Higher Expression of Annexin A2 in Metastatic Bladder Urothelial Carcinoma Promotes Migration and Invasion

Simple Summary Bladder urothelial carcinoma (BLCA) arises from basal cells that develop dysplasia and carcinoma in situ and, if unchecked, progress to invasion and metastases. Major players underlying the molecular mechanisms of invasive and metastatic BLCA are yet to be determined. Annexin A2 (AnxA2), a Ca++-dependent phospholipid-binding protein, is overexpressed in various cancers and facilitates cell migration and invasion. This study established a correlation between AnxA2 and BLCA using existing BLCA data from the Cancer Genome Atlas (TCGA). In addition, we determined its role in the proliferation, migration, and invasion of metastatic bladder cancer cells. Abstract In this study, we aim to evaluate the significance of AnxA2 in BLCA and establish its metastatic role in bladder cancer cells. Analysis of TCGA data showed that AnxA2 mRNA expression was significantly higher in BLCA tumors than in normal bladder tissues. High mRNA expression of AnxA2 in BLCA was significantly associated with high pathological grades and stages, non-papillary tumor histology, and poor overall survival (OS), progression-free survival (PFS), and diseases specific survival (DSS). Similarly, we found that AnxA2 expression was higher in bladder cancer cells derived from high-grade metastatic carcinoma than in cells derived from low-grade urothelial carcinoma. AnxA2 expression significantly mobilized to the surface of highly metastatic bladder cancer cells compared to cells derived from low-grade tumors and associated with high plasmin generation and AnxA2 secretion. In addition, the downregulation of AnxA2 cells significantly inhibited the proliferation, migration, and invasion in bladder cancer along with the reduction in proangiogenic factors and cytokines such as PDGF-BB, ANGPT1, ANGPT2, Tie-2, bFGF, GRO, IL-6, IL-8, and MMP-9. These findings suggest that AnxA2 could be a promising biomarker and therapeutic target for high-grade BLCA.


Introduction
Bladder cancer is the 6th most common cancer in the US, with 81,180 estimated new cases and 17,100 estimated deaths in 2022 [1,2]. More than 90% of these cases are bladder urothelial carcinoma (BLCA). The prognosis and treatment options for BLCA are highly variable. Non-muscle invasive bladder cancers include stages 0 and 1. They comprise non-invasive papillary Ta, in situ Tis, tumors that invade lamina propria T1 [3]. These make up about 70% of all bladder cancers, are treated with localized approaches, and have an overall good prognosis but recur frequently [4,5]. This high risk of recurrence necessitates The membrane was then washed and incubated with the biotinylated antibody cocktail. The immunoblot signals were captured using the alpha-imager Fluoretech HD2. The protein signals were measured using NIH ImageJ software and analyzed using AAH-ANG-1/2 software (RayBiotech, Inc.). The results were normalized to the positive and negative controls on the array.

Cell Proliferation Assay
Cell proliferation assay was performed as described previously [30]. Briefly, bladder cancer cells were seeded in a 96-well plate at the density of 500 cells/well in Mc-Coy's 5a medium. After incubation, 10 µL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium Bromide (MTT) solution (5 mg/mL in PBS) was added to each well and incubated for additional 4 h at 37 • C. The medium was then aspirated from each well. The formazan crystals formed in the well were dissolved by adding Dimethylsulfoxide (100 µL) to each well. The absorbance of formazan at 570 nm was measured by a microplate reader (Synergy 2, BioTek Instruments, Inc., Winooski, VT, USA) at different time points.

Cell Migration Assay
Cell migration assay was performed as described previously [30]. Bladder cancer cells were seeded in 6-well plates and grew into full confluency. A sterile pipette tip was used to create a clear line in each well. The growth medium was replaced with a fresh medium. The migration toward the center of the wound was determined at indicated time points.

Transwell Invasion Assay
Bladder cancer cells were seeded onto the upper chamber of a transwell membrane coated with Matrigel (BD Biosciences) in a serum-free medium [31]. The lower chamber was filled with the complete serum-containing medium as a chemoattractant. The transwell plates were cultured in a CO 2 incubator for 24 h, and then cells inside the upper chamber were removed with cotton swabs. The invaded cells that remained on the lower surface of the filter were fixed with 1% paraformaldehyde, permeabilized with methanol, and then stained with crystal violet (0.1%). The number of stained cells in six randomly selected fields of each chamber were manually counted and analyzed.

