Expression of transcription factor SALL4 in bladder urothelial carcinoma and its relationship with epithelial mesenchymal transformation


 Purpose

To evaluate the expression levels of spalt-like transcription factor 4 (SALL4) in bladder urothelial carcinoma, and determine its role and underlying mechanism of action in mediating the proliferation, migration and invasion of bladder cancer cells.
Methods

SALL4 expression was examined in 170 bladder patient urothelial carcinoma tissue samples by immunohistochemistry. Using a SALL4 overexpression plasmid and siRNA-SALL4, the effects of overexpressing and silencing SALL4 on the proliferation, migration and invasion of T24 and 5637 bladder cancer cells was examined using CCK8, migration and invasion assays. Western blot analysis was performed to detect epithelial mesenchymal transition (EMT)-related protein expression.
Results

The expression rate of SALL4 in low-grade and high-grade urothelial carcinomas was found to be 10% and 49.12%, respectively (P < 0.001), while SALL4 expression was not observed in the normal urothelium. SALL4 protein expression was positively correlated with histological grade, depth of invasion, lymphatic metastasis and vascular invasion of bladder urothelial carcinoma (P < 0.05). In addition, a shorter overall survival time and poor prognosis was observed in the SALL4 protein expression group. Overexpression of SALL4 led to significantly increased cell proliferation, migration and invasion, while knockdown of SALL4 had the opposite effect. In the SALL4 overexpression group, N-cadherin, vimentin, Snail and β-catenin expression were significantly increased, while E-cadherin expression was significantly decreased (P < 0.05). Promotion of EMT was also observed in SALL4-overexpressing cells. In contrast, in the SALL4-siRNA-treated group, EMT was reversed and β-catenin expression was reduced.
Conclusions

Our data show that the SALL4 gene is associated with the proliferation, invasion and poor prognosis of bladder urothelial carcinoma, and may mediate its effects via the Wnt/β-catenin signaling pathway, which regulates the EMT pathway. Thus, down-regulation of SALL4 may provide a novel therapeutic strategy for the treatment of bladder urothelial carcinoma.


Introduction
Bladder urothelial carcinoma is the second most common malignant urogenital tumor, while bladder cancer is the tenth most commonly diagnosed cancer, with the fourth highest incidence rate in males worldwide. The incidence rate of bladder urothelial carcinoma has been increasing over the past forty years [1,2]. Bladder urothelial carcinoma can be categorized as low-grade and high-grade, as well as noninvasive and invasive. Low-grade urothelial carcinoma of the bladder has a better prognosis, while highgrade urothelial carcinoma has a higher risk of progression, high invasion and metastatic rates, and poor prognosis. The precise molecular mechanisms of bladder urothelial carcinoma remain unclear, and there is no effective targeted therapy. At present, an increasing number of studies have focused on targeted therapies [3], such as the broblast growth factor receptor (FGFR) inhibitors erda tinib and rogaratinib, which have been shown to have a promising role in advanced urothelial carcinoma treatment. In addition, everolimus [3], a mTOR inhibitor, is also clinically bene cial in the treatment of urothelial carcinoma. Thus, patients would bene t from the development of targeted therapeutic strategies for bladder cancer.
EMT is a cellular biological process in which epithelial cells transform into mesenchymal phenotypes.
During EMT, the polarity of epithelial cells disappears and the expression of E-cadherin decreases, while the expression of N-cadherin, vimentin and other mesenchymal markers increases. EMT is associated with tumor progression, invasion, metastasis and chemotherapy resistance [19,20,21]. EMT was recently shown to be important during the progression of bladder cancer metastasis and invasion [22]. EMT is also associated with the tumor stage and grading of bladder cancer [23]. The most signi cant feature of EMT is the decrease or deletion of E-cadherin expression, which is negatively correlated with tumor invasion and metastasis [24]. A role for the SALL4 gene in EMT has previously been described in many types of tumor. Zhang et al, for example, have demonstrated that SALL4 induces EMT and promotes gastric cancer metastasis by activating the TGF-β/SMAD signaling pathway [25]. In addition, SALL4 has been shown to have a positive regulatory effect on the zinc nger E-box binding homeobox 1 (ZEB1), and inhibits E-cadherin transcription. SALL4 has also been associated with cell dispersion and expression of interlobular gene phenotypes, as well as enhancing the in ltration and migration of breast cancer cells [26]. However, the mechanism of SALL4 in bladder urothelial carcinoma remains unknown.
In the present study, we aimed to evaluate the role of SALL4 and EMT regulation in bladder urothelial carcinoma using SALL4 overexpression and RNA interference assays. Furthermore, we assessed SALL4 expression levels in patient samples and determined the correlation between SALL4 expression and clinical parameters. Our ndings indicate that SALL4 may be a potential therapeutic biomarker in patients with bladder urothelial carcinoma.

