Investigation of mechanism of methionine aminopeptidase-2 as a molecular target against cancer-induced bone destruction

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Osteoclastic bone resorption plays an important role on cancer-induced bone diseases like bone metastases or cancer bone invasion. We previously demonstrated that the angiogenesis inhibitor, TNP-470, inhibited not only tumor growth but also osteoclastic bone resorption. We also presented that TNP-470 has therapeutic use for bone metastasis, bone invasion and hormonal hypercalcemia in vivo models. Recently, it is reported that the candidate-target molecule of TNP-470 is methionine aminopeptidase-2 (MetAP-2), however, the relationship between TNP-470 and MetAP-2 has been still well unknown. Also, there is no information about the role of MetAP-2 on osteoclastogenesis.In the present study, we examined the physiological function of MetAP-2 on osteoclastic bone resorption to know whether we could use MetAP-2 as a molecular target against the cancer-induced bone diseases. The expression of MetAP-2 was continuously observed as same level during the osteoclast formation and differentiation on spleen cells/RAW264.7 treated with RANKL and M-CSF. In contrast, TNP-470 markedly inhibited the osteoclast formation, but increased the expression of MetAP-2. The up-regulation of MetAP-2 may inhibit the osteoclast formation. Therefore, we transfected the expression vector with the target sequence against MetAP-2 gene into RAW264.7 using the siRNA system to decrease MetAP-2 expression. The transfectants with knock-down of MetAP-2 gene formed the large and matured osteoclasts with treatment of RANKL/M-CSF, and osteoclast number was lesser than that of control RAW cells. This indicated that the function of MetAP-2 might inhibit the osteoclast maturation. Next, we examined the effect of TNP-470 on the signal transduction of RANK/RANKL pathway. TNP-470 did not inhibits the expression of TRAF6, I-κBα and pI-κBα, but increased the transcriptional factor, NFATcl. This indicates the TNP-470 may not affect the RANK/RANKL→NF-κB pathway.