Immunoprecipitation
The bladder cancer cells (2.5 × 10 6 ) were plated in a 100 mm Petri dish overnight in a complete medium and then replaced with a serum-free medium for another 24 h. After incubation, the serum-free medium was collected and incubated with anti-AnxA2 (BD Pharmingen #610069) antibody at 4 • C overnight. Protein A/G-PLUS agarose beads (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were then added to the medium and incubated for 2 h at 4 • C. The agarose beads were collected, washed, and resuspended in a 2× sample buffer. After boiling, the supernatant was collected and then analyzed by immunoblot analysis.

Elution of Cell Surface AnxA2
The confluent bladder cancer cells were washed with PBS and then incubated with Versene (0.53 mM EDTA in PBS) for 10 min at 37 • C. The Versene-wash fractions were collected and subjected to SDS-PAGE and immunoblot analysis. In addition, the Versenewash fractions were examined for lack of cytosolic proteins by immunoblotting with an anti-3-phosphoglycerate kinase antibody.

Cell Surface Biotinylation
The bladder cancer cells were washed with serum-free medium and then biotinylated with sulfo-N-hydroxysulfosuccinimide (NHS) biotin (0.5 mg/mL; Cayman Chemicals) for 30 min at 37 • C in a cell culture incubator [32]. After several PBS washes, the cells were lysed in Triton-X-100 lysis buffer, and then surface-biotinylated proteins were purified by incubation with streptavidin-conjugated Sepharose (Cell signaling) overnight at 4 • C. After washing with lysis buffer, Laemmli sample buffer (2×) was added to dissociate the surface-biotinylated proteins from the beads, and they were subjected to SDS-PAGE and immunoblot analysis.

Plasmin Generation Assay
Chromogenic plasmin generation assay was performed in the presence of a competitive AnxA2 hexapeptide inhibitor (LCKLSL) or control peptide (LGKLSL). The bladder cancer cells were seeded equally in 12-well plates in a complete serum medium. After overnight incubation, the cell culture medium was replaced with serum-free phenol red-free medium containing either an AnxA2 hexapeptide or a control peptide or in the absence of both for an additional 4 h. The reaction was initiated by adding 100 nm Glu-plasminogen (Diapharma Group, Inc., West Chester Township, OH, USA). After 24 h, supernatant from all the wells was obtained, and after centrifugation, 100 µL of supernatant was added to a 96-well plate in triplicates. Freshly prepared 1 mM chromogenic substrate S-2403 (Diapharma Group, Inc.) was added to each well, and absorbance was measured at 410 nm (Synergy HT-BioTek) at different time intervals. The fold change in plasmin generation was calculated by normalizing the amount of plasmin generated in the untreated group.

RNA Expression Data for BLCA Patients
The University of California Santa Cruz browser was used to download BLCA RNAseq data (Illumina HiSeq) from The Cancer Genome Atlas (TCGA) [33]. The Log2 transformed normalized RSEM count data for BLCA tumor samples (n = 409), and normal bladder samples (n = 19) were extracted for further analysis. The corresponding clinical information such as stage, grade, tumor histology, overall survival (OS) status, OS time (days), progression-free survival (PFS) status, PFS time (days), diseases specific survival (DSS) status, and DSS time (days) was also obtained from the BLCA-TCGA database. Survival analyses were based on Cox proportional hazard regression model to assess the effect of high vs. low AnxA2 on survival and if the effect is significant. We have reported the hazard ratio (high vs. low) and the corresponding confidence interval (CI). For each dataset, we have performed the Schoenfeld residual test for potential violation of the proportional hazards (PH) assumption, and in each case, the assumption is not violated. These analyses determined the impacts of the annexins on OS, PFS, and DSS.

