BMPR2 inhibition induced apoptosis and autophagy via destabilization of XIAP in human chondrosarcoma cells

Abstract

Bone morphogenetic proteins (BMPs) are multifunctional proteins, and their receptors (BMPRs) have crucial roles in the process of signaling. However, their function in cancer is somewhat inconsistent. It has been demonstrated that more prevalent expression of bone morphogenetic protein receptor 2 (BMPR2) has been detected in dedifferentiated chondrosarcomas than conventional chondrosarcomas. Here, we find that BMPR2 inhibition induces apoptosis and autophagy of chondrosarcoma. We found that BMPR2 expression was correlated with the clinicopathological features of chondrosarcomas, and could predict the treatment outcome. Knockdown of BMPR2 by small interfering RNA results in growth inhibition in chondrosarcoma cells. Silencing BMPR2 promoted G2/M cell cycle arrest, induced chondrosarcoma cell apoptosis through caspase-3-dependent pathway via repression of X-linked inhibitor of apoptosis protein (XIAP) and induced autophagy of chondrosarcoma cells via XIAP-Mdm2-p53 pathway. Inhibition of autophagy induced by BMPR2 small interfering RNA (siBMPR2) sensitized chondrosarcoma cells to siBMPR2-induced apoptotic cell death, suggesting that autophagy has a protective role for chondrosarcoma cells in context of siBMPR2-induced apoptotic cell death. In vivo tumorigenicity assay in mice indicated that inhibition of BMPR2 reduced tumor growth. Taken together, our results suggest that BMPR2 has a significant role in the tumorigenesis of chondrosarcoma, and could be an important prognostic marker for chondrosarcoma. BMPR2 inhibition could eventually provide a promising therapy for chondrosarcoma treatment.

Figure 1

BMPR2 expression is correlated with clinicopathological features of chondrosarcomas, and predicts treatment outcome. (a) Western blot analysis showed that BMPR2 was expressed in chondrosarcomas but not in the normal articular cartilage tissues. (b) Average expression levels of BMPR2 of normal articular cartilage and chondrosarcoma tissues. Data are presented as mean±S.D. (n=12). **P<0.001. (c) The expression of BMPR2 in a cohort of 78 human chondrosarcoma specimens was determined by IHC staining of BMPR2. Representative images of BMPR2 IHC staining in normal and chondrosarcoma specimens are shown. (d) Kaplan–Meier curves for relapse-free survival in chondrosarcoma patients with or without positive BMPR2 staining. Positive BMPR2 staining (>10% cells stained with BMPR2) in chondrosarcoma specimens is significantly associated with a poorer relapse-free survival (P=0.030)

Figure 2

Knockdown of BMPR2 by siRNA results in the inhibitory growth of chondrosarcoma cells. (a) Expression of mRNA levels of BMPR2 in HCS-2/8 and SW1353 cells were significantly repressed at 48 h after BMPR2 siRNA transfection. The siBMPR2 transfection efficiency was measured by RT-PCR. (b) The chondrosarcoma cell viabilities decreased sharply when treated with siBMPR2 for 48 h, as assayed by MTS. (c and d) Suppression of BMPR2 significantly decreased capacities of colony formation of HCS-2/8 and SW1353 cells, as analyzed by colony formation assay. (e) Expression of protein levels of BMPR2 in HCS-2/8 and SW1353 cells were suppressed significantly at 48 h after siBMPR2 transfection. Inhibition of BMPR2 resulted in the dephosphorylation of Smad1/5, as assayed by western blot analysis. Data are presented as mean±S.D. (n=3). **P<0.001

Figure 3

siRNA-mediated knockdown of BMPR2 silencing promoted G2/M cell cycle arrest. (a) Cells ware stained with PI following treatment with or without siBMPR2 transfection for 48 h. Cell cycle was analyzed using flow cytometry. (b) The bar chart shows the percentage of cells in cell cycle. The BMPR2 siRNA transfection resulted in a significant increase in the proportion of cells in the G2/M phase in HCS-2/8 and SW1353 cells. (c) Expression of p-Rb and cyclin B1 were inhibited in chondrosarcoma cell lines when treated with siBMPR2 for 48 h, as assayed by western blot analysis. Representative data from one of three independent experiments are shown in (a–c)

Figure 4

BMPR2 siRNA promoted chondrosarcoma cell apoptosis via destabilizing of XIAP. (a) Cells transfected with siBMPR2 were stained with Annexin-V-FITC (20 mg/ml) and PI (20 mg/l). Apoptosis analysis was performed by flow cytometry. (b) The bar graph showed a significant increase in the early and late apoptotic index. (c) Three BMPR2 siRNA sequences were used to repress BMPR2 in SW1353 cells, and the level of XIAP was sharply reduced when treated with siBMPR2s. (d) A BMP signaling inhibitor, LDN-193189, was used to block the BMP/Smads pathway. LDN-193189 did not reduce the level of XIAP, whereas knockdown of BMPR2 did reduce the protein level of XIAP. (e) Expression of XIAP decreased, and cleavages of caspase-3 and PARP enhanced in both HCS-2/8 and SW1353 cells when treated with BMPR2 siRNA for 48 h, as assayed by western blot. (f) Annexin-V-FITC (20 mg/ml) and PI (20 mg/l) dual staining were performed when SW1353 cells were transfected with siXIAP for 48 h. Apoptosis analysis was performed by flow cytometry. Representative data from one of three independent experiments are shown in (a and c–f)

