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Low Expression of Ephb2, Ephb3, and Ephb4 in Bladder Cancer: Novel Potential Indicators of Muscular Invasion

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Low Expression of Ephb2, Ephb3, and Ephb4 in Bladder Cancer: Novel Potential Indicators of Muscular Invasion Original Article Yonsei Med J 2021 Aug;62(8):679-690 https://doi.org/10.3349/ymj.2021.62.8.679 pISSN: 0513-5796 · eISSN: 1976-2437 Low Expression of EphB2, EphB3, and EphB4 in Bladder Cancer: Novel Potential Indicators of Muscular Invasion Tae Ho Lee1, Jin Hyung Heo2, Ju-Yeon Jeong3, Gee Hoon Lee2, Dong Soo Park1, and Tae Hoen Kim2,3 Departments of 1Urology, 2Pathology, and 3Clinical Research Institute, CHA Bundang Medical Center, CHA University, Seongnam, Korea. Purpose: Eph receptors are differentially expressed in numerous malignant tumors. This study intended to analyze the roles of EphB receptors (EphB2, B3, and B4) in urinary bladder cancer. Materials and Methods: Tissue microarray-based immunohistochemical analysis was used to investigate the expression patterns of EphB2, EphB3, and EphB4 in 154 bladder cancer specimens. Immunohistochemical staining was conducted examining the extent of stained cells and staining intensity. EphB was considered to be highly expressed when the intensity of staining was more than moderate in >25% of cells in the tissue section. Small interfering RNA (siRNA) was used to knock down EphB expression in bladder cancer cell lines (T24, 5637) to determine the effects of EphB on tumor cell invasion, proliferation, and migration. Results: EphB receptors (B2, B3, and B4) were detected in 40.9% (EphB2, 63/154), 71.4% (EphB3, 110/154), and 53.2% (EphB4, 82/154) of bladder cancer specimens. Low expression of EphB2, B3, and B4 receptors were significantly associated with higher tu- mor grade (EphB2, p<0.001; EphB3, p=0.032; EphB4, p<0.001) and muscular invasion (EphB2, p=0.002; EphB3, p=0.009; EphB4, p<0.001). No obvious correlation was observed with other clinicopathological variables, such as age, sex, recurrence, lymph node involvement, metastasis, and overall survival. Inactivation of EphB receptors by siRNA transfection increased cell viability, tumor cell invasion, proliferation, and migration in comparison with untransfected cancer cells. Conclusion: Low expression of EphB receptors (B2, B3, and B4) can be a predictive marker for muscular invasion of bladder cancer. Key Words: ‌EphB2, EphB3, EphB4, bladder cancer, muscle invasion, predictive marker INTRODUCTION ing with muscle-invasive bladder cancer, including metastatic cancer. Non-muscle-invasive bladder cancer patients are ini- Urinary bladder cancer is the most lethal urologic malignancy tially treated with transurethral resection and half of these pa- worldwide. It is estimated to affect nearly 75000 people in the tients experience recurrence of the disease. About 5–30% of United States, with 15000 people dying from the disease every these patients progress to muscle-invasive bladder cancer.2 In year. 1 About 75% of bladder cancer patients present with non- spite of an overall 5-year survival rate for bladder cancer of 82%, muscle-invasive bladder cancer, with the remaining present- the 5-year survival rate for metastatic cancer only 6%: the 5-year survival rate for localized cancers is 94%.3 Since most initial di- Received: January 19, 2021 Revised: May 12, 2021 agnoses are made from transurethral bladder biopsies, it can Accepted: June 3, 2021 Corresponding author: Tae Hoen Kim, MD, PhD, Department of Pathology, CHA difficult to determine the tumor is invasive. Therefore, there is Bundang Medical Center, CHA University, 59 Yatap-ro, Bundang-gu, Seongnam a clear need for both predictors of muscular invasion and ef- 13496, Korea. fective targets for novel systemic therapies. Tel: 82-31-780-5438, Fax: 82-31-780-5476, E-mail: [email protected] Eph/ephrin signaling mediates angiogenesis and has been •The authors have no potential conflicts of interest to disclose. shown to be associated with regulation of cell growth, morphol- © Copyright: Yonsei University College of Medicine 2021 ogy, migration, and survival. Additionally, Eph/ephrin signal- This is an Open Access article distributed under the terms of the Creative Com- mons Attribution Non-Commercial License (https://creativecommons.org/licenses/ ing has also been found to be associated with the regulation of 4 by-nc/4.0) which permits unrestricted non-commercial use, distribution, and repro- cytoskeleton organization. Eph receptors are differentially ex- duction in any medium, provided the original work is properly cited. pressed in various cancers, promoting cancer growth or sup- www.eymj.org 679 Low Expression of EphB in Bladder Cancer pressing cancer progression depending on the cancer type.5 tients: TURB and repeated TURB; 7 patients: TURB and radical EphB expression has been shown to be related to poor progno- cystectomy; 3 patients: TURB and partial cystectomy; 1 pa- sis in osteosarcoma, endometrial cancer, and melanoma.6,7 tient: TURB and repeated TURB twice; 1 patient: two times of A retrospective study indicated that EphB4, EphB6, EphA2, TURB and radical cystectomy). When possible, adjacent nor- EphA4, and EphA7 were significantly