Statistical Analysis
GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) was used for statistical analyses, and results were presented as mean ± SEM. Comparison between two groups was performed using Student's t-test, while the comparison for more than two groups was performed using one-way ANOVA. Statistical significance was two-tailed and considered significant if the p-value was at least ≤ 0.05: (*), p < 0.05; (**), p < 0.01; (***), p < 0.001; (****), p < 0.0001.

AnxA2 mRNA Expression Is Associated with BLCA Patients
To investigate the role of AnxA2 in bladder urothelial carcinoma, we analyzed the expression of AnxA2 mRNA in normal bladder tissue samples (n = 19) and BLCA tissue samples (n = 409) from the TCGA database [34]. The expression of AnxA2 was significantly upregulated in BLCA tumor tissues compared to normal bladder tissues (p < 0.0001, Figure 1A). In addition, our analysis revealed that high expression of AnxA2 is significantly associated with aggressive features of BLCA. The high expression of AnxA2 was accompanied by high tumor stage and grade ( Figure 1B,C). AnxA2 expression was also significantly high in non-papillary tumors (p < 0.001) compared to papillary tumors ( Figure 1D). These observations suggest that AnxA2 expression is significantly associated with aggressive high-grade tumor phenotypes in BLCA patients. using Student's t-test, while the comparison for more than two groups was performed using one-way ANOVA. Statistical significance was two-tailed and considered significant if the p-value was at least ≤ 0.05: (*), p < 0.05; (**), p < 0.01; (***), p < 0.001; (****), p < 0.0001.

AnxA2 mRNA Expression Is Associated with BLCA Patients
To investigate the role of AnxA2 in bladder urothelial carcinoma, we analyzed the expression of AnxA2 mRNA in normal bladder tissue samples (n = 19) and BLCA tissue samples (n = 409) from the TCGA database [34]. The expression of AnxA2 was significantly upregulated in BLCA tumor tissues compared to normal bladder tissues (p < 0.0001, Figure 1A). In addition, our analysis revealed that high expression of AnxA2 is significantly associated with aggressive features of BLCA. The high expression of AnxA2 was accompanied by high tumor stage and grade ( Figure 1B,C). AnxA2 expression was also significantly high in non-papillary tumors (p < 0.001) compared to papillary tumors (Figure 1D). These observations suggest that AnxA2 expression is significantly associated with aggressive high-grade tumor phenotypes in BLCA patients. Statistical analysis was performed using a two-tailed Student's t-test. **** indicates p < 0.0001. Scatter plot analysis of AnxA2 mRNA expression in TCGA-BLCA patients based on stage (B), grade (C), and tumor histology (D). Statistical analyses were performed using either student's t-test or one-way ANOVA followed by Tukey's multiple comparison test. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

High Expression of AnxA2 Is Correlated with Poor Prognosis in BLCA Patients
We further analyzed AnxA2 mRNA expression association with survival in BLCA patients [34]. AnxA2 mRNA levels were dichotomized into low and high based on the median of logarithmized expression values. We observed a significant poor overall survival (OS) in BLCA patients with high AnxA2 mRNA expression [hazard ratio = 1.452; 95% confidence interval (CI) = 1.022−1.958, p = 0.0144; Figure 2A] compared to low AnxA2 mRNA expression. In addition, we also observed that high mRNA expression of AnxA2 was significantly associated with worse diseases specific survival (DSS; hazard ratio = 1.562; 95% CI = 1.085−2.249; log-rank p = 0.0165; Figure 2B) and worse progression-free survival (PFS; hazard ratio = 1.433; 95% CI = 1.06−1.937; log-rank p = 0.0193; Figure 2C) in BLCA patients. This analysis confirms that high mRNA expression of AnxA2 in tumor tissues results in poor survival in BLCA patients and suggests AnxA2 as a potential prognostic predictor of BLCA patients. Furthermore, the expression level of AnxA2 alone was able to distinguish the tumor from normal tissue, with an area under the curve (AUC) of 0.6346 ± 0.0576 (95% CI 0.5217-0.7475, p = 0.0472) ( Figure 2D). ) and tumor tissue (n = 409) samples obtained from the TCGA-BLCA RNAseq database tical analysis was performed using a two-tailed Student's t-test. **** indicates p < 0.0001. Scat analysis of AnxA2 mRNA expression in TCGA-BLCA patients based on stage (B), grade ( tumor histology (D). Statistical analyses were performed using either student's t-test or o ANOVA followed by Tukey's multiple comparison test. (*, p < 0.05; **, p < 0.01; ***, p < 0.001

