Background
Bladder cancer is a common malignant tumor with high mortality. The morbidity of bladder cancer has steadily increased during these years [
1]. A total of 60,490 new diagnosed bladder cancer patients in the United States, and about 12,240 of these patients died of the disease in 2017 [
2]. Besides traditional surgical treatment, targeted chemotherapy and immunotherapy have gained importance in treating bladder cancer [
3‐
6]. However, 25% of bladder cancer is reported to be muscle invasive, and approximately 50% of these patients die from a life-threatening metastasis within 2 years [
6,
7]. Generally, bladder cancer is characterized by hematuria, dysuria, and urination. However, few specific biomarkers of bladder cancer have been identified to date.
Overnutrition with modern lifestyle is a crucial risk factor for cancer, and
O-GlcNAcylation reflects the glucose status of cells [
8]. About 2–3% of glucose entering the cell passes through the hexosamine biosynthetic pathway (HBP), which regulates the
O-GlcNAcylation of intracellular proteins [
9]. Analogous to phosphorylation,
O-linked
N-acetylglucosaminylation (
O-GlcNAcylation) is a invertible post-translational modification that influences almost all cellular processes [
9‐
11]. OGT is critical for
O-GlcNAcylation, it adds the
O-GlcNAc moiety to the free hydroxyl group of selected serine and threonine residues on proteins, which is removed by
O-GlcNAc-selective
N-acetyl-β-D-glucosaminidase (
O-GlcNAcase, OGA) [
12].
O-GlcNAcylation has been reported to modulate cell functions, and multiple proteins have been demonstrated with O-GlcNAc modification over these years [
11]. The deregulation of
O-GlcNAcylation has been implicated in various diseases [
13,
14]. Moreover, various studies indicated that
O-GlcNAcylation was upregulated in various cancers and might be related to various hallmarks of cancer, such as cell proliferation, survival, metastasis, invasion, and so forth [
15]. Rozanski et al. reported mRNA expression of OGT in the urine of 51.7% patients with bladder cancer, but not in the urine of healthy individuals [
16]. However, no further studies about OGT and
O-GlcNAcylation in bladder cancer have been reported to date.
Autophagy is a cellular defensive pathway under unfavorable circumstances. It is imperative to cell survival [
17]. However, autophagy induces cell death under certain conditions [
18,
19]. Extensive evidence is available on the role of autophagy in the progression of various diseases such as cancer [
20,
21]. Autophagy may prevent tumor progression and improve the efficacy of cancer therapy [
22]. Increased
O-GlcNAcylation has been reported to inhibit autophagy [
23]. In addition, the inhibition of
O-GlcNAcylation seems to facilitate autophagosome formation and increase autophagic flux [
23,
24].
Surgery combined with chemotherapy and/or radiotherapy is the current strategy for treating bladder cancer. Cisplatin-based chemotherapy is the mainstay of both muscle-invasive and metastatic bladder cancer [
25]. Cisplatin causes DNA damage, leading to apoptosis and cell death [
26]. OGT was found to mediate histone
O-GlcNAcylation, regulating DNA damage response (DDR) [
27]. Moreover, Miura et al. demonstrated that O-GlcNAc modification affected the ATM-mediated DDR. Whether DDR is regulated by
O-GlcNAcylation in bladder cancer needs to be further explored.
This study aimed to demonstrate the expression and function of OGT and O-GlcNAc modification in bladder cancer. The results advanced the understanding of the tumor-promoting effect of OGT and O-GlcNAcylation in bladder cancer.
Methods
Materials and reagents
Anti-OGT antibody was obtained from Bioworld Technology, Co. (Nanjing, China). Other primary antibodies were purchased from Abcam (Cambridge, MA, USA). Fluorescent-labeled secondary antibodies were procured from Jackson Immuno Research. Cisplatin was obtained from Selleckchem (TX, USA). Autophagy inhibitor chloroquine (CQ) was obtained from Sigma–Aldrich (MO, USA).
Tumor microarray and immunohistochemical analysis
The paraffin sections were taken from 169 patients with bladder cancer (85 patients with NMIBC and 84 with MIBC) for tissue microarray. All these samples were obtained from the Department of Urology, Shanghai Tenth People’s Hospital, Tongji University (Shanghai, China) from 2013 to 2016. Tumor-rich areas were board-certified by the pathologist. After constructing the tissue microarray, the samples were stained for
O-GlcNAcylation (RL2) and OGT. The intensity of the staining was scored using the H-score method (3D HISTECH, H-SCORE = ∑(PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3) [
28].
