UBC cell lines (5637, SW780, T24, UMUC-3, and SCaBER) and nonmalignant urothelial cell line (SV-HUC-1) were cultured in RPMI 1640 medium (Life Technologies) supplemented with penicillin/streptomycin (Invitrogen, United States) and 10% FBS at 37°C with 5% CO₂. Reagents and chemicals used in this study were listed in Supplementary Table S1.

Formalin-fixed, paraffin-embedded UBC tissues and relevant clinicopathological variables were acquired from Shanghai General Hospital. The informed consent of all patients was obtained, and the research protocol was formally approved by the ethics committee of Shanghai General Hospital.

Gene expression and clinicopathologic information of The Cancer Genome Atlas Urothelial Bladder Carcinoma (TCGA-BLCA) were downloaded from Genomic Data Commons Data Portal. Other public microarray data were retrieved from the Gene Expression Omnibus (GEO) database.

The PDE4B and CBX7 overexpressing cell lines were obtained by infecting with virus expressing Flag-tagged PDE4B (pCDH-3xFlag-PDE4B) and CBX7 (pCDH-3xFlag-CBX7), respectively. For knockdown assays, two siRNA targeting PDE4B were transfected into cells using Lipofectamine 3000 (Thermo Fisher Scientific). Two CBX7 shRNA were transfected into cells using the lentiviral pLKO.1 backbone. The siRNA and shRNA sequences used were listed in Supplementary Table S2.

Total protein was isolated by lysis buffer with protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Western blotting was performed following the standard methods. Antibodies used in this study were listed in Supplementary Table S3.

2,000 cells/well were seeded into a 96-well plate. If cells adhered to the bottom, 10 μL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] was added to each well for 4 h at 37°C and it was identified as 0 h. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO) at 37°C for 15 min and the absorbance at 490 nm was examined. After 24, 48, and 72 h, the similar procedure was carried out.

Confluent cultures in 6-well plates were scratched with a 200 μL pipette tip, washed with PBS and then cultured in medium containing 1% FBS. Images were captured after 0, 24, and 48 h respectively to estimate wound closure (%).

Cell invasion was measured using 8 μm pore-size transwell inserts (Millipore, United States). 1 × 10⁵ cells were seeded into the top chamber pre-coated with 1:8 diluted Matrigel, then 500 μL medium containing 10% FBS was added to the bottom chamber. After the incubation for 24 h, cells were fixed in formaldehyde, stained with crystal violet and counted.

GSEA software was applied to explore the correlation of PDE4B expression on the progression of UBC. Gene set collections were downloaded from MSigDB database. Nominal p-value <0.05 and FDR <0.25 were considered to a statistically significant gene set.

5 μm thick paraffin-embedded bladder tumor sections were prepared and stained according to the previously described procedure (Yan et al., 2020). Antibodies used were listed in Supplementary Table S3.

The ChIP assay was performed as previously described (Chang et al., 2018). Briefly, cells were fixed in 1% paraformaldehyde for 10 min at room temperature, following quenched using 0.125 mol/L glycine for 5 min. The formaldehyde-fixed cells pellets were lysed with cell lysis buffer and sonicated using Diagenode Bioruptor Pico Sonicator for 30 cycles (30 s/90 s on/off) to obtain a DNA range of 200–1,000 bp. Samples were incubated with protein A/G (Millipore, #17-371) and antibody (anti-CBX7, Abcam, ab21873; anti-ubH2AK119, Cell Signaling Technology, #8240S) overnight at 4°C. Samples were washed 4 times with low salt, high salt, LiCl, and TE wash buffers, respectively. Elution of protein/DNA complexes were reversed cross-link to obtain free DNA at 62°C for 2 h with shaking and incubated at 95°C for 10 min. DNA was purified using spin columns (Millipore, #17-10085). Individual ChIP samples were analyzed by qRT-PCR using PED4B promoter primers. The primers used were listed in Supplementary Table S2.

All statistical analyses were performed using GraphPad Prism 8.0. Data were presented as means ± standard deviation (SD). Student’s t test was performed to compare statistical significance between groups. Pearson correlation was used to analyze the association between genes expression. A chi-square test was employed to assess the association between PDE4B expression and clinicopathological variables. Kaplan-Meier (Log rank test) method was performed to estimate the overall survival. A value of p < 0.05 was considered statistically significant.