High Expression of AnxA2 Is Correlated with Poor Prognosis in BLCA Patients
We further analyzed AnxA2 mRNA expression association with survival in patients [34]. AnxA2 mRNA levels were dichotomized into low and high based median of logarithmized expression values. We observed a significant poor overa vival (OS) in BLCA patients with high AnxA2 mRNA expression [hazard ratio = 95% confidence interval (CI) = 1.022−1.958, p = 0.0144; Figure 2A] compared to low A mRNA expression. In addition, we also observed that high mRNA expression of A was significantly associated with worse diseases specific survival (DSS; hazard 1.562; 95% CI = 1.085−2.249; log-rank p = 0.0165; Figure 2B) and worse progressio survival (PFS; hazard ratio = 1.433; 95% CI = 1.06−1.937; log-rank p = 0.0193; Figure  BLCA patients. This analysis confirms that high mRNA expression of AnxA2 in tissues results in poor survival in BLCA patients and suggests AnxA2 as a potentia nostic predictor of BLCA patients. Furthermore, the expression level of AnxA2 alon able to distinguish the tumor from normal tissue, with an area under the curve (A 0.6346 ± 0.0576 (95% CI 0.5217-0.7475, p = 0.0472) ( Figure 2D).

AnxA2 Expression in Bladder Cancer Cell Lines
The BLCA RNAseq analysis demonstrated that AnxA2 mRNA expression was significantly elevated in high-grade tumors compared to low-grade tumors in BLCA patients. therefore, the expression of AnxA2 protein was compared by immunoblot analysis in lysates prepared from RT4 and T24 bladder cancer cell lines, which were derived from a grade I urothelial carcinoma (also called papilloma) patient [35] and poorly differentiated (grade III) bladder carcinoma with metastatic profile [36], respectively. The immunoblot presented in Figure 3A,B showed that AnxA2 protein expression was significantly higher in T24 bladder cancer cells than in RT4 cells. Our results further confirm that high expression of AnxA2 is significantly associated with the aggressive phenotype of bladder cancer. In addition, we have further examined whether increased expression of AnxA2 in bladder cancer mobilizes it to the outer membrane. To elute cell surface AnxA2, we used Versene (0.53 mM EDTA in PBS) solution to wash the cells. Because AnxA2 is a Ca ++ -dependent phospholipid-binding protein, the AnxA2 protein should be dissociated from the cell surface after Versene wash. Therefore, we collected the Versene-wash fractions and analyzed them with immunoblotting. The blots in Figure 3C,D show that a large pool of AnxA2 is present in the Versene wash fraction of T24 cells compared to RT4 cells. We also analyzed the cell surface levels of AnxA2 in bladder cancer cells by conjugating all cell surface proteins with membrane-impermeable biotin and immobilizing them with streptavidin-linked beads. Immunoblot analysis of biotin-conjugated and streptavidinimmobilized cell surface extracts revealed that AnxA2 expression at the cell surface of T24 cells was 3-fold higher compared to RT4 cells ( Figure 3E,F). Coomassie staining was used as protein loading controls for both Versene-wash fractions and cell surface biotinylated extracts. These results suggest that high cell surface expression of AnxA2 is associated with aggressive phenotypes of bladder cancer cells.
tients. therefore, the expression of AnxA2 protein was compared by immunoblot ana in lysates prepared from RT4 and T24 bladder cancer cell lines, which were derived a grade I urothelial carcinoma (also called papilloma) patient [35] and poorly differ ated (grade III) bladder carcinoma with metastatic profile [36], respectively. The munoblot presented in Figures 3A,B showed that AnxA2 protein expression was sig cantly higher in T24 bladder cancer cells than in RT4 cells. Our results further confirm high expression of AnxA2 is significantly associated with the aggressive phenotyp bladder cancer. In addition, we have further examined whether increased expressio AnxA2 in bladder cancer mobilizes it to the outer membrane. To elute cell surface An we used Versene (0.53 mM EDTA in PBS) solution to wash the cells. Because AnxA2 Ca ++ -dependent phospholipid-binding protein, the AnxA2 protein should be dissoci from the cell surface after Versene wash. Therefore, we collected the Versene-wash tions and analyzed them with immunoblotting. The blots in Figure 3C,D show that a l pool of AnxA2 is present in the Versene wash fraction of T24 cells compared to RT4 We also analyzed the cell surface levels of AnxA2 in bladder cancer cells by conjuga all cell surface proteins with membrane-impermeable biotin and immobilizing them streptavidin-linked beads. Immunoblot analysis of biotin-conjugated and streptav immobilized cell surface extracts revealed that AnxA2 expression at the cell surface o cells was 3-fold higher compared to RT4 cells ( Figure 3E,F). Coomassie staining was as protein loading controls for both Versene-wash fractions and cell surface biotinyl extracts. These results suggest that high cell surface expression of AnxA2 is associ with aggressive phenotypes of bladder cancer cells.  The cell surface biotinylation was performed as described in the material and methods. The extracts were immunoblotted with an anti-AnxA2 antibody. Coomassie Blue staining was performed for loading control. A ∼14-kD, Coomassie-stained band, whose levels seemed invariant in both cell lines, was used to account for differences in protein loading. (F) The intensity of AnxA2 was performed by densitometry, and fold change in AnxA2 expression was determined. **, p < 0.01. Uncropped Western Blots of (A,C,E) are available in File S1.