Figure 4

BMPR2 siRNA promoted chondrosarcoma cell apoptosis via destabilizing of XIAP. (a) Cells transfected with siBMPR2 were stained with Annexin-V-FITC (20 mg/ml) and PI (20 mg/l). Apoptosis analysis was performed by flow cytometry. (b) The bar graph showed a significant increase in the early and late apoptotic index. (c) Three BMPR2 siRNA sequences were used to repress BMPR2 in SW1353 cells, and the level of XIAP was sharply reduced when treated with siBMPR2s. (d) A BMP signaling inhibitor, LDN-193189, was used to block the BMP/Smads pathway. LDN-193189 did not reduce the level of XIAP, whereas knockdown of BMPR2 did reduce the protein level of XIAP. (e) Expression of XIAP decreased, and cleavages of caspase-3 and PARP enhanced in both HCS-2/8 and SW1353 cells when treated with BMPR2 siRNA for 48 h, as assayed by western blot. (f) Annexin-V-FITC (20 mg/ml) and PI (20 mg/l) dual staining were performed when SW1353 cells were transfected with siXIAP for 48 h. Apoptosis analysis was performed by flow cytometry. Representative data from one of three independent experiments are shown in (a and c–f)

Figure 5

BMPR2 silencing treatment induced autophagy via XIAP-Mdm2-p53 pathway. (a) TEM images showing autophagic vacuoles (arrows) observed in siBMPR2-transfected chondrosarcoma cells for 48 h (middle and right). No or few autophagic vacuoles are observed in siNC-transfected cells (left). (b) Cells were transfected with BMPR2 siRNA for 48 h, then were incubated with rabbit polyclonal anti-LC3 Ab (1 : 200) overnight at 4 °C and then reacted with anti-rabbit IgG conjugated with Dylight 488 (1 : 400) for 1 h at room temperature. The cells were then visualized using confocal microscopy. (c) Quantification of the number of LC3-II punctas in the siNC- and siBMPR2-transfected cells, plotted as LC3-II punctas per cell. Data are presented as mean±S.D. (n=3). **P<0.001. The P-values were calculated using a two-sided Student's t-test. (d) Downregulation of BMPR2 induced accumulation of LC3-II and degradation of p62 (a known autophagy substrate) when treated with BMPR2 siRNA for 48 h, as assayed by western blot. (e) Silencing BMPR2 resulted in the degradation of XIAP, and subsequently enhancement of Mdm2 led to repression of p53 and eventually induced accumulation of LC3-II and degradation of p62, as assayed by western blot analysis. (f) Cleaved caspase-3 and PARP were increased when SW1353 cells were treated with siXIAP for 48 h, whereas p62 and LC3 remained unchanged. When cells were transfected with both siXIAP and siBMPR2, siRNAs significantly increased the expression of Mdm2, LC3-II, cleaved PARP and caspase-3, and decreased the expressions of p53 and p62, compared with when transfected with siBMPR2 alone, as assayed by western blot analysis. Representative data from one of three independent experiments are shown in (a, b, d, e and f)

Figure 6

Inhibition of siBMPR2-induced autophagy sensitized chondrosarcoma cells to siBMPR2-induced apoptotic cell death. (a) Pretreatment of cells with 3-MA or siBeclin-1 sharply reduced the number of viable siBMPR2-treated cells, as assayed by MTS. (b) Colony formation of chondrosarcoma cells was decreased more sharply by Beclin-1 and BMPR2 siRNA transfection than by BMPR2 siRNA. (c) SW1353 cells were stained with Annexin-V-FITC (20 mg/ml) and PI (20 mg/l) following siBMPR2 treatment with or without siBeclin-1 transfection for 48 h. Apoptosis was analyzed by flow cytometry. (d) Compared with siBMPR2 transfection group, the bar graph showed a significant increase in apoptosis rate in co-transfection of siBMPR2 and siBeclin-1. (e) Apoptosis- and autophagy-related proteins were examined by western blot following siBMPR2 transfection with or without siBeclin-1 for 48 h. (f) Compared with siBMPR2 transfection group, the bar graph showed a significant increase in cleaved PARP in co-transfection of siBMPR2 and siBeclin-1 (P<0.01). (g) The bar graph showed a great decrease in LC3-II in co-transfection of siBMPR2 and siBeclin-1 compared with siBMPR2 transfection group (P<0.01). Representative data from one of three independent experiments are shown in (b–e)

Figure 7

BMPR2 inhibition suppressed chondrosarcoma tumor growth in vivo. (a) Tumor volume growth curve after intratumor injection of siBMPR2 or siNC. BMPR2 siRNA treatment resulted in significantly decreased tumor growth, compared with the siNC group (Dunnett's t-test, P<0.01). (b) Representative images of xenograft tumors. (c) Representative images ( × 400) of IHC analysis of BMPR2, Ki-67, p-Rb, XIAP, cleaved caspase-3 and p62 in tumors. (d) Representative western blot analysis elucidating a significant repression of BMPR2 protein level in the BMPR2 siRNA treatment group compared with the siNC group. Changes of XIAP, PARP, P62 and LC3-II proteins followed the same trend as in vitro, assayed by western blot analysis. (e) Proposed mechanisms responding to BMPR2 siRNA-induced effects in chondrosarcoma cells