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

骨吸收在骨转移或癌骨浸润等癌性骨病中起重要作用。我们以前证明，血管生成抑制剂，TNP-470，不仅抑制肿瘤生长，但也骨细胞骨吸收。我们还介绍了TNP-470在体内模型中对骨转移、骨侵袭和激素性高钙血症具有治疗用途。近年来，TNP-470的候选靶分子是甲硫氨酸氨基肽酶-2（MetAP-2），但二者之间的关系尚不清楚。在本研究中，我们检测了MetAP-2在骨细胞骨吸收中的生理功能，以了解是否可以将MetAP-2用作对抗癌症诱导的骨疾病的分子靶点。在RANKL和M-CSF处理的脾细胞/RAW264.7上，在破骨细胞形成和分化过程中持续观察到MetAP-2的表达水平相同。相反，TNP-470显著抑制破骨细胞的形成，但增加MetAP-2的表达。MetAP-2的上调可能抑制破骨细胞的形成。因此，我们使用siRNA系统将具有针对MetAP-2基因的靶序列的表达载体转染到RAW 264. 7中以降低MetAP-2的表达。敲除MetAP-2基因的转染细胞在RANKL/M-CSF作用下形成了较大的成熟破骨细胞，破骨细胞数量少于对照RAW细胞。这表明MetAP-2的功能可能抑制破骨细胞的成熟。接下来，我们检查了TNP-470对RANK/RANKL通路的信号转导的影响。TNP-470不抑制TRAF 6、I-κBα和pI-κBα的表达，但增加转录因子NFATcl的表达。这表明TNP-470可能不影响RANK/RANKL→NF-κB通路。

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.