High Expression of AnxA2 at the Cell Surface of Bladder Cancer Cells Promotes Plasmin Generation
AnxA2-mediated plasmin generation assay was performed to examine whether an increased cell surface pool of AnxA2 in bladder cancer cells is associated with an extensive production of plasmin. First, we compared the biochemical conversion of plasminogen to plasmin in RT4 and T24 cell lines ( Figure 4A). We found that plasmin production is approximately 18-fold higher in T24 cells than in RT4 cells. To further confirm that the high production of plasmin in T24 cells is due to the increased cell surface pool of AnxA2, we treated the cells with a competitive hexapeptide inhibitor (LCKLSL) of tPA, which binds to AnxA2 or control peptide (LGKLSL) which does not possess any plasmin inhibitory effects [37,38]. As shown in Figure 4B, treating T24 cells with LCKLSL peptide resulted in a 2.3-fold reduction in plasmin generation compared with the control peptide, LGKLSL. These results suggest that the increased cell surface levels of AnxA2 in T24 cells directly contribute to the high production of AnxA2-mediated cell surface generation of plasmin.
massie Blue staining was performed for loading control. A ∼14-kD, Coomassie-stained band, whos levels seemed invariant in both cell lines, was used to account for differences in protein loading. (F The intensity of AnxA2 was performed by densitometry, and fold change in AnxA2 expression wa determined. **, p < 0.01. Uncropped Western Blots of (A,C,E) are available in Figure S1.

High Expression of AnxA2 at the Cell Surface of Bladder Cancer Cells Promotes Plasmin Generation
AnxA2-mediated plasmin generation assay was performed to examine whether a increased cell surface pool of AnxA2 in bladder cancer cells is associated with an extensiv production of plasmin. First, we compared the biochemical conversion of plasminogen t plasmin in RT4 and T24 cell lines ( Figure 4A). We found that plasmin production is ap proximately 18-fold higher in T24 cells than in RT4 cells. To further confirm that the hig production of plasmin in T24 cells is due to the increased cell surface pool of AnxA2, w treated the cells with a competitive hexapeptide inhibitor (LCKLSL) of tPA, which bind to AnxA2 or control peptide (LGKLSL) which does not possess any plasmin inhibitor effects [37,38]. As shown in Figure 4B, treating T24 cells with LCKLSL peptide resulted i a 2.3-fold reduction in plasmin generation compared with the control peptide, LGKLSL These results suggest that the increased cell surface levels of AnxA2 in T24 cells directl contribute to the high production of AnxA2-mediated cell surface generation of plasmin