Patient samples
A total of 170 para n-embedded bladder cancer specimens and 20 normal bladder tissue samples were collected. The patients underwent surgical treatment between 2012 and 2019 at the Fujian Medical University A liated Second Hospital (Quanzhou, China). None of the patients received neoadjuvant radiotherapy before surgery. Written informed consent was obtained from all patients. All cases were con rmed by pathology. The pathological grading was based on the World Health Organization (WHO) grading standard. A further 20 cases of normal bladder mucosa adjacent to the cancer were sampled. Follow up was conducted by calling or browsing the electronic pathology system and electronic medical record system of our hospital. The deadline for follow-up was November 1, 2019. This study was approved by the Ethical Committee of Fujian Medical University A liated Second Hospital (Fujian, China).

Cell culture and plasmids
Human T24 and 5637 bladder cancer cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences and grown in RPMI-1640 supplemented with 10% FBS in a 37˚C humidi ed chamber in the presence of 5% CO 2 . The SALL4 eukaryotic expression plasmid (PLVX-SALL4-puro) was constructed and its sequence was identi ed by Genepharma (Shanghai, China).

Immunohistochemical staining
Immunohistochemical staining of SALL4 was performed using a two-step Elivision plus staining technique. Immunohistochemical staining was performed on 4 μm tissue microarray sections of formalin-xed and para n-embedded tissue samples. Antigen repair was performed using EDTA antigen repair solution, and placing the tissue sections into a boiling pressure cooker and low heat for 20 min. Samples were incubated with a rabbit polyclonal antibody against SALL4 (ab29112, Abcam, Cambridge, MA, USA, 1:100) at room temperature for 2 h in a humidi ed chamber. Endogenous peroxidase was removed with 3% hydrogen peroxide at room temperature for 10 min, followed by incubation with an HRPconjugated secondary antibody for 30 min at room temperature. Antibody binding was visualized by DAB staining, and the reaction was stopped by immersing tissue sections into distilled water once the brown color was visible. Tissue sections were counterstained by hematoxylin, dehydrated in graded ethanol and mounted. Stained samples were scored by two experienced pathologists according to the semiquantitative method. Five representative areas were randomly selected and examined at 400 times eld of vision by microscopy. The score of each eld was determined by the proportion of positive cells and staining intensity. The proportion of positive cells was scored as follows: 0 (0%), 1 (1-5%), 2 (6-25%), 3 (26-50%), and 4 (50-100%). The staining intensity was scored as follows: 0 (negative), 1 (weak), 2 (medium) or 3 (strong). To obtain a nal score, we multiplied the two scores, and the average score of the ve selected elds was calculated. A score of ≥2 was positive for expression.

Cell transfection
During their log phase of growth, 5637 and T24 bladder cancer cell lines were diluted to a density of 1×10 6 cells/mL, and 1 mL of cell suspension was placed in each well of a six-well plate. When the cell density reached 40% to 50%, the cell culture medium was changed to 1640 serum-free medium for 6 h. USA) respectively were diluted with 1 mL 1640 medium and mixed. After incubation for 15 mins in the dark, the mixture was added to the cells. After 6 h, the medium was changed to complete medium.
Cell counting kit-8 (CCK8) assay The proliferation of 5637 and T24 cells was assessed using the CCK8 assay kit (Dojindo, Shanghai, China). Cells were seeded onto 96-well plates at a density of 1 × 10 3 cells/well and incubated in a humidi ed 5% CO 2 incubator for 0, 12, 24, 36 and 48 h. Then, 100 μL of CCK8 reagent was added to each well and incubated for a further 2 h. Absorbances were measured at 450 nm in a multiplate reader (Promega, Madison, WI, USA).

