, 8 and 12 h at 37°C. Subsequently, the cells were collected for western blotting. Subsequently, ImageJ v1.48 was used to calculate FOXM1 protein levels at different time points.

The experiments were independently repeated three times. Data are presented as the mean ± standard deviation. Unpaired Student's t-test was used for comparisons between unpaired groups, paired Student's t-test was used for comparisons between paired groups, and one-way ANOVA followed by Tukey's post-hoc test was used for comparisons among multiple groups. Prior to statistical analysis, the normality of data was verified by the Shapiro-Wilk test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism v5.0 (Dotmatics).

To clarify the role of OTUD7B in breast carcinogenesis, first the expression levels of OTUD7B in breast cancer tissues and cells were examined. Western blotting showed that the expression levels of OTUD7B were higher in breast cancer tissues than those in adjacent normal tissues (Fig. 1A). Next, the mRNA and protein expression levels of OTUD7B were analyzed in several breast cancer cell lines. OTUD7B mRNA and protein expression levels were increased in breast cancer cell lines compared with those in MCF-10A cells (Fig. 1B and C). Among the breast cancer cell lines, MDA-MB-468, MDA-MB-453 and MCF-7 cells showed relatively higher OTUD7B expression levels; therefore, these three cell lines were selected for further analysis.

A previous study has shown that OTUD7B maintains the stemness of neural progenitor cells by removing polyubiquitin conjugates from SOX2 protein, increasing its stability (20). Thus, whether OTUD7B affects the stemness of MDA-MB-468 cells was investigated. Western blotting showed that shOTUD7B was successfully transfected into MDA-MB-468 cells and reduced the protein expression levels of OTUD7B in the cells (Fig. 2A). The protein expression levels of SOX2 and Nanog, two stemness-associated proteins, were markedly suppressed by OTUD7B knockdown (Fig. 2A). Furthermore, the mean fluorescence intensity of CD44 and EpCAM were also significantly decreased in MDA-MB-468 cells with stable knockdown of OTUD7B (Fig. 2B and C). Sphere formation assays showed that the tumorsphere diameters of MDA-MB-468 cells were significantly reduced by OTUD7B knockdown (Fig. 2D). The impact of OTUD7B on cell proliferation was investigated and it was found that OTUD7B knockdown inhibited the proliferation and colony formation abilities of MDA-MB-468 cells (Fig. 2E and F). These data indicated that OTUD7B knockdown may suppress the stemness and proliferation of MDA-MB-468 cells.

FOXM1 is a transcriptional activator that is involved in the stemness of several types of human cancer cells (21), and its degradation rates are accelerated in OTUD7b-depleted cells (22). Whether FOXM1 is involved in the effects of OTUD7B on the stemness of breast cancer cells was explored in the present study, as was whether OTUD7B interacts with FOXM1 in breast cancer cells. HA-OTUD7B and Flag-FOXM1 plasmids were co-transfected into breast cancer cells and the interaction between OTUD7B and FOXM1 was detected by co-IP experiments. FLAG-tagged FOXM1 was detected in the HA-OTUD7B immunoprecipitates and vice versa (Fig. 3A).

OTUD7B is a deubiquitinase; therefore, whether OTUD7B affects FOXM1 ubiquitination and stability was explored. As shown in Fig. 3B, in the immunoprecipitate pulled down by the Flag antibody, the His-Ub level in the group with overexpression of HA-OTUD7B was markedly lower than that in the group without overexpression of HA-OTUD7B, indicating that OTUD7B reduced the ubiquitination level of Flag-FOXM1. Protein biosynthesis was pharmacologically inhibited using cycloheximide in MDA-MB-468 cells and FOXM1 protein levels over 12 h with or without OTUD7B overexpression were evaluated. It was found that Flag-FOXM1 protein levels consistently decreased from 0 to 12 h after cycloheximide treatment and this decrease was significantly alleviated by OTUD7B overexpression in MDA-MB-468 cells (Fig. 3C). These findings suggested that OTUD7B stabilizes FOXM1 protein by reducing its polyubiquitination.

To examine the role of FOXM1 in the OTUD7B-mediated effects on the proliferation and stemness of breast cancer cells, the effects of FOXM1 overexpression on the expression of stemness markers were analyzed. It was revealed that the protein expression levels of SOX2, Nanog, CD44, CD24 and EpCAM were increased by FOXM1 overexpression in OTUD7B-knockdown breast cancer cells (Figs. 4 and 5). The reduced tumorsphere diameter of OTUD7B-knockdown breast cancer cells was also reversed by FOXM1 overexpression (Fig. 6).

To determine the role of FOXM1 in OTUD7B knockdown-mediated cell proliferation inhibition, FOXM1 was overexpressed in OTUD7B-knockdown breast cancer cells and the proliferation of the cells was analyzed by CCK8 assay. As shown in Fig. 7A-C, OTUD7B knockdown inhibited the proliferation of MDA-MB-468, MDA-MB-453 and MCF-7 cells, whereas the overexpression of FOXM1 blocked the inhibitory effect induced by OTUD7B knockdown. Similar results were observed in colony formation assays, which showed that overexpression of FOXM1 alleviated the inhibitory effect of OTUD7B silencing on the colony formation ability of MDA-MB-468, MDA-MB-453 and MCF-7 cells (Fig. 7D).