AnxA2 Secretion from Bladder Cancer Cells
Although AnxA2 does not have signals for secretion, other investigators reported i as a secretory protein [22,25,27,[39][40][41]. Therefore, we examined the secretion of AnxA2 i Figure 4. AnxA2-mediated plasmin generation. (A) Chromogenic plasmin generation assay was performed in RT4 and T24 bladder cancer cell lines as described in the materials and methods. To serve as a control, Glu-plasminogen was omitted from the reaction mixture. The fold increase in plasmin generation was determined. (B) Chromogenic plasmin generation assay was performed in T24 in the presence of either AnxA2 inhibitory hexapeptide (LCKLSL; 1 mM) or control peptide (LGKLSL; 1 mM). The fold change in plasmin generation was obtained. The data are means ±SEM (n = 3). ns , non-significant; *, p < 0.05.

AnxA2 Secretion from Bladder Cancer Cells
Although AnxA2 does not have signals for secretion, other investigators reported it as a secretory protein [22,25,27,[39][40][41]. Therefore, we examined the secretion of AnxA2 in the condition media of RT4 and T24 bladder cancer cells. Secreted AnxA2 protein was immunoprecipitated from the serum-free condition media of RT4 and T24 cells using an anti-AnxA2 antibody. On immunoblotting with anti-AnxA2 antibody, high expression of AnxA2 was predominantly observed in conditioned media of T24 cells. In contrast, deficient AnxA2 expression was detected in RT4 cells ( Figure 5), indicating that the cells derived from high-grade tumors secrete more AnxA2 than those derived from low-grade tumors.

Depletion of AnxA2 Inhibits the Proliferation, Migration, and Invasion of Bladder Cancer Cells
To elucidate the biological functions of AnxA2, we down-regulated the expression of AnxA2 in the bladder cancer T24 cells. The depletion of AnxA2 was detected by immunoblot analysis. We found that the stable transfection of AnxA2 shRNA effectively reduces the expression of AnxA2 in T24 cells ( Figure 6A,B). The expression level of AnxA2 was only 9% of the cells transfected with non-targeting shRNA, indicating that AnxA2 shRNA significantly reduced the AnxA2 levels in T24 cells. On this basis, we explore the effects of AnxA2 on bladder cancer cell proliferation by performing the MTT assay. The results revealed that AnxA2 knockdown significantly impaired the proliferation of T24 cells. The cell proliferation was considerably lower in AnxA2-depleted cells compared to the non-targeting shRNA-transfected cells after 2, 3, and 4 days of time intervals (p < 0.0001) ( Figure 6C). The role of AnxA2 in T24 bladder cancer cell migration was assessed using the wound healing assay. Significant closure of the scratch wound was observed in the T24 cells transfected with non-targeting shRNA after 12 h. However, scratch wound closure was significantly inhibited in AnxA2-depleted T24 cells (p < 0.01) compared to non-targeting shRNA transfected cells ( Figure 6D,E). Transwell assays were performed to examine the role of AnxA2 in bladder cancer cell invasion. The number of T24 cells that had migrated through to the bottom of the transwell membrane was significantly lower in the AnxA2-depleted T24 cells compared to the T24 cells transfected with non-targeting shRNA (p < 0.01; Figure 6F,G). These results have demonstrated that AnxA2 plays a crucial role in bladder cancer cell proliferation, migration, and invasion. the condition media of RT4 and T24 bladder cancer cells. Secreted AnxA2 protein was immunoprecipitated from the serum-free condition media of RT4 and T24 cells using an anti-AnxA2 antibody. On immunoblotting with anti-AnxA2 antibody, high expression of AnxA2 was predominantly observed in conditioned media of T24 cells. In contrast, deficient AnxA2 expression was detected in RT4 cells ( Figure 5), indicating that the cells derived from high-grade tumors secrete more AnxA2 than those derived from low-grade tumors. Figure 5. AnxA2 secretion from bladder cancer cell lines. The RT4 or T24 cells (2.5 × 10 6 ) were plated in a 100 mm Petri dish overnight and then replaced with a serum-free medium. AnxA2 protein was immunoprecipitated from the serum-free medium after 24 h incubation using an anti-AnxA2 antibody (BD Biosciences #610069). The amount of secreted AnxA2 in the medium was analyzed by immunoblotting using an anti-AnxA2 antibody. Uncropped Western Blot is available in Figure S1.

