RANKL acts directly on RANK-expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Osteoclast differentiation is impaired in the absence of inhibitor of kappa B kinase alpha.

Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.

A RANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function.

Signaling through receptor activator of nuclear factor-kappaB (RANK) is essential for the differentiation and activation of osteoclasts, the cell principally responsible for bone resorption. Animals genetically deficient in RANK or the cognate RANK ligand are profoundly osteopetrotic because of the lack of bone resorption and remodeling. RANK provokes biochemical signaling via the recruitment of intracellular tumor necrosis factor receptor-associated factors (TRAFs) after ligand binding and receptor oligomerization. To understand the RANK-mediated signal transduction mechanism in osteoclastogenesis, we have designed a system to recapitulate osteoclast differentiation and activation in vitro by transfer of the RANK cDNA into hematopoietic precursors genetically deficient in RANK. Gene transfer of RANK constructs that are selectively incapable of binding different TRAF proteins revealed that TRAF pathways downstream of RANK that affect osteoclast differentiation are functionally redundant. In contrast, the interaction of RANK with TRAF6 is absolutely required for the proper formation of cytoskeletal structures and functional resorptive activity of osteoclasts. Moreover, signaling via the interleukin-1 receptor, which also utilizes TRAF6, rescues the osteoclast activation defects observed in the absence of RANK/TRAF6 interactions. These studies are the first to define the functional domains of the RANK cytoplasmic tail that control specific differentiation and activation pathways in osteoclasts.