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Introduction

Bladder cancer is one of the most common malignant neoplasms in genitourinary system (1,2). More than 350,000 individuals are diagnosed with incident bladder cancer per year worldwide, including more than 100,000 per year in China (3). Epithelial to mesenchymal transition (EMT) and generation of cancer initiating cells (CICs) have been proposed to play important roles in recurrence of bladder cancer (4,5). Understanding EMT and formation of CICs requires first and foremost the understanding of their molecular mechanisms.

Human HLA-F adjacent transcript 10 (FAT10), an 18-kDa protein, is related to crucial cellular physiology and pathophysiology, including regulation of cell-cycle progression, apoptosis and immune-mediated inflammation (6,7). Knockdown of FAT10 inhibits proliferation and growth in bladder cancer (8). FAT10 protein expression is upregulated in bladder cancer and increased expression of FAT10 is correlated with progression and poor prognosis of bladder cancer (8). However, functional contribution of FAT10 to EMT and formation of CICs remains unclear in bladder cancer.

Materials and methods

Bladder cancer patients and tumor tissues

Patients with bladder cancer were recruited from the First Affiliated Hospital of Soochow University (Suzhou, China) from January 2000 to December 2008. Tumor samples were obtained from 98 patients with primary bladder cancer. The patients consisted of 42 women (42.9%) and 56 men (57.1%) with a median age of 70 years old (range: 42–90 years). All tumors and control tissues were reviewed by two pathologists. Normal bladder epithelium (NBE) samples were obtained from patients with prostate cancer who had undergone radical prostatectomy (9,10). NBE samples were also reviewed by two pathologists. The Ethics Committee of the First Affiliated Hospital of Soochow University approved the study. The study was performed according to the Helsinki Declaration and after ethical approval was obtained from the local ethics committee. Written informed consent was obtained from each subject before inclusion into the trial.

Immunohistochemistry (IHC)

Detection of FAT10 was performed on 5 mm paraffin sections of tumor tissues with primary FAT10 antibody (ab192581) and secondary antibody (ab191866; both Abcam, Cambridge, UK). The materials and methods for IHC were described previously (11).

Bladder cancer cell lines

Human invasive bladder cancer (T24, HT-1376, J82, UMUC-3, RT4 and TCCSUP) cell lines were obtained from Tiangen Biotech Co., Ltd. (Beijing, China) and described previously (12,13). Briefly, cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and penicillin/streptomycin (Sigma, Inc., St. Louis, MO, USA). All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.

FAT10-expressing plasmids/empty vectors (pcDNA3.1) and transfection

FAT10-expressing plasmids and empty vectors (pcDNA3.1) were purchased from Tiangen Biotech Co., Ltd. For transfection experiments, the cells were cultured in serum-free medium without antibiotics at 60% confluence for 24 h, and then transfected with transfection reagent (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. After incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 h.

Western blot analysis

It was performed as described previously (14). Mainly, after incubation with anti-FAT10 antibody (ab134077; 1:500), anti-E-Cadherin antibody (ab40772; 1:500), anti-Vimentin antibody (ab92547; 1:500), anti-ALDH1A1 antibody (ab23375; 1:500), anti-N-cadherin antibody (ab76011; 1:500) and anti-β-actin antibody (ab5694; 1:500) overnight at 4°C. Anti-rabbit secondary antibody (ab191866; 1:10,000; all Abcam) were used for 30 min at room temperature. The specific proteins were visualized by Odyssey™ Infrared Imaging System (Gene Company Lincoln, NE, Shanghai, China). β-actin was a loading control.

Migration and invasion assay

For Transwell migration assays, 2.5×104 cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences, San Jose, CA, USA). For invasion assays, 1.25×105 cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). In both assays, cells were plated in medium without serum, and medium supplemented with serum was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h and cells that did not migrate or invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with the Diff-Quick Staining Set (Medion Diagnostics, Düdingen, Switzerland) and counted.

Sphere growth

It was performed as described previously (15). Cells (103/ml) in serum-free RPMI1640/1 mM Na-pyruvate were seeded on 0.5% agar precoated 6-well plates. After 10 days, half the medium was exchanged every third day. Single spheres were picked and counted. The results are reported as the number of formed spheres per 10,000 cells seeded.

Immunofluorescence analyses

It was performed as described previously (14). After transfection with expressing plasmids, the cells were fixed in 4% paraformaldehyde for 15 min, and then blocked with goat serum blocking solution for 20 min at room temperature. Then, rabbit antibody against vimentin (1:200 dilution; Abcam) were added, and the mixtures were incubated in a humid chamber overnight. After washing three times with NaCl/Pi, cells were incubated with secondary antibodies (1:10,000 dilution; Abcam) for 30 min at 37 °C. After washing with NaCl/Pi, the samples were observed under a laser scanning confocal microscope (Olympus, Tokyo, Japan). 4′6-diamidino-2-phenylindole (DAPI) staining (blue) was used to highlight nuclei.

