Abstract
Breast cancer is the most common malignant tumor and the leading cause of cancer-related death in women. The ubiquitin-proteasome system regulates the stability of most proteins controlling various biological processes in human cells. PSMD7, as a core component of the 26S proteasome, is critical for the degradation of ubiquitinated proteins in the proteasome. Currently, PSMD7 expression and its roles in the progression of breast cancer remain largely unknown. In this study, we assessed the level of PSMD7 in breast cancer tissues and investigated the underlying molecular events by which PSMD7 could play a role in tumor progression. The results showed that the PSMD7 level was significantly upregulated in breast cancer tissues. PSMD7 expression was closely associated with tumor subtype, tumor size, lymph node invasion, and TNM stage. A high PSMD7 level predicted poor overall survival (OS) and disease-free survival (DFS) in breast cancer patients. Furthermore, univariate Cox regression analysis indicated that lymph node invasion, distant metastasis, and PSMD7 expression were associated with OS and DFS. Multivariate regression analysis indicated that PSMD7 was an independent predictor of OS (HR=1.310, 95% CI=1.038-1.652). Importantly, PSMD7 knockdown induced cell cycle arrest in the G0/G1 phase, leading to cell senescence and apoptosis. PSMD7 knockdown inhibited the expression of key cell cycle-related proteins and promoted the stability of p21 and p27 in breast cancer cells. PSMD7 may be a valuable prognostic indicator and potential therapeutic target for breast cancer.
Keywords: Deubiquitinase, PSMD7, breast cancer, prognosis, cell cycle-related proteins
Introduction
Breast cancer is a complex malignant tumor derived from breast epithelial tissue. It is for the most common malignant cancer and the leading cause of cancer-related death in women. According to the Global Cancer Statistics of 2018, nearly every one in four cancer cases was breast cancer, and almost one in six cancer deaths was breast cancer related [1]. Four main subtypes of breast cancer are normally defined based on different hormone receptor levels: estrogen receptor (ER)-positive or progesterone receptor (PR)-positive and human epidermal growth factor receptor 2 (HER-2)-negative breast cancer is defined as luminal A; ER- or PR-positive and HER-2-positive breast cancer is defined as luminal B; HER-2-positive only breast cancer is defined as luminal HER-2; and breast cancer negative for all three receptors is defined as basal [2]. Currently, the main treatments for breast cancer are surgery, radiotherapy, cytotoxic drugs, and targeted therapy. With early screening, diagnosis, and intervention for breast cancer, the mortality and recurrence of breast cancer have been significantly reduced. However, many breast cancer patients do not benefit from adjuvant therapy due to the absence of well-defined molecular targets [3]. New molecular targets are urgently needed to assess the progression and improve the treatment of the disease.
The ubiquitin-proteasome system plays an essential role in a variety of biological processes. More than 80% of cellular proteins are degraded through this important pathway, which is involved in regulating cellular function and maintaining homeostasis. The 26S proteasome is a multisubunit complex with a molecular weight of 2.5 MDa and is composed of two 19S regulatory particles and a 20S core particle [4]. Importantly, the 26S proteasome is critical for the degradation of substrates marked by polyubiquitin chains. Many studies have shown that the 26S proteasome is involved in cell cycle progression, apoptosis, transcription, DNA repair, protein quality control, and antigen presentation [5,6]. Due to the critical role of the 26S proteasome in cell biological processes, especially in tumor cell growth and survival, inhibition of proteasome function has become a powerful strategy for anticancer therapy. Bortezomib, a proteasome inhibitor, has emerged as an effective drug target for clinical treatment. It can induce cell cycle arrest and apoptosis by disrupting various signaling pathways and NF-kB function in multiple myeloma [7].
Proteasome 26S subunit, non-ATPase 7 (PSMD7), also known as Rpn8 or Mov34, is an ATP-independent component of the 19S regulatory subunit and interacts with PSMD14 to form heterodimers as a functional complex that is extremely critical for ubiquitinated substrate degradation in the proteasome [8,9]. Our recent study revealed that an upregulated PSMD14 level was associated with progressive disease status in lung adenocarcinoma via the regulation of p21 stability [10]. Although the role of PSMD14 as a component of the proteasome has been widely reported in many studies [11-13], the function of PSMD7 remains largely unknown. A previous study revealed that PSMD7 regulated the G2/M phase cell cycle transition during HIV infection [14]. The polymorphism rs17336700 in the PSMD7 gene is thought to be associated with ankylosing spondylitis [15]. PSMD7 knockdown induced cell apoptosis and inhibited tumorigenesis in esophageal squamous cell carcinoma [13]. However, PSMD7 expression in breast cancer tissues and its role in tumorigenesis remain to be determined. In this study, we explored the expression of PSMD7 and its potential role in modulating cell fate in breast cancer. We found that PSMD7 was significantly overexpressed in cancer tissues and that a high PSMD7 level was associated with poor clinical stages, OS, and DFS in breast cancer. The knockdown of PSMD7 can lead to cell cycle arrest, senescence, and apoptosis via regulation of key cell cycle proteins in breast cancer cells. These findings indicate that PSMD7 could be a prognostic indicator and promising therapeutic target in breast cancer.