To investigate whether PDE4B was involved in UBC development, multiple databases containing tumor clinicopathological information were analyzed. As shown in Figures 1A–F, higher PDE4B mRNA level was observed in advanced stages patients from TCGA-BLCA and GEO databases (p < 0.01). Moreover, PDE4B mRNA level was elevated in high-grade patients than that in low-grade patients (p < 0.05, Figures 2A–D). Consistently, our IHC data also showed that high-grade patients expressed higher level of PDE4B protein compared to low-grade patients (Figure 2E). In addition, PDE4B protein level was significantly associated with tumor grade (p = 0.036; Table 1) and T stage (p = 0.003; Table 1). In summary, we concluded that the PDE4B expression was closely correlated with clinicopathological characteristics.

Next, we performed Kaplan-Meier survival analysis to examine the clinical value of PDE4B in UBC. In GSE32894 database, higher PDE4B mRNA expression heralded worse prognosis (p = 0.0009, Figure 3A). To validate the results from public database, we collected 59 UBC specimens for IHC and made survival analysis. Consistently, higher PDE4B protein level indicated shorter overall survival time (p = 0.0118, Figure 3B). The representative images of IHC staining of different PDE4B staining intensities were displayed in Figure 3C. Collectively, these findings revealed that upregulation of PDE4B was remarkably correlated with unfavorable prognosis in patients with UBC.

Above data indicated that PDE4B may function as an oncogene in UBC progression. Furthermore, we found that most UBC cell lines expressed higher PDE4B level compared with nonmalignant urothelial cell line SV-HUC-1 (Figure 4A and Supplementary Figure S1). Interestingly, we also observed that higher expression of PDE4B was specifically in basal/squamous subtype, which was the UBC subtypes with poor prognosis (Supplementary Figure S2). To investigate potential oncogenic role of PDE4B, we stably ectopically expressed PDE4B in T24 cells, which expressed relatively lower endogenous PDE4B (Figures 4A,B). Firstly, we explored the effects of PDE4B on cell proliferation. The results showed that ectopic expression of PDE4B notably promoted T24 cells viability (Figure 4C). In line with this, overexpression of PDE4B markedly enhanced cancer cells invasiveness (Figures 4D,E). Wound healing assays also demonstrated that cells overexpressing PDE4B significantly facilitated cell migration (Figures 4F,G). Taken together, these results indicated that overexpression of PDE4B facilitated UBC cells proliferation and invasion.

We then conducted the loss-of-function approach to further explore the function of PDE4B. SCaBER and SW780 cells were selected for knockdown as the relatively higher level of endogenous PDE4B protein (Figure 4A). PDE4B expression was decreased after two independent siRNAs to PDE4B were transfected into cells (Figure 5A). As shown in Figures 5B,C, knockdown of PDE4B markedly inhibited SCaBER and SW780 cells viability. Consistently, PDE4B deficiency notably suppressed cells invasive abilities (Figures 5D,E). Wound healing also demonstrated that PDE4B-silenced SCaBER and SW780 cells filled the gap much slowly than control cells (Figures 5F–I). Taken together, these functional data implied that depletion of PDE4B reduced the capability of UBC cells to proliferate and invade.

The Chromobox protein homolog 7 (CBX7) belongs to the Polycomb Group (PcG) family, which is a component of canonical polycomb repressive complex1 (PRC1), contributing to transcriptional target genes repression by prompting the RING protein in PRC1 to monoubiquitinate H2AK119 (Simon and Kingston, 2009; Musselman et al., 2012). In our previous study, downregulation of PDE4B expression was observed in CBX7 knockdown cells. When we looked up the RNAseq dataset (GSE158224) in our previous study, we observed that PDE4B was downregulated in CBX7 overexpressing cells, compared to control T24 cell group (Huang et al., 2021b). Thus, we speculated that CBX7 may be a potential regulator of PDE4B.

