Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

Abstract Bladder cancer (BLCA) is a common genitourinary cancer in patients, and tumour angiogenesis is indispensable for its occurrence and development. However, the indepth mechanism of tumour angiogenesis in BLCA remains elusive. According to recent studies, the tight junction protein family member occludin (OCLN) is expressed at high levels in BLCA tissues and correlates with a poor prognosis. Downregulation of OCLN inhibits tumour angiogenesis in BLCA cells and murine xenografts, whereas OCLN overexpression exerts the opposite effect. Mechanistically, the RT‐qPCR analysis and Western blotting results showed that OCLN increased interleukin‐8 (IL8) and p‐signal transducer and activator of transcription 3 (STAT3) levels to promote BLCA angiogenesis. RNA sequencing analysis and dual‐luciferase reporter assays indicated that OCLN regulated IL8 transcriptional activity via the transcription factor STAT4. In summary, our results provide new perspectives on OCLN, as this protein participates in the development of BLCA angiogenesis by activating the IL8/STAT3 pathway via STAT4 and may serve as a novel and unique therapeutic target.


| INTRODUC TI ON
Bladder cancer (BLCA) is the most common malignant cancer of the urinary system. The incidence rate of BLCA in males is 3-4 times that in women, and it is the fourth most common malignancy in men. 1 Non-muscle-invasive BLCA (NMIBC) accounts for approximately 75% of all BLCA cases, while other cases are considered muscleinvasive bladder cancer (MIBC). The 5-year survival rate of patients with NMIBC is more than 85%, but upon relapse, easy progression to MIBC, which invades the basement membrane and spreads from the bladder to visceral organs, is likely and eventually leads to tumour metastasis and a low survival rate. 2 Neovascularization contributes to growth and development, tissue and organ regeneration and numerous pathological conditions of tumors. 3 In addition, neovascularization is indispensable for the development of malignant tumors 4 and provides opportunities for distant metastasis. 5 Tumour vascularization involves a variety of biological processes that are regulated by a wide range of secreted factors and signalling pathways.
Among them, antibodies and tyrosine kinase inhibitors play an important role in antiangiogenic treatment for several types of cancer. 6 BLCA is a highly vascularized disease, and although angiogenesis has been reported to participate in the development of BLCA, 7,8 its molecular mechanism and related signalling pathways are generally still unclear. Therefore, the biological function and specific mechanism of tumour angiogenesis in BLCA remain to be fully elaborated.
Occludin (OCLN) is an important tight junction protein that regulates cytoskeletal remodeling, 9 it is reported to be aberrantly expressed in many malignant cancers during tumour progression and to contribute to apoptosis and metastasis. In lung cancer, downregulation of OCLN inhibits activation of the AKT/PI3K signalling pathway and cell proliferation, thus increasing tumour apoptosis in vitro and in vivo. 10 In addition, OCLN levels in the peripheral blood have been used as a tumorigenic biomarker of cerebral oedema caused by blood-brain barrier (BBB) damage in patients with brain tumors. 11 Other tight junction proteins, such as tight junction protein 1 (TJP1) and Claudin-5, have been indicated to regulate vascular abnormalities during tumour progression. [12][13][14] A few studies have also shown roles of OCLN in modulating tumour angiogenesis and tube-forming activity for tumour invasion. 15,16 OCLN S490 phosphorylation mediates VEGF to induce retinal endothelial cell proliferation and neovascularization. 17 Exosomal miR-25-3p promotes vascular permeability and angiogenesis by regulating OCLN expression in endothelial cells. 18 However, researchers have not clearly determined whether and how OCLN regulates BLCA angiogenesis.
Signal transducers and activators of transcription signalling (STATs) have been proven to be molecules that connect cytokine signalling with the regulation of important cellular mechanisms such as tumour cell survival and infiltration, inflammation and immunity. 19 Recently, one of the STAT proteins, STAT3, was reported to play critical roles in BLCA cell viability and invasiveness. 20,21 Furthermore, miR-153 mediates BLCA vascular remodelling and tryptophan metabolism by targeting IL6/STAT3/VEGF signaling. 22 Another STAT, STAT4, has also emerged as a tumorigenic gene that triggers tumour metastasis and progression. 23,24 STAT4 was reported to activate IL8 transcription and the production of inflammatory mediators. 25 STAT3 has also been reported to participate in and activate IL8regulated tumour angiogenesis. 26 IL8, a member of the chemokine family, was proven to be involved in tumour angiogenesis and metastasis in BLCA and other malignant cancers. [27][28][29] Currently, researchers have not determined whether STAT3/4 is involved in IL8-mediated modulation of BLCA angiogenesis.
Here, we observed markedly upregulated OLCN expression in BLCA that was significantly associated with tumour angiogenesis in both patients with BLCA and murine xenograft models. Furthermore, OCLN overexpression enhanced the activity of the IL8/STAT3 signalling pathway by activating STAT4, ultimately contributing to tumour vascular remodelling and metastasis. Therefore, this study focused on the biological mechanism of OCLN in BLCA angiogenesis and provides potential targets for tumour treatment.