Migration and invasion assays
Cell migration and invasion assays were performed in Transwell chambers (Corning Incorporated, Corning, NY, USA). For the invasion assays, the Transwell inserts were coated with 15 μg/μL Matrigel.
After transfection, 5637 and T24 cells (1×10 5 ) were seeded into the upper chamber, which contained 200 μL of serum-free medium. The lower chambers contained 500 μl RPMI-1640 medium. After incubation for 24 h at 37°C, cells that passed through the lter were xed with 4% (4 mol/L) PFA and stained with 0.1% (0.05 g/L) crystal violet for 30 min. Five elds were randomly selected, counted and photographed using a microscope (Olympus Corporation, Japan). The assay was performed in triplicate.

Western blot analysis
Treated cells were collected and lysed in ice cold cell lysis buffer for protein extraction. Cell lysates were centrifuged at 13,000 rpm for 5 min at 4˚C. The concentration of the protein samples was determined using a BCA assay kit (Thermo Scienti c, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene uoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk for 1 h and incubated at 4˚C overnight with the primary antibodies outlined below, followed by incubation for 1 h with the appropriate secondary antibody. Positive protein bands were visualized by enhanced chemiluminescence (ECL) staining (Millipore). Proteins of interest were detected by incubating membranes with the following primary antibodies: anti-SALL4 (1:1000, rabbit monoclonal, ab29112),

SALL4 expression and clinicopathological characteristics in bladder urothelial carcinoma
In a total of 170 bladder cancer samples, immunohistochemical staining revealed that SALL4 expression was positive in 64 cases (37.6%) and located predominantly in the nucleus (Fig. 1). No staining was observed in the normal bladder tissue. Table 1 summarizes the correlation between SALL4 expression and the clinicopathological characteristics of the tumor samples. In low-grade urothelial carcinoma of the bladder, negative or weak SALL4 staining was observed (10%), and was signi cantly lower than highgrade urothelial carcinoma SALL4 staining (49.12%, P<0.001). We found that SALL4 expression was positively correlated with in ltration depth (P=0.008). Similarly, SALL4 was signi cantly upregulated in patients with lymphatic metastasis or vascular invasion. In addition, the Kaplan-Meier survival curve revealed that the overall survival (OS) in SALL4-positive patients was signi cantly shorter than in SALL4negative patients (P<0.05, Fig. 2).

Con rmation of SALL4 overexpression and knockdown in bladder cancer cell lines
To elucidate the role of SALL4 in bladder cancer, we ectopically overexpressed or knocked down SALL4 in T24 or 5637 cell lines, respectively. T24 cells were transfected with PLVX-SALL4-puro, while 5637 cells were transfected with SALL4 siRNAs. After transfection for 48 h, SALL4 mRNA and protein levels were detected by qPCR and western blot analysis, respectively. We found that SALL4 mRNA and protein expression levels were signi cantly up-regulated in T24 cells transfected with PLVX-SALL4-puro (Fig 3A,  Fig 3B), and decreased by 70% in 5637 cells transfected with SALL4 siRNA compared with the NC groups ( Fig 3C, Fig 3D).

SALL4 promotes the proliferation of bladder cancer cells
The CCK8 assay revealed that SALL4 overexpression signi cantly promoted proliferation in bladder cancer cells compared with the NC and empty vector groups (P<0.05, Fig 4A). As shown in Fig. 4B, cell proliferation was signi cantly inhibited in the SALL4-siRNA-treated group compared with the NC and siRNA-control groups (P < 0.05).

