Expression of receptor activator of nuclear factor kappa B ligand in bladder cancer

DOI: 10.1111/iju.13756 The bone is a frequent site of metastasis in various malignancies, such as prostate and mammary cancers. In contrast, the incidence of bone metastasis in patients with advanced bladder cancer was reported to be 40%, and approximately 13% of bladder cancer patients undergoing radical cystectomy were shown to develop bone metastasis within 5 years after surgery. Importantly, skeletal-related events consisting of pathological fracture, spinal cord compression and intractable pain reduce the quality of life. Meanwhile, denosumab (XGEVA; Amgen, Thousand Oaks, CA, USA), a human monoclonal antibody against RANKL, was found to be a new therapeutic option for bone metastasis. Cancer cells penetrate the bone marrow and induce osteoblasts to produce cytokines, which promotes the secretion of RANKL. The accelerated expression of RANKL induces osteoclast hyperplasia and facilitates bone resorption. Denosumab controls these mechanisms by inhibiting RANK. Previous studies showed that primary prostate cancer cells expressed the RANK and RANKL genes, and that their expression was further elevated in lesions of bone metastasis. Thus, denosumab is expected to exert its antitumor effect by inhibiting RANKL. To the best of our knowledge, no studies have assessed the expression of RANKL in bladder cancer. The institutional review board of Yokohama City University Medical Center (Yokohama, Japan) approved this study (no. D1507018). Sections from a bladder tissue microarray composed of 136 malignant tissues, 40 nonmalignant tissues and 36 bladder cancer tissues with prognosis information (US Biomax, Rockville, MD, USA) were subjected to immunohistochemical staining using a primary antibody to RANKL (MAB626-100, diluted to 1:200; RSD, Minneapolis, MN, USA) and RANK (sc-9072, diluted to 1:100; Santa Cruz Biotechnology, Dallas, TX, USA). Two genitourinary pathologists blinded to the identity of the sample then examined the slides. Overall, the expression levels of RANKL in bladder cancer tissue specimens were significantly (P < 0.001) higher than those in non-cancerous tissue specimens (Table 1; Fig. S1). Although the higher RANKL expression group showed a worse prognosis, no significant associations between the RANK/RANKL expression and the clinicopathological features, including the prognosis, tumor grade and pT stage, were observed (Table 1; Tables S1, S2; Fig. S2). We also carried out an in vitro study using UMUC-3 bladder cancer cells with a cell invasion assay (Fig. S3), cell migration assay (Fig. S4), cell proliferation assay (Fig. S5) and cell cycle analysis using flow cytometry (Table S3) with Si-Control and Si-RANKL RNA. The protocol has already been described in detail. When we knocked down RANKL, the cell migration of UMUC-3 decreased, and their apoptosis increased significantly. However, no significant differences were observed in the invasion. In contrast, RANKL knockdown in UMUC-3 increased the cell proliferation. Further studies are required to confirm the progression of bladder cancer through the RANKL pathway. Several studies have determined the levels of RANKL expression in urological malignancies, including renal and prostate cancers. Mikami et al. showed that the elevated expression of RANKL and RANK in renal cell carcinoma was an independent predictor of tumor recurrence and bone metastasis. Chen et al. reported that the increased expression of RANKL/RANK was correlated with higher Gleason score, TNM stage or serum prostate-specific antigen levels, as well as androgen dependence in prostate cancer patients. In the present study, we found that RANKL expression was upregulated in bladder cancers compared with non-malignant urothelial specimens. However, there were no significant correlations between RANKL expression and clinicopathological features available for the patient cohort. In summary, the present study showed that RANKL was overexpressed in bladder cancer. However, its functional role in bladder cancer, especially tumor progression and metastasis, remains uncertain. Further investigation in a larger study population is required to validate the current results. Urological Notes