| Matrigel plug assay
BALB/c nude mice (male, 6-8 weeks) were randomly divided into 4 groups (n = 5 mice/group), and 2 × 10 6 tumour cells were injected subcutaneously into the backs of mice. The cell suspension and 150 µl of Matrigel (#356231, Corning, NY, USA) were mixed at a ratio of 1:3. After two weeks, the Matrigel plugs were collected and photographed, followed by IHC staining and a pathological examination.
All animal experimental procedures were approved by the Animal Experimental Ethics Committee of Sun Yat-Sen University.

| Plasmids and transient transfection procedures
The primer sequences for the Flag-OCLN (NCBI Gene ID:

| siRNAs and transient transfection procedures
All OCLN-specific RNAs (siRNAs) and the negative control siRNA were designed by and purchased from RiboBio (stB0018520A/B-1-5, siN0000002-1-5, Guangzhou, China). The siRNAs were transfected into 293T cells using Lipofectamine™ 3000. 24 hours after the transfection of siRNAs, the cells were lysed for Western blotting or RT-qPCR assays.

| Enzyme-linked immunosorbent assay (ELISA)
Cells were transfected with the corresponding plasmids, and the supernatant was collected and centrifuged to completely remove cell debris after 48 h. A human IL8 ELISA kit (EK0413, BOSTER, Wuhan, China) was used to detect the IL8 concentration in CM according to the manufacturer's instructions. Levels of the secreted protein were measured from a standard curve.

| Clinical patients and immunohistochemistry (IHC) assay
All BLCA specimens (n = 120) were collected from the Fifth Affiliated Hospital of Sun Yat-Sen University and were used after obtaining written consent from the patients. The collected tissues were first embedded in paraffin and cut into 4μm-thick slices before the IHC assay. Sections were baked at 65°C for 3 h and then deparaffinized in xylene and alcohol. After dewaxing, the samples were washed with water and incubated with a citrate buffer solution (pH 9.0) in a pressure cooker for 2.5 min for antigen retrieval. Sections were treated with 3% hydrogen peroxide in methanol to block endogenous peroxidase activity and then incubated with goat serum for 30 min to block nonspecific staining. The sections were incubated with OCLN (ab216327, rabbit, Abcam, Cambridge, MA, USA, 1:100) and CD31 (ARG52748, rabbit, Arigobio, Hsinchu City, Taiwan, 1:100) primary antibodies followed by secondary antibodies. A biotinylated secondary antibody (PV-9000, ZSGB-BIO, Beijing, China) was applied, followed by an incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB). Finally, sections were counterstained with haematoxylin and sealed with neutral gum. Five fields were randomly selected from each sample to calculate expression. Scale bar = 100 µm. Staining/expression was scored as described in a previous study. 31

| Dual-luciferase reporter assay
The IL8 promoter was used to drive firefly luciferase activity in the   The firefly and Renilla luciferase activities were detected with a microplate reader. Firefly luciferase activity was normalized to Renilla luciferase activity.

| Quantitative real-time PCR assay
Total RNA was extracted from cells using TRIzol reagent (Invitrogen,  (Table 1).
Collectively, these results indicated that OCLN expression is strongly associated with BLCA clinical features, and the protein may function as an oncogene in the development and progression of BLCA.

| OCLN promotes BLCA angiogenesis in vitro and in vivo
BLCA is a highly vascularized tumour, and we investigated whether  (Figure 2I,J). We also detected CD31 levels in clinical patients with BLCA carcinoma and paraneoplastic tissue and found that CD31 was expressed at higher levels in high-grade BLCA samples than in low-grade samples ( Figure 2K). Furthermore, OCLN expression was positively correlated with angiogenesis phenotypes ( Table 2). All of these findings verified that OCLN regulates BLCA angiogenesis. Taken together, OCLN promotes BLCA angiogenesis in vitro and in vivo.