Materials and methods
Patients
To determine PSMD7 expression in breast cancer, we purchased human breast invasive ductal carcinoma (IDC) tissue microarrays (#HBreD090CS01, Shanghai Outdo Biotech Company, Shanghai, China). The demographic and clinicopathological features of the patients in this study are shown in Table S1. We also used another cohort of 8 breast cancer patients enrolled at the Affiliated Zhangjiagang Hospital of Soochow University from September 2018 to November 2018. All of the patients underwent curative surgery for breast cancer without any previous adjuvant therapy. The diagnoses were confirmed by at least two experienced histological pathologists at the hospital. All of the oncology stages were determined according to the NCCN Breast Cancer Guidelines (http://www.NCCN.org). Breast cancer tissues were carefully isolated during surgery, and matched noncancerous breast tissues were collected at least one centimeter away from the edge of the lesion. The tissue samples were immediately stored at -80°C for further extraction of proteins and immunohistochemistry. The demographic, clinical, and pathological features of the patients are shown in Table S2. The research on human specimens was approved by Zhangjiagang Hospital’s Institutional Review Board (No. 2019001). Written permission was requested and obtained from all of the patients in this study.
Bioinformatics analysis
We used publicly available clinical information from the cBioportal for Cancer Genomics (TCGA) database (http://www.cbioportal.org/) [16,17], including basic demographic and clinical information as well as survival status after surgery to assess the correlations between PSMD7, clinical pathological status, and patient survival in breast cancer. We retrieved clinical information from 1,082 breast cancer patients for correlation analysis. We selected the tumor patients in the upper and lower quartiles of PSMD7 expression and obtained 540 patients for OS analysis and 464 patients for DFS analysis. Patients with complete demographic and clinical information were also used for univariate and multivariate Cox regression analyses for OS and DFS, respectively.
Cell culture
The breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, Corning, NY, USA) supplemented with 10% FBS and antibiotics consisting of 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere at 37°C with 5% CO2. The cells were passaged at 75-80% confluency every 2 to 3 days.
Western blotting and antibodies
Total proteins from the tissues and cells were isolated using FLAG lysis buffer (50 mM Tris·HCl, pH 7.9, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, and 10% glycerol) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were sonicated for 10 s and then incubated on ice for 30 min. Supernatants were collected by centrifuging at 12000 × g at 4°C for 30 min. The protein concentrations were measured using a BCA protein assay kit (23227, Pierce, Rockford, IL, USA). All of the protein samples were equalized for total protein after protein quantitation and subjected to 10% SDS/PAGE. Nitrocellulose membranes (GE Healthcare, Munich, Germany) were used to transfer the proteins via Western blotting. The membranes were blocked in 5% skim milk for 1 h at room temperature and then incubated overnight at 4°C with gentle shaking in diluted primary antibodies (1:500) against PSMD7 (sc-390705), Cyclin D1 (sc-450), Cyclin B1 (sc-7393), Cdk4 (sc-23896), Cdk1 (sc-54), Cdc25c (sc-13138), p53 (sc-126), p27 (sc-1641), p21 (sc-53870), Rb (sc-47562) (all obtained from Santa Cruz Biotechnology, Dallas, TX, USA), phospho-Rb (9308, Cell Signaling Technology, Danvers, MA, USA), caspase-3 (19677, Proteintech, Rosemont, IL, USA), cleaved caspase-3 (9661, Cell Signaling Technology, Danvers, MA, USA), and β-actin (A5441, Sigma-Aldrich). The membranes were incubated with horseradish peroxidase-labeled secondary antibodies after extensive washes and detected using an enhanced chemiluminescent kit (ECL, WBKLS0500, Millipore, Bedford, MA, USA) with a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) detection system. The signals were analyzed and quantified via ImageJ software (National Institutes of Health, Bethesda, MD, USA) with β-actin as a loading control.
Immunohistochemistry (IHC)
The frozen tissues were sectioned at -20°C to a thickness of 10 µm and fixed in 4% paraformaldehyde for 15 min at room temperature. The slides were washed twice in phosphate-buffered saline (PBS) containing 0.3% Triton X-100 for 10 min each, followed by preincubation in 10% normal goat serum for 1 h. They were then washed 3 times with PBS and incubated overnight at 4°C with primary PSMD7 antibody at 1:100. Endogenous peroxidases were blocked with 0.3% H2O2 for 15 min. Biotinylated secondary antibody was added to the sections after extensive washing. Solutions containing streptavidin and biotin-conjugated HRP (Beyotime, China) was then added to the sections after washing 3 times. After incubation with the solution for 1 h, the sections were developed in 3’-diaminobenzidine (DAB, Beyotime, China) for 30 min after extensive washing. The reaction was stopped by rinsing in H2O for 5 min. The sections were counterstained in hematoxylin, dehydrated, cleared, and mounted in neutral resin. A Leica upright microscope (DM4000B, Leica Microsystems, Heidelberg, Germany) was used to observe the immunohistochemical staining. For tissue microarray IHC staining, 4 µm paraffin embedded tissue sections were stained with PSMD7 primary antibody at a dilution of 1:25 after deparaffinization and antigen retrieval. The protein staining percentage was scored from 0 to 3 for each section. A score of 0 was given to samples with no positive cells or a percentage of positive cells ≤5%. A score of 1 was given to cases whose percentage of positive cells was 6%-25%. If the average percentage of positive cells was >25% but ≤50%, then the expression was scored as 2, whereas the percentage of positive cells >50% but ≤75% was scored as 3. Finally, >75% positive staining percentage was scored as 4. Protein staining density was scored from 0 to 2 as follows: no staining was scored as 0, light brown as 1 and dark brown as 2. The final scores were calculated as the product of the staining percentage and intensity scores for each section. All of the sections were independently assessed by two pathologists. A mean score was used if controversial results were obtained.
siRNA transfection
siRNA duplexes targeting PSMD7 mRNA and one negative control scramble sequence were designed and synthesized to knock down the expression of target genes. MCF-7 and MDA-MB-231 cells were transfected by an Amaxa Cell Line Nucleofector Kit V (VCA-1003, Lonza, Cologne, Germany) according to the manufacturer’s instructions. The specific sequences of the siRNAs were as follows: siPSMD7-1: 5’-GCCCUAAACUACACAAGAAUU-3’ and 5’-UUCUUGUGUAGUUUAGGGCUU-3’; siPSMD7-2: 5’-UGACAUUGCCAUCAACGAAUU-3’ and 5’-UUCGUUGAUGGCAAUGUCAUU-3’; and NC: 5’-UUCUCCGAACGUGUCACGUTT-3’ and 5’-ACGUGACACGUUCGGAGAATT-3’.
Cell proliferation analysis
Cell proliferation assays were conducted using a kit containing WST-8 (CCK-8, CK04, Dojindo, Kumamoto, Japan). MCF-7 and MDA-MB-231 cells were seeded in 96-well plates at 1,500 cells per well after siRNA transfection. Cell proliferation was detected over the next 4 days following the manufacturer’s protocol. The absorbance of each well was measured at 450 nm as the optical density (OD) of CCK-8 under a microplate reader (Bio-Rad Laboratories). The results were analyzed and are shown as the mean ± SD.
Cell viability
A crystal violet assay was used to measure the cell viability after siRNA transfection in breast cancer cells. MCF-7 and MDA-MB-231 cells were seeded in 6-well plates at a density of 50,000 cells per well after transfection. The cells were fixed with methanol and then stained with 0.1% crystal violet for a specific time. Various time points were selected to compare and show the difference. MCF-7 cells were stained on the third day, while MDA-MB-231 cells were stained on the fifth day after siRNA transfection.
Flow cytometric analysis
MCF-7 and MDA-MB-231 cells were harvested 72 h after transfection. Both floating and adherent cells were collected using centrifugation at 1200 × g for 10 min. Then, 70% ice cold ethanol diluted with PBS was used to fix the cells. The cells were stained with PI/RNase staining buffer (BD Biosciences, San Diego, CA, USA) and incubated at 37°C for 30 min. A Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA) was used to analyze the results.
Senescence-associated β-galactosidase (SA-β-Gal) assay
Senescent cells were detected using a SA-β-Gal assay kit (C0602, Beyotime, Nantong, China) 72 h after transfection in accordance with the manufacturer’s instructions. Specific cells stained by β-galactosidase were viewed under an inverted fluorescence microscope (DMI4000B, Leica Microsystems). Representative areas were captured under different conditions. The SA-β-Gal-positive senescent cells were calculated and expressed as a percentage of the total number of cells in five separate fields. Two independent experiments were conducted to calculate the average value and standard deviation of the SA-β-Gal-positive cells.
Quantitative RT-PCR (real-time PCR)
Total RNA was extracted from the harvested cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Subsequently, cDNAs were synthesized using a Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis kit (K1632, Thermo Fisher, Waltham, MA, USA). RT-PCR was performed on a QuantStudio Dx Real-Time PCR instrument (Applied Biosystems) using standard SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) following the following cycle conditions: 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The results were analyzed by the comparative cycle threshold method, and β-actin was used as an internal control. The sequences of primers (Sangon, Shanghai, China) were as follows: PSMD7 F: 5’-TTGGAGCAGAGGAAGCTGAG-3’, R: 5’-CTGACATCTGGCAGCAGGTT-3’; Cyclin D1, F: 5’-ACGAAGGTCTGCGCGTGTT-3’, R: 5’-CCGCTGGCCATGAACTACCT-3’; Cdk4, F: 5’-CCTGGCCAGAATCTACAGCTA-3’, R: 5’-ACATCTCGAGGCCAGTCATC-3’; Cyclin B1, F: 5’-AAGAGCTTTAAACTTTGGTCTGGG-3’, R: 5’-CTTTGTAAGTCCTTGATTTACCATG-3’; Cdk1, F: 5’-TGGATCTGAAGAAATACTTGGATTCTA-3’, R: 5’-CAATCCCCTGTAGGATTTGG-3’; Cdc25C, F: 5’-GATGTCCCTAGAACTCCAGTG-3’, R: 5’-AGTTATCTCCCCACTGCTAAGA-3’; Rb, F: 5’-AGGATCAGATGAAGCAGATGG-3’, R: 5’-TGCATTCGTGTTCGAGTAGAAG-3’; p21, F: 5’-CCATGTGGACCTGTCACTGTCTT-3’, R: 5’-CGGCCTCTTGGAGAAGATCAGCCG-3’; p27, F: 5’-TTTGACTTGCATGAAGAGAAGC-3’, R: 5’-AGCTGTCTCTGAAAGGGACATT-3’ and β-actin, F: 5’-AGAGCTACGAGCTGCCTGAC-3’, R: 5’-AGCACTGTGTTGGCGTACAG-3’.
P21 and p27 stability assay
MCF-7 cells were treated with 50 µg/ml cycloheximide (CHX) to block protein synthesis 48 h after transfection. The cells were harvested at specific time points and the p21 and p27 levels were determined via Western blotting. β-Actin was used as an internal reference.
Statistical analysis
The data are shown as the mean ± SD. ImageJ (version 1.8.0_112), GraphPad Instat software (GraphPad Prism 5.01, GraphPad Software Inc., San Diego, CA, USA), and SPSS statistics 17.0 were used to conduct the statistical analysis. The results were calculated by Student’s t-test for comparisons between two groups and one-way ANOVA for multiple groups. Chi-squared or Fisher’s exact test was used to analyze the correlation between PSMD7 and demographic and clinical features. Survival curves were compared using the log-rank test. Univariate and multivariate Cox regression were also used for survival analysis. A P value <0.05 was considered statistically significant.
Results
PSMD7 expression is upregulated in breast cancer tissues
PSMD7 is a key functional component of the 26S proteasome for protein degradation. To explore PSMD7 expression in breast cancer, we conducted an IHC analysis using tissue microarrays in a cohort of IDC patients. IHC staining data from 3 patients was missing from the microarray. PSMD7 expression was detected in both tumor and paired noncancerous tissues from 42 breast cancer patients (Figures 1A and S1). The results demonstrated that PSMD7 expression was upregulated in 40 (95.2%), unchanged in 1 (2.4%) and downregulated in 1 (2.4%) out of 42 patients. Thus, the PSMD7 level was significantly elevated in breast cancer tissues compared with noncancerous tissues (P<0.001, chi-squared test; Figure 1B). PSMD7 staining was observed in both the cytoplasm and nucleus in cells from tumor and noncancerous tissues. Stronger staining was detected in acinar cells and epithelial cells, and weaker staining was observed in fibroblasts and fat cells from the noncancerous tissues. PSMD7 staining in the tumor cells was much stronger than that in the controls. We also used frozen tissues from another cohort of 8 patients to perform IHC staining and Western blot analysis. PSMD7 staining was consistently stronger in the tumor tissues than in the controls (Figure S2). PSMD7 expression increased in 6 of 8 tumor tissues in comparison with the controls, as demonstrated by Western blotting (Figure 1C).
Figure 1.
PSMD7 expression is upregulated in breast cancer tissues. A. Typical IHC staining of PSMD7 in paired tumor and adjacent noncancerous tissues. IHC staining was performed in 45 pairs of tissues, and a representative image of three pairs is shown. The bar represents 100 µm. B. PSMD7 expression by IHC staining from a tissue microarray was scored in 42 pairs of breast cancer tissues (T) and adjacent noncancerous tissues (N) as referenced in the Materials and Methods section. Three pairs of tissues (A9 and A10; A11 and A12; B11 and B12) were excluded from the analysis due to missing staining data. C. PSMD7 protein expression in 8 paired tumor and adjacent noncancerous tissues from breast cancer patients was determined by Western blotting. β-Actin was used as an internal control. IHC: immunohistochemistry.
PSMD7 level is associated with clinical features and prognosis in breast cancer
We searched the TCGA database to analyze the relationship between the expression of PSMD7 and demographic and clinicopathological characteristics. The results showed that PSMD7 expression was associated with age, tumor subtype, tumor size, lymph node invasion, and tumor TNM stage (Table 1). A higher level of PSMD7 indicated a more progressive disease status in breast cancer. To explore the prognostic significance of PSMD7 expression in breast cancer patients, we conducted a long-term outcome analysis by data mining in the TCGA database. Our results demonstrated that patients with higher PSMD7 expression showed a significantly poorer OS and DFS compared to patients with a lower PSMD7 expression level (Figure 2). Furthermore, tumor subtype, lymph node invasion, distant metastasis, TNM stage, and PSMD7 expression were statistically associated with OS in the univariate Cox regression analysis. The multivariate Cox regression analysis indicated that prior malignancy history, lymph node invasion, distant metastasis, and PSMD7 expression were independent prognostic factors of OS in these patients (Table 2). Age, lymph node invasion, distant metastasis, and PSMD7 expression were associated with DFS in the univariate Cox regression analysis. However, age was the only independent predictor of DFS in the multivariate model. Distant metastasis and PSMD7 expression showed a marginally significant difference for DFS in breast cancer (Table 3).
Table 1.
The expression of PSMD7 correlated with demographic and clinical pathological characteristics in breast cancer patients
| PSMD7 High | PSMD7 Low | P value | |
|---|---|---|---|
| Age | |||
| ≥50 | 372 | 417 | 0.002* |
| <50 | 169 | 124 | |
| Prior malignancy history | |||
| Yes | 22 | 35 | 0.102 |
| No | 518 | 506 | |
| Tumor subtype | |||
| Normal | 17 | 19 | <0.001* |
| LumA | 137 | 362 | |
| LumB | 117 | 80 | |
| HER2 | 67 | 11 | |
| Basal | 153 | 18 | |
| Tumor size | |||
| T1 | 116 | 160 | 0.002* |
| T2-T4 | 424 | 379 | |
| Lymph node invasion | |||
| Negative | 235 | 277 | 0.010* |
| Positive | 296 | 254 | |
| Distant metastasis | |||
| Negative | 445 | 449 | 0.332 |
| Positive | 16 | 11 | |
| Tumor TNM stage | |||
| I | 67 | 113 | <0.001* |
| II-IV | 464 | 419 | |
| Radiation treatment | |||
| Yes | 212 | 214 | 0.818 |
| No | 47 | 45 |
The clinical information from 1,082 breast cancer patients was retrieved from the cBioportal for the Cancer Genome Atlas (TCGA) database for correlation analysis. The chi-square test was used to compare differences;
P<0.05 was considered significant.
Figure 2.
High PSMD7 expression indicates poor OS and DFS in breast cancer patients. Kaplan-Meier survival curves of upper and lower PSMD7 expression for OS and DFS in breast cancer patients. The patients were stratified into upper and lower quartiles for PSMD7 expression, resulting in 540 patients used for OS analysis and 464 patients for DFS analysis. The patient clinical information and PSMD7 expression were retrieved from the cBioportal for Cancer Genomics (TCGA) database and analyzed by a log-rank test. P<0.05 was considered statistically significant. OS: overall survival; DFS: disease-free survival.
Table 2.
Univariate and multivariate Cox regression analyses of prognostic factors of overall survival (OS) in breast cancer patients
| Parameters | n | Univariate regression analysis | Multivariate regression analysis (n=363) | ||
|---|---|---|---|---|---|
|
|
|
||||
| HR (95% CI) | P value | HR (95% CI) | P value | ||
| Age | 502 | 1.328 (0.786-2.244) | 0.289 | - | - |
| Prior malignancy history | 501 | 2.246 (0.893-5.644) | 0.085 | 3.945 (1.104-14.094) | 0.035* |
| Tumor subtype | 452 | 0.097 | 0.978 | ||
| LumA | 1.250 (0.159-9.799) | 0.832 | 0.000 (0.000-1.057E+278) | 0.974 | |
| LumB | 1.162 (0.571-2.363) | 0.679 | 1.202 (0.418-3.457) | 0.733 | |
| HER2 | 1.331 (0.539-3.288) | 0.536 | 1.002 (0.338-2.970) | 0.997 | |
| Basal | 3.007 (1.243-7.274) | 0.015* | 1.410 (0.424-4.691) | 0.575 | |
| Tumor size | 501 | 1.276 (0.749-2.175) | 0.369 | - | - |
| Lymph node invasion | 491 | 2.933 (1.725-4.988) | <0.001* | 2.652 (1.157-6.084) | 0.021 |
| Distant metastasis | 426 | 7.700 (3.629-16.341) | <0.001* | 5.832 (2.239-15.190) | <0.001* |
| TNM stage | 494 | 2.220 (1.060-4.647) | 0.034* | 1.483 (0.389-5.654) | 0.564 |
| Radiation treatment | 478 | 0.653 (0.393-1.084) | 0.099 | 0.556 (0.289-1.073) | 0.080 |
| PSMD7 expression | 502 | 1.205 (1.056-1.375) | 0.006* | 1.310 (1.038-1.652) | 0.023* |
Data from a total of 540 breast cancer patients with demographic and clinical information were extracted from the cBioportal for Cancer Genomics (TCGA) database for univariate and multivariate Cox regression analyses. Normal subtype was used as a contrast indicator (reference category). HR: hazard ratio, CI: confidence interval.
P<0.05 was considered significant.
Table 3.
Univariate and multivariate Cox regression analyses of prognostic factors of disease-free survival (DFS) in breast cancer patients
| Parameters | n | Univariate regression analysis | Multivariate regression analysis (n=369) | ||
|---|---|---|---|---|---|
|
|
|
||||
| HR (95% CI) | P value | HR (95% CI) | P value | ||
| Age | 449 | 0.435 (0.232-0.816) | 0.010* | 0.367 (0.180-0.746) | 0.006* |
| Prior malignancy history | 448 | 1.168 (0.280-4.872) | 0.832 | - | - |
| Tumor subtype | 407 | 0.939 | - | - | |
| LumA | 0.000 (0.000-3.213E+302) | 0.975 | - | - | |
| LumB | 0.851 (0.367-1.972) | 0.706 | - | - | |
| HER2 | 0.628 (0.189-2.089) | 0.449 | - | - | |
| Basal | 1.134 (0.300-4.289) | 0.854 | - | - | |
| Tumor size | 449 | 1.740 (0.799-3.788) | 0.163 | - | - |
| Lymph node invasion | 444 | 2.130 (1.077-4.215) | 0.030* | 1.288 (0.594-2.792) | 0.521 |
| Distant metastasis | 373 | 9.585 (1.293-71.046) | 0.027* | 6.442 (0.794-52.244) | 0.081 |
| TNM stage | 445 | 2.564 (0.907-7.242) | 0.076 | 2.254 (0.621-8.184) | 0.217 |
| Radiation treatment | 430 | 0.897 (0.476-1.690) | 0.736 | - | - |
| PSMD7 expression | 449 | 1.197 (1.002-1.430) | 0.048* | 1.227 (0.996-1.511) | 0.054 |
Data from a total of 464 breast cancer patients with demographic and clinical information were extracted from the cBioportal for Cancer Genomics (TCGA) database for univariate and multivariate Cox regression analyses. The normal subtype was used as a contrast indicator (reference category). HR: hazard ratio, CI: confidence interval.
P<0.05 was considered significant.
PSMD7 knockdown induces cell cycle arrest, senescence, and apoptosis in breast cancer cells
To investigate the effects of PSMD7 on the growth of breast cancer cells, we knocked down the expression of PSMD7 in both MCF-7 and MDA-MB-231 cell lines via siRNA interference. Cell proliferation was examined by CCK-8 assays on the next four consecutive days after siRNA transfection. The results showed that PSMD7 knockdown significantly inhibited cell proliferation in both cell lines (Figure 3A). We further confirmed the results using crystal violet staining, where PSMD7 knockdown cells showed significantly fewer colonies than the controls (Figure 3B). To further clarify the mechanisms involved in proliferation inhibition, we conducted a flow cytometric analysis by PI staining to detect the cycle profile after PSMD7 was knocked down in the cells. Our results demonstrated that PSMD7 knockdown induced cell cycle arrest at the G0/G1 phase in MCF-7 cells and led to a slight increase in the number of apoptotic cells (sub-G1 phase). Although cell apoptosis was markedly increased in MDA-MB-231 cells after PSMD7 knockdown, the proportion of cells in the G0/G1 phase remained unchanged (Figure 3C). Finally, we detected cell senescence via SA-β-Gal staining after PSMD7 knockdown. Both breast cancer cell lines were stained with SA-β-Gal 72 h after transfection. We found that the cell morphology changed from a spindle shape to an enlarged, flattened, and irregular shape after PSMD7 was knocked down in MCF-7 cells but not MDA-MB-231 cells (Figure 4A). PSMD7 knockdown induced a significant increase in the SA-β-Gal-positive cells among MCF-7 cells, whereas we did not observe obvious SA-β-Gal staining either in the controls or in PSMD7 knockdown MDA-MB-231 cells (Figure 4A and 4B).
Figure 3.
PSMD7 knockdown induces cell cycle arrest and apoptosis in breast cancer cells. A. The viability of MCF-7 and MDA-MB-231 cells transfected with NC and two siRNA sequences against PSMD7 (si-PSMD7-1 and si-PSMD7-2) was assessed by CCK-8 assays. The OD value was determined at 1, 2, 3 and 4 days post-transfection. The assays were performed at four replicates for each group. The experiments were repeated twice, and a representative result is shown. B. Crystal violet staining was conducted to assess the survival of MCF-7 and MDA-MB-231 cells transfected with NC, si-PSMD7-1 or si-PSMD7-2 for 72 hours. The experiments were repeated twice, and a representative staining is shown. C. MCF-7 and MDA-MB-231 cells were treated with NC, si-PSMD7-1 or si-PSMD7-2 for 72 hours. Cell cycle and apoptosis (sub-G1 phase) were determined by PI staining followed by FACS analysis (left panel). The percentage of cells in each phase of the cell cycle is also demonstrated (right panel). The experiments were repeated twice, and a representative result is shown. **represents P<0.01 by Student’s t-test. NC: scrambled control siRNA; PI: propidium iodide.
Figure 4.
PSMD7 knockdown induces cellular senescence in MCF-7 but not MDA-MB-231 cells. A, B. Exponentially growing MCF-7 and MDA-MB-231 cells were transfected with NC, si-PSMD7-1 or si-PSMD7-2. After 72 hours, the cells were fixed and incubated with SA-β-Gal staining solution overnight. The senescence-like phenotype was imaged by microscopy (100-fold). The number of SA-β-Gal-positive cells was counted and is expressed as a percentage of the total number of cells in five separate fields. The means and standard deviation of SA-β-Gal-positive cells were derived from two independent experiments. **represents P<0.01 by Student’s t-test; ns: not significant. SA-β-Gal: senescence-associated β-galactosidase.
PSMD7 knockdown regulates the expression of cell cycle proteins
We investigated the underlying molecular mechanism by which PSMD7 regulates the cell cycle, apoptosis, and senescence in breast cancer cells. PSMD7 forms heterodimers with PSMD14 to modulate protein degradation in the 26S proteasome. Previous research demonstrated that PSMD14 knockdown inhibited cell growth by regulating cell cycle proteins [18]. Thus, disruption of the PSMD14-PSMD7 complex may have a profound effect on these proteins. We evaluated Cyclin D1, Cdk4, Cyclin B1, Cdk1, Cdc25c, p27, p21, Rb, pRb, caspase-3, and cleaved caspase-3 protein expression after PSMD7 was depleted from breast cancer cells. Our analysis demonstrated that the expression of the cell cycle proteins Cyclin D1, Cdk4, Cyclin B1, Cdk1, and Cdc25c was significantly reduced in MCF-7 cells, whereas the expression of Cdk1 and Cdc25c was decreased in MDA-MB-231 cells when PSMD7 was knocked down (Figure 5A). Moreover, p27 and p21 levels were markedly increased in both cell lines, whereas cleaved caspase-3 showed a slight increase in MCF-7 cells and a dramatic increase in MDA-MB-231 cells (Figure 5A). Additionally, the levels of Rb and pRb showed a significant reduction in both cell lines (Figure 5A). To examine whether the knockdown of PSMD7 affects the transcriptional regulation of the cell cycle proteins, the mRNA expression of these proteins was also determined after PSMD7 was knocked down in both cell lines. The results indicated that the mRNA levels of Cyclin D1, Cdk4, Cyclin B1, Cdk1, and Cdc25c were significantly reduced 48 to 72 h after PSMD7 knockdown in both cell lines (Figure 5B and 5C), suggesting that PSMD7 may play a role in transcriptional repression. Although p21 and p27 protein levels were markedly elevated after PSMD7 silencing, we did not observe an increase in p21 or p27 mRNA expression in either cell type (Figure 5).
Figure 5.
A. Whole-cell lysates from MCF-7 and MDA-MB-231 cells were extracted after the cells were transfected with NC, si-PSMD7-1 or si-PSMD7-2 for 72 hours. Aliquots of 30 µg whole-cell lysates were subjected to SDS-PAGE followed by Western blot analysis of the indicated antibodies. β-Actin was used as a loading control. B, C. MCF-7 and MDA-MB-231 cells were transfected with NC or si-PSMD7-2 for 48 and 72 hours. The mRNA expression of cell cycle-related proteins was detected by RT-PCR. P values were calculated by Student’s t-test (*P<0.05, **P<0.01, and ***P<0.001). NC: scrambled control siRNA; RT-PCR: real-time PCR.
PSMD7 knockdown increases the stability of p21 and p27
P21 and p27 are the founding members of the Cip/Kip family of CKIs, which regulate cell cycle progression and cell senescence [19-23]. The stability of both proteins is regulated by ubiquitination and proteasome-mediated degradation [24]. Depletion of PSMD7 may cause proteasome malfunction, so it is of interest to explore whether knockdown of PSMD7 affects the stability of p21 and p27. We blocked protein synthesis using CHX 48 h after transfection and harvested the cells at a series of specific time points. PSMD7 was effectively knocked down by the siRNA sequence after transfection. The PSMD7 level remained steady even after the cells were treated with CHX for 5 h (Figure 6A). P21 and p27 protein expression gradually and markedly decreased after CHX treatment. Importantly, the stability of both proteins significantly increased after PSMD7 knockdown in breast cancer cells in comparison with that of the controls (Figure 6A and 6B). These results suggested that disruption of the proteasome by depleting PSMD7 may have a profound effect on the stability of cell cycle proteins.
Figure 6.
Knockdown of PSMD7 promotes p21 and p27 stability in breast cancer cells. A. MCF-7 cells were transfected with NC or si-PSMD7-2. After 24 hours, cells were treated with 50 µg/ml CHX and collected at the indicated time points. Western blot analysis was performed with p21, p27 and PSMD7 antibodies. β-Actin was used as an internal control. B. Quantification of the p21 and p27 levels relative to β-actin expression at indicated time points was determined by ImageJ software, and the degradation curve is shown. NC: scrambled control siRNA; CHX: cycloheximide.