Cell culture and transient transfection
Human bladder epithelial permanent cell (sv-huc-1) and human bladder cancer cell lines (T24, UMUC-3, 5637 and EJ) were obtained from the Institute of Cell Research of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute 1640 media/Dulbecco’s Modified Eagle Medium/F12 K (Invitrogen, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. Lipofectamine 2000 (Invitrogen, CA, USA) was used for transfection in accordance with the manufacturer’s protocol. si-OGT and si-NC were all obtained from GenePharma (Shanghai, China). The sequence was 5’-GAAGAAAGUUCGUGGCAAA-3′ for si-OGT. The transfection efficiency was detected using Western blot analysis after cultivation for 48 h.
Western blot analysis
Total protein of cells or tissues was extracted using precooled radio-immunoprecipitation assay lysis buffer with protease inhibitor. An equal amount of protein was separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. Afterward, the membranes were incubated overnight with specific primary antibodies at 4 °C. Then, the membranes were incubation with secondary antibodies for 2 h. The signals were identified by electro chemiluminescent detection.
Cell proliferation in vitro
Cell proliferation was measured using the MTT assay as previously described [
29]. The lentivirus expressing shRNAs against OGT was produced by Jiman Co. (Shanghai, China). UMUC-3 cells were infected with LV-sh-OGT or LV-sh-NC and then selected using puromycin (Sigma–Aldrich). The expression of OGT and
O-GlcNAc was examined at RNA and protein levels. For colony formation assays, LV-sh-OGT or LV-sh-NC cells were plated in six-well plates with a density of 1 × 10
3/well. After cultivation for 10 days, the plates were methanol-fixed, and then stained with 0.1% crystal violet.
Xenograft assays in nude mice
After generating stably transfected LV-sh-NC and LV-sh-OGT cell lines, the cells (3 × 106 in 0.2 mL of PBS) were implanted subcutaneously into the dorsal flanking sites of male BALB/c nude mice (N = 10 in each group, 6 weeks). The tumorigenic potential was evaluated 3 weeks after inoculation. The mice were sacrificed using pentobarbital overdose (1%), and tumors were weighed and excised. The animal care and experiments were carried out under the National Institutes of Health Guide for Care and Use of Laboratory Animals. All animal studies were approved by the Institutional Animal Care and Use Committee of the Shanghai Tenth People’s Hospital of Tongji University.
Flow cytometry
For the apoptosis assay, Annexin V–fluorescein isothiocyanate (FITC) detection kit (BD Biosciences, Erembodegem, Belgium) was used according to the manufacturer’s introduction. The cells were collected, washed twice with PBS, resuspended in Annexin V–FITC and propidium iodide (PI), and stained in the dark for 15 min at room temperature [
29]. Subsequently, the cell apoptosis rate was analyzed using flow cytometry (fluorescence-activated cell sorting, BD Biosciences). For cell cycle distribution analysis, the cells were harvested and fixed in 70% ice-cold ethanol overnight. Then, the cells were centrifuged and resuspended in PBS containing PI (BD Biosciences) and RNase (100 μg/mL) as well as Triton X-100 (0.2%) for 30 min. Finally, flow cytometry was used to analyze cell cycle distribution [
29]. The tests were performed three times for each sample.
GFP-LC3 puncta assay
The GFP-LC3 plasmid was used in this study to monitor autophagy. In brief, the cells were cultured and transfected with GFP-LC3 plasmid for 24 h. Then, they were treated, cell images were chosen randomly under a confocal microscope, and the number of puncta was calculated.
Statistical analysis
The statistical analysis was performed using GraphPad Prism 5 (GraphPad Prism Software Inc., CA, USA) and SPSS 23 (SPSS, Inc., IL, USA). The data were presented as mean ± standard deviation (SD). The differences between two groups were analyzed using the Mann–Whitney U test or two-tailed unpaired Student t test. In all analyses of this study, a P value < 0.05 was considered statistically significant.
Discussion
Nutritional conditions can regulate tumor development by affecting the signaling pathways involved in tumor cell growth [
9,
39]. Increased glucose intake in cancer cells contributes to increased HBP flux. Thus,
O-GlcNAcylation levels rise in response to elevated UDP-GlcNAc, as the product of HBP flux. Recent studies reported that increased
O-GlcNAcylation is a common feature of various tumors and contributes to transformed phenotypes [
9,
10,
15]. Hyper-
O-GlcNAcylation has been reported to be correlated with the excessive growth of cancer cells by regulating key proteins that modulate cell cycle progression [
40]. In addition, hyper-
O-GlcNAcylation was verified to have an anti-apoptotic influence in cancer cells. Moreover, previous studies also showed that hyper-
O-GlcNAcylation was associated with cancer cell invasion, metastasis, and angiogenesis [
30,
32,
33]. Therefore, it is believed that the suppression of hyper-
O-GlcNAcylation may be a therapeutic target for various types of cancers.
