br Methods br Results br Discussion One of
Discussion One of the hallmarks of cancer is the ability of cancer equol to evade apoptosis. Inhibition of apoptosis not only enhances the survival of cancer cells but also facilitates their escape from chemotherapy . Therefore, apoptosis regulatory mechanisms are promising targets for anti-cancer therapy. Inhibitors of apoptosis proteins (IAPs) are able to abrogate death-inducing signals, by direct inhibition of caspases and modulation of NF-κB . IAP antagonists are being tested in the clinical setting for their ability to restore apoptosis in cancer cells. In this work we confirmed that MDA-MB-231 breast cancer cells are sensitive to AT-406 by restoring apoptosis in this cell line at nanomolar concentrations . It was previously shown by us and other authors that RANK (receptor activator of NF-κB) is expressed in vitro by several human breast cancer cell lines, including MDA-MB-231 [15–18,24]. In RANK-positive cells, RANKL (RANK ligand) induces the activation of RANK-dependent pathway, increases migration and invasion, and induces epithelia–mesenchymal transition, favoring an invasive phenotype [14–16,19,20,25]. This fact is extremely relevant in the context of bone metastases since the molecular triad RANK–RANKL–OPG (osteoprotegerin) controls bone remodeling. In bone metastatic disease there is an unbalanced bone remodeling. Tumor cells interact with the bone microenvironment and stimulate osteoclastogenesis by releasing important factors like PTHrP (parathyroid hormone-related protein). This leads to an increased amount of growth factors released from bone matrix that further promote tumor growth, in a “vicious cycle” [3,26]. In this work we show that RANK-positive breast cancer cells show an up-regulation of cIAP-2 when activated by RANKL, and are more sensitive to AT-406, when using non-limiting concentrations of the drug. In fact, it was previously reported that cIAP2 is one of the most-upregulated genes in PC-3 prostate cancer cell line, which expresses the highest levels of RANK, when stimulated with RANKL . Pharmacokinetics analysis of AT-406 on SCID mice bearing MDA-MB-231 tumors has shown that AT-406 achieves a maximum concentration in plasma between 1.5μM and 9.9μM, and a maximum concentration in tumor tissue between 0.5μM and 18.2μM, depending on the dose and administration route (intravenous versus oral gavage) . Therefore, the described effects of AT-406 concentrations ranging from 100nM to 1μM may be further increased in vivo. It was formerly shown in an animal model of breast cancer bone metastases that RANKL inhibition inhibits osteolysis induced by MDA-MB-231 cells, but also enhances the ability of rhApo2L/TRAIL to reduce skeletal tumor burden in vivo. Therefore, it may be important to address if AT-406 could be used in combination with an anti-RANKL therapy in the context of bone metastatic disease. It is known that IAPs also play a role in osteoclast differentiation and survival. Osteoclast differentiation and function are stimulated by activation of the alternative NF-κB pathway . In monocytes, cIAP1 and 2, that have E3 ligase activity, are recruited by TRAF3 upon binding to NIK (NF-κB inducing kinase), targeting it to the proteosome [29,30]. RANK activation induces release of NIK and activation of the non-canonical NF-κB pathway, leading to the up-regulation of osteogenic transcription factors like Nfatc1 and osteoclast differentiation [29,30]. On the other hand, activation of the canonical NF-κB pathway, via RANKL–RANK–TRAF6, up-regulates cIAP1 and 2 and inhibits cell death [23,31]. This dual effect of IAPs in osteoclastogenesis and osteoclast survival led us to assess the effect of AT-406 on osteoclastogenesis. We found that AT-406 increases osteoclast differentiation in a dose-dependent manner, but independent of IAPs up-regulation. However, AT-406 induced a 2.5-fold increase in Nfactc1, at the early stages of osteoclast differentiation. Upon removal of RANKL mature osteoclasts enter apoptosis, which was increased by AT-406. We also demonstrate that breast cancer cells exposed to AT-406 are less osteogenic. Although the conditioned medium from breast cancer cells treated with AT-406 still increased osteoclastogenesis (3-fold), it was significantly lower than the effect of the conditioned medium from untreated cells. This effect is most probably due to breast cancer derived factors, and not to AT-406, since mouse monocytes were cultured with RANKL, impairing AT-406-induced apoptosis.