RANKL Immunisation Inhibits Prostate Cancer Metastasis by Modulating EMT Through A RANKL-Dependent Pathway

RANKL Immunisation Inhibits Prostate Cancer Metastasis by Modulating EMT Through A RANKL-Dependent Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research RANKL Immunisation Inhibits Prostate Cancer Metastasis by Modulating EMT Through A RANKL-Dependent Pathway Mineon Park, Yong Jin Cho, Bora Kim, Young Jong Ko, Yuria Jang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-105442/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Method: In this study, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa. Results: RANKL activation was observed in human PCa tissue specimens. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Conclusion: Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa Trial registration: Chosun University Hospital, CHOSUN 2020-06-001. Registered 01 June 2020-prospectevely registered, https://hosp.chosun.ac.kr/medi_depart/ site=hospital&mn=151&type=view&catename=IRB Cellular & Molecular Neuroscience RANKL prostate cancer metastasis EMT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Prostate cancer (PCa) is the second most common cancer in males and the fifth leading cause of death worldwide [ 1 ]. Metastasis of PCa cells to the skeleton occurs in a predictable manner, with lesions tending to appear first in the axial skeleton, followed by appendicular tissues [ 2 ]. Considering the effects of PCa on both haematopoiesis and bone structure, bone metastasis is a major cause of morbidity in patients with advanced disease. Replacement of haematopoietic tissue by metastatic PCa cells is associated with anaemia and increased morbidity (mortality). The 5-year survival of most patients with PCa is almost 100%; however, that of PCa patients with metastasis to distant sites is as low as 28%. Thus, PCa is one of the deadliest cancers [ 3 ]. Experimental and clinical observations reveal that treatment with anti-resorptive agents suppresses or even prevents PCa metastasis [ 4 – 7 ], and that accelerated bone turnover stimulates progression of skeletal secondary tumours [ 8 – 10 ]. During bone metastasis of PCa, cancer cell-derived cytokines stimulate expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which in turn activates bone resorption. RANKL, also known as tumour necrosis factor-related activation-induced cytokine (TRANCE) [ 11 ], osteoprotegerin ligand (OPGL) [ 12 , 13 ] and osteoclastic differentiation factor (ODF) [ 14 ], interacts with RANK and is involved in all the steps related to tumour development, from initial tumour formation to migration of cancer cells and subsequent metastasis [ 15 ]. RANKL is expressed in several tissues, including brain, skin, intestine, skeletal muscle, kidney, liver, lung and mammary tissue; however, expression is very high in bone [ 16 ], lymphoid organs and the vascular system [ 17 ]. RANKL binds to RANK on the surface of pre-osteoclasts, activating them and inducing formation of osteoclasts [ 18 ]. Recent studies report expression of RANK and RANKL by various solid tumours, including breast cancer. RANKL accelerates migration and metastasis of cancer cells expressing RANK [ 19 , 20 ]. Furthermore, it protects breast cancer cells from apoptosis in response to DNA damage and controls self-renewal and anchorage-independent growth of tumour-initiating cells [ 21 ]. However, it is unclear how RANKL signalling triggers metastasis of PCa. Epithelial-to-mesenchymal transition (EMT), a rapid and often reversible phenotypic change in epithelial cells, is an important phenomenon underlying cancer metastasis. Originally, EMT was described in the context of developmental processes such as heart morphogenesis and mesoderm and neural crest formation. Epithelial cells lose structures involved in cell–cell adhesion (e.g., adherens junctions and desmosomes), modulate their polarity and rearrange their cytoskeleton, which is consistent with the typical switch of intermediate filaments from cytokeratins to vimentin [ 22 ]. A recent report shows that the oncogenic c-MYC, Wnt signalling and β-catenin pathways activate the Snail/glycogen synthase kinase-3 (GSK-3) axis and induce EMT [ 23 ]. The concept of exploiting the host’s immune system to treat cancer relies on the ability of immune cells to eliminate malignant cells at the early transformation stage in a process called immune surveillance [ 24 ]. Passive anti-cytokine immunotherapy with specific high-affinity antibodies has been tested in animal models and clinical trials of rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma, Crohn’s disease, psoriasis and other articular autoimmune disorders. This strategy facilitates production of anti-auto-cytokine antibodies by the immune system in response to active vaccination. However, development of a desired antibody response to self-proteins necessitates suppression of immune resistance. Common anti-cytokine vaccines are prepared from autologous proteins that are converted to derivatives that lack biological activity following treatment with glutaraldehyde or formaldehyde [ 25 ]. A previous study developed a mouse RANKL mutant (mRANKL-MT) protein and confirmed its ability to inhibit osteoporosis. Here, we used mRANKL-MT as an immunogen for RANKL-targeting immunotherapy of bone disease and investigated its potential as a cancer vaccine in a mouse model of metastatic cancer. We confirmed the activity of mRANKL-MT and clarified its effects on RANK/RANKL signalling-mediated EMT in transient RANKL-overexpressing cell lines and animal models. We found that mRANKL-MT suppressed RANKL-dependent β-catenin signalling. We also found that mRANKL-MT immunotherapy altered the characteristics of cancer cells and effectively suppressed RANKL-dependant cancer metastasis. Methods Patient samples Three patient tissue samples from PCa patients not receiving chemotherapy, hormone therapy, or radiotherapy were obtained before surgery from Chosun University Hospital during 01 June 2020 and 31 July 2020 (Chosun University Hospital (CHOSUN 2020-06-001)). Tumours and normal tissues were harvested and immediately fixed in a formalin solution (neutral buffered, 10%; Sigma-Aldrich) for 24 h. Tissue specimens were successively dehydrated in ethanol and treated with xylene. Paraffin embedded tissues were sliced into 5 μm-thick sections, deparaffinised with xylene and rehydrated through graded alcohol solutions. The tissues were stained with H&E for histological analysis. Immuno-histochemical studies were performed using the Novolink TM Polymer Detection System kit (Leica Biosystems) as per the manufacturer’s instructions. Antibodies specific for IL-6 (1:200; Cell Signaling Technology) and RANKL (1:200, Cell Signaling Technology) were used. Cell lines and cultures Human prostate adenocarcinoma luciferase-labelled PC3 luc cells were obtained from Professor Park and maintained at 37°C/5% CO 2 in Rowell Park Memorial Institute (RPMI) 1640 medium (Welgene, Korea) supplemented with 10% heat-inactivated foetal bovine serum (certified, GIBCO, USA) and a 10% antibiotic solution (Welgene). Cloning of hRANKL The RNA used to clone hRANKL cDNA was extracted from MG63 cells (ATCC ® CRL-1427 ™ ) expressing RANKL. The quality of the extracted RNA was verified by agarose gel electrophoresis. The cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer’s instructions. Amplification and cloning of the hRANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM magnesium chloride (MgCl 2 ), 10 μM primers (hRANKL- Bcl I: 5'-TGATCAAAGCTTGAAGCTCAGCCTTTTGC-3' and hRANKL - Xho I: 5'-CTCGAGATCTATATCTCGAACTTTAAAAGCCCC-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 μL of the RANKL gene construct (template). The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 70°C for 30 s. The polymerase chain reaction (PCR) product was cloned into the Bam H1/ Xho I sites of a pMX vector (CELL BIOLABS, USA). Sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA). Retroviral hRANKL transduction Plat-E cells were seeded at a density of 3 × 10 5 cells/well in a six-well plate for 24 h and then transiently transfected with hRANKL/pMX using 0.2 μg plasmid and 0.6 μL of the FuGENE HD transfection reagent (Promega, Madison, WI, USA), according to the manufacturer’s protocol. After incubation, the DNA/FuGENE mixture was added drop-wise onto Plat-E cells. Viral supernatants were recovered from the culture medium at 48 h after transfection. Virus-containing supernatants were filtered through 0.45 μm non-pyrogenic filters and supplemented with 10 μg/mL polybrene (Sigma-Aldrich). Reverse-transcription quantitative PCR (RT-qPCR) Total RNA was extracted from PCa cells using Trizol (Invitrogen) and 1 μg was used for RT-qPCR along with oligo-dT primers (10 μg) and dNTPs (10 mM). Next, qRT-PCR was performed to analyse cDNA using SYBR Green SuperMix (BIORAD, USA) on a CFX Connect Real-Time System (BIORAD, USA). All target gene primers were purchased from Bioneer Co. (Daejeon, Korea) and the cDNA was amplified using the following primer sets: E-cadherin (h): 5'-TGGAGGAATTCTTGCTTTGC-3' (forward) and 5'-TGGAGGAATTCTTTTGC-3' (reverse); vimentin (h): 5'-GACGCCATCAACACCGAGTT-3' (forward) and 5'-GACGCCATC AACACCGAGTT-3' (reverse); β-catenin (h): 5'-ACAAACTGTTTTGAAAATCCA-3' (forward) and 5'-CGAGTCATTGCATACTGTCC-3'(reverse); MMP-9 (h): 5'-TCCAGTACCAAGACAAAG-3' (forward) and 5'-TTGCACTGCACGGTTGAA-3' (reverse); RANK (h): 5'-CAAATGCAGACCCTGGA CCA-3' (forward) and 5'-AAACGCCAAAGATGATGGCA-3' (reverse); RANKL (h), 5'-CCTGTAT GCCAACATTTGCTTTC-3' (forward) and 5'-TTCCTCTCCAGACCGTAACTTAAA-3' (reverse); IL -6 (h): 5'-AGCAAAGAGGCACTGGCAGA-3' (forward) and 5'-GTACTCATCTGCACAGCTCTGG C-3' (reverse); TCF-4 (h): 5'-GCTCAGGGTATGGAACCGGC-3' (forward) and 5'-CCCTGTAGTC CTGGTGGCATG-3' (reverse); c-MYC (h): 5'-CCTGGTGCTCCATGAGGAGAC-3' (forward) and 5'-AGACTCTGACCTTTTGCCAGG-3' (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5'-TCAAGAAGGTGGTGAAGCAG-3' (forward) and 5'-AGTGGGAGTTGCTGTTGAAG T-3' (reverse). Values on the vertical axis represent 2(−ΔCt); ΔCt is the discrepancy between the target gene Ct and GAPDH Ct. Western blot analysis The cells were washed twice with phosphate-buffered saline (PBS; pH 7.4) and total proteins were extracted using radio immunoprecipitation assay buffer supplemented with 1% protease inhibitors, phosphatase inhibitors and phenylmethylsulfonyl fluoride (PMSF). The protein concentration was measured using the BCA Protein Assay Kit (Thermo Pierce TM ). The membrane was blocked with a solution containing 5% skim milk in TBS-T for 30 min and then washed in TBS-T. The membrane was incubated for overnight at 4°C with the following primary antibodies: E-cadherin (sc-7870, Santa Cruz Biotechnology), N-cadherin (ab76011, Abcam), β-catenin (#29822 94, Millipore), MMP-9 (#13667, Cell Signaling), IL-6 (#12153, Cell Signaling Technology), c-MYC (9E10, Santa Cruz Biotechnology), TCF-4 (#2565, Cell Signaling Technology), RANK (#4845, Cell Signaling Technology), P-ERK (#9101, Cell Signaling Technology), ERK (#9102, Cell Signaling Technology), GAPDH (#2118, Cell Signaling Technology), P-AKT (#9271, Cell Signaling Technology), AKT (#9272, Cell Signaling Technology), P-SRC (#2105, Cell Signaling Technology), SRC (#2108, Cell Signaling), GSK-3B (#9315, Cell Signaling Technology) and P-GSK-3B (#9336, Cell Signaling Technology). Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H + L) and HRP-conjugated AffiniPure goat anti-mouse IgG (H + L) were obtained from Proteintech Group, Inc (Jackson) and used as secondary antibodies. Cell migration Cells were seeded in 6-well plates for the cell migration assay. After each treatment, a confluent monolayer was wounded using a 200 µL pipette tip. Images of wound closure were obtained under an inverted microscope after 48 h. The wound area was calculated using NIH ImageJ software. Cell invasion assay A total of 1 × 10 5 transfected cells were seeded into the top chamber of a 24-well polycarbonate Transwell chamber (8.0 µm pore size; Corning Incorporated, Glendale, AZ, USA) and then treated for 24 h with hRANKL or RANKL. The number of trypan blue-stained cells in five random fields was counted using an inverted microscope. Luciferase reporter assay to assess Wnt/β -catenin activity Cells were seeded into a 24-well plate 24 h prior to transient transfection with either 2 µg of TOPflash or FOPflash reporter plasmid along with 1 µg DNA using Lipofectamine 3000 (Thermo Fisher). The TOPflash luciferase reporter plasmid contains TCF-4-binding sites upstream of the luciferase gene, resulting in luciferase activity in the presence of active Wnt/β-catenin signalling. The FOPflash reporter plasmid, on the other hand, carried mutated TCF-4-binding sites. Total cell extracts were assayed for luciferase activity according to the manufacturer’s instructions (Promega). Immunoprecipitation Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.6–8.0, 100 mM sodium chloride NaCl, 300 mM sucrose, 3 mM MgCl 2 [buffer A]; and 20 mM Tris pH 8.0, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid [buffer B]). Whole cell lysates obtained by centrifugation were incubated with antibodies specific for active β-catenin (Millipore) and TCF-4 (Cell Signaling Technology) (dilution 1:100) and protein A Sepharose beads (Amersham Biosciences) for 2 h at room temperature. The immune complexes were washed three times using wash buffer and examined by western blotting. Site-directed mutagenesis and production and purification of mRANKL-MT The RNA used for the cloning of RANKL cDNA was extracted from MC3T3-E1 cells (Korean Cell Line Bank, Seoul, Korea) expressing RANKL. The quality of the extracted RNA was verified by agarose gel electrophoresis and cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer’s instructions. Amplification and cloning of the RANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM MgCl 2 , 10 μM primers (mRANKL- Nde I: 5'-CATATGAAGCCTGAGGCCCAGCC ATTTGC-3'; mRANKL- Xho I: 5'-CTCGAGGTCTATGTCCTGAACTTTGAAAGCC-3'; mRANKL (K180R)-F: 5'-CCCATCGGGTTCCCATCGAGTCACTCTGTCCTCTTG-3'; mRANKL (K180R)-R: 5'-CAAGAGGACAGAGTGACTCGATGGGAACCCGATGGG-3'; mRANKL (D189I, R190K)-F: 5'-CTCTTGGTACCACATCAAGGGCTGGGCCAAGAT-3'; mRANKL (D189I, R190K)-R: 5'-ATC TTGGCCCAGCCCTTGATGTGGTACCAAGAG-3'; mRANKL-MT (H223F, H224Y)-F: 5'-AA CA TTTGCTTTCGGTTTTATGAAACATCGGGAAGCG-3'; or mRANKL-MT (H223F, H224Y)-R: 5'-CGCTTCCCGATGTTTCATAAAACCGAAAGCAAATGTT-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 μL of RANKL gene construct as the template. The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 70°C for 30 s. The PCR product obtained was cloned into the Nde I/ Xho I site of the GST-30a vector (Novagen, Madison, WI, USA). Mutations at positions 180, 189–190 and 223–224 were introduced using megaprimers [26]. The PCR product was transformed into Escherichia coli BL21-CodonPlus (DE3)-RIPL (Novagen) by electroporation (5 ms, 12.5 kV/cm) and the transformed cells were cultivated in Luria-Bertani broth containing kanamycin (50 μg/mL, T&I, Daejeon, Korea). Plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA). The cloned product was confirmed by sequencing. All sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA). The recombinant plasmid carrying mRANKL-MT was expressed from a single E. coli BL21-CodonPlus (DE3)-RIPL colony using previously described methods [26]. Purification of mRANKL-MT E. coli cells expressing mRANKL-MT were cultivated in 1 L of an auto-induction medium supplemented with kanamycin (50 μg/mL), as previously described. After centrifugation at 6000 × g for 20 min at 4°C, the pelleted cells were resuspended in 10 mL of lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4) supplemented with 0.1 mg/mL lysozyme and 0.1 mM PMSF. Glycerol (20% v/v; CARLO ERBA, France) was added to the cell suspension and the cells were sonicated and centrifuged at 15,000 × g for 10 min at 4°C. The supernatants were passed through 0.2 μm paper filters and applied to Ni 2+ -affinity chromatography HisTrap FF columns (1 mL; GE Healthcare Life Science, Piscataway, NJ, USA) equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 5 mM dithiothreitol, pH 7.4). The columns were subsequently washed using binding buffer supplemented with 20 mM imidazole. After washing, bound protein was eluted using elution buffer (Qiagen). The eluted protein was dialysed against a dialysis buffer (20% v/v glycerol in PBS) in a 10,000 MW Slide-A-Lyzer Dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). The purified protein was vacuum concentrated (Savant Instruments, Holbrook, NY, USA) and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were calculated using the Bradford assay. For endotoxin removal, an additional washing step was introduced after the initial wash for chromatography. Animal study The animal experimental protocol was approved by the Institutional Animal Care and Use Committee, Chosun University, Gwangju, Korea (CIACUC2019-A0015). All experiments were performed in accordance with relevant guidelines and regulations. Five-week-old male athymic nude mice (BALB-c/nu, Orient Bio Co. LTD, Seoul, Korea) were used to generate a xenograft model by intracardiac injection of PC3 Wild , PC3 +RANKL (RANKL overexpression), or PC3 +RANKL + IM (immunisation) cells. Following immunisation, mice were divided into an immunisation group and a non-immunisation group. The Sham group was immunised by a subcutaneous injection of PBS, while the immunisation group was injected subcutaneously with mRANKL-MT (100 µg/kg three times every 2 weeks). Mouse sera and tissue samples were collected according to indicated schedule. In vivo bioluminescence measurement Tumour-bearing tissues were subjected to in vivo bioluminescence imaging using a Living Image® 4.5.4 IVIS Imaging System (Perkin Elmer). For luciferase imaging, D-luciferin (Promega) was injected intraperitoneally before imaging. Quantitative detection of luciferase was performed as follows: regions of interest (ROIs) were drawn to capture detected fluorescence, and auto-regions ROIs were used to precisely outline the target region. Quantitative analysis of RANKL The amount of RANKL in mouse serum was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, USA) according to the manufacturer’s protocol. Absorbance was measured in a colorimetric microplate reader (BioTek, USA) at 450 nm. Measurement of anti-RANKL antibody titers Serum samples obtained from immunised mice were serially diluted with PBS containing 0.02% sodium azide and 2% bovine serum albumin (BSA), and then applied to ELISA plates (Sigma-Aldrich) coated with mouse recombinant tumour necrosis factor ligand superfamily member 11 (TNFSF11; 10 μg/mL, R&D Systems). Reactivity of serum antibodies to the target protein was determined using an HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific) at a dilution of 1/1000 in PBS/0.02% sodium azide/2% BSA. After development with 1,2- phenylenediamine dihydrochloride (0.4 mg/mL in 0.066 M disodium phosphate, 0.035 M citric acid and 0.01% hydrogen peroxide), absorbance was measured in an ELISA plate reader at 450 nm. Statistical analysis Data are expressed as the mean ± standard deviation (SD) from three independent experiments. GraphPad Prism version 6.0 software for windows was used to analyse in vitro and in vivo data . Statistical significance for pairwise comparison was evaluated using an unpaired t -test or one-way analysis of variance (ANOVA) with Turkey’s post-hoc test. Results were considered significant at *p < 0.05. Results Expression of RANKL in human PCa metastasis tissue specimens To investigate the association between RANKL and PCa metastasis in human patients, we examined expression of RANKL and metastasis markers interleukin-6 (IL-6) in human PCa metastatic tissue specimens. Immunohistochemical analysis of tissue specimens from patients with PCa metastasis harboured irregularly shaped and dysplastic cells, whereas normal tissues did not. In addition, RANKL expression was elevated, along with that of IL-6, relative to that in normal tissue. (Fig. 1 ). Thus, RANKL expression was closely related to PCa metastasis, suggestive of an important role in this process. Effects of human RANKL (hRANKL) on EMT and metastasis of PC3 cells To explore the relationship between RANKL and EMT of PC3 cells, we performed cell migration and invasion assays with PC3 cells treated with hRANKL. In the invasion assay, wound healing in hRANKL-treated PC3 cells was significantly better than that in control cells (Fig. 2 A). Cell invasion also increased significantly following hRANKL treatment (Fig. 2 B). EMT is closely related to tumour metastasis and progression. Therefore, to determine changes at the molecular level, we measured EMT markers in PC3 cells at the mRNA (Supp. 1) and protein (Fig. 2 C) levels following treatment with hRANKL. Expression of the EMT marker E-cadherin in hRANKL-treated PC3 cells fell significantly, but that of vimentin and β-catenin increased. Expression of MMP-9 and IL-6 (markers of metastasis) were significantly higher in hRANKL-treated cells than in control cells. Expression of E-cadherin protein was significantly lower in hRANKL-treated PC3 cells than in control cells. By contrast, expression of vimentin, β-catenin and MMP-9 proteins was significantly upregulated following treatment with hRANKL. The Wnt/β-catenin pathway regulates PCa metastasis and plays an important role in cancer progression; therefore, we examined the effects of RANKL on Wnt signalling using a TOP/FOP reporter assay (Fig. 2 D). No changes in TOP/FOP reporter luciferase activity were observed in hRANKL-treated PC3 cells. Next, we examined phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), SRC and GSK-3B to investigate the effects of RANKL on mitogen-activated protein kinase (MAPK) and Wnt signalling in PC3 cells (Fig. 2 E). PC3 cells treated with hRANKL showed a significant and time-dependent reduction in the level of phosphorylated GSK-3B. A time-dependent increase in SRC and AKT phosphorylation levels was also observed. Next, we performed co-immunoprecipitation of β-catenin and TCF-4 to investigate the status of TCF-associated signalling in hRANKL-treated PC3 cells (Fig. 2 F). The results showed a slight increase in TCF-4 levels in hRANKL-treated PC3 cells. Thus, RANKL treatment may trigger metastasis of PC3 cells by suppressing GSK-3B phosphorylation and facilitating EMT. Overexpression of RANKL modulates EMT and metastasis of PC3 cells To investigate whether RANKL overexpression stimulates PC3 cell growth in vitro , cells were transiently transfected with an overexpression plasmid containing RANKL. GFP expression by PC3 RANKL cells was monitored by fluorescence microscopy (Supp. 2A). We found a significant increase in expression of RANKL mRNA and protein (Supple 2B and 2C). Next, we performed migration and invasion assays using PC3 RANKL cells to evaluate the effects of RANKL overexpression on EMT and metastasis. Wound healing was significantly better in PC3 RANKL cells than in control cells (Fig. 3 A). Also, PC3 RANKL cells were significantly more invasive than control cells (Fig. 3 B). Analysis of mRNA encoding EMT- and metastasis-related factors in PC3 RANKL cells revealed significant downregulation of the gene encoding E-cadherin (Supp. 2D). Furthermore, expression of genes encoding vimentin, MMP-9, IL-6 and β-catenin was upregulated significantly in PC3 RANKL cells. Protein expression analysis revealed that PC3 RANKL cells expressed significantly lower levels of E-cadherin than PC3 Wild cells (Fig. 3 C). By contrast, expression of N-cadherin, MMP-9, IL-6, c-MYC and β-catenin was significantly higher in PC3 RANKL cells. PC3 RANKL cells showed a significant increase in TOP/FOP luciferase reporter activity (Fig. 3 D). Immunoprecipitation of β-catenin was carried out to investigate the signal transduction pathway associated with TCF in PC3 RANKL cells (Fig. 3 E). PC3 RANKL cells overexpressing RANKL showed a significant increase in activation of the MAPK and β-catenin/TCF-4 signalling pathways owing to stronger binding between β-catenin and TCF-4 than in PC3 Wild cells. Thus, ectopic overexpression of RANKL may increase EMT and the metastatic properties of PC3 cells via the β-catenin/TCF-4 signalling pathway, suggesting the therapeutic potential of RANKL targeting for prevention of PCa metastasis. Therapeutic Effects Of Mrankl-mt In Pc3 Cell-inoculated Mice Alignment of the mRNA sequence of mRANKL-MT with that of mRANKL-WT identified a region that could be amplified using selected primers. The recombinant mRANKL-WT sequence encoded the full-length 158 amino acid target region, which includes residues 158 to 316 (Fig. 4 A). To create point mutations, Lys180, Asp189-Arg190 and His223-His224 were transformed to Arg180, Ile189-Lys190 and Phe223-Tyr224, respectively. The resulting hRANKL, mRANKL-WT and mRANKL-MT molecules had similar molecular weights (Supp. 3). Male BALB-c/nu mice were injected subcutaneously with hRANKL, mRANKL-WT, or mRANKL-MT (100 µg/kg; three times every 2 weeks). After immunisation, 1 ⋅ 10 6 PC3 Wild or PC3 RANKL+ cells were injected into the left ventricle of Sham or immunised mice. Serum and tumour-bearing tissues were collected after 16 weeks (Fig. 4 B). To observe bone metastasis, luciferase activitiy in tumour-bearing tissues of Sham, PC3 Wild , PC3 RANKL+ and PC3 RANKL+ + IM mice was detected by IVIS. In PC3 RANKL+ mice, large and strong bioluminescence spots were detected throughout the body at 16 weeks post-cancer cell injection (Fig. 4 C). However, no bioluminescence signals were detected in PC3 RANKL+ + IM mice. The photon flux values were significantly higher in PC3 RANKL+ mice than in PC3 RANKL+ + IM mice (Fig. 4 D). Survival rate analysis revealed a significant decrease in the survival of animals in the PC3 RANKL+ groups compared with that of animals from the PC3 Wild group. The survival rate improved higher in the PC3 RANKL+ + IM group than that in the PC3 RANKL+ group (Fig. 4 E). The metastasis rate in the PC3 RANKL+ group was higher than that in the PC3 Wild group; however, that in the PC3 RANKL+ + IM group was significantly less than that in the PC3 RANKL+ group (Fig. 4 F). Therapeutic Effects Of Anti-rankl Antibodies Induced By Rankl Immunisation To examine the histological characteristics of metastatic tumour-bearing tissues, metastatic lesions from each mouse were stained with haematoxylin and eosin Y (H&E). As shown in Fig. 5 A, gross examination of the excised tibiae from PC3 RANKL+ mice revealed a tumour mass in the primary spongiosum (trabecular epiphysis) and bone marrow cells; this was not observed in PC3 RANKL+ + IM mice. In particular, expressions of IL-6 and RANKL increased markedly in the trabecular epiphysis region of bones from PC3 RANKL+ mice, but were undetectable in PC3 RANKL+ + IM mice. Next, we investigated whether mRANKL-MT induces production of anti-RANKL antibodies. The concentration of RANKL (Fig. 5 B) was highest in PC3 RANKL+ , and production of antibodies (Fig. 5 C) was highest in PC3 RANKL+ + IM mice. Also, we measured anti-RANKL antibody levels in the PC3 RANKL + + IM group with bone metastasis to investigate generation of anti-RANKL antibodies after immunisation with mRANKL-MT (Fig. 5 D). The anti-RANKL titer in mice with bone metastasis was significantly higher than that in mice without bone metastasis. Also, serum RANKL levels in the PC3 RANKL+ + IM group without metastasis were significantly lower than those in mice with metastasis (Fig. 5 E). These observations suggest that anti-RANKL antibodies generated by RANKL immunisation suppress metastasis of PCa cells. Effect Of Immunisation On Emt And Metastasis To investigate the effects of mRANKL-MT on PCa metastasis, sera obtained from immunised mice were used to treat RANKL-overexpressing PC3 cells. The results of cell migration assays showed that wound healing was inhibited significantly in PC3 RANKL+ cells treated with immune serum (Fig. 6 A). In addition, the invasive ability of PC3 RANKL+ cells declined following treatment with immune sera (Fig. 6 B). Analysis of mRNA encoding EMT- and metastasis-related factors in PC3 RANKL+ cells treated with immune sera revealed significant upregulation of E-cadherin expression (Supp. 4). By contrast, expression of vimentin, MMP-9, IL-6 and β-catenin was downregulated significantly in immune serum-treated PC3 RANKL+ cells. Protein expression analysis showed that E-cadherin expression was higher in immune serum-treated PC3 RANKL+ cells than in control serum-treated PC3 RANKL+ cells (Fig. 6 C). By contrast, expression of N-cadherin, MMP-9, IL-6 and β-catenin was significantly lower in immune serum-treated PC3​​ RANKL+ cells, as was expression of c-MYC. The luciferase activity of the TOP/FOP reporter in immunised serum-treated PC3 RANKL+ cells fell significantly (Fig. 6 D). Immunoprecipitation analysis revealed a significant decrease in binding between β-catenin and TCF-4 in cells treated with immune serum (Fig. 6 E). Finally, we measured expression of phosphorylated ERK, AKT, SRC and GSK-3B to investigate the effect of immune serum on MAPK and Wnt signalling in PC3 RANKL+ cells (Fig. 6 F). Cells treated with immune serum showed a significant and time-dependent increase in GSK-3B phosphorylation and a time-dependent decrease in SRC phosphorylation. These results indicate that the EMT and metastatic properties of PC3 RANKL+ cells were inhibited by treatment with immune serum. Discussion PCa, which is common among men in the western world, is associated with high mortality and morbidity with respect to advanced metastasis to the bone. Evidence suggests that the RANKL signalling cascade plays a key role in proliferation, metastasis, migration and invasion of PCa [ 6 ]. The RANKL–RANK interaction plays a pivotal role in PCa metastasis; indeed, RANKL expression induces osteoclast hyperplasia and bone destruction during PCa metastasis [ 27 ]. RANKL activates RANK directly on tumour cells, as evidenced by dysregulation of several biochemical signalling pathways in PCa cells. High expression of RANKL facilitates PCa metastasis, an idea consistent with previous studies showing that signalling through the RANK/RANKL axis is related to bone metastases of solid tumours [ 20 , 28 ]. Here, we demonstrated RANKL immunoreactivity in bone metastatic lesions of PCa patients. We also found that hRANKL-treated or hRANKL-overexpressing PCa cells showed a significant increase in expression of metastasis markers such as IL-6. In particular, in vitro experiments show that RANKL stimulation markedly increases the migration and invasion of PC3 cells, downregulates expression of the epithelial marker E-cadherin and upregulates the mesenchymal marker vimentin. EMT correlates with tumour metastasis and progression, which is consistent with impaired cell–cell adhesion following the loss of E-cadherin expression [ 29 , 30 ]. Furthermore, we show that GSK-3B phosphorylation was reduced significantly following RANKL treatment of PC3 cells due to the effect of RANKL on MAPK and Wnt signalling. Also, we observed altered expression of β-catenin and TCF-4 in PC3 RANKL+ cells, which resulted in a highly conserved developmental signalling pathway that includes the major effector protein β-catenin. Wnt signalling is an essential pathway involved in cell development, proliferation and differentiation; indeed, regulatory abnormalities in Wnt signalling are associated with metastasis of many cancers [ 31 ]. In particular, RANKL overexpression in PC3 cells led to a significant increase in expression of Wnt3a, suggesting that RANKL is a potential target of Wnt signalling in cancer cells [ 32 ]. RANKL plays a fundamental role in osteoclastogenesis by interacting with the RANK receptor on osteoclast progenitors during bone destruction by metastatic breast cancer, thereby driving osteoclast cell lineage commitment, monocyte cell fusion and osteoclast maturation via regulation of NF-κB-mediated gene expression; therefore, we were intrigued to find out whether catabolic Wnt signalling mechanisms exist alongside anabolic Wnt pathways to regulate osteoclast formation in bone. β-catenin, the critical effector of the Wnt pathway, regulates a number of key processes during development, including proliferation, differentiation and cell fate determination [ 33 ]. Normally, β-catenin is localised to the cell adhesion junctions in epithelial cells and its abnormal cytoplasmic/nuclear stabilisation drives uncontrolled transcription of target genes (including c-jun, cyclin D1, c-myc, survivin and MMP-7) that regulate cell proliferation, survival and adhesion [ 34 ]. In view of cancer cell fate, it is not surprisingly that overexpression of RANKL by PC3 cells led to increased binding of β-catenin to TCF4 and to increased TOP activity. Regulation of β-catenin is linked to the pathogenesis of a number of human cancers, particularly those with an epithelial cell origin. Supporting its putative role as a Wnt signalling target, we confirmed that RANKL overexpression led to transcriptional activation of β-catenin in PC3 cells. Over the past decades, it has become clear that the RANK/RANKL axis exerts a broad range of functions during cancer cell fate. In the cancer setting, the RANK-RANKL pathway plays a role in every stage of tumorigenesis. Therefore, inhibition of RANKL by anti-RANKL antibodies is expected to be more far-reaching than simple inhibition of cancer cell activation. Denosumab, a drug used to treat metastatic prostate bone loss, has received FDA approval; this drug inhibits the RANK-RANKL pathway [ 35 ]. Denosumab is an effective and safe drug, which is superior to zoledronic acid in terms of skeletal-related events prevention; this was borne out by a combined analysis that included three randomised phase III trials with a similar set-up [ 36 ]. These trials included patients with bone metastases due to advanced breast cancer [ 37 ], prostate cancer [ 38 ], other solid tumours or multiple myeloma [ 39 ]. However, despite medical and commercial success, passive anti-cytokine drugs such as OPG-Fc and denosumab have several limitations, including high production costs, the need for regular infusion and a limited half-life [ 40 ]. Thus, the aim is to develop a RANKL vaccine. In comparison with antibodies and other biologics, vaccines are better models for treatment of chronic disease because they are relatively cheap and small doses of protein can have a strong and long-lasting effect [ 41 , 42 ]. Here, we developed a novel vaccine targeting RANKL and examined its efficacy in a murine model of prostate cancer metastasis. To circumvent the problem of the immunogen triggering cytokine activity, mutants of RANKL were generated to prevent its interaction with RANK. A previous study shows that immunisation with mutant RANKL molecules generates anti-RANKL antibodies that block the interaction between RANKL and its receptor in an animal model of osteoporosis, thereby preventing proliferation and differentiation of osteoclasts and improving bone density [ 43 ]. Therefore, to block RANKL activation during PCa metastasis, we immunised mice with mRANKL-MT followed by intracardiac injection of PC3 cells. Inhibiting RANKL in animal models of metastases exerts therapeutic effects by inhibiting cancer cell metastasis. Currently, in vivo tumour models that are most commonly used to study the process of cancer metastasis rely on introduction of tumour cells directly into the systemic circulation by injection into the left ventricle of laboratory rodents [ 44 , 45 ]. Thus, we employed a mouse model of PCa metastasis that more accurately reflects the metastatic process of this type of cancer. Studies on the effects of mRANKL-MT in PC3 RANKL+ mice showed that tumour growth was completely inhibited. Immunisation with mRANKL-MT effectively inhibited metastasis of tumour cells by generating anti-RANKL antibodies. To further confirm the action of RANKL immunisation, we assessed the effects of immune serum from immunised mice on PCa cells. Anti-RANKL antibodies blocked the RANKL-mediated chemotaxis of tumour cells. Furthermore, anti-RANKL antibodies inactivated RANKL on tumour cells directly. Treatment of RANKL-overexpressing PC3 cells with immune serum almost entirely abolished cancer cell migration and invasion. Wu et al. showed that a recombinant inactive RANKL vaccine (Y234pNO2Phe) induced high antibody titers and protected mice from collagen-induced arthritis by inhibiting osteoclast function and by preventing bone erosion [ 46 ]. However, no study has reported that these types of RANKL vaccine have been used to inhibit cancer metastasis. In addition, we found that EMT and metastasis-related genes were downregulated following treatment of RANKL-overexpressing PC3 cells with immune serum. The antisera obtained from mice immunised with mRANKL-MT almost entirely inhibited the EMT process in RANKL-overexpressing PC3 cells. EMT is characterised by the loss of cell–cell adhesion and by an increase in cell motility; it is a key process in cancer progression and metastasis, making EMT inhibition an attractive therapeutic strategy [ 47 , 48 ]. Deregulation of Wnt/β-catenin signalling is a hallmark of PCa metastasis [ 49 , 50 ] and β-catenin is a critical end component of the Wnt signalling pathway, which regulates cell growth, apoptosis and migratory behaviour in response to intercellular adhesion molecules [ 51 ]. Activation of β-catenin in PCa cells leads to transactivation of Wnt signalling target genes, including cyclin D1, HEF1 and matrix metalloproteinase 9 [ 52 ]. Also, previous studies show that expression of Wnt-1 and β-catenin is increased in invasive PCa cell lines and in primary prostate cancer specimens [ 53 ]. In line with these previous reports, we demonstrated that treatment of PC3 cells with immune serum led to a marked decrease in RANK/SRC and GSK-3β signalling and β-catenin/TCF-4 transcription (Fig. 7 ). β-catenin forms a cell adhesion complex with E-cadherin, raising the possibility that loss of expression or a change in β-catenin distribution in the cell alters downstream signalling, decreases intercellular adhesion and promotes metastasis. These results suggest that the inhibitory effect of immune serum on PCa cell metastasis may involve suppression of the Wnt/β-catenin signalling pathway. In summary, this study demonstrates the protective role of mRANKL-MT against RANKL-induced PCa in mice. This effect was mediated via induction of a high-titer antibody response, inhibition of EMT and metastatic functions. Our results highlight the potential application of an anti-RANKL vaccine for treatment of metastatic RANKL-induced PCa. Moreover, the results suggest that mutant RANKL may be a potential RANKL vaccine that prevents and/or treats RANKL targeting in patients with metastatic PCa prostate cancer. Abbreviations PCa Prostate cancer; NF-κB:Nuclear factor kappa-light-chain-enhancer of activated B cells; RANKL:receptor activator of NF-kB ligand; EMT:epithelial-mesenchymal transition; TCF-4:Transcription factor 4; TRANCE:tumour necrosis factor-related activation-induced cytokine; OPGL:osteoprotegerin