A previous study demonstrated that a high mRNA level of OGT was associated with poor differentiation of bladder cancer cells [
16]. However, further studies about
O-GlcNAcylation in bladder cancer are lacking. The
O-GlcNAcylation level in cell lines and clinical tissues was examined to explore the potential role of
O-GlcNAcylation in bladder cancer. The present study testified that hyper-
O-GlcNAcylation was associated with the upregulated OGT level in bladder cancer cells. Meanwhile, it was demonstrated that the
O-GlcNAcylation level was higher in clinical bladder cancer tissues than in normal bladder tissues. Notably, the
O-GlcNAcylation level was higher in MIBC tissues than in NMIBC tissues. Hyper-
O-GlcNAcylation and overexpression of OGT have been described in various cancer types, including lung, breast, colon, liver, prostate, and endometrial [
30,
32,
34,
35,
41,
42]. Therefore,
O-GlcNAcylation has been suggested as a new cancer hallmark.
The hyper-O-GlcNAcylation of bladder cancer cells was reduced by OGT knockdown and its effects on phenotypes were examined. The OGT knockdown–induced reduction of hyper-O-GlcNAcylation suppressed the proliferation of bladder cancer cells in vitro and subcutaneous xenograft tumor growth in nude mice. The present study reported that OGT knockdown–induced cell proliferation inhibition might be due to apoptosis increasing and cell cycle arrest. These data suggested that the inhibition of OGT might be a potential therapeutic strategy in bladder cancer.
Hyper-
O-GlcNAcylation has been suggested to blunt autophagy [
23]. The present study reported that the reduction of
O-GlcNAcylation by OGT knockdown potently increased autophagic flux in T24 and UMUC-3 cells. Autophagy had dual roles in cancer modulation. The study demonstrated the pro-survival role of
O-GlcNAcylation reduction–induced autophagy in bladder cancer cells. Further experiments are needed to explore the mechanism underlying this phenotype.
The interaction between apoptosis and autophagy elicited physiopathological changes in different cell types and stresses. Chen et al. reported that autophagy suppression enhanced PT-induced apoptosis and cell death in bladder cancer cells [
22], indicating a pro-survival role of interdependence between apoptosis and autophagy. On the contrary, the inhibition of autophagy attenuated FTY720-induced apoptosis [
43]. The present study also revealed that the downregulation of
O-GlcNAcylation by OGT knockdown–induced apoptosis and autophagy (Fig.
4a and b, and Fig.
5g and h). Cell apoptosis increased during the inhibition of autophagy, and cell viability was not restored after autophagy inhibition (Fig.
5i). These results suggested that autophagy exerted a tumor-promoting effect during the downregulation of
O-GlcNAcylation in bladder cancer cells.
O-GLcNAcylation was reported to be involved in regulating protein function, stability, or localization during post-translational modification. DNA damage induced by chemicals or x-rays led to changes in the
O-GlcNAcylation of cellular proteins. A previous study proved that
O-GlcNAcylation affected DDR by modulating key proteins involved in DDR [
27,
38]. The project of cisplatin/gemcitabine (GC project) is the first-line therapy for locally advanced or metastatic bladder cancer [
44]. This study demonstrated that OGT knockdown pronouncedly increased the chemosensitivity of bladder cancer cells to cisplatin. This finding indicated that hyper-
O-GlcNAcylation in bladder cancer might promote chemoresistance of cells to DNA-damaging agents, and the chemosensitivity might be rescued by reducing hyper-
O-GlcNAcylation. Although
O-GlcNAcylation might be involved in DDR, the details of this involvement need further investigation.
In summary, this novel study reported the role of O-GlcNAcylation in bladder cancer. It elucidated hyper-O-GlcNAcylation and deregulated expression of OGT in bladder cancer cells and clinical samples. It also demonstrated that cell proliferation was inhibited by the OGT knockdown–induced downregulation of O-GlcNAcylation in vitro and in vivo. In addition, autophagic flux was increased by OGT knockdown, and autophagy had a pro-survival role. The study further showed that the chemosensitivity of cells to DNA-damaging agent cisplatin was increased by OGT knockdown.
A limitation of the present study was that the mechanism underlying the biological effects of OGT and O-GlcNAcylation in bladder cancer was not explored. Future studies should aim to investigate the potential mechanism.