SALL4 promotes the migration and invasion of bladder cancer cells
Cell invasion and migration is an important step during tumor metastasis. We examined the migratory and invasive abilities of SALL4-overexpressing or SALL4-silenced cells by Transwell assay. T24 bladder cancer cells transfected with PLVX-SALL4-puro had higher invasive and migratory capabilities than the NC and PLVX-puro groups (P<0.01, Fig. 5A). In contrast, the invasion and migration of 5637 cells was decreased after knockdown of SALL4 (P< 0.05, Fig. 5B). Our ndings con rmed that SALL4 may contribute to tumor invasion and migration in bladder cancer.

SALL4 affects the expression of β-catenin and EMT-related proteins
To determine the potential mechanism of action of SALL4 in bladder cancer, we examined the expression levels of β-catenin and components of the EMT signaling pathway by western blot analysis. The protein expression levels of N-cadherin, vimentin, Snail and β-catenin were signi cantly increased in SALL4overexpressing cells compared with the NC and PLVX-puro groups (P<0.05), while E-cadherin expression was signi cantly decreased (P < 0.05, Fig 6A). In the SALL4-silenced group, a signi cant decrease in Ncadherin, vimentin, Snail and β-catenin protein expression was observed (P < 0.05), together with a signi cant increase in E-cadherin (P < 0.05, Fig 6B). Thus, our data indicate that SALL4 may be associated with the Wnt/β-catenin signaling pathway.

