Abstract 1426: Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis.

Abstract The primary site of prostate cancer metastasis is the skeleton with the resultant metastases containing areas of extensive osteogenesis and osteolysis. The latter is a process controlled by multinucleated osteoclasts and receptor activator of nuclear kappa B ligand (RANKL) is critical for osteoclastogenesis. RANKL is a type II transmembrane molecule that forms a trimer on the cell surface and mediates its effects via RANK. We have shown that matrix metalloproteinases-3 and -7 (MMP-3, MMP-7) can process RANKL to a soluble form, sRANKL. The objective of this study is to determine the functionality of membrane bound RANKL versus sRANKL in promoting osteoclastogenesis. Full-length murine RANKL cDNA was isolated and inserted into the pcDNA 3.1(+) expression construct. In vitro translation experiments demonstrated that RANKL was being generated and could be processed by MMP-3 and MMP-7. N-terminal amino acid sequencing revealed that the site of cleavage occurs in the juxtamembrane region of RANKL between amino acids 145 and 146. We hypothesized that cleavage in the juxtamembrane region could generate an active soluble form of sRANKL. Osteoclastogenesis and resorption assays with myeloid precursors demonstrated the functionality of sRANKL. To determine whether RANKL or sRANKL is the major mediator of osteoclastogenesis in vivo we have generated RANKL mutants that are resistant to cleavage by MMPs and other proteases. Using a site directed mutagenesis kit we have designed a number of mutants and found that Δ158, (deletion of amino acids 71-157) and, Δ148, deletion of amino acids 72-147), were resistant to proteolytic cleavage. We anticipate that the while Δ158 and Δ148 are resistant to cleavage, they will be functional. We will therefore test the functionality of these two deletion mutants in-vitro by co-culturing myeloid precursor cells with Cos-7/Cos-1 cells expressing wild type and mutant RANKL proteins. In conclusion, we have demonstrated that RANKL is proteolytically cleaved to generate a soluble active form. We have generated non-cleavable mutant forms of RANKL that will allow us to assess the role of membrane bound versus soluble RANKL in-vitro and in-vivo. The results of these experiments will further elucidate the roles of RANKL and sRANKL in prostate to bone metastases in addition to being informative for the engineering of future RANKL based therapies. Citation Format: Jeremy J. McGuire, Mitsuru Futakuchi, Conor Lynch. Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2013-1426