Wound healing assay

It was performed as described previously (15). One day before transfection, equal numbers of UMUC-3 cells (5.0×104) were seeded into 24-well plates without antibiotics. Cells were then transfected with FAT10-expressing plasmids or empty vectors using Lipofectamine 2000 (Invitrogen). When the cell confluence reached ~90% at 48 h post-transfection, an artificial homogenous wound was created onto the monolayer with a sterile plastic 100 ll micropipette tip. After wounding, the debris was removed by washing the cells with serum-free medium. Migration of cells into the wound was observed at different time points. Cells that migrated into the wounded area or cells with extended protrusion from the border of the wound were visualized and photographed under an inverted microscope (magnification, ×40; Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for microRNAs

It was performed as described previously (16). For miR-200 detection, 1 µg total RNA extracted from UMUC-3 cells was converted to cDNA using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction condition was as follows: Denaturation at 95°C for 30 sec, and followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec, and extension at 95°C for 15 sec. The plates were read using the ABI PRISM 7900HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Cq values were calculated using the SDS version 2.4 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). miR-200 expression level was normalized to that of U6 using the 2−ΔΔCq method. The TaqMan primers for miR-200 and U6 were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.). The following PCR primers were used in the following PCR reactions: miR-200 forward, 5′-GCCGTCTAACACTGTCTGGTA-3′ and reverse, 5′-CCTACGCCACAATTAACAAGCC-3′ (17); U6 forward, 5′-CGCTTCGGCAGCACATATACTA-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCA-3′ (17). The assay was performed in triplicate.

RT-qPCR for mRNA

It was performed as described previously (14). The primers used for FAT10 mRNA are: Forward, 5′-CAATGCTTCCTGCCTCTGTG-3′ and reverse, 5′-TGCCTCTTTGCCTCATCACC-3′; the primers used for GAPDH are: Forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′.

Statistical analysis

Results were analyzed using SAS software v9.4 (SAS Institute Inc., Cary, NC, USA). Data were presented as mean ± standard error of the mean (SEM) of separate experiments (n=3). Statistical significance was determined by Student's t-test and two-tailed P<0.05 were considered to indicate a statistically significant difference. Overall survival and progression-free survival were analyzed by Kaplan-Meier methods (18–20).

Results

Expression of the FAT10 protein is dysregulated in bladder cancer cells and over-expression of FAT10 promotes EMT in UMUC-3 cells

To investigate FAT10 protein expression in NBE samples and bladder carcinoma cell lines (T24, HT-1376, J82, UMUC-3, RT4 and TCCSUP), we performed western blot analysis. We observed that FAT10 protein was increased in bladder carcinoma cell lines, compared with NBE samples (Fig. 1A). To examine the role of FAT10 expressing plasmids in formation of CICs and EMT, we performed western blot to determine whether FAT10 protein was upregulated by FAT10 expressing plasmids in UMUC-3 cells. Western blot analysis showed that FAT10 protein expression was upregulated by FAT10 expressing plasmids (Fig. 1B). To examine whether FAT10 could promote EMT, we transfected UMUC-3 cells with FAT10 expressing plasmids and observed that overexpression of FAT10 promoted EMT in the cells morphology (Fig. 1C). To identify that changes in morphology were associated with EMT, we performed western blot analysis to determine expression of epithelial and mesenchymal markers in UMUC-3 cells transfected with FAT10 expressing plasmids and empty vectors. We observed that E-cadherin (epithelial marker) expression was inhibited and Vimentin and N-cadherin (mesenchymal markers) expression were induced by FAT10 in UMUC-3 cells (Fig. 1D). To further analyze whether overexpression of FAT10 could promote Vimentin protein expression, we used immunofluorescence analysis to determine its expression in UMUC-3 cells transfected with empty vectors or FAT10 expressing plasmids (Fig. 1E). But due to the lack of E-cadherin and N-cadherin antibodies for immunofluorescence analysis, we did not determine their expressions by immunofluorescence analysis.

Figure 1. FAT10 is dysregulated in bladder cancer cell lines and FAT10 overexpression promotes EMT in UMUC-3 cells. (A) Western blot analysis for FAT10 protein in NBEs samples and bladder cancer cell lines (T24, HT-1376, J82, RT4, UMUC-3 and TCCSUP). (B) Western blot analysis for FAT10 protein in UMUC-3 cells transfected with empty vector (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). (C) UMUC-3 cells were transfected with empty vector (pcDNA3.1, mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). The photographs for morphology of the cells were obtained following 72 h of transfection (scale bar, 50 µm). (D) Western blot analysis for Vimentin, E-cadherin and N-cadherin protein in UMUC-3 cells transfected with empty vector (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). (E) Immunofluorescence analysis for Vimentin protein in UMUC-3 cells transfected with empty vector (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). n=3 (scale bar, 20 µm). FAT10, HLA-F adjacent transcript 10; EMT, epithelial to mesenchymal transition; NBE, normal bladder epithelium; DAPI, 4′6-diamidino-2-phenylindole.