ligand; ODF:osteoclastic differentiation factor; GSK-3:glycogen synthase kinase-3; mRANKL-MT:mouse RANKL mutant; IL-6:Interleukin-6; RPMI:Roswell Park Memorial Institute; FBS:Fetal bovine serum; HRP:Horseradish peroxidase; PCR:Ploymerase chain reaction; IHC:Immunohistochemistry; Declarations Authors’ contributions M. P., Y. C. and W. L. designed and performed the research, analysed and interpreted the data, and wrote the paper; B. K., Y. K., Y. J. and Y. J. analysed and interpreted the in vitro data, and helped to draft some of the figures; W. L. performed and supervised RT-PCR and western blot data analysis; B. K. and Y. K. assisted with the ELISA assays; M. P., Y. C , B. K., Y. C., M. P., Y. K. and Y. J. performed in vivo studies under the supervision of W. L.; M. P. and Y. J. performed histologic analysis under the supervision of H. H.; M. P. and Y. C. performed pathological review of the samples; and W. L. analysed publicly available datasets and supervised the study. Funding This study was supported by research funding from Chosun University (awarded in 2020). Ethics approval and consent to participate Patient study was approved and supervised by Chosun University Hospital’s institutional review board. (Chosun University Hospital, Gwangju, Korea (CHOSUN 2020-06-001)). And animal experimental protocol was approved by the Institutional Animal Care and Use Committee, Chosun University, Gwangju, Korea (CIACUC2019-A0015) and Consent for publication Not applicable. Competing interests The authors declare no financial conflicts of interest. Author details 1 Laboratory of Orthopaedic Research, Chosun University Hospital, Dong-Gu, Gwangju, 61452, Republic of Korea 2 Department of Orthopaedic Surgery, Chosun University Hospital, Dong-Gu, Gwangju, 61452, Republic of Korea 3 Department of Biomedical Sciences Chonnam National University Medical School, Gwangju 61469, Republic of Korea 4 Department of Premedical Science, College of Medicine, Chosun University, Dong-Gu, Gwangju 61452, Republic of Korea References Rawla P: Epidemiology of Prostate Cancer. World J Oncol 2019, 10:63-89. Logothetis CJ, Lin SH. Osteoblasts in prostate cancer metastasis to bone. Nat Rev Cancer. 2005;5:21-28. Jacobs SC: Spread of prostatic cancer to bone. Urology. 1983;21:337-344. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-105442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":4544308,"identity":"1cf9100c-32b9-474a-b602-5ac56baa8879","order_by":0,"name":"Mineon Park","email":"","orcid":"","institution":"Chosun University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mineon","middleName":"","lastName":"Park","suffix":""},{"id":4544309,"identity":"cf0efab2-fc43-4ca4-a3fa-3bb19aeb0f15","order_by":1,"name":"Yong Jin Cho","email":"","orcid":"","institution":"Chosun University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Jin","lastName":"Cho","suffix":""},{"id":4544310,"identity":"e266fcc5-36cb-4b7c-8fc8-eaed527819c3","order_by":2,"name":"Bora Kim","email":"","orcid":"","institution":"Chosun University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bora","middleName":"","lastName":"Kim","suffix":""},{"id":4544311,"identity":"2ea30666-33fb-4d61-adb3-b36b3c854737","order_by":3,"name":"Young Jong Ko","email":"","orcid":"","institution":"Chosun University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Young","middleName":"Jong","lastName":"Ko","suffix":""},{"id":4544312,"identity":"32b1cf4a-1e59-42e7-a624-0a90a0e8fb02","order_by":4,"name":"Yuria Jang","email":"","orcid":"","institution":"Chosun University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuria","middleName":"","lastName":"Jang","suffix":""},{"id":4544313,"identity":"683a3119-23db-4529-97bb-2172ee48523f","order_by":5,"name":"Hoon Hyun","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hoon","middleName":"","lastName":"Hyun","suffix":""},{"id":4544314,"identity":"8b2c0a12-aada-482f-8cfb-23d3c5d4f83d","order_by":6,"name":"Wonbong Lim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYHACNhAhxyCBKkBYizHpWhIbiNZicLz32IOPO2rT+2c3H/7Mu4NBnr+BLe0DXi1nzqUbzjxzPHfGnWNp0rxnGAxnHGA7PAOfFrMbOWbSvG3HcjdI5Jgx87YxMG5gYG/G6zCYlnQDiRzjz0At9sRqqUkAajEAMhgSNzCwHcarxf7MGXPDmW0HDGfcSEuTnNsmkTzjMFsyXi2S7T1mDz621cnzz0g+/OFtm41tf3ubMV4tUAB3CjB2mInRwMBQR5yyUTAKRsEoGJkAAOx7RBQ2BMprAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6337-6599","institution":"Chosun University College of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wonbong","middleName":"","lastName":"Lim","suffix":""}],"badges":[],"createdAt":"2020-11-09 23:31:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-105442/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-105442/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3640917,"identity":"5788c19a-05bf-42e1-a359-1f1b77c89949","added_by":"auto","created_at":"2020-11-17 18:46:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242207,"visible":true,"origin":"","legend":"Histopathology of metastatic prostate cancer tissues. \nRepresentative H\u0026E stained and immunostained images showing expression of RANKL and IL-6 in normal (no histopathological change) or metastasis tissues. Magnification: 200×. Scale = 100 μm.","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/96f72c5fdfb6307c5363a84e.jpg"},{"id":3640918,"identity":"c0c5abea-f845-49a6-a8b1-eec7b4248b37","added_by":"auto","created_at":"2020-11-17 18:46:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267163,"visible":true,"origin":"","legend":"EMT and metastatic properties of PC3 cells following hRANKL treatment.\n(A) Treatment with hRANKL led to a significant increase in the migratory capacity of PC3 cells, as evident from the decrease in the wound gap distance at 48 h. Magnification, 100×; scale bar, 100 μm. The data in the associated graphs are expressed as the mean ± SD. (B) Transwell invasion assays were performed to compare the invasiveness of hRANKL-treated PC3 cells and untreated cells. Treatment with hRANKL led to a significant reduction in the invasiveness of PC3 cells. Magnification, 100×; scale bar, 100 μm. Data in the associated graphs are expressed as the mean ± SD. (C) Expression of EMT and metastasis-related proteins E-cadherin, N-cadherin, β-catenin, MMP-9, IL-6, c-MYC, TCF-4 and β-actin in hRANKL-treated PC3 cells was measured by western blotting. β-actin was used as a loading control. (D) TOP/FOP luciferase reporter assays in PC3Wild and PC3RANKL+ cells. (E) Western blot analysis of MAPK phosphorylation in PC3 cells incubated with 1 g/mL hRANKL for 0, 5, 15 and 30 min. (F) Co-immunoprecipitation for β-catenin and TCF-4 in hRANKL-treated PC3 cells. Results are representative of three separate experiments with comparable results.","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/a1f62c31f0b564c138a73490.jpg"},{"id":3640919,"identity":"ddadcec8-8aea-4fc7-98ce-a5bf3e1add80","added_by":"auto","created_at":"2020-11-17 18:46:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":291945,"visible":true,"origin":"","legend":"Modulation of the EMT and metastatic properties of PC3 cells following overexpression of RANKL.\n(A) A cell migration assay was performed to compare wound healing in PC3Wild and PC3RANKL+ cells. PC3RANKL+ cells showed a significant increase in migratory capacity, as evident from the decreased wound gap at 48 h. Magnification, 100X; scale bar, 100 μm. The data in the associated graphs are expressed as the mean ± SD. (B) Transwell invasion assays were performed to compare the invasiveness of PC3Wild and PC3RANKL+ cells. PC3RANKL+ cells showed a significant increase in invasiveness. Magnification, 100X; scale bar, 100 μm. Data in the associated graphs are expressed as the mean ± SD. (C) Expression of E-cadherin, N-cadherin, β-catenin, MMP-9, IL-6 and β-actin in PC3Wild and PC3RANKL+ cells, as measured by western blotting. β-actin was used as a loading control. (D) TOP/FOP luciferase reporter assays in PC3Wild and PC3RANKL+ cells. Significant differences were observed at *p \u003c 0.05, compared with the control. (E) Immunoprecipitation for β-catenin and TCF-4 in PC3Wild and PC3RANKL+ cells. Each blot was obtained under the same experimental conditions and the data were representative of three separate experiments with comparable results.","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/65684f3139d539c7e71d35a0.jpg"},{"id":3640920,"identity":"69c5233a-f0fe-49fd-ad2d-00def160d865","added_by":"auto","created_at":"2020-11-17 18:46:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304894,"visible":true,"origin":"","legend":"Effects of immunisation with mutant RANKL.\n(A) Comparison of the sequences of hRANKL, mRANKL-WT and mRANKL-MT. (B) In vivo experimental flow. Male BALB-c/nu mice (6-weeks-old) were immunised with a subcutaneous injection of test agent (20 µg/kg). (C) Tumour growth and metastases were monitored via in vivo bioluminescence imaging of the reporter activity induced by PC3Wild, PC3RANKL+ and PC3RANKL+ + IM treatment. Data per representative mouse are shown. A large hot-spot of bioluminescence was observed in vivo following inoculation of mice with PC3RANKL+ cells. Multiple localised and distant metastases were observed in vivo after the injection of PC3RANKL+ cells into the hearts of nude mice. Coloured bars indicate the bioluminescence signal intensity (photon/s/cm2/steradian). (D) The bioluminescence photon flux that appeared after inoculation of cells into mice is shown graphically. Data from related graphs are displayed as the mean ± SD. (E) Survival and (F) metastasis rate in mice from the SHAM, PC3Wild, PC3RANKL+ and PC3RANKL+ + IM groups.","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/1eafcb11f9a8baac2b8c8a67.jpg"},{"id":3640921,"identity":"8644a4fb-280a-4e35-92d5-23a0117b1817","added_by":"auto","created_at":"2020-11-17 18:46:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":322785,"visible":true,"origin":"","legend":"Effects of immunisation with mutant RANKL on PC3-innoculated metastatic model. (A) representative H\u0026E stained and immunostained images of RANKL and IL-6 in the PC3Wild, PC3RANKL+ and PC3RANKL+ + IM groups. Magnification: 200×. Scale bar = 100 μm. (B) Concentration of RANKL in mouse serum. The mean ± SD values were obtained by densitometry, as shown in the analysis. Significant differences were observed at *p \u003c 0.05 and **p \u003c 0.01 vs. control. (C) Serum samples from mice were obtained after immunisation. Anti-RANKL values in PC3Wild, PC3RANKL+ and PC3RANKL+ + IM groups. The mean  SD values were obtained by densitometry, as shown in the analysis. Significant differences were observed at *p \u003c 0.05 and **p \u003c 0.01 vs. the control. (D) Anti-RANKL values and (E) RANKL concentration in the serum of RANKL-immunized mice with metastasis (+) or metastasis (-). Bar graphs show the mean ± standard deviation (SD). Significant differences were observed at *p \u003c 0.05, metastasis (+) vs. no metastasis (-).","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/7fb646f40fb9f9d1f6b9de8f.jpg"},{"id":3640922,"identity":"8ec7688b-2e0e-43cc-aa8c-6d5d60e07637","added_by":"auto","created_at":"2020-11-17 18:46:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":344129,"visible":true,"origin":"","legend":"EMT and metastatic properties of PC3 cells treated with immune sera.