Anti-β-actin and anti-lamin antibodies were used as the internal standard. (E) Quantification of the amount of NF-κB p65, normalized to the amounts of the corresponding proteins, respectively. The results are representative of 5 independent experiments. *p < 0.01, as compared to controls (ANOVA with Dunnett’s test). Discussion In this study, we demonstrated that RANKL induces EMT through the upregulation of Snail and Twist expression levels in GS-9973 normal breast epithelial cells and breast cancer cells. We also found that RANKL-induced EMT accelerated cell migration and invasion

in normal breast epithelial cells and breast cancer cells. It has been indicated that aberrant RANK signaling promotes breast tumorigenesis AZD6738 manufacturer [20]. It has also been reported that RANKL induces the migration and metastasis of RANK-expressing cancer cells [16–18]. In addition, high RANK

expression levels in primary tumors of patients have been correlated with poor prognoses and higher risk of developing bone metastasis [21]. Collectively, the findings suggest that the RANKL/RANK Berzosertib research buy system promotes cell migration, invasion, and metastasis by EMT in RANK-expressing cancer cells. RANKL/RANK signaling activates a variety of downstream pathways. RANK assembles into functional trimers. Various tumor necrosis factor receptor-associated factor proteins associate with the cytoplasmic domain of RANK and mediate ligand-induced signaling. RANKL/RANK induces the activation

of NF-κB mediated by the I-κB kinase complex [22, 23]. Members of the mitogen-activated protein kinase family, including JNK and ERK, are activated downstream of RANK [24, 25]. RANK also induces the activation of the phosphoinositol 3-kinase/Akt/mTOR pathway and the Janus kinase 2/STAT3 pathway [26, 27]. Our results clearly demonstrate that RANKL induces activation of NF-κB but not of ERK1/2, Akt, mTOR, JNK, and STAT3. It has been reported that the activation of NF-κB upregulated the expression levels of Snail and fibronectin and Elongation factor 2 kinase induced EMT [28, 29]. It has also been indicated that NF-κB activation promotes cell migration and invasion by stabilization of Snail in breast cancer cells [30]. Furthermore, it has been reported that NF-κB-induced Twist expression required EMT in normal breast epithelial cells and breast cancer cells [31]. Collectively, these results suggest that RANKL/RANK signaling induces EMT by NF-κB activation and upregulation of Snail and Twist in normal breast epithelial cells and breast cancer cells. Moreover, we observed that DMF, a NF-κB inhibitor, inhibited RANKL-induced EMT and enhanced the expressions of Snail and Twist, cell migration, and invasion. A previous report has shown that NPI-0052, a proteasome inhibitor, suppresses EMT via the inhibition of NF-κB activation and Snail expression [32].