Up-regulation of soluble RANKL at tumor-bone interface is critical for mammary tumor-induced osteolysis

2795 Bone metastases are most common in patients with advanced breast cancer. The incidence is very high with 65 -75% patients suffer from bone metastasis. Bone metastatic lesions in breast cancer patients are predominantly osteolytic. We hypothesize that tumor-bone interaction is critical for tumor-induced osteolysis and establishment as bone metastasis. We have developed a murine model to examine tumor-stromal interaction in osteolytic bone metastasis. Using this model, we analyzed whether tumor-bone microenvironment regulates expression of genes critical for tumor-induced osteolysis. We used microarray analysis for gene expression profiling at the tumor bone interface versus the tumor alone area from syngenic mice injected with three different mammary tumor cell lines that differ in their metastatic potential (4T1 being the aggressive, Cl66 is moderately metastatic and Cl66M2 is poorly metastatic). Microarray analysis revealed the up-regulation of receptor activator of nuclear factor κB ligand (RANKL) in all three cell lines at the tumor bone interface as compared to tumor alone area. We did not observe significant difference in RANK expression. This observation was further validated by quantitative real time RT-PCR and immunohistochemistry. In addition, the levels of soluble RANKL was significantly elevated at TB-interface as compared to tumor alone area. Inhibition of RANKL using antisense oligonucleotides (AS-RANKL) in syngenic mice injected with Cl66 cells, abrogated mammary-tumor-induced osteolysis. mRNA analysis showed significantly decreased expression of RANKL at TB-interface in AS-RANKL-treated groups compared to control-oligonucleotide treated group. The levels of sRANKL and osteoprotegrin (OPG), a decoy receptor for RANKL, ratios significantly decreased following inhibition of RANKL expression. Together, these results demonstrate that soluble RANKL is an important mediator of mammary tumor-induced osteolysis and the inhibition of RANKL expression using antisense oligonucleotide might be a novel therapeutic strategy for breast cancer bone metastasis.