\n(A) Cell migration assays were performed to compare the wound healing capacity of immune serum-treated PC3RANKL+ cells and untreated cells. Immune serum treatment decreased the migration capacity of PC3 cells, as evident from the increase in the wound healing gap at 48 h. Magnification, 100; scale bar, 100 μm. Data from related graphs are displayed as the mean ± SD. (B) Transwell invasion assays were performed to compare the invasiveness of immunised serum-treated PC3RANKL+ and untreated cells. Serum-treated PC3RANKL+ cells showed a significant decrease in invasiveness. Magnification, 100; scale bar, 100 μm. Data in the associated graphs are expressed as the mean ± SD. (C) Expression of E-cadherin, N-cadherin, β-catenin, MMP-9, IL-6, c-MYC, TCF-4 and β-actin in immune serum-treated PC3RANKL+ cells, as measured by western blotting. β-actin was used as a loading control. Similar results were obtained in three independent experiments. (D) TOP/FOP luciferase reporter assays in immune serum-treated PC3RANKL+ cells. Significant differences were observed at *p \u003c 0.05 vs. the control. (E) Co-immunoprecipitation of β-catenin and TCF-4 from PC3RANKL+ and immune serum-treated PC3RANKL+ cells. Each blot was obtained under the same experimental conditions. (F) Western blot analysis of MAPK phosphorylation levels in serum-treated PC3RANKL+ cells. Similar results were obtained in three independent experiments.","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/79f637dfd14569461dd93f31.jpg"},{"id":3640923,"identity":"d4d7465a-0306-4fa7-a7c5-d31e8e6c1de0","added_by":"auto","created_at":"2020-11-17 18:46:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":208230,"visible":true,"origin":"","legend":"Schematic diagram illustrating anti-RANKL-mediated inhibition of RANK signaling. Activation of RANK by RANKL activates AKT and ERK, thereby liberating the active form of β-catenin. Generation of anti-RANKL antibodies by mRANKL-MT inactivates RANKL, thereby downregulating AKT/ERK activity and preventing downstream of β-catenin.","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/e75420d7e4cf6f18dd3a94f2.jpg"},{"id":13615587,"identity":"a9741b5b-06b9-421f-b063-d0499d0485f7","added_by":"auto","created_at":"2021-09-17 06:46:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1372869,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/4b39bdba-1aa2-4135-b04c-43604e763a7c.pdf"},{"id":3640924,"identity":"47ac0461-f8b5-4ad9-9391-17cd7ce8295c","added_by":"auto","created_at":"2020-11-17 18:46:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15420,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryLegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/2eecab076826943385b30ed7.docx"},{"id":3640925,"identity":"602f6d2b-6591-465b-a9ae-e7d83532133f","added_by":"auto","created_at":"2020-11-17 18:46:58","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":184876,"visible":true,"origin":"","legend":"","description":"","filename":"supple1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/c816498cf2f6eea1618efe7e.jpg"},{"id":3640926,"identity":"77659110-61d4-4124-ae29-cf22bf799312","added_by":"auto","created_at":"2020-11-17 18:46:58","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":256512,"visible":true,"origin":"","legend":"","description":"","filename":"supple2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/d2ad9d35fb87406658ffcbc0.jpg"},{"id":3640927,"identity":"7093f51d-76ec-4a6c-9ca5-66b25fbbaccc","added_by":"auto","created_at":"2020-11-17 18:46:58","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":56701,"visible":true,"origin":"","legend":"","description":"","filename":"supple3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/45af3eb60f2814769b06f962.jpg"},{"id":3640928,"identity":"37d4bbc7-b04a-4a02-ac92-265fbef8bc50","added_by":"auto","created_at":"2020-11-17 18:46:59","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":152462,"visible":true,"origin":"","legend":"","description":"","filename":"supple4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105442/v1/aba0eea1087f5f78d2e61eb4.jpg"}],"financialInterests":"","formattedTitle":"\u003cp\u003eRANKL Immunisation Inhibits Prostate Cancer Metastasis by Modulating EMT Through A RANKL-Dependent Pathway\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eProstate cancer (PCa) is the second most common cancer in males and the fifth leading cause of death worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Metastasis of PCa cells to the skeleton occurs in a predictable manner, with lesions tending to appear first in the axial skeleton, followed by appendicular tissues [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Considering the effects of PCa on both haematopoiesis and bone structure, bone metastasis is a major cause of morbidity in patients with advanced disease. Replacement of haematopoietic tissue by metastatic PCa cells is associated with anaemia and increased morbidity (mortality). The 5-year survival of most patients with PCa is almost 100%; however, that of PCa patients with metastasis to distant sites is as low as 28%. Thus, PCa is one of the deadliest cancers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Experimental and clinical observations reveal that treatment with anti-resorptive agents suppresses or even prevents PCa metastasis [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and that accelerated bone turnover stimulates progression of skeletal secondary tumours [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring bone metastasis of PCa, cancer cell-derived cytokines stimulate expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which in turn activates bone resorption. RANKL, also known as tumour necrosis factor-related activation-induced cytokine (TRANCE) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], osteoprotegerin ligand (OPGL) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and osteoclastic differentiation factor (ODF) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], interacts with RANK and is involved in all the steps related to tumour development, from initial tumour formation to migration of cancer cells and subsequent metastasis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. RANKL is expressed in several tissues, including brain, skin, intestine, skeletal muscle, kidney, liver, lung and mammary tissue; however, expression is very high in bone [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], lymphoid organs and the vascular system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. RANKL binds to RANK on the surface of pre-osteoclasts, activating them and inducing formation of osteoclasts [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies report expression of RANK and RANKL by various solid tumours, including breast cancer. RANKL accelerates migration and metastasis of cancer cells expressing RANK [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, it protects breast cancer cells from apoptosis in response to DNA damage and controls self-renewal and anchorage-independent growth of tumour-initiating cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, it is unclear how RANKL signalling triggers metastasis of PCa. Epithelial-to-mesenchymal transition (EMT), a rapid and often reversible phenotypic change in epithelial cells, is an important phenomenon underlying cancer metastasis. Originally, EMT was described in the context of developmental processes such as heart morphogenesis and mesoderm and neural crest formation. Epithelial cells lose structures involved in cell\u0026ndash;cell adhesion (e.g., adherens junctions and desmosomes), modulate their polarity and rearrange their cytoskeleton, which is consistent with the typical switch of intermediate filaments from cytokeratins to vimentin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A recent report shows that the oncogenic c-MYC, Wnt signalling and β-catenin pathways activate the Snail/glycogen synthase kinase-3 (GSK-3) axis and induce EMT [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe concept of exploiting the host\u0026rsquo;s immune system to treat cancer relies on the ability of immune cells to eliminate malignant cells at the early transformation stage in a process called immune surveillance [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Passive anti-cytokine immunotherapy with specific high-affinity antibodies has been tested in animal models and clinical trials of rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma, Crohn\u0026rsquo;s disease, psoriasis and other articular autoimmune disorders. This strategy facilitates production of anti-auto-cytokine antibodies by the immune system in response to active vaccination. However, development of a desired antibody response to self-proteins necessitates suppression of immune resistance. Common anti-cytokine vaccines are prepared from autologous proteins that are converted to derivatives that lack biological activity following treatment with glutaraldehyde or formaldehyde [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A previous study developed a mouse RANKL mutant (mRANKL-MT) protein and confirmed its ability to inhibit osteoporosis.\u003c/p\u003e \u003cp\u003eHere, we used mRANKL-MT as an immunogen for RANKL-targeting immunotherapy of bone disease and investigated its potential as a cancer vaccine in a mouse model of metastatic cancer. We confirmed the activity of mRANKL-MT and clarified its effects on RANK/RANKL signalling-mediated EMT in transient RANKL-overexpressing cell lines and animal models. We found that mRANKL-MT suppressed RANKL-dependent β-catenin signalling. We also found that mRANKL-MT immunotherapy altered the characteristics of cancer cells and effectively suppressed RANKL-dependant cancer metastasis.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree patient tissue samples from PCa patients not receiving chemotherapy, hormone therapy, or radiotherapy were obtained before surgery from Chosun University Hospital during 01 June 2020 and 31 July 2020 (Chosun University Hospital (CHOSUN 2020-06-001)). Tumours and normal tissues were harvested and immediately fixed in a formalin solution (neutral buffered, 10%; Sigma-Aldrich) for 24 h. Tissue specimens were successively dehydrated in ethanol and treated with xylene. Paraffin embedded tissues were sliced into 5 \u0026mu;m-thick sections, deparaffinised with xylene and rehydrated through graded alcohol solutions. The tissues were stained with H\u0026amp;E for histological analysis. Immuno-histochemical studies were performed using the Novolink\u003csup\u003eTM\u003c/sup\u003e Polymer Detection System kit (Leica Biosystems) as per the manufacturer\u0026rsquo;s instructions. Antibodies specific for IL-6 (1:200; Cell Signaling Technology) and RANKL (1:200, Cell Signaling Technology) were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman prostate adenocarcinoma luciferase-labelled PC3\u003csup\u003eluc\u003c/sup\u003e cells were obtained from Professor Park and maintained at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e in Rowell Park Memorial Institute (RPMI) 1640 medium (Welgene, Korea) supplemented with 10% heat-inactivated foetal bovine serum (certified, GIBCO, USA) and a 10% antibiotic solution (Welgene).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning of hRANKL \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA used to clone hRANKL cDNA was extracted from MG63 cells (ATCC\u003csup\u003e\u0026reg;\u003c/sup\u003e CRL-1427\u003csup\u003e\u0026trade;\u003c/sup\u003e) expressing RANKL. The quality of the extracted RNA was verified by agarose gel electrophoresis. The cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer\u0026rsquo;s instructions. Amplification and cloning of the hRANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM magnesium chloride (MgCl\u003csub\u003e2\u003c/sub\u003e), 10 \u0026mu;M primers (hRANKL-\u003cem\u003eBcl\u003c/em\u003eI: 5'-TGATCAAAGCTTGAAGCTCAGCCTTTTGC-3' and hRANKL\u003cstrong\u003e-\u003c/strong\u003e\u003cem\u003eXho\u003c/em\u003eI: 5'-CTCGAGATCTATATCTCGAACTTTAAAAGCCCC-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 \u0026mu;L of the RANKL gene construct (template). The thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 5 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 30 s and extension at 70\u0026deg;C for 30 s. The polymerase chain reaction (PCR) product was cloned into the \u003cem\u003eBam\u003c/em\u003eH1/\u003cem\u003eXho\u003c/em\u003eI sites of a pMX vector (CELL BIOLABS, USA). Sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRetroviral hRANKL transduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlat-E cells were seeded at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in a six-well plate for 24 h and then transiently transfected with hRANKL/pMX using 0.2 \u0026mu;g plasmid and 0.6 \u0026mu;L of the FuGENE HD transfection reagent (Promega, Madison, WI, USA), according to the manufacturer\u0026rsquo;s protocol. After incubation, the DNA/FuGENE mixture was added drop-wise onto Plat-E cells. Viral supernatants were recovered from the culture medium at 48\u0026thinsp;h after transfection. Virus-containing supernatants were filtered through 0.45 \u0026mu;m non-pyrogenic filters and supplemented with 10 \u0026mu;g/mL polybrene (Sigma-Aldrich).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse-transcription quantitative PCR\u003c/strong\u003e\u003cstrong\u003e (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from PCa cells using Trizol (Invitrogen) and 1 \u0026mu;g was used for RT-qPCR along with oligo-dT primers (10 \u0026mu;g) and dNTPs (10 mM). Next, qRT-PCR was performed to analyse cDNA using SYBR Green SuperMix (BIORAD, USA) on a CFX Connect Real-Time System (BIORAD, USA). All target gene primers were purchased from Bioneer Co. (Daejeon, Korea) and the cDNA was amplified using the following primer sets:\u003c/p\u003e\n\u003cp\u003eE-cadherin (h): 5'-TGGAGGAATTCTTGCTTTGC-3' (forward) and 5'-TGGAGGAATTCTTTTGC-3' (reverse); vimentin (h): 5'-GACGCCATCAACACCGAGTT-3' (forward) and 5'-GACGCCATC AACACCGAGTT-3' (reverse); \u0026beta;-catenin (h): 5'-ACAAACTGTTTTGAAAATCCA-3' (forward) and 5'-CGAGTCATTGCATACTGTCC-3'(reverse); MMP-9 (h): 5'-TCCAGTACCAAGACAAAG-3' (forward) and 5'-TTGCACTGCACGGTTGAA-3' (reverse); RANK (h): 5'-CAAATGCAGACCCTGGA CCA-3' (forward) and 5'-AAACGCCAAAGATGATGGCA-3' (reverse); RANKL (h), 5'-CCTGTAT GCCAACATTTGCTTTC-3' (forward) and 5'-TTCCTCTCCAGACCGTAACTTAAA-3' (reverse); IL -6 (h): 5'-AGCAAAGAGGCACTGGCAGA-3' (forward) and 5'-GTACTCATCTGCACAGCTCTGG C-3' (reverse); TCF-4 (h): 5'-GCTCAGGGTATGGAACCGGC-3' (forward) and 5'-CCCTGTAGTC CTGGTGGCATG-3' (reverse); c-MYC (h): 5'-CCTGGTGCTCCATGAGGAGAC-3' (forward) and 5'-AGACTCTGACCTTTTGCCAGG-3' (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5'-TCAAGAAGGTGGTGAAGCAG-3' (forward) and 5'-AGTGGGAGTTGCTGTTGAAG T-3' (reverse). Values on the vertical axis represent 2(\u0026minus;\u0026Delta;Ct); \u0026Delta;Ct is the discrepancy between the target gene Ct and GAPDH Ct.