To confirm this hypothesis, qRT-PCR and Western blotting were applied to determine whether CBX7 can negatively regulate the expression of PDE4B. As shown in Figures 6A,B, PDE4B was decreased in CBX7-overexpressed T24 and UMUC-3 cells, but increased in CBX7-silenced 5,637 cells at both mRNA and protein levels. We further detected the association between PDE4B and CBX7 expression levels in UBC patients. The IHC data showed that CBX7 mainly expressed in the nuclei of low grade UBC cells, but weakly positive signal in high grade, while PDE4B-positive signals were barely detected in low grade UBC cells, but strong positive signals in cytosol were observed in high grade UBC cells (Figure 6C). Of note, the expression of CBX7 in UBC samples was negatively associated with the expression of PDE4B (n = 32, p = 0.0075, Figure 6D). To further determine whether CBX7 suppresses the transcription of PDE4B in a canonical PRC1-dependent manner, we carried out ChIP assays and the results revealed that CBX7 was recruited to the promoter region of PDE4B gene and consequently inhibited the PDE4B transcription by increasing the abundance of mono-ubiquitinated H2A at K119 (ubH2AK119) repressive mark at its promoter region (Figures 6E,F). Collectively, these findings indicated that PDE4B was transcriptionally repressed by CBX7 in a PRC1-dependent manner.

With PDE4B overexpression or depletion, we observed significant changes in cell migration and invasion capabilities. These cell phenotypic changes have the same characteristics as EMT. It is universally recognized that EMT is highly correlated with tumor invasion and metastasis, in which cancer cells acquire mobility with phenotype changes (Nieto et al., 2016; Dongre and Weinberg, 2019). Thus, we suspected that EMT may be responsible for PDE4B-mediated changes in migration and invasion. As shown in Figure 7A, the GESA results revealed that high PDE4B expression was significantly correlated with EMT gene sets in TCGA-BLCA, GSE13507, and GSE32894. Accordingly, we then detected the mRNA expression of EMT-related factors, as well as transcription regulators in PDE4B-overexpressing T24 cells. The results showed that the epithelial marker E-cadherin was decreased upon PDE4B overexpression (Figure 7B). Moreover, ectopic PDE4B increased the expression of two EMT-related transcription factors TWIST1 and TWIST2 (Figure 7C). Next, we analyzed the correlation between PDE4B and E-cadherin, TWIST1 and TWIST2 in TCGA-BLCA and GSE13507 databases. Consistent with our findings, PDE4B expression showed significantly negative correlation with E-cadherin expression, but notably positive correlation with TWIST1 and TWIST2 expression (Figures 7D,E). Western blotting further proved the reduction of E-cadherin and the elevation of Twist1/2 upon PDE4B overexpression (Figure 7G). Consequently, these results revealed the role of PDE4B in EMT induction.

As a pivotal mediator in Wnt/β-catenin signaling pathway, β-catenin exerts a pivotal role in tumor invasion and metastasis (Nusse and Clevers, 2017; Zhang and Wang, 2020). Furthermore, increased expression of β-catenin could induce EMT (Yang et al., 2017; Zhu et al., 2020). To better understand the mechanism of PDE4B-mediated EMT, we performed GSEA in GSE13507 database and drew conclusion that high PDE4B expression group was enriched in the Wnt/β-catenin pathway (Figure 7F). Consistently, with PDE4B overexpression, the protein levels of active β-catenin in T24 cells were upregulated (Figure 7G). Collectively, these data implied that PDE4B modulated EMT by targeting the β-catenin signaling.

Next, we investigated the potential therapeutic value of PDE4B in UBC. Rolipram (Rol) was reported to be an effective PDE4B inhibitor, which exerted important tumor suppressive effects by inhibiting the PDE4B/mTOR/Myc axis in colorectal cancer (Kim et al., 2019). Thus, we explored whether pharmacological inhibition of PDE4B by rolipram could rescue the tumor-promoting effects induced by PDE4B overexpression. The results of invasion assays showed that rolipram at 20 μM significantly reduced the number of invaded cells in PDE4B-overexpressing T24 cells (Figures 8A,B). Consistently, treatment with rolipram partly rescued the protein level of E-cadherin, Twist1/2 and β-catenin (Figure 8C). Altogether, we concluded that pharmacological repression of PDE4B by rolipram could partly rescue the tumor-promoting effects induced by PDE4B overexpression.