Overexpression of FAT10 promotes migration and invasion in UMUC-3 cells

Next, we performed would healing assay to examine motility of UMUC-3 cells transfected with empty vector or FAT10 expressing plasmids. We found that overexpression of FAT10 promoted motility (Fig. 2A). Moreover, overexpression of FAT10 did not affect proliferation in vitro (data not shown), but did promote invasion by 1.9-fold and migration by 2.8-fold (Fig. 2B). These effects were specifically attributable to the biological activities of FAT10, as indicated by the fact that equivalent overexpression of empty vectors, failed to influence invasion or motility (data not shown).

Figure 2. Overexpression of FAT10 promotes motility in UMUC-3 cells. (A) Wound healing assay for UMUC-3 cells transfected with empty vector (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). The photographs for morphology of the cells were obtained following 72 h of transfection (scale bar, 100 µm). (B) Invasion and migration assays in UMUC-3 cells transfected (scale bar, 10 µm). P<0.05, as indicated (n=3). FAT10, HLA-F adjacent transcript 10.

Overexpression of FAT10 promotes formation of CICs in UMUC-3 cells

To determine whether FAT10 could promote formation of CICs, we performed sphere forming assay to evaluate formation of CICs. We observed that formations of spheres were increased in UMUC-3 cells transfected with FAT10 expressing plasmids (Fig. 3A). ALDH1A1 is a marker for CICs and predictor of bladder cancer patients' outcome (5). ALDH1A1 protein expression was examined by Western blot analysis. Western blot analysis showed that ALDH1A1 protein was upregulated by FAT10 in UMUC-3 cells (Fig. 3B).

Figure 3. Overexpression of FAT10 is associated with the increased formation of cancer initiating cells. (A) Sphere growth assay for UMUC-3 cells transfected with empty vectors (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). Scale bar, 20 µm. (B) Western blot analysis for ALDH1A1 protein in UMUC-3 cells transfected with empty vectors (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). P<0.01, as indicated (n=3). FAT10, HLA-F adjacent transcript 10; ALDH1A1, aldehyde dehydrogenase 1 family member A1.

FAT10 protein overexpression is associated with poor outcome in Chinese patients

Because we found FAT10 promoted EMT and formation of CICs in bladder cancer. We therefore next tested whether FAT10 protein expression was associated with bladder cancer disease progression and recurrence in patient samples. We performed IHC analyses to determine FAT10 protein in 98 primary tumors. The low-FAT10 expression group was defined as <50th percentile (Fig. 4A) and high-FAT10 expression group was defined as ≥50th percentile (Fig. 4B). Kaplan-Meier analysis was used to estimate overall survival. Differences in overall survival between high-FAT10 expression group (≥50th percentile) and the low-FAT10 expression group (<50th percentile) were analyzed by the log-rank test. We found that decreased FAT10 expression in bladder cancer was associated with prolonged survival and less frequent metastasis (Fig. 4C and D). To identify FAT10 protein and mRNA expression in bladder cancer tissues and adjacent normal tissues, we used western blot analysis and RT-PCR. Protein and mRNA were isolated from 20 pairs of bladder cancer tissues and normal tissues. We found that FAT10 protein and mRNA were significantly increased in bladder cancer tissues (Fig. 4E and F).

Figure 4. Overexpression of FAT10 is associated with poor survival outcomes in Chinese patients. (A) IHC for FAT10, the low-FAT10 expression group (magnification, ×40). (B) IHC for FAT10, the high-FAT10 expression group (magnification, ×40). (C) Kaplan-Meier for overall survival curves. (D) Kaplan-Meier for progression-free survival curves. Low FAT10 indicates patients with low FAT10 protein expression and High FAT10 indicates patients with high FAT10 protein expression. (E) Western blot analysis for FAT10 protein expression in bladder cancer and adjacent normal tissues. (F) Reverse transcription-quantitative polymerase chain reaction for FAT10 mRNA in bladder cancer and adjacent normal tissues. P<0.01, as indicated (n=20). FAT10, HLA-F adjacent transcript 10; IHC, immunohistochemistry; HR, hazard ratio; CI, confidence intervals; C group, bladder cancer; N group, adjacent normal tissues.