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were washed twice with phosphate-buffered saline (PBS; pH 7.4) and total proteins were extracted using radio immunoprecipitation assay buffer supplemented with 1% protease inhibitors, phosphatase inhibitors and phenylmethylsulfonyl fluoride (PMSF). The protein concentration was measured using the BCA Protein Assay Kit (Thermo Pierce\u003csup\u003eTM\u003c/sup\u003e). The membrane was blocked with a solution containing 5% skim milk in TBS-T for 30 min and then washed in TBS-T. The membrane was incubated for overnight at 4\u0026deg;C with the following primary antibodies: E-cadherin (sc-7870, Santa Cruz Biotechnology), N-cadherin (ab76011, Abcam), \u0026beta;-catenin (#29822 94, Millipore), MMP-9 (#13667, Cell Signaling), IL-6 (#12153, Cell Signaling Technology), c-MYC (9E10, Santa Cruz Biotechnology), TCF-4 (#2565, Cell Signaling Technology), RANK (#4845, Cell Signaling Technology), P-ERK (#9101, Cell Signaling Technology), ERK (#9102, Cell Signaling Technology), GAPDH (#2118, Cell Signaling Technology), P-AKT (#9271, Cell Signaling Technology), AKT (#9272, Cell Signaling Technology), P-SRC (#2105, Cell Signaling Technology), SRC (#2108, Cell Signaling), GSK-3B (#9315, Cell Signaling Technology) and P-GSK-3B (#9336, Cell Signaling Technology). Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H + L) and HRP-conjugated AffiniPure goat anti-mouse IgG (H + L) were obtained from Proteintech Group, Inc (Jackson) and used as secondary antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 6-well plates for the cell migration assay. After each treatment, a confluent monolayer was wounded using a 200 \u0026micro;L pipette tip. Images of wound closure were obtained under an inverted microscope after 48 h. The wound area was calculated using NIH ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell invasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e transfected cells were seeded into the top chamber of a 24-well polycarbonate Transwell chamber (8.0 \u0026micro;m pore size; Corning Incorporated, Glendale, AZ, USA) and then treated for 24 h with hRANKL or RANKL. The number of trypan blue-stained cells in five random fields was counted using an inverted microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase reporter assay to assess Wnt/\u0026beta;\u003c/strong\u003e\u003cstrong\u003e-catenin activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded into a 24-well plate 24 h prior to transient transfection with either 2 \u0026micro;g of TOPflash or FOPflash reporter plasmid along with 1 \u0026micro;g DNA using Lipofectamine 3000 (Thermo Fisher). The TOPflash luciferase reporter plasmid contains TCF-4-binding sites upstream of the luciferase gene, resulting in luciferase activity in the presence of active Wnt/\u0026beta;-catenin signalling. The FOPflash reporter plasmid, on the other hand, carried mutated TCF-4-binding sites. Total cell extracts were assayed for luciferase activity according to the manufacturer\u0026rsquo;s instructions (Promega).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in lysis buffer (20 mM Tris-HCl pH 7.6\u0026ndash;8.0, 100 mM sodium chloride NaCl, 300 mM sucrose, 3 mM MgCl\u003csub\u003e2\u003c/sub\u003e [buffer A]; and 20 mM Tris pH 8.0, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid [buffer B]). Whole cell lysates obtained by centrifugation were incubated with antibodies specific for active \u0026beta;-catenin (Millipore) and TCF-4 (Cell Signaling Technology) (dilution 1:100) and protein A Sepharose beads (Amersham Biosciences) for 2 h at room temperature. The immune complexes were washed three times using wash buffer and examined by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSite-directed mutagenesis and production and purification of \u003c/strong\u003emRANKL-MT\u003c/p\u003e\n\u003cp\u003eThe RNA used for the cloning of RANKL cDNA was extracted from MC3T3-E1 cells (Korean Cell Line Bank, Seoul, Korea) expressing RANKL. The quality of the extracted RNA was verified by agarose gel electrophoresis and cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer\u0026rsquo;s instructions. Amplification and cloning of the RANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026mu;M primers (mRANKL-\u003cem\u003eNde\u003c/em\u003eI: 5'-CATATGAAGCCTGAGGCCCAGCC ATTTGC-3'; mRANKL-\u003cem\u003eXho\u003c/em\u003eI: 5'-CTCGAGGTCTATGTCCTGAACTTTGAAAGCC-3'; mRANKL (K180R)-F: 5'-CCCATCGGGTTCCCATCGAGTCACTCTGTCCTCTTG-3'; mRANKL (K180R)-R: 5'-CAAGAGGACAGAGTGACTCGATGGGAACCCGATGGG-3'; mRANKL (D189I, R190K)-F: 5'-CTCTTGGTACCACATCAAGGGCTGGGCCAAGAT-3'; mRANKL (D189I, R190K)-R: 5'-ATC TTGGCCCAGCCCTTGATGTGGTACCAAGAG-3'; mRANKL-MT (H223F, H224Y)-F: 5'-AA CA TTTGCTTTCGGTTTTATGAAACATCGGGAAGCG-3'; or mRANKL-MT (H223F, H224Y)-R: 5'-CGCTTCCCGATGTTTCATAAAACCGAAAGCAAATGTT-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 \u0026mu;L of RANKL gene construct as the template.\u003c/p\u003e\n\u003cp\u003eThe thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 5 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 30 s and extension at 70\u0026deg;C for 30 s. The PCR product obtained was cloned into the \u003cem\u003eNde\u003c/em\u003eI/\u003cem\u003eXho\u003c/em\u003eI site of the GST-30a vector (Novagen, Madison, WI, USA). Mutations at positions 180, 189\u0026ndash;190 and 223\u0026ndash;224 were introduced using megaprimers [26]. The PCR product was transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21-CodonPlus (DE3)-RIPL (Novagen) by electroporation (5 ms, 12.5 kV/cm) and the transformed cells were cultivated in Luria-Bertani broth containing kanamycin (50 \u0026mu;g/mL, T\u0026amp;I, Daejeon, Korea). Plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA). The cloned product was confirmed by sequencing. All sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA). The recombinant plasmid carrying mRANKL-MT was expressed from a single \u003cem\u003eE. coli\u003c/em\u003e BL21-CodonPlus (DE3)-RIPL colony using previously described methods [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of mRANKL-MT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e cells expressing mRANKL-MT were cultivated in 1 L of an auto-induction medium supplemented with kanamycin (50 \u0026mu;g/mL), as previously described. After centrifugation at 6000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 20 min at 4\u0026deg;C, the pelleted cells were resuspended in 10 mL of lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4) supplemented with 0.1 mg/mL lysozyme and 0.1 mM PMSF.\u003c/p\u003e\n\u003cp\u003eGlycerol (20% v/v; CARLO ERBA, France) was added to the cell suspension and the cells were sonicated and centrifuged at 15,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. The supernatants were passed through 0.2 \u0026mu;m paper filters and applied to Ni\u003csup\u003e2+\u003c/sup\u003e-affinity chromatography HisTrap FF columns (1 mL; GE Healthcare Life Science, Piscataway, NJ, USA) equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 5 mM dithiothreitol, pH 7.4). The columns were subsequently washed using binding buffer supplemented with 20 mM imidazole.\u003c/p\u003e\n\u003cp\u003eAfter washing, bound protein was eluted using elution buffer (Qiagen). The eluted protein was dialysed against a dialysis buffer (20% v/v glycerol in PBS) in a 10,000 MW Slide-A-Lyzer Dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). The purified protein was vacuum concentrated (Savant Instruments, Holbrook, NY, USA) and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were calculated using the Bradford assay. For endotoxin removal, an additional washing step was introduced after the initial wash for chromatography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experimental protocol was approved by the Institutional Animal Care and Use Committee, Chosun University, Gwangju, Korea (CIACUC2019-A0015). All experiments were performed in accordance with relevant guidelines and regulations. Five-week-old male athymic nude mice (BALB-c/nu, Orient Bio Co. LTD, Seoul, Korea) were used to generate a xenograft model by intracardiac injection of PC3\u003csup\u003eWild\u003c/sup\u003e, PC3\u003csup\u003e+RANKL \u003c/sup\u003e(RANKL overexpression), or PC3\u003csup\u003e+RANKL \u003c/sup\u003e+ IM (immunisation) cells. Following immunisation, mice were divided into an immunisation group and a non-immunisation group. The Sham group was immunised by a subcutaneous injection of PBS, while the immunisation group was injected subcutaneously with mRANKL-MT (100 \u0026micro;g/kg three times every 2 weeks). Mouse sera and tissue samples were collected according to indicated schedule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e bioluminescence measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumour-bearing tissues were subjected to \u003cem\u003ein vivo\u003c/em\u003e bioluminescence imaging using a Living Image\u0026reg; 4.5.4 IVIS Imaging System (Perkin Elmer). For luciferase imaging, D-luciferin (Promega) was injected intraperitoneally before imaging. Quantitative detection of luciferase was performed as follows: regions of interest (ROIs) were drawn to capture detected fluorescence, and auto-regions ROIs were used to precisely outline the target region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative analysis of RANKL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of RANKL in mouse serum was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R\u0026amp;D Systems, USA) according to the manufacturer\u0026rsquo;s protocol. Absorbance was measured in a colorimetric microplate reader (BioTek, USA) at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of anti-RANKL antibody titers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum samples obtained from immunised mice were serially diluted with PBS containing 0.02% sodium azide and 2% bovine serum albumin (BSA), and then applied to ELISA plates (Sigma-Aldrich) coated with mouse recombinant tumour necrosis factor ligand superfamily member 11 (TNFSF11; 10 \u0026mu;g/mL, R\u0026amp;D Systems). Reactivity of serum antibodies to the target protein was determined using an HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific) at a dilution of 1/1000 in PBS/0.02% sodium azide/2% BSA. After development with 1,2- phenylenediamine dihydrochloride (0.4 mg/mL in 0.066 M disodium phosphate, 0.035 M citric acid and 0.01% hydrogen peroxide), absorbance was measured in an ELISA plate reader at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean \u0026plusmn; standard deviation (SD) from three independent experiments. GraphPad Prism version 6.0 software for windows was used to analyse \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo \u003c/em\u003edata\u003cem\u003e. \u003c/em\u003eStatistical significance for pairwise comparison was evaluated using an unpaired \u003cem\u003et\u003c/em\u003e-test or one-way analysis of variance (ANOVA) with Turkey\u0026rsquo;s post-hoc test. Results were considered significant at *p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":" \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eExpression of RANKL in human PCa metastasis tissue specimens\u003c/h2\u003e \u003cp\u003eTo investigate the association between RANKL and PCa metastasis in human patients, we examined expression of RANKL and metastasis markers interleukin-6 (IL-6) in human PCa metastatic tissue specimens. Immunohistochemical analysis of tissue specimens from patients with PCa metastasis harboured irregularly shaped and dysplastic cells, whereas normal tissues did not. In addition, RANKL expression was elevated, along with that of IL-6, relative to that in normal tissue. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, RANKL expression was closely related to PCa metastasis, suggestive of an important role in this process.\u003c/p\u003e\u003cp\u003e \u003cb\u003eEffects of human RANKL (hRANKL) on EMT and metastasis of PC3 cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the relationship between RANKL and EMT of PC3 cells, we performed cell migration and invasion assays with PC3 cells treated with hRANKL.\u003c/p\u003e \u003cp\u003eIn the invasion assay, wound healing in hRANKL-treated PC3 cells was significantly better than that in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cell invasion also increased significantly following hRANKL treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eEMT is closely related to tumour metastasis and progression. Therefore, to determine changes at the molecular level, we measured EMT markers in PC3 cells at the mRNA (Supp.