Depletion of AnxA2 inhibits the proliferation, migration, and invasion of bladder cancer cells
To elucidate the biological functions of AnxA2, we down-regulated the expression of AnxA2 in the bladder cancer T24 cells. The depletion of AnxA2 was detected by immunoblot analysis. We found that the stable transfection of AnxA2 shRNA effectively reduces the expression of AnxA2 in T24 cells ( Figure 6A,B). The expression level of AnxA2 was only 9% of the cells transfected with non-targeting shRNA, indicating that AnxA2 shRNA significantly reduced the AnxA2 levels in T24 cells. On this basis, we explore the effects of AnxA2 on bladder cancer cell proliferation by performing the MTT assay. The results revealed that AnxA2 knockdown significantly impaired the proliferation of T24 cells. The cell proliferation was considerably lower in AnxA2-depleted cells compared to the non-targeting shRNA-transfected cells after 2, 3, and 4 days of time intervals (p < 0.0001) ( Figure 6C). The role of AnxA2 in T24 bladder cancer cell migration was assessed using the wound healing assay. Significant closure of the scratch wound was observed in the T24 cells transfected with non-targeting shRNA after 12 h. However, scratch wound closure was significantly inhibited in AnxA2-depleted T24 cells (p < 0.01) compared to non-targeting shRNA transfected cells ( Figure 6D,E). Transwell assays were performed to examine the role of AnxA2 in bladder cancer cell invasion. The number of T24 cells that had migrated through to the bottom of the transwell membrane was significantly lower in the AnxA2-depleted T24 cells compared to the T24 cells transfected with non-targeting shRNA (p < 0.01; Figure 6F,G). These results have demonstrated that AnxA2 plays a crucial role in bladder cancer cell proliferation, migration, and invasion. AnxA2 secretion from bladder cancer cell lines. The RT4 or T24 cells (2.5 × 10 6 ) were plated in a 100 mm Petri dish overnight and then replaced with a serum-free medium. AnxA2 protein was immunoprecipitated from the serum-free medium after 24 h incubation using an anti-AnxA2 antibody (BD Biosciences #610069). The amount of secreted AnxA2 in the medium was analyzed by immunoblotting using an anti-AnxA2 antibody. Uncropped Western Blot is available in File S1.

Discussion
Bladder urothelial carcinoma is a highly heterogeneous disease with varying clinical outcomes. Patients with the same tumor stage may have different survival outcomes [2][3][4][5]42]. The survival rates correlate directly with the cancer cell proliferation, migration, and invasion of bladder cancer. Thus, it is essential to identify molecular markers to detect bladder cancer early before cancer cells become invasive and metastatic. Annexins display different expression patterns in bladder cancer and play an essential role in the pathogenesis by triggering various downstream signaling pathways and functional regulatory effects [19,20,[43][44][45][46][47][48]. Increasing evidence indicates that AnxA2 is upregulated in several types of human cancer [13,17,21,22,24,[26][27][28][29]. Annexin A2 is a potential biomarker for predicting drug resistance and recurrence of bladder cancers [19,20]. The TCGA data suggests that AnxA2 was significantly overexpressed in BLCA tumors. A high abundance of AnxA2 was noticeably associated with high grade and stage and aggressive histologic grade bladder cancer and revealed poor prognosis in bladder cancer patients. We found that the AnxA2 protein was upregulated in bladder cancer cells derived from high-grade

