Repositioning Bazedoxifene as a novel IL-6/GP130 signaling antagonist for human rhabdomyosarcoma therapy

Abstract

Interleukins-6 (IL-6)/GP130 signaling pathway represents a promising target for cancer therapy due to its critical role in survival and progression of multiple types of cancer. We have identified Bazedoxifene, a Food and Drug Administration (FDA)-approved drug used for the prevention of postmenopausal osteoporosis, with novel function as inhibitor of IL-6/GP130 interaction. In this study, we investigate the effect of Bazedoxifene in rhabdomyosarcoma and evaluate whether inhibiting IL-6/GP130 signaling is an effective therapeutic strategy for rhabdomyosarcoma. The inhibitory effect of Bazedoxifene was assessed in rhabdomyosarcoma cell lines in vitro and RH30 xenograft model was used to further examine the suppressive efficacy of Bazedoxifene on tumor growth in vivo. Rhabdomyosarcoma cells showed their sensitivity to GP130 inhibition using gene knockdown or neutralized antibody, suggesting IL-6/GP130 as therapeutic target in rhabdomyosarcoma cells. Bazedoxifene decreased the signal transducer and activator of transcription3 (STAT3) phosphorylation, blocked STAT3 DNA binding, and down-regulated the expression of STAT3 downstream genes. Bazedoxifene also induced cell apoptosis, reduced cell viability, and inhibited colony formation in rhabdomyosarcoma cells. The inhibition of colony formation, STAT3 phosphorylation, or cell viability following Bazedoxifene treatment was partially reversed by addition of excess IL-6 or overexpression of constitutive STAT3, respectively, supporting Bazedoxifene acted through IL-6/GP130 signaling. In addition, Bazedoxifene repressed cell invasion and angiogenesis in vitro. Furthermore, oral administration of Bazedoxifene significantly suppressed tumor growth and expression of STAT3 phosphorylation in nude mice bