Depletion of PSMD14 suppresses bladder cancer proliferation by regulating GPX4

Tissue samples and clinic data

From 2016 to 2018, 181 patients diagnosed with primary BC at the Affiliated Hospital of Qingdao University were recruited. All patients underwent surgical resection. The paraffin-embedded BC specimens and adjacent non-tumor tissues from these patients were collected. Clinicopathological data were collected as previously described (Zhang et al., 2018). Patients were followed up until July 2022, and data relative to patient prognosis were recorded. The protocol for this study and the written consent forms were approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (QYFYWZLL 27227). All patients/guardians gave their written consent.

Plasmid construction and lentivirus transduction

The lentiviral vector containing the PSMD14 sequence encoding PSMD14 overexpression (PSMD14-OE), PSMD14 knockdown (PSMD14-KD), and the control vector (PSMD14-Ctrl) were purchased from Hanheng BioChem Corporation (Shanghai, China). The plasmids were transfected into T24 and 5637 cells according to the manufacturer’s instructions. The transfection efficiency was monitored by western blotting. Cells were then infected with lentiviruses and selected by puromycin (p8230; Solarbio, Beijing, China).

Cell lines and cultures

Human BC T24, 5637, J82, and UM-UC3 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells (except for 5637 cells) were cultured with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA; C11995500BT; Thermo Fisher, Waltham, MA, USA) supplemented with 10–20% fetal bovine serum (FBS; Gibco, Waltham, MA, USA; 10099-141c; Thermo Fisher, Waltham, MA, USA). The 5637 cells were cultured with PRIM1640 (Gibco, Waltham, MA, USA; C11875500BT; Thermo Fisher, Waltham, MA, USA). All cells were housed in a humidified atmosphere at 37 °C with 5% CO₂. The ferroptosis inhibitor ferrostatin-1 and ferroptosis activator RSL3 were purchased from Med ChemExpress (MCE).

Quantitative real-time PCR analysis

Cells were lysed with TRIzol reagent (CW0580S; CWBIO, Beijing, China), and total RNA was extracted. RNA (1000 ng) was reversely transcribed and amplified using a SuperRT one-step real-time PCR kit (CW0742S; CWBIO, Beijing, China) and a real-time PCR detection system (ABI, 7500, Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. GAPDH was considered the standard internal reference. Real-time PCR conditions were as follows: initial denaturation at 95 °C for 5 min and 40 cycles of 95 °C for 15 s, 65 °C for 30 s, 72 °C for 30 s, and a final extension of 1 min. The experiment was repeated three times independently. Relative quantitative method (2^−ΔΔCt) was utilized to calculate the results, which were presented as fold changes. The designed primers were as follows: GPX4: 5′-GAGGCAAGACCGAAGTAAACTAC-3′ (F) and 5′-CCGAACTGGTTACACGGGAA-3′(R); GADPH: 5′-CTGACTTCAACAGCGACACC-3′ (F), and 5′-TGCTGTAGCCAAATTCGTTGT-3′(R).

Western blotting analysis

Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) (R0020, Solarbio, Beijing, China) supplemented with a protease inhibitor cocktail (CW2200S, CWBIO, Beijing, China) and phosphatase inhibitors (CW2383S, CWBIO, Beijing, China) for 30 min. Protein concentration was determined using a BCA protein assay kit (Solarbio, Beijing, China). Total proteins (30 μg) were separated using 12.5% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (PVDF) (IPVH00010; Millipore, Burlington, MA, USA) at 110 voltage for 90 min. After blocking with 5% fat-free milk for 2 h at room temperature, the membranes were incubated at 4 °C overnight with primary antibodies against PSMD14 (1:1,000; clone: 12059-1-AP; Proteintech, Wuhan, China), GPX4 (1:1,000; clone: 67763-1-lg; Proteintech, Wuhan, China), and anti-actin rabbit polyclonal (1:5,000; clone: E-AB-2058; Elabscience, Wuhan, China).

Cell proliferation assay

Cells were seeded at a density of 1 × 10³ cells per well in 96-well plates. Cell proliferation was evaluated using the CCK-8 (cell counting kit-8) assay (Dojindo, Dalian, China) at indicated time points according to the manufacturer’s instructions. Absorbance at 450 nm was measured after incubation with 10 μL CCK-8 regent and 90 μL cell culture medium for 1.5 h at 37 °C. Light absorbance was measured using an automated microplate reader (Infinit f200; Tecan, Port Melbourne, Australia).

Colony formation assay

Cells (8 × 10² cells/well) were seeded in 6-well plates. The medium was replaced every 3 days. After culturing for 14 days, the plates were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 1 min and counted.

Immunohistochemistry

Immunohistochemistry (IHC) was performed on paraffin-embedded BC tissues and adjacent tissues (n = 181). After deparaffinization and rehydration by xylene and a series of graded ethanol washes, sections were treated with Tris-EDTA (pH = 9.0) at 100 °C for 10 min. The sections were then treated with 3% hydrogen peroxidase (10 min, room temperature), followed by incubation with primary antibody targeting PSMD14 (1:600; clone: 12059-1-AP; Proteintech, Wuhan, China) and GPX4 (1:800; clone: 67763-1-lg; Proteintech, Wuhan, China), for 1 h at 37 °C. Sections were incubated with HRP-conjugated secondary antibody for 60 min and a 3,3-diaminobenzidine (DAB) kit (ZL1-9017; ZSGB-Bio, Beijing, China) for 1 min. Finally, the sections were counterstained with hematoxylin.

The scoring of the IHC staining was based on the extent of positive tumor cells and the intensity of the staining (0 = negative, 1 = weak, 2 = medium, or 3 = strong). The percentage of positive tumor cells was scored as 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). The two scores were multiplied and the resulting immune-reactive score (IRS) (values from 0–12) were used to classify the samples into two categories: high (5–12 score) and low (1–4 score) (Zhang et al., 2018).

Animal experiments

Twenty female BALB/c nude mice (4–6-weeks old, 15 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under specific pathogen-free conditions in 12/12 cycle of light at room temperature (24–26 °C). Mice were fed a full fat diet and autoclaved water. The number of mice did not exceed five per cage. A total of 1 × 10⁷ infected 5637 cells were suspended in 100 μL PBS and injected into the shoulder of the mice. Tumor length (L) and width (W) were observed for 4 weeks. Tumor volume (V) was monitored by measuring the length and width of the tumor using the following equation: V = (L × W²) × 0.5. The mice were euthanized by cervical dislocation after inhalational of CO₂ when the maximum diameter of any tumor was near 1.5 cm. Tumor tissues were excised and embedded in paraffin for ematoxylin and eosin (HE) or IHC staining. Animal experiments were reviewed and approved by the Ethics Committee on Animal Experiments of the affiliated hospital of Qingdao University (AHQU-MAL20220715).

Statistical analysis

The results were analyzed with SPSS 19.0.0 (SPSS, Chicago, IL, USA). Chi-square (χ2) or Fisher’s exact test were used to compare frequencies between groups. Survival curves for disease-free survival (DFS) and overall survival (OS) were calculated using Kaplan-Meier analysis with the log-rank test. Multivariate analysis was performed using the Cox proportional hazard model. A P-value < 0.05 was considered statistically significant.

PSMD14 status and association with clinicopathological characteristics

We evaluated the expression of PSMD14 in 181 pairs of BC and corresponding normal tissues by IHC. IHC staining showed that the PSMD14 protein was expressed at significantly higher levels in BC (138/181) compared to normal tissues (43/181) (76.24% vs 23.76%, P = 0.02) (Fig. 1). The correlation between PSMD14 expression and clinicopathological characteristics is presented in Table 1. PSMD14 showed significantly higher expression in tumors with larger diameters (tumor diameter >3 cm vs tumor diameter ≤3 cm; 85.14% vs 70.09%, P = 0.019). Patients with a family history of cancer were more likely to have high PSMD14 expression (yes vs no; 92.16% vs 70.00%, P = 0.002). Although high expression of PSMD14 was more frequent in patients with tumor vascular invasion, there was no statistical significance (92.86% vs 74.85%, P > 0.05). No significant differences were found between PSMD14 expression and other clinicopathological characteristics in this study (Table 1).

Prognostic value of PSMD14 expression in primary BC

Kaplan-Meier survival analysis and log rank test showed that factors significantly associated with DFS were sex (P = 0.002), invasive cancer (P = 0.001), tumor stage (P = 0.001), invasion of the intestinal wall (P = 0.001), lymph node metastasis (P = 0.001), vessel carcinoma embolus (P = 0.001), vascular invasion (P = 0.001), alcohol intake (P = 0.001), smoking history (P = 0.0012), family history of cancer (P = 0.0436), and PSMD14 expression (P = 0.0232) (Table 2).

Patients with high expression of PSMD14 showed shorter DFS (P = 0.0232) (Fig. 2A). To determine prognostic values independent of sex, we entered invasive cancer, tumor stage, intestinal wall invasion, lymph node metastasis, vessel carcinoma embolus, vascular invasion, history of alcohol intake, smoking history, cancer family history and expression information of PSMD14 in a Cox regression model. We found that PSMD14 expression was an independent predictor of worse DFS, and tumors with high expression of PSMD14 were associated with a 2.89-fold increase in the risk of cancer recurrence or metastasis (HR = 2.89, 95% CI [1.247–6.711], P = 0.013) (Table 3). In addition, PSMD14 was not a statistically significant risk factor for OS in the univariate analysis (Table 3).

PSMD14 promoted BC cell proliferation in vitro and in vivo

To assess the functional role of PSMD14 in the development and progression of BC, we first examined endogenous levels of PSMD14 in several BC cell lines. We found high endogenous expression of PSMD14 in T24, UM-UC3 and J82, and low expression levels in 5637 cells (Fig. 3A). We performed a knockdown of PSMD14 expression in T24 (Figs. 3B and 3E) and 5637 cells (Figs. 3C and 3F) using the PSMD14-KD vector. BC cell proliferation with low expression of PSMD14 was significantly suppressed compared to control cells (Figs. 4A and 4B). Similarly, the colony formation assay showed that PSMD14 depletion inhibited colony number formation (Figs. 4D and 4E). Conversely, PSMD14 was overexpressed in 5637 cells (Figs. 3D and 3G). Compared to PSMD14-Ctrl cells, the overexpression of PSMD14 promoted BC cell growth according to the CCK8 assay (Fig. 4C). The colony formation assay showed that PSMD14 overexpression increased the colony number of 5637 cells (Fig. 4F). Therefore, the reciprocal effects of knockdown and overexpression of PSMD14 in vitro suggested that PSMD14 promoted the proliferation of BC cells.

A total of 20 mice were used to evaluate the proliferation potential of PSMD14 in vivo. Briefly, 5637 cells transfected with different vectors (PSMD14-OE and PSMD14-KD) were injected subcutaneously into the nude mice. Cells transfected with the PSMD14-Ctrl vector were injected as a control. The tumors were formed on day 6 after injection and were collected on day 24. As shown in Fig. 5, the tumors formed by PSMD14-KD cells were significantly smaller than those formed by the control cells (824.4 ± 160.67 mm³ vs 349.2 ± 151.14 mm³, P = 0.001). Tumor volume (463.4 ± 222.27 mm³ vs 1,026.4 ± 351.65 mm³, P = 0.002) and weight (0.766 ± 0.12 g vs 1.304 ± 0.21 g, P = 0.002) were markedly increased in the PSMD14-OE group compared to the control group. The tumors were collected and further analyzed using IHC. IHC analysis demonstrated that PSMD14 expression increased in the PSMD14-OE group and decreased in the PSMD14-KD group compared to the control group (Fig. 6).

Depletion of PSMD14 antagonizes BC growth by decreasing the expression of the GPX4 protein

GPX4, a ferroptosis-related gene in mammals, plays a pivotal role in the inhibition of various tumors. To determine whether PSMD14 acts on GPX4 in BC, PCR, western blotting, and IHC assays were performed to evaluate the status of GPX4 in PSMD14-OE and PSMD14-KD BC cells. We found that GPX4 was markedly down-regulated in PSMD14-KD BC cells, and GPX4 was markedly up-regulated in PSMD14-OE BC cells (Figs. 3B–3D). To verify whether PSMD14 regulated BC proliferation through the GPX4 pathway, we treated PSMD14-OE BC cells with RSL3 and PSMD14-KD cells with ferrostatin-1. As expected, the promotion of cell proliferation induced by PSMD14 overexpression was reversed when BC cells were treated with RSL3, as shown by the CCK-8 assay (Fig. 7C). The inhibition of PSMD14-KD-induced cell proliferation was also reversed when treated with ferrostatin-1 (Figs. 7A and 7B). Western blotting assays showed that GPX4 expression was significantly increased in ferrostatin-1 treated cells, and GPX4 was significantly decreased in RSL3 treated cells (Fig. 7). Together, these results indicated that the depletion of PSMD14 inhibited BC tumor growth in vitro and in vivo by targeting GPX4.