RANKL Acts Directly on RANK-Expressing Prostate T umor Cells and Mediates Migration and Expression of T umor Metastasis Genes

Department of Hematology, Amgen Inc., Seattle, Washington 98119, USA.
The Prostate (Impact Factor: 3.57). 01/2008; 68(1):92-104. DOI: 10.1002/pros.20678
Source: PubMed


Metastases to bone are a frequent complication of human prostate cancer and result in the development of osteoblastic lesions that include an underlying osteoclastic component. Previous studies in rodent models of breast and prostate cancer have established that receptor activator of NF-kappaB ligand (RANKL) inhibition decreases bone lesion development and tumor growth in bone. RANK is essential for osteoclast differentiation, activation, and survival via its expression on osteoclasts and their precursors. RANK expression has also been observed in some tumor cell types such as breast and colon, suggesting that RANKL may play a direct role on tumor cells.
Male CB17 severe combined immunodeficient (SCID) mice were injected with PC3 cells intratibially and treated with either PBS or human osteprotegerin (OPG)-Fc, a RANKL antagonist. The formation of osteolytic lesions was analyzed by X-ray, and local and systemic levels of RANKL and OPG were analyzed. RANK mRNA and protein expression were assessed on multiple prostate cancer cell lines, and events downstream of RANK activation were studied in PC3 cells in vitro.
OPG-Fc treatment of PC3 tumor-bearing mice decreased lesion formation and tumor burden. Systemic and local levels of RANKL expression were increased in PC3 tumor bearing mice. PC3 cells responded to RANKL by activating multiple signaling pathways which resulted in significant changes in expression of genes involved in osteolysis and migration. RANK activation via RANKL resulted in increased invasion of PC3 cells through a collagen matrix.
These data demonstrate that host stromal RANKL is induced systemically and locally as a result of PC3 prostate tumor growth within the skeleton. RANK is expressed on prostate cancer cells and promotes invasion in a RANKL-dependent manner.

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Available from: Allison Jacob, Jun 17, 2014
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    • "(B) The same cells used for the above tumor xenografts were grown in vitro and processed for flow cytometry. For anti-RANK staining, the pattern of staining by both test antibodies N-1H8 and N-2B10 was compared with a previously identified mAb (M331) useful for flow cytometry applications (Armstrong [31]). Solid grey line: Unstained; Red line: Secondary control¼Goat antimouse APC; Blue line: Isotype control 4D2 (anti-AGP3 muIgG1), 1 μg/mL; Purple line: M331 (anti-huRANK muIgG1), 1 μg/mL; Green line: N-1H8 (anti-huRANK muIgG1), 1 μg/mL; Black line: N-2B10 (anti-huRANK muIgG1), 1 μg/mL. "
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    ABSTRACT: Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential mediator of osteoclast formation, function and survival. In patients with solid tumor metastasis to the bone, targeting the bone microenvironment by inhibition of RANKL using denosumab, a fully human monoclonal antibody (mAb) specific to RANKL, has been demonstrated to prevent tumor-induced osteolysis and subsequent skeletal complications. Recently, a prominent functional role for the RANKL pathway has emerged in the primary bone tumor giant cell tumor of bone (GCTB). Expression of both RANKL and RANK is extremely high in GCTB tumors and denosumab treatment was associated with tumor regression and reduced tumor-associated bone lysis in GCTB patients. In order to address the potential role of the RANKL pathway in another primary bone tumor, this study assessed human RANKL and RANK expression in human primary osteosarcoma (OS) using specific mAbs, validated and optimized for immunohistochemistry (IHC) or flow cytometry.
    Journal of Bone Oncology 07/2015; 125(3). DOI:10.1016/j.jbo.2015.06.002 · 1.21 Impact Factor
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    • "RANK also played a key role in tumor cell migration and invasion [20], [21]. Moreover, it has been demonstrated that RANKL promotes migration, and invasion of several types of human tumor cells expressing its receptor RANK [22], [23], [24]. However, the role of RANKL-RANK axis in modulating the behaviors of HCC cells is mostly unknown. "
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    ABSTRACT: Background Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown. Methods Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability. Results We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells. Conclusions RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.
    PLoS ONE 09/2014; 9(9):e108507. DOI:10.1371/journal.pone.0108507 · 3.23 Impact Factor
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    • "RANKL expressed by osteoblasts and stromal cells in bone tissue may represent an important chemoattractant or “soil” factor that underlies preferential metastasis of certain tumors to the bone. RANKL stimulation of prostate cancer cells can induce multiple signaling pathways which stimulate cellular migration, chemotaxis, and invasion through collagen matrix (58). RANKL, RANK, and OPG expression was greater in bone metastases than lymph-node metastases in prostate cancer (43). "
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    ABSTRACT: The role of the receptor activator of nuclear factor-κB ligand (RANKL)/RANK system is well characterized within bone, where RANKL/RANK signaling mediates osteoclastogenesis and bone resorption. However, this system has also been shown to influence biologic processes beyond the skeletal system, including in the immune system and in cancer. RANKL/RANK signaling is important in lymph-node development, lymphocyte differentiation, dendritic cell survival, T-cell activation, and tolerance induction. The RANKL/RANK axis may also have direct, osteoclast-independent effects on tumor cells. Indeed, activity of the RANKL/RANK pathway in cancer cells has been correlated with tumor progression and advanced disease. Denosumab, a fully human monoclonal antibody against RANKL, inhibits osteoclastogenesis and is widely used not just for the treatment of osteoporosis, but for the prevention of skeletal-related events from bone metastases in solid malignancies such as breast and prostate cancer. The potential effects of denosumab on the immune system have been largely ignored. Nevertheless, with the emergence of immunotherapies for cancer, denosumab may impact the effectiveness of these therapies, especially if they are given in combination. In this article, we review the role of RANKL/RANK in bone, immunity, and cancer. Examining the potential effects of routine treatment with denosumab beyond the bone represents an important area of investigation.
    Frontiers in Oncology 01/2014; 3:329. DOI:10.3389/fonc.2013.00329
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