\u0026nbsp;1) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) levels following treatment with hRANKL. Expression of the EMT marker E-cadherin in hRANKL-treated PC3 cells fell significantly, but that of vimentin and β-catenin increased. Expression of MMP-9 and IL-6 (markers of metastasis) were significantly higher in hRANKL-treated cells than in control cells. Expression of E-cadherin protein was significantly lower in hRANKL-treated PC3 cells than in control cells. By contrast, expression of vimentin, β-catenin and MMP-9 proteins was significantly upregulated following treatment with hRANKL.\u003c/p\u003e \u003cp\u003eThe Wnt/β-catenin pathway regulates PCa metastasis and plays an important role in cancer progression; therefore, we examined the effects of RANKL on Wnt signalling using a TOP/FOP reporter assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). No changes in TOP/FOP reporter luciferase activity were observed in hRANKL-treated PC3 cells.\u003c/p\u003e \u003cp\u003eNext, we examined phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), SRC and GSK-3B to investigate the effects of RANKL on mitogen-activated protein kinase (MAPK) and Wnt signalling in PC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). PC3 cells treated with hRANKL showed a significant and time-dependent reduction in the level of phosphorylated GSK-3B. A time-dependent increase in SRC and AKT phosphorylation levels was also observed.\u003c/p\u003e \u003cp\u003eNext, we performed co-immunoprecipitation of β-catenin and TCF-4 to investigate the status of TCF-associated signalling in hRANKL-treated PC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The results showed a slight increase in TCF-4 levels in hRANKL-treated PC3 cells. Thus, RANKL treatment may trigger metastasis of PC3 cells by suppressing GSK-3B phosphorylation and facilitating EMT.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of RANKL modulates EMT and metastasis of PC3 cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether RANKL overexpression stimulates PC3 cell growth \u003cem\u003ein vitro\u003c/em\u003e, cells were transiently transfected with an overexpression plasmid containing RANKL. GFP expression by PC3\u003csup\u003eRANKL\u003c/sup\u003e cells was monitored by fluorescence microscopy (Supp.\u0026nbsp;2A). We found a significant increase in expression of RANKL mRNA and protein (Supple 2B and 2C).\u003c/p\u003e \u003cp\u003eNext, we performed migration and invasion assays using PC3\u003csup\u003eRANKL\u003c/sup\u003e cells to evaluate the effects of RANKL overexpression on EMT and metastasis. Wound healing was significantly better in PC3\u003csup\u003eRANKL\u003c/sup\u003e cells than in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Also, PC3\u003csup\u003eRANKL\u003c/sup\u003e cells were significantly more invasive than control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Analysis of mRNA encoding EMT- and metastasis-related factors in PC3\u003csup\u003eRANKL\u003c/sup\u003e cells revealed significant downregulation of the gene encoding E-cadherin (Supp.\u0026nbsp;2D). Furthermore, expression of genes encoding vimentin, MMP-9, IL-6 and β-catenin was upregulated significantly in PC3\u003csup\u003eRANKL\u003c/sup\u003e cells. Protein expression analysis revealed that PC3\u003csup\u003eRANKL\u003c/sup\u003e cells expressed significantly lower levels of E-cadherin than PC3\u003csup\u003eWild\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). By contrast, expression of N-cadherin, MMP-9, IL-6, c-MYC and β-catenin was significantly higher in PC3\u003csup\u003eRANKL\u003c/sup\u003e cells.\u003c/p\u003e\u003cp\u003ePC3\u003csup\u003eRANKL\u003c/sup\u003e cells showed a significant increase in TOP/FOP luciferase reporter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Immunoprecipitation of β-catenin was carried out to investigate the signal transduction pathway associated with TCF in PC3\u003csup\u003eRANKL\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). PC3\u003csup\u003eRANKL\u003c/sup\u003e cells overexpressing RANKL showed a significant increase in activation of the MAPK and β-catenin/TCF-4 signalling pathways owing to stronger binding between β-catenin and TCF-4 than in PC3\u003csup\u003eWild\u003c/sup\u003e cells. Thus, ectopic overexpression of RANKL may increase EMT and the metastatic properties of PC3 cells via the β-catenin/TCF-4 signalling pathway, suggesting the therapeutic potential of RANKL targeting for prevention of PCa metastasis.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eTherapeutic Effects Of Mrankl-mt In Pc3 Cell-inoculated Mice\u003c/h2\u003e\n \u003cp\u003eAlignment of the mRNA sequence of mRANKL-MT with that of mRANKL-WT identified a region that could be amplified using selected primers. The recombinant mRANKL-WT sequence encoded the full-length 158 amino acid target region, which includes residues 158 to 316 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To create point mutations, Lys180, Asp189-Arg190 and His223-His224 were transformed to Arg180, Ile189-Lys190 and Phe223-Tyr224, respectively. The resulting hRANKL, mRANKL-WT and mRANKL-MT molecules had similar molecular weights (Supp.\u0026nbsp;3). Male BALB-c/nu mice were injected subcutaneously with hRANKL, mRANKL-WT, or mRANKL-MT (100 \u0026micro;g/kg; three times every 2 weeks). After immunisation, 1 \u0026sdot; 10\u003csup\u003e6\u003c/sup\u003e PC3\u003csup\u003eWild\u003c/sup\u003e or PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells were injected into the left ventricle of Sham or immunised mice. Serum and tumour-bearing tissues were collected after 16 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo observe bone metastasis, luciferase activitiy in tumour-bearing tissues of Sham, PC3\u003csup\u003eWild\u003c/sup\u003e, PC3\u003csup\u003eRANKL+\u003c/sup\u003e and PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM mice was detected by IVIS. In PC3\u003csup\u003eRANKL+\u003c/sup\u003e mice, large and strong bioluminescence spots were detected throughout the body at 16 weeks post-cancer cell injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, no bioluminescence signals were detected in PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM mice. The photon flux values were significantly higher in PC3\u003csup\u003eRANKL+\u003c/sup\u003e mice than in PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eSurvival rate analysis revealed a significant decrease in the survival of animals in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e groups compared with that of animals from the PC3\u003csup\u003eWild\u003c/sup\u003e group. The survival rate improved higher in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM group than that in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The metastasis rate in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e group was higher than that in the PC3\u003csup\u003eWild\u003c/sup\u003e group; however, that in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM group was significantly less than that in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \n\u003ch2\u003eTherapeutic Effects Of Anti-rankl Antibodies Induced By Rankl Immunisation\u003c/h2\u003e\n \u003cp\u003eTo examine the histological characteristics of metastatic tumour-bearing tissues, metastatic lesions from each mouse were stained with haematoxylin and eosin Y (H\u0026amp;E). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, gross examination of the excised tibiae from PC3\u003csup\u003eRANKL+\u003c/sup\u003e mice revealed a tumour mass in the primary spongiosum (trabecular epiphysis) and bone marrow cells; this was not observed in PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM mice. In particular, expressions of IL-6 and RANKL increased markedly in the trabecular epiphysis region of bones from PC3\u003csup\u003eRANKL+\u003c/sup\u003e mice, but were undetectable in PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM mice.\u003c/p\u003e\u003cp\u003eNext, we investigated whether mRANKL-MT induces production of anti-RANKL antibodies. The concentration of RANKL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) was highest in PC3\u003csup\u003eRANKL+\u003c/sup\u003e, and production of antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) was highest in PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM mice. Also, we measured anti-RANKL antibody levels in the PC3\u003csup\u003eRANKL +\u003c/sup\u003e + IM group with bone metastasis to investigate generation of anti-RANKL antibodies after immunisation with mRANKL-MT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The anti-RANKL titer in mice with bone metastasis was significantly higher than that in mice without bone metastasis. Also, serum RANKL levels in the PC3\u003csup\u003eRANKL+\u003c/sup\u003e + IM group without metastasis were significantly lower than those in mice with metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These observations suggest that anti-RANKL antibodies generated by RANKL immunisation suppress metastasis of PCa cells.\u003c/p\u003e \n\u003ch2\u003eEffect Of Immunisation On Emt And Metastasis\u003c/h2\u003e\n \u003cp\u003eTo investigate the effects of mRANKL-MT on PCa metastasis, sera obtained from immunised mice were used to treat RANKL-overexpressing PC3 cells. The results of cell migration assays showed that wound healing was inhibited significantly in PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells treated with immune serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, the invasive ability of PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells declined following treatment with immune sera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAnalysis of mRNA encoding EMT- and metastasis-related factors in PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells treated with immune sera revealed significant upregulation of E-cadherin expression (Supp.\u0026nbsp;4). By contrast, expression of vimentin, MMP-9, IL-6 and β-catenin was downregulated significantly in immune serum-treated PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells. Protein expression analysis showed that E-cadherin expression was higher in immune serum-treated PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells than in control serum-treated PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). By contrast, expression of N-cadherin, MMP-9, IL-6 and β-catenin was significantly lower in immune serum-treated PC3​​\u003csup\u003eRANKL+\u003c/sup\u003e cells, as was expression of c-MYC.\u003c/p\u003e \u003cp\u003eThe luciferase activity of the TOP/FOP reporter in immunised serum-treated PC3\u003csup\u003eRANKL+\u003c/sup\u003ecells fell significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Immunoprecipitation analysis revealed a significant decrease in binding between β-catenin and TCF-4 in cells treated with immune serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Finally, we measured expression of phosphorylated ERK, AKT, SRC and GSK-3B to investigate the effect of immune serum on MAPK and Wnt signalling in PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Cells treated with immune serum showed a significant and time-dependent increase in GSK-3B phosphorylation and a time-dependent decrease in SRC phosphorylation. These results indicate that the EMT and metastatic properties of PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells were inhibited by treatment with immune serum.\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003ePCa, which is common among men in the western world, is associated with high mortality and morbidity with respect to advanced metastasis to the bone. Evidence suggests that the RANKL signalling cascade plays a key role in proliferation, metastasis, migration and invasion of PCa [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The RANKL\u0026ndash;RANK interaction plays a pivotal role in PCa metastasis; indeed, RANKL expression induces osteoclast hyperplasia and bone destruction during PCa metastasis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. RANKL activates RANK directly on tumour cells, as evidenced by dysregulation of several biochemical signalling pathways in PCa cells.