Overexpression of FAT10 promotes cisplatin resistance in UMUC-3 cells

To identify whether overexpression of FAT10 decreases the efficacy of cisplatin in UMUC-3 cells, we performed MTT assay in UMUC-3 cells treated with the indicated concentrations (µg/ml) of cisplatin (Fig. 5A). We observed that over-expressing FAT10 promoted cisplatin resistance (Fig. 5A). Downregulation of miR-200 has been proposed as an important step in tumor progression (21,22). Thus, we performed real time PCR to examine miR-200 expression in UMUC-3 cells transfected with FAT10 expressing plasmids or empty vectors. We observed that miR-200 expression was increased by overexpression of FAT10 in UMUC-3 cells (Fig. 5B).

Figure 5. Overexpression of FAT10 promotes cisplatin resistance in UMUC-3 cells. (A) MTT for cell viability in UMUC-3 cells. UMUC-3 cells transfected with empty vectors (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10) were treated with different doses of cisplatin. **P<0.01 vs. UMUC-3-Mock. (B) Reverse transcription-quantitative polymerase chain reaction for miR-200 in UMUC-3 cells transfected with empty vectors (pcDNA3.1; mock) or FAT10 expressing plasmids (pcDNA3.1-FAT10). P<0.01, as indicated (n=3). FAT10, HLA-F adjacent transcript 10; miR, microRNA.

Discussion

FAT10, a member of the UBL family, is involved in regulation of aberrant mitosis, cell cycle, chromosomal stability and apoptosis (6,23–25). Recent evidence indicates that aberrant FAT10 expression is common in cancers and it may serve as a new biomarker for the diagnosis and treatment of cancers (26,27). FAT10 promotes bladder cancer progression by different mechanisms (e.g., stabilizing surviving) (8). But, functional contribution of FAT10 to EMT and formation of CICs remains unclear in bladder cancer.

EMT facilitates tissue remodeling during embryonic development and is viewed as an essential early step in tumor invasion, migration and metastasis (21,28–30). Moreover, EMT plays an important role in the formation of CICs and metastasis of bladder cancer (30–32). In this study, we observed that FAT10 protein expression was dysregulated in bladder cancer cell lines, which was consistent with a previous report (8). We showed that overexpression of FAT10 was associated with increased formation of CICs and promoted motility in UMUC-3 cells. ALDH1A1-positive bladder cancer cells are associated with increased formation of CICs (5). It has been proposed as a useful proxy for monitoring the progression of bladder cancer and as a useful marker for identifying bladder cancer patients with poor prognosis (5). For the first time, we observed that ALDH1A1 protein were upregulated by FAT10 in bladder cancer cells.

Bladder cancer is highly chemo-sensitive and cisplatin is one of the most effective drug (33). However, one of major limitations is that many tumors acquire cisplatin resistance after an initial complete response. Accumulating evidence suggests that formations of CICs lead to cisplatin resistance (34,35). Thus, understanding molecular mechanisms of cisplatin resistance is a critical goal for the treatment of bladder cancer. We observed that overexpression of FAT10 promoted formation of CICs and cisplatin resistance. Numerous studies support the contention that EMT and formations of CICs play important roles in the progression of cancer (36). In line with a previous report (8), we observed that increased FAT10 protein expression in tumor tissues is associated with poor outcome in Chinese patients.

Downregulation of miR-200 has been proposed as an important step in tumor progression (37). Enforced expression of miR-200 is sufficient to reverse EMT and miR-200 expression is negatively associated with drug resistance in bladder cancer (21,22). We found that miR-200 expression was decreased by overexpression of FAT10 in UMUC-3 cells. Thus, FAT10 might promote EMT by downregulating miR-200 expression. These results suggest FAT10 may be a novel target for the treatment of bladder cancer.

Several limitations should be noted in this study. Because of the absence of normal bladder cell lines, we used NBE samples as controls. Although we used the antibody anti-E-cadherin and anti-N-cadherin in western blot analysis, the anti-bodies could not be used for immunofluorescence analyses. some stem cell surface markers (e.g., CD44) have been proposed as proxies for formation of CIC (38); molecular mechanisms of cisplatin resistance are related to inhibition of apoptosis (39,40). However, we did not have chance to detect stem cell surface markers and expression of apoptosis related proteins by flow cytometry (FACS), because there was no FACS available in our lab. Despite these limitations, this paper provides the first insight into the potential role of FAT10 in relation to EMT and formations of CICs in bladder cancer.

Acknowledgements

Not applicable.

Funding

The present study was supported by Key Discipline Medicine of Jiangsu Province [grant no. (201151) XK 201151].

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

JH and CL conceived the study, performed the majority of the experimental work, and wrote the draft of the manuscript. ZW, NF, JD, XD, YY and YG performed the remainder of the experimental work, and assisted with the preparation of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The Ethics Committee of the First Affiliated Hospital of Soochow University approved the study. Written informed consent was obtained from each patient.

Consent for publication

Written informed consent for publication was obtained from each patient.

Competing interests

The authors declare that they have no competing interests.

References