| OCLN mediated BLCA angiogenesis by regulating IL8 expression
We performed RNA sequencing to identify changes in gene expression in T24 OCLN-silenced cells and to further investigate the mechanism by which OCLN regulates angiogenesis. The two T24 OCLN-shRNA groups presented 1547 and 172 downregulated genes, respectively, and 190 genes were common to the two groups ( Figure 3A). We performed an enrichment analysis of these downregulated genes and found that they were enriched in pathways such as protein processing in the endoplasmic reticulum, cytokine-cytokine receptor interactions, and abnormal transcriptional regulation in the cancer pathway ( Figure S1a). More interestingly, the functional annotation analysis identified many extracellular gene sets, including those related to exosomes, extracellular spaces and the extracellular matrix ( Figure S1b). According to previous reports, tumour cells generally secrete large amounts of angiogenic factors into the extracellular matrix, promoting the formation of new blood vessels and tumour metastasis, 32 which provides further support for the regulation of tumour angiogenesis by OCLN in BLCA. As expected, 22 of these genes were associated with the promotion of angiogenesis, and 16 of these genes expression were decreased in OCLN-silenced cells compared with control cells (Figure 3B), suggesting that OCLN may regulate BLCA angiogenesis by regulating genes associated with angiogenesis.
In addition, we performed an RT-qPCR assay to detect the expression of angiogenic factors after OCLN knockdown; IL8 was the most substantially downregulated gene among them ( Figure 3C), and this finding was further validated by ELISA in OCLN knockdown BLCA cells ( Figure 3D). Consistently, OCLN overexpression increased IL8 expression ( Figure 3E). As the chemokine, IL8 has been shown to induce angiogenesis in multiple vascular-rich malignancies. 33 We restored IL8 expression in OCLN-silenced cells and collected CM to monitor the tube formation of endothelial cells and further confirm whether IL8 was involved in the mechanism by which OCLN regulates BLCA angiogenesis. IL8 enhanced the tube formation of EA.hy926 cells induced by the downregulation of OCLN ( Figure 3F,G). Collectively, IL8 is involved in OCLN-mediated regulation of BLCA angiogenesis.
F I G U R E 4 IL8/ STAT3 is involved in the process of Occludin (OCLN)-mediated angiogenesis in bladder cancer (BLCA). (A) p-STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells. (B) 5637 and T24 cells were transfected with the OCLN plasmid and treated with or without the STAT3 inhibitor Stattic, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed by performing staining with Calcein AM and imagining using a fluorescence microscope. Scale bar = 100 µm.
(C) The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. D, p-STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells after IL8 supplementation. (E) p-STAT3 and STAT3 protein levels were detected in OCLN-overexpressing 5637 and T24 cells after blocking IL8. (F) 5637 and T24 cells were transfected with the OCLN plasmid or cultured with the IL8-neutralizing antibody; tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. G, The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01 | 2373 YANG et Al.

| IL8 is involved in the process of OCLNmediated STAT3 angiogenesis in BLCA
We next explored the detailed mechanism by which OCLN regulates

| OCLN regulates the IL8/STAT3 pathway to mediate BLCA angiogenesis through STAT4
In the enrichment analysis of proangiogenic genes in OCLN knockdown cells shown in Figure 3B, we identified another significantly downregulated STAT family protein, STAT4, which has been reported to be involved in the occurrence and development of multiple tumours and tissue angiogenesis. [35][36][37] In addition, STAT4 has been reported to promote angiogenesis in pancreatic cancer by inducing IL8 transcription. 38 However, researchers have not yet clarified the potential association between STAT4 and IL8 in BLCA angiogenesis. First, we detected IL8 expression in 5637 and T24 cells overexpressing STAT4, and the results indicated that STAT4 overexpression upregulated IL8 levels ( Figure 5A). In addition, tube formation assays indicated an  41 In the present study, OCLN was expressed at high levels and correlated strongly with the TNM stage in patients with F I G U R E 5 STAT4 is involved in the mechanism by which Occludin (OCLN) regulates IL8/STAT3 signalling to mediate bladder cancer angiogenesis. (A) Relative IL8 mRNA levels were detected in control and STAT4-overexpressing 5637 and T24 cells using RT-qPCR. (B) The STAT4 plasmid or the indicated vector was transfected into 5637 and T24 cells, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (C) The segment lengths were analysed, and the meshes were quantified using ImageJ software. (D) The relative STAT4 mRNA levels were detected in control and OCLN shRNA-transfected 5637 and T24 cells. (E) Cells were transfected with the indicated siRNAs or plasmids for 24 h. The relative knockdown and overexpression efficiency was determined using Western blotting and RT-qPCR. (F) 293T cells were cotransfected with the indicated plasmids for 24 h, and the relative activity of the IL8 promoter was evaluated by performing a dual luciferase reporter assay. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01 BLCA (Figure 1 Figures 3C and 5D). In addition, STAT4 promoted tube formation by endothelial cells cultured with CM derived from BLCA cells ( Figure 5B,C), moreover, OCLN mediated IL8 transcription through STAT4 ( Figure 5F), further confirming the role of STAT4 in BLCA angiogenesis. In addition, we showed that STAT3 participates in OCLNmediated regulation of tumour angiogenesis in BLCA ( Figure 4B,C).
OCLN altered p-STAT3 levels but not total STAT3 levels ( Figure 4A), potentially because STAT3 phosphorylation and transport into the nucleus is involved in the regulation of STAT4 transcriptional activity by OCLN. JAK phosphorylation is required for STAT4 activation. 49,50 Most likely, the regulation of these signalling pathways and subsequent transcription mainly depends on the posttranslational modifications, especially phosphorylation. Therefore, studies focusing on changes in the phosphorylation of proteins in STAT-related pathways will be very important to identify the key factors contributing to OCLN-regulated BLCA angiogenesis.
In summary, we discovered that OCLN expression is strongly

ACK N OWLED G EM ENTS
We sincerely thank Zhao-Xia Dong and Sze-Hoi Chan for their as-

CO N FLI C T O F I NTE R E S T
All authors agree to submit the article and have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets analysed during the current study are available from the corresponding author on reasonable request.