New drugs that inhibit the
osteoprotegerin (OPG)/receptor activator of NF-κB
ligand (RANKL)/RANK pathway have demonstrated efficacy for the treatment of bone
metastasis. Toxicities induced by these drugs, however, including
osteonecrosis of the jaw and
hypocalcemia, may adversely affect
therapy. The aim of this study was to identify additional therapeutic targets that can be combined with OPG/RANKL/RANK pathway inhibition in the treatment of
prostate cancer bone
metastasis. We established a stable transfectant that produces high levels of OPG
mRNA and
protein from PC-3 human
prostate cancer cells (PC3-OPG). The culture medium of PC3-OPG cells significantly inhibited the differentiation of mouse monocytes into mature osteoclasts. Furthermore, when PC3-OPG cells were injected into the bones of nude mice, bone destruction and
tumor-induced osteoclast formation were reduced. Injection into bone of the mixtures containing equal amounts of
green fluorescent protein (GFP)-expressing PC-3 cells (PC3-GFP) and PC3-OPG cells also reduced bone destruction, compared to the control mixture. PC3-GFP cells were subsequently isolated from bone
tumors and used for microarray analysis to assess changes in gene expression following osteolytic
tumor growth inhibition by OPG. We selected the top 10 upregulated genes based on results from microarrays and confirmed
mRNA expression of each gene by RT-PCR. The expression patterns of
retinol-binding protein 4 (RBP4) and placenta-specific 8 (PLAC8) were consistent with microarray results. Expression of these genes was also increased in the bone
tumors of PC3-GFP/PC3-OPG-injected mice. Knockdown of both RBP4 and PLAC8 by
siRNA inhibited the growth of PC-3 cells in vitro. Thus, RBP4 and PLAC8 may become new therapeutic targets for
prostate cancer bone
metastasis, in combination with OPG/RANKL/RANK pathway inhibition.