Discussion
Bladder urothelial carcinoma is a highly heterogeneous disease with varying clinical outcomes. Patients with the same tumor stage may have different survival outcomes [2][3][4][5]42]. The survival rates correlate directly with the cancer cell proliferation, migration, and invasion of bladder cancer. Thus, it is essential to identify molecular markers to detect bladder cancer early before cancer cells become invasive and metastatic. Annexins display different expression patterns in bladder cancer and play an essential role in the pathogenesis by triggering various downstream signaling pathways and functional regulatory effects [19,20,[43][44][45][46][47][48]. Increasing evidence indicates that AnxA2 is upregulated in several types of human cancer [13,17,21,22,24,[26][27][28][29]. Annexin A2 is a potential biomarker for predicting drug resistance and recurrence of bladder cancers [19,20]. The TCGA data suggests that AnxA2 was significantly overexpressed in BLCA tumors. A high abundance of AnxA2 was noticeably associated with high grade and stage and aggressive histologic grade bladder cancer and revealed poor prognosis in bladder cancer patients. We found that the AnxA2 protein was upregulated in bladder cancer cells derived from high-grade tumors and also associated with higher secretion in the media. In vitro assays demonstrated that depletion of AnxA2 significantly inhibited the proliferation, metastasis, and invasion of bladder cancer cells by downregulating the expression of proangiogenic proteins and cytokines. Our results provide a strong case for AnxA2 as a potential prognostic predictor with poor clinical outcomes for bladder cancer patients.
Upregulation of AnxA2 has been observed in gastric cancer, endometrial cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, breast cancer, kidney renal clear cell carcinoma, high-grade prostate cancer, and glioblastoma. It has a significant correlation with advanced stage and unfavorable prognosis [13,17,21,22,24,[26][27][28][29]. Previous studies suggested that AnxA2 was upregulated in bladder urothelial carcinoma tumor tissues and associated with lymph node metastasis and distant metastasis [19,20]. Based on these findings, we conducted a bioinformatics analysis to explore the expression of AnxA2 in the BLCA-TCGA database. Our analysis confirmed that a high expression level of AnxA2 was noticeably associated with high tumor stage and grade and an unfavorable prognosis of bladder cancer. Furthermore, the high expression of AnxA2 was significantly observed in non-papillary carcinoma, which is usually high-grade and invasive, compared to papillary carcinoma, which is usually low-grade and non-invasive [49,50]. Furthermore, Kaplan-Meier curves analysis indicated that AnxA2 was a prognostic factor for BLCA. Together, these results further established that high mRNA expression of AnxA2 might contribute to the tumorigenesis of BLCA and be an essential risk factor for the poor clinical outcome of bladder cancer patients.
AnxA2 is primarily present in the cytoplasm and cell membrane of the cells, and a small fraction is also present in the nucleus [12,13,15,21]. AnxA2 exists either as a monomer or a heterotetramer complex formed by two of p11 and two AnxA2 monomers [11]. Upon translocation to the outer cell membrane, the AnxA2 heterotetramer complex serves as a receptor for tPA, converts plasminogen into plasmin, and promotes angiogenesis. Our study indicated that cell surface expression of AnxA2 is very high in T24 cells derived from a high-grade tumor. In addition, our results also showed that high cell surface expression of AnxA2 in T24 cells produces a large amount of plasmin which plays an important role in angiogenesis and metastasis of high-grade bladder urothelial carcinoma [51,52]. Blocking the activity of the AnxA2 using LCKLSL hexapeptide can have potential therapeutic implications on inhibiting the pathological processes associated with enhanced plasmin generation in high-grade aggressive BLCA [18,37,38]. Furthermore, Lu et al. identified secretory AnxA2 as a potential urine biomarker for urothelial carcinoma of the upper urinary tract [53]. Our results are consistent with these findings in urothelial carcinoma of the bladder. The high secretion of AnxA2 from T24 cells was significantly observed with aggressive subtypes of bladder cancer.
However, our study has some potential limitations. First, we must collect the tumor tissue samples from bladder urothelial carcinoma patients and relevant prognoses to confirm further the AnxA2 expression and its association with clinical outcomes. Secondly, we must determine how AnxA2 promotes bladder cancer cell proliferation, migration, and invasion. These limitations will be addressed in future studies.

Conclusions
In conclusion, our results demonstrated that high expression of AnxA2 promotes proliferation, migration, and invasion in bladder cancer cells, and AnxA2 mRNA expression was significantly associated with the poor prognosis of BLCA patients. To our knowledge, this is the first report that demonstrated the biological function of AnxA2 in Bladder urothelial carcinoma. Our study provides evidence that AnxA2 might be a potential therapeutic target and prognostic marker for Bladder Urothelial Carcinoma.