Discussion
The prognosis of bladder cancer is associated with histological grading, staging, lymphatic invasion and lymph node metastasis [27]. Multiple studies have reported that SALL4 is overexpressed in various tumors and is involved in tumor progression. SALL4 is positively correlated with lymph node metastasis, tumor stage, and poor prognosis. Although SALL4 is reportedly expressed in high-grade, but not low-grade bladder urothelial carcinoma, its speci c mechanism remains unclear [7]. Consistent with previous studies, our immunohistochemical data revealed high SALL4 expression levels in high-grade urothelial carcinoma, while low-grade urothelial carcinoma was associated with negative or scattered SALL4 expression. Our results also demonstrated that SALL4 protein expression was positively correlated with histological grade, depth of invasion, lymph node metastasis and vascular invasion of bladder urothelial carcinoma. In addition, the SALL4-positive group was associated with poor prognosis and short OS.
Thus, our ndings indicate that SALL4 may be closely associated with tumor invasion and progression rather than tumorigenesis. SALL4 is expressed aberrantly in bladder cancer cells and has diverse effects in different types of tumors, for example, in endometrial carcinoma, HCC, lung cancer and gastric cancer [28][29][30][31]. Lymphatic invasion increases the risk of recurrence and disease progression in non-muscle invasive bladder cancer (NMIBC), and is associated with disease invasiveness in muscle invasive bladder cancer (MIBC), and may therefore predict recurrence and OS [32]. Since overexpression of SALL4 in bladder urothelial carcinoma has a worse prognosis, it has been hypothesized that SALL4 may be involved in the progression of bladder urothelial carcinoma.
To further investigate the effect of SALL4 on the growth of bladder carcinoma cells, we used a SALL4 overexpression plasmid and siRNA interference to examine the effects of SALL4 on the proliferation, migration and invasion of bladder carcinoma cells. We demonstrate that the proliferative, migratory and invasive abilities of bladder carcinoma cells were signi cantly enhanced after up-regulation of SALL4, and signi cantly decreased after down-regulation of SALL4. Our results are consistent with previous studies, which have shown that the SALL4 gene is associated with the proliferation, migration and invasion of various tumors. For example, overexpression of SALL4 in lung cancer promoted the proliferation of tumor cells, while deletion of the SALL4 gene led to G1 cell cycle arrest and inhibition of cancer cell growth [33]. In esophageal, colon and endometrial cancers, overexpression of SALL4 was associated with stronger migratory and invasive abilities, while inhibition of SALL4 resulted in less migration and invasion [28,34,35]. Our data demonstrate a role for SALL4 in mediating proliferation, migration and invasion of bladder cancer cells, and further indicate that SALL4 could be used as a potential target gene for bladder cancer treatment.
To further explore the possible mechanism of SALL4 in bladder cancer, we detected the expression levels of β-catenin and several key components of the EMT signaling pathway by western blot analysis in vitro. The Wnt/β-catenin signaling pathway is associated with EMT and involved in tumor invasion and metastasis [36,37]. Here, we show that SALL4 overexpression was associated with increased N-cadherin, vimentin and Snail expression, and decreased E-cadherin expression. In contrast, SALL4 silencing resulted in down-regulation of N-cadherin, vimentin and Snail expression and increased E-cadherin expression. In endometrial carcinoma [11] and gastric cancer [31], up-regulation of SALL4 can lead to upregulation of N-cadherin and down-regulation of E-cadherin, as well as promote EMT and enhance invasive and metastatic abilities. Consistent with these reports, our data also indicate that SALL4 is involved in the regulation of EMT, and subsequent regulation of migration, invasion and metastasis in bladder cancer.
In addition, we found that β-catenin expression was signi cantly increased in the SALL4-overexpression group compared with the control group, while β-catenin expression was signi cantly lower in the SALL4-siRNA group. SALL4-mediated activation of the Wnt/β-catenin signaling pathway promotes tumor proliferation and invasion in a variety of tumors, such as acute myeloid leukemia (AML) [38] and cervical cancer [39]. Previously, β-catenin was found to be up-regulated in human bladder cancer specimens compared to normal urothelial cells [40]. Urakami et al concluded that the β-catenin signaling pathway was involved in the pathological progression of bladder cancer, by promoting tumor cell proliferation, migration and invasion, and inhibiting apoptosis [41]. Therefore, SALL4 may activate the Wnt/β-catenin signaling pathway, and the resulting up-regulation of β-catenin may further promote bladder cancer cell proliferation, migration and invasion. Based on our ndings, we speculate that SALL4 may activate the Wnt/β-catenin signaling pathway in bladder cancer, and that the resulting up-regulation in β-catenin expression may be involved in the regulation of EMT, and promotion of proliferation, migration and invasion.
There are also some limitations to our study. First, immunocoprecipitation studies are required to examine the direct effect of SALL4 on β-catenin, and con rm that SALL4 is involved in the Wnt/β-catenin signaling pathway and associated with EMT in bladder cancer cells. In addition, future studies should examine the relationship between SALL4 and tumor growth in vivo. For example, the effectiveness of SALL4-targeted drugs to treat tumors in animal tumor models could be measured to determine whether targeting SALL4 leads to inhibition of tumor growth.  Overexpression and knockdown of SALL4 in bladder urothelial carcinoma.
A and B, T24 cells were transfected with control PLVX-puro or PLVX-SALL4-puro, and SALL4 mRNA and protein expression levels were determined by real time-PCR and western blot analysis. C and D, Real time-PCR and western blot analysis were used to examine SALL4 mRNA and protein expression levels in 5637 cells transfected with control siRNA or SALL4-siRNA. β-actin was used as an internal control (* P < 0.05, *** P < 0.001) Figure 4 Cell growth curves of SALL4-overexpressing T24 and SALL4-knockdown 5637 cell lines were determined by CCK8 assay. A, T24 cells; B, 5637 cells. (Scale bar = 100 μm) (* P < 0.05, ** P < 0.01) Figure 6 SALL4 affects the expression of β-catenin and EMT-related proteins.
Western blot analysis was used to assess β-catenin and EMT-related (N-cadherin, vimentin, Snail and Ecadherin) protein expression levels after SALL4 overexpression or knockdown. A, The effects of SALL4 overexpression on β-catenin, N-cadherin, vimentin, Snail and E-cadherin protein expression levels in T24 cells; B, The effects of SALL4 knockdown on β-catenin, N-cadherin, vimentin, Snail and E-cadherin protein expression levels in 5637 cells. Data are expressed as mean±SD from three independent experiments (* P < 0.05, ** P < 0.01).