Discussion
Breast cancer remains one of the most serious public health issues worldwide. With an annual incidence of more than 500,000 deaths, breast cancer is the most common cause of cancer-related death in women [25]. Although new drugs and treatment regimens have significantly relieved disease progression, a number of patients showed a poor prognosis and a higher risk of disease relapse [26]. Conducting personalized therapies on specific molecular subtypes is of considerable value [27]. Thus, identifying and evaluating new biomarkers and therapeutic targets is a high priority. We found that PSMD7 was significantly upregulated in breast cancer tissues. A high PSMD7 level was associated with a worse tumor subtype, larger tumor size, lymph node invasion, and advanced TNM stage. The increased PSMD7 expression in tumor tissues predicted poor OS and DFS in breast cancer patients. Thus, PSMD7 may be a potential prognostic marker for breast cancer patients.
Proteasome inhibitors have recently shown significant anticancer value [28-32]. PSMD7 and PSMD14 are the core components of the 26S proteasome and are closely connected. PSMD7 interacts with PSMD14 to activate proteasome function to regulate ubiquitinated substrate degradation. Although many studies have demonstrated that PSMD14 is an antiproteasome target for tumor therapies of different cancer types [11,12], the role and function of PSMD7 in tumorigenesis remains largely unknown. We found that knockdown of PSMD7 inhibited cell proliferation by inducing cell cycle arrest, senescence, and apoptosis in breast cancer cells. PSMD7 knockdown mainly induced G1 phase arrest and senescence in MCF-7 cells but promoted cell apoptosis in MDA-MB-231 cells, suggesting that the effects of PSMD7 on cell growth and survival were cell-type specific. The absence of p53 may result in a reduction in DNA damage repair and directly lead to cell apoptosis in MDA-MB-231 cells. In fact, we observed a slight increase in apoptotic cells in MCF-7 cells. The cleaved caspase 3 level was consistently elevated after PSMD7 was depleted in both cell lines. Further investigation is needed to clarify the underlying molecular events by which PSMD7 regulates cell apoptosis.
Cell cycle progression is tightly regulated by cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors. Cyclin D/Cdk4 regulates the transition from the G1 phase to the S phase, whereas Cyclin B/Cdk1 regulates the transition from the G2 phase to the M phase [33-35]. Rb, a G1 phase cell cycle inhibitor, is phosphorylated and activated, which disables the inhibition of transcription factors and allows the transcription of cell cycle genes, thus promoting the transition of cells from the G1 phase to the S phase. Cdc25c (cell division cycle 25), a tyrosine phosphatase dephosphorylated Cdk1, triggers mitosis [36,37]. We observed that both the mRNA and protein levels of these cell cycle-related proteins were markedly inhibited in PSMD7 knockdown cells, suggesting that PSMD7 may regulate gene transcription in a direct or indirect manner.
P21 and p27 are members of the Cdk inhibitor (CKI) family and negatively regulate the cell cycle [38,39]. P21 and p27 inhibit Cdk activity and induce cell cycle arrest, senescence, and apoptosis in response to various stimuli [40-43]. Both proteins are involved in tumor progression in breast cancer [44]. We determined that p21 and p27 significantly increased after PSMD7 was knocked down in breast cancer cells, implying that PSMD7-mediated CKI regulation may play a critical role in the tumorigenesis of breast cancer. Previous studies indicated that p21 and p27 caused Rb dephosphorylation and depletion [45]. We observed a consistent increase in p21 and p27 levels and a concomitant decrease in Rb and pRb in PSMD7-depleted cells. Although PSMD7 knockdown inhibited gene transcription, we found that elevated p21 and p27 levels were the result of the increased stability of both proteins. Previous studies indicated that the degradation of both proteins was highly dependent on an intact proteasome [46,47]. Depletion of PSMD7 may disrupt the proteasome, leading to malfunction of the protein degradation pathway in cells.
Conclusions
In summary, our results showed that the PSMD7 level was significantly upregulated in breast cancer tissues and closely related to clinical features. A higher PSMD7 level was associated with progressive disease and predicted poor survival in breast cancer patients. Knockdown of PSMD7 induced cell cycle arrest, senescence, and apoptosis by regulating cell cycle proteins in breast cancer cells. Our results indicate that PSMD7 may be a strong prognostic indicator and potential therapeutic target for breast cancer.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (81972624), Suzhou Science and Technology Development Program (SYSD2019001), and the Research Foundation for Advanced Talents by the Affiliated Zhangjiagang Hospital of Soochow University (ZKY201900) to Dawei Li.
Disclosure of conflict of interest
None.
Abbreviations
- PSMD7
proteasome 26S subunit, non-ATPase 7
- Rb
retinoblastoma protein
- Cdks
cyclin-dependent kinases
- CKI
Cdk inhibitor
- OS
overall survival
- DFS
disease-free survival
- TCGA
The Cancer Genome Atlas
- DMEM
Dulbecco’s Modified Eagle Medium
- FBS
fetal bovine serum
- PI
propidium iodide
- SA-β-Gal
senescence-associated β-galactosidase
- RT-PCR
real-time PCR
- CHX
cycloheximide
- ECL
enhanced chemiluminescence
- IHC
immunohistochemistry
- PBS
phosphate-buffered saline
Supporting Information
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