associated with poor mal tissue was also collected. Human specimens were collect- overall survival in breast cancer patients.8 Their results demon- ed under approval from the CHA Bundang Medical Center In- strated that several Eph receptors may be important targets for stitutional Review Board (IRB2017-09-052). treatment in breast cancer. Another study demonstrated that Analysis and grading of tissues were performed by a blinded EphB4 was overexpressed in bladder cancer cells and mediat- reviewer. A manual tissue arrayer (Quick-Ray Manual Tissue ed cell survival.9 Additionally, several studies have demon- Microarrayer; Unitma Co. Ltd., Seoul, Korea) was used to con- strated that EphB4 is overexpressed in a few different cancers, struct issue microarrays from archival formalin-fixed, paraffin- including head and neck, breast, prostate, and uterine can- embedded tissue blocks. Tissue cylinders (diameter: 3 mm) cer.10-13 In breast cancer, Eph receptor expression was found to were punched from the tumor region of the donor block and be significantly associated with cancer aggressiveness and in- were re-embedded into the recipient block. Four μm sections vasiveness. In an animal model of breast cancer, overexpres- were made from tissue microarrays. sion of EphA2 induced tumor progression and promoted me- tastasis.14 Notwithstanding, the clinical significance of EphB Immunohistochemistry expression in cancer is controversial. Loss of EphB2 expression Tissue microarray sections were deparaffinized in xylene and has been shown to be correlated with more advanced colorec- rehydrated in alcohol. Endogenous peroxidase activity was tal cancer and poorer overall survival.15 In bladder cancer, re- suppressed by 10 min of immersion in 3% hydrogen peroxide. searchers indicated that loss of EphB2 expression and overex- For antigen retrieval, each section was heated in 0.01 mol/L sodi- pression of EphB4 could possibly affect tumor progression.16 um citrate buffer (pH 6.0) for 30 min. The sections were washed Meanwhile, EphB3 knockout mice exhibited an increased fre- three times in phosphate-buffered saline (PBS) for 5 min and quency of retinal ganglion cell axon guidance errors to the optic incubated for 1 h at room temperature with mouse monoclonal disc, where EphB3 is involved in the formation of topographic antibodies to human EphB2, EphB3, and EphB4 (1:100; Epito- maps.17 However, in malignant tumors, the function of EphB3 mics, Burlingame, CA, USA). The sections were then incubat- remains unknown. Moreover, while some researchers have re- ed with horseradish peroxidase-labeled rabbit anti-mouse ported a suppressive effect for EphB receptors (B2, B3, and B4) immunoglobulin (DAKO; 1 h at room temperature) and de- in colorectal cancer,18 other noted tumor-promoting properties veloped with diaminobenzidine at room temperature to re- for EphB3 in lung cancer.19 veal staining. The Eph family comprises a group of receptor protein tyro- sine kinases that are involved in parenchyma tissue integrity Interpretation of immunostaining and cell-cell communication during embryogenesis. Since Positive ephrin receptor (EphB2, B3, and B4) staining was de- the disorganization of tissue architecture is critical for tumor in- fined as brown granules in the cytoplasm or nuclei. Staining in- vasiveness, we aimed to examine the expression of EphB family tensity was evaluated as no staining, weak intensity, moderate receptors during bladder cancer progression. To our knowl- intensity, or strong intensity. The extent of staining was catego- edge, EphB (B2, B3, and B4) expression in urinary bladder can- rized as follows: 0–4%, 5–25%, 26–50%, 51–75%, and 76–100%. cer has not been well investigated, and to elucidate the roles of A case was considered to highly express EphB when the inten- EphB receptors in bladder cancer, we examined the expression sity of staining was more than moderate in >25% of cells in the of EphB receptors in surgical bladder specimens and performed tissue section. functional studies using bladder cancer cell lines. Cell lines and culture The human bladder cancer cell lines (T24, 5637) were obtained MATERIALS AND METHODS from the American Type Culture Collection (Rockville, MD, USA). T24 and 5637 cells were cultured in Roswell Park Memo- Patients and tissue samples for EphB (B2, B3, and B4) rial Institute (RPMI)-1640 medium supplemented with 10% immunohistochemistry fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL Cases of bladder cancer treated at the CHA Bundang Medical streptomycin, and incubated at 37°C in a humidified atmo- Center, School of Medicine, CHA University from 2006 to 2012 sphere consisting of 5% CO2. were reviewed. A total of 154 specimens of bladder cancer were obtained from 134 patients who underwent transurethral EphB siRNA transfection resection of the bladder (TURB) or partial cystectomy or radi- The siRNA used to silence EphB (B2, B3, and B4) and the nega- cal cystectomy. Eighteen patients had repeated surgery (6 pa- tive control vector (cat. no. SN-1013) were purchased from Bi- 680 https://doi.org/10.3349/ymj.2021.62.8.679 Tae Ho Lee, et al. oneer, Inc. (Daejeon, Korea) Cells were plated at 70% conflu- Hercules, CA, USA).
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