Aurora-B expression was negatively correlated with prognosis in OS.
To investigation the relationship of Aurora-B expression with prognosis in OS. We examined the expression of Aurora-B by immunohistochemical staining and collected the follow-up information from OS patients. We found that the expression of Aurora-B protein is closely correlated with Enneking stage (Table1; Fig. 1A-B) (P༜0.01). Moreover, Kaplan-Meier analysis showed that high levels of Aurora-B expression were negative correlated with poor overall survival (Fig. 1C) (P༜0.01) and lower metastasis-free survival (Fig. 1D) (P༜0.01) in these OS patients. These results collectively indicate that the expression of Aurora-B protein was negatively correlated with prognosis in OS.
Aurora-B knockdown induces autophagy in OS.
To investigate the correlation between Aurora-B and autophagy, we first applied immunohistochemical staining to examine the Aurora-B and LC3 expression in OS tissues. In the tissues with Aurora-B-positive expression, LC3 protein was poorly expressed, and the opposite was observed in the tissue samples that tested negative for Aurora-B expression (Table1; Fig. 2A and B) (P༜0.05). These results suggested that a potential correlation between Aurora-B and autophagy in OS. Further investigate the effect of Aurora-B on autophagy in OS cells, we knocked down endogenous Aurora-B with shRNA (short-hairpin RNA) in 143B and HOS cells by lentiviral infection (Fig. 2C and 2D). The RFP-GFP-LC3 fusion assay was performed to observe the formation of autophagosomes and autolysosomes in OS cells. The number of yellow LC3 puncta (representing autophagosomes) and red LC3 puncta (representing autolysosomes) were both higher in Aurora-B silenced cells than in cells infected with scrambled lentivirus, which represented the negative control (Fig. 2E). Similar results were observed by transmission electron microscopy (Fig. 2F). Furthermore, the ratio of LC3-II to LC3-I was significantly increased, whereas p62 expression was decreased, in cells with Aurora-B-knockdown relative to that in cells infected with scrambled lentiviruses (Fig. 2G). The results indicate that inhibition of Aurora-B enhanced autophagy in OS cells. Furthermore, to distinguish whether the accumulation of LC3 is due to the induction of autophagy or the depletion of autophagy flux, cells were treated with the specific autophagy inhibitor chloroquine (CQ), which inhibits autophagosome-lysosome fusion, thus preventing LC3 degradation. As shown in Fig. 1F, the LC3 II protein level was significantly increased following treatment with CQ in both control and Aurora-B-silenced cells compared with that in cells that were not treated with CQ (Fig. 2H). These results indicate that Aurora-B knockdown more likely enhances autophagic flux than inhibits autophagy in OS cells.
Silencing Aurora-B induces OS cells autophagy by suppressing the mTOR/ULK1 pathway.
The mTOR/ULK1 pathway is known to regulate autophagy, and the inactivation of this pathway has been shown to enhance autophagy levels(21, 22). AMPK is a critical sensor of stress and energy metabolism that negatively regulates mTOR, activating autophagy initiation factor ULK1 and promoting autophagic flux(23, 24). To explore the molecular mechanisms involved in the enhancement of autophagy by Aurora-B knockdown, we examined the expression of proteins involved in the mTOR/ULK1 pathway in Aurora-B-knockdown 143B and HOS cells. Interestingly, as shown in Fig. 3A, the phosphorylation levels of AMPK and ULK1 were upregulated and those of mTOR were downregulated in Aurora-B knockdown 143B and HOS cells, which was consistent with the observed changes in total AMPK, ULK1, and mTOR levels. These results suggest that Aurora-B knockdown enhances autophagy by decreasing mTOR/ULK1 pathway activity. Recent evidence has additionally demonstrated that the suppression of the malignant phenotype of OS cells by Aurora-B silencing might be achieved via autophagy-mediated activation of the mTOR/ULK1 signaling pathway. Thus, to further investigate the role of the mTOR/ULK1 pathway during Aurora-B silencing-induced autophagy, we investigated the expression of the autophagy-related protein p62 after treatment with MHY-1485, a well-known mTOR activator(25), or not, in Aurora-B-knockdown or scrambled OS cells. In the Aurora-B-knockdown OS cells, MHY-1485 treatment activated mTOR/ULK1 signaling and increased p62 levels. In the scrambled groups, after treatment with MHY-1485, the expression of p62 and phosphorylation of mTOR increased (Fig. 3B). These results reveal that inhibition of Aurora-B induced autophagy in OS cells via decreasing the mTOR/ULK1 pathway.
Aurora-B knockdown-induced autophagy inhibits migration and invasion in OS cells.
Autophagy is known to be involved in tumor progression (26, 27). In order to elucidate the effect of Aurora-B inhibition induced autophagy on the metastasis of OS cells, wound healing, transwell migration and invasion assays were performed to detect migration and invasion ability of OS cells. We surprisingly found that Aurora-B inhibition significantly decreased the motility and invasive ability of 143B and HOS cells. After treatment with CQ, the attenuated ability could be reversed. (Fig. 4A-F). These findings indicate that autophagy plays an essential role in the process of Aurora-B affecting the ability of motility and invasive in OS cells. Autophagy is reported to inhibit cell migration by accelerating the degradation of MMP family proteins(26, 27), therefore, we sought to determine the MMP2 protein levels in Aurora-B-knockdown OS cells with or without CQ treatment. Results showed that the suppression of MMP2 expression due to Aurora-B knockdown could be restored by CQ treatment (Fig. 4G). These findings demonstrate that inhibitio of Aurora B suppresses the migration and invasion of OS cells by stimulating autophagy.
Activation of the mTOR/ULK1 pathway reverses the effect of Aurora-B inhibition on migration and invasion of OS cells
To confirm whether the mTOR/ULK1 pathway contributes to Aurora-B-induced suppression of the malignant phenotype in OS cells, we performed wound healing, transwell migration, and invasion assays to evaluate the migration and invasion ability of Aurora-B-knockdown OS cells treated with MHY1485 or not. The results revealed that the inhibition of migration and invasion ability induced by Aurora-B knockdown could be reversed by MHY1485 treatment (Fig. 5A-F). And it indicates that activation of the mTOR/ULK1 pathway reverses the induction of Aurora-B knockdown-induced autophagy, which inhibits migration and invasion in OS cells.
Aurora-B knockdown inhibits metastasis via mTOR-mediated autophagy in vivo.
In order to investigate how the inhibition of Aurora-B affects the metastasis of OS, we established an orthotopic xenograft model using the luciferase-labeled 143B cell line (Fig. 6A). Bioluminescent imaging analysis revealed that the number of metastatic foci in the lung was significantly decreased when treated with AZD2811, a specific Aurora-B inhibitor in these mice models. Furthermore, 3BDO, a new mTOR activator, or CQ injection reversed these effects. H༆E staining and lung anatomy confirmed this result (Fig. 6B-D). These results suggested that Aurora-B knockdown inhibits OS metastasis via mTOR-mediated autophagy in vivo.