\u003c/p\u003e \u003cp\u003eHigh expression of RANKL facilitates PCa metastasis, an idea consistent with previous studies showing that signalling through the RANK/RANKL axis is related to bone metastases of solid tumours [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Here, we demonstrated RANKL immunoreactivity in bone metastatic lesions of PCa patients. We also found that hRANKL-treated or hRANKL-overexpressing PCa cells showed a significant increase in expression of metastasis markers such as IL-6. In particular, \u003cem\u003ein vitro\u003c/em\u003e experiments show that RANKL stimulation markedly increases the migration and invasion of PC3 cells, downregulates expression of the epithelial marker E-cadherin and upregulates the mesenchymal marker vimentin. EMT correlates with tumour metastasis and progression, which is consistent with impaired cell\u0026ndash;cell adhesion following the loss of E-cadherin expression [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, we show that GSK-3B phosphorylation was reduced significantly following RANKL treatment of PC3 cells due to the effect of RANKL on MAPK and Wnt signalling. Also, we observed altered expression of β-catenin and TCF-4 in PC3\u003csup\u003eRANKL+\u003c/sup\u003e cells, which resulted in a highly conserved developmental signalling pathway that includes the major effector protein β-catenin. Wnt signalling is an essential pathway involved in cell development, proliferation and differentiation; indeed, regulatory abnormalities in Wnt signalling are associated with metastasis of many cancers [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In particular, RANKL overexpression in PC3 cells led to a significant increase in expression of Wnt3a, suggesting that RANKL is a potential target of Wnt signalling in cancer cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. RANKL plays a fundamental role in osteoclastogenesis by interacting with the RANK receptor on osteoclast progenitors during bone destruction by metastatic breast cancer, thereby driving osteoclast cell lineage commitment, monocyte cell fusion and osteoclast maturation via regulation of NF-κB-mediated gene expression; therefore, we were intrigued to find out whether catabolic Wnt signalling mechanisms exist alongside anabolic Wnt pathways to regulate osteoclast formation in bone. β-catenin, the critical effector of the Wnt pathway, regulates a number of key processes during development, including proliferation, differentiation and cell fate determination [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Normally, β-catenin is localised to the cell adhesion junctions in epithelial cells and its abnormal cytoplasmic/nuclear stabilisation drives uncontrolled transcription of target genes (including c-jun, cyclin D1, c-myc, survivin and MMP-7) that regulate cell proliferation, survival and adhesion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In view of cancer cell fate, it is not surprisingly that overexpression of RANKL by PC3 cells led to increased binding of β-catenin to TCF4 and to increased TOP activity. Regulation of β-catenin is linked to the pathogenesis of a number of human cancers, particularly those with an epithelial cell origin. Supporting its putative role as a Wnt signalling target, we confirmed that RANKL overexpression led to transcriptional activation of β-catenin in PC3 cells.\u003c/p\u003e \u003cp\u003eOver the past decades, it has become clear that the RANK/RANKL axis exerts a broad range of functions during cancer cell fate. In the cancer setting, the RANK-RANKL pathway plays a role in every stage of tumorigenesis. Therefore, inhibition of RANKL by anti-RANKL antibodies is expected to be more far-reaching than simple inhibition of cancer cell activation. Denosumab, a drug used to treat metastatic prostate bone loss, has received FDA approval; this drug inhibits the RANK-RANKL pathway [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Denosumab is an effective and safe drug, which is superior to zoledronic acid in terms of skeletal-related events prevention; this was borne out by a combined analysis that included three randomised phase III trials with a similar set-up [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These trials included patients with bone metastases due to advanced breast cancer [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], other solid tumours or multiple myeloma [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, despite medical and commercial success, passive anti-cytokine drugs such as OPG-Fc and denosumab have several limitations, including high production costs, the need for regular infusion and a limited half-life [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, the aim is to develop a RANKL vaccine. In comparison with antibodies and other biologics, vaccines are better models for treatment of chronic disease because they are relatively cheap and small doses of protein can have a strong and long-lasting effect [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Here, we developed a novel vaccine targeting RANKL and examined its efficacy in a murine model of prostate cancer metastasis. To circumvent the problem of the immunogen triggering cytokine activity, mutants of RANKL were generated to prevent its interaction with RANK. A previous study shows that immunisation with mutant RANKL molecules generates anti-RANKL antibodies that block the interaction between RANKL and its receptor in an animal model of osteoporosis, thereby preventing proliferation and differentiation of osteoclasts and improving bone density [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, to block RANKL activation during PCa metastasis, we immunised mice with mRANKL-MT followed by intracardiac injection of PC3 cells. Inhibiting RANKL in animal models of metastases exerts therapeutic effects by inhibiting cancer cell metastasis. Currently, \u003cem\u003ein vivo\u003c/em\u003e tumour models that are most commonly used to study the process of cancer metastasis rely on introduction of tumour cells directly into the systemic circulation by injection into the left ventricle of laboratory rodents [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Thus, we employed a mouse model of PCa metastasis that more accurately reflects the metastatic process of this type of cancer. Studies on the effects of mRANKL-MT in PC3\u003csup\u003eRANKL+\u003c/sup\u003e mice showed that tumour growth was completely inhibited. Immunisation with mRANKL-MT effectively inhibited metastasis of tumour cells by generating anti-RANKL antibodies.\u003c/p\u003e \u003cp\u003eTo further confirm the action of RANKL immunisation, we assessed the effects of immune serum from immunised mice on PCa cells. Anti-RANKL antibodies blocked the RANKL-mediated chemotaxis of tumour cells. Furthermore, anti-RANKL antibodies inactivated RANKL on tumour cells directly. Treatment of RANKL-overexpressing PC3 cells with immune serum almost entirely abolished cancer cell migration and invasion. Wu et al. showed that a recombinant inactive RANKL vaccine (Y234pNO2Phe) induced high antibody titers and protected mice from collagen-induced arthritis by inhibiting osteoclast function and by preventing bone erosion [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, no study has reported that these types of RANKL vaccine have been used to inhibit cancer metastasis. In addition, we found that EMT and metastasis-related genes were downregulated following treatment of RANKL-overexpressing PC3 cells with immune serum. The antisera obtained from mice immunised with mRANKL-MT almost entirely inhibited the EMT process in RANKL-overexpressing PC3 cells. EMT is characterised by the loss of cell\u0026ndash;cell adhesion and by an increase in cell motility; it is a key process in cancer progression and metastasis, making EMT inhibition an attractive therapeutic strategy [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Deregulation of Wnt/β-catenin signalling is a hallmark of PCa metastasis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and β-catenin is a critical end component of the Wnt signalling pathway, which regulates cell growth, apoptosis and migratory behaviour in response to intercellular adhesion molecules [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Activation of β-catenin in PCa cells leads to transactivation of Wnt signalling target genes, including cyclin D1, HEF1 and matrix metalloproteinase 9 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Also, previous studies show that expression of Wnt-1 and β-catenin is increased in invasive PCa cell lines and in primary prostate cancer specimens [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In line with these previous reports, we demonstrated that treatment of PC3 cells with immune serum led to a marked decrease in RANK/SRC and GSK-3β signalling and β-catenin/TCF-4 transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). β-catenin forms a cell adhesion complex with E-cadherin, raising the possibility that loss of expression or a change in β-catenin distribution in the cell alters downstream signalling, decreases intercellular adhesion and promotes metastasis. These results suggest that the inhibitory effect of immune serum on PCa cell metastasis may involve suppression of the Wnt/β-catenin signalling pathway.\u003c/p\u003e\u003cp\u003eIn summary, this study demonstrates the protective role of mRANKL-MT against RANKL-induced PCa in mice. This effect was mediated via induction of a high-titer antibody response, inhibition of EMT and metastatic functions. Our results highlight the potential application of an anti-RANKL vaccine for treatment of metastatic RANKL-induced PCa. Moreover, the results suggest that mutant RANKL may be a potential RANKL vaccine that prevents and/or treats RANKL targeting in patients with metastatic PCa prostate cancer.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstate cancer; NF-κB:Nuclear factor kappa-light-chain-enhancer of activated B cells; RANKL:receptor activator of NF-kB ligand; EMT:epithelial-mesenchymal transition; TCF-4:Transcription factor 4; TRANCE:tumour necrosis factor-related activation-induced cytokine; OPGL:osteoprotegerin ligand; ODF:osteoclastic differentiation factor; GSK-3:glycogen synthase kinase-3; mRANKL-MT:mouse RANKL mutant; IL-6:Interleukin-6; RPMI:Roswell Park Memorial Institute; FBS:Fetal bovine serum; HRP:Horseradish peroxidase; PCR:Ploymerase chain reaction; IHC:Immunohistochemistry;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. P., Y. C. and W. L. designed and performed the research, analysed and interpreted the data, and wrote the paper; B. K., Y. K., Y. J. and Y. J. analysed and interpreted the in vitro data, and helped to draft some of the figures; W. L. performed and supervised RT-PCR and western blot data analysis; B. K. and Y. K. assisted with the ELISA assays; M. P., Y. C , B. K., Y. C., M. P., Y. K. and Y. J. performed \u003cem\u003ein vivo\u003c/em\u003e studies under the supervision of W. L.; M. P. and Y. J. performed histologic analysis under the supervision of H. H.; M. P. and Y. C. performed pathological review of the samples; and W. L. analysed publicly available datasets and supervised the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by research funding from Chosun University (awarded in 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatient study was approved and supervised by Chosun University Hospital\u0026rsquo;s institutional review board. (Chosun University Hospital, Gwangju, Korea (CHOSUN 2020-06-001)). And animal experimental protocol was approved by the Institutional Animal Care and Use Committee, Chosun University, Gwangju, Korea (CIACUC2019-A0015) and\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no financial conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eLaboratory of Orthopaedic Research, Chosun University Hospital, Dong-Gu, Gwangju, 61452, Republic of Korea\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Orthopaedic Surgery, Chosun University Hospital, Dong-Gu, Gwangju, 61452, Republic of Korea\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Biomedical Sciences Chonnam National University Medical School, Gwangju 61469, Republic of Korea\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eDepartment of Premedical Science, College of Medicine, Chosun University, Dong-Gu, Gwangju 61452, Republic of Korea\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRawla P: Epidemiology of Prostate Cancer. 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PLoS Genet. 2013;9:e1003180.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RANKL, prostate cancer, metastasis, EMT","lastPublishedDoi":"10.21203/rs.3.rs-105442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-105442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethod:\u003c/strong\u003e In this study, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e RANKL activation was observed in human PCa tissue specimens. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTrial registration:\u003c/strong\u003e Chosun University Hospital, CHOSUN 2020-06-001. Registered 01 June 2020-prospectevely registered, https://hosp.chosun.ac.kr/medi_depart/ site=hospital\u0026amp;mn=151\u0026amp;type=view\u0026amp;catename=IRB\u003c/p\u003e","manuscriptTitle":"RANKL Immunisation Inhibits Prostate Cancer Metastasis by Modulating EMT Through A RANKL-Dependent Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-17 18:46:54","doi":"10.21203/rs.3.rs-105442/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf1a539e-1cc3-4c9d-994b-9e4ad3e31ad0","owner":[],"postedDate":"November 17th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1104534,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2020-11-18T12:09:17+00:00","versionOfRecord":[],"versionCreatedAt":"2020-11-17 18:46:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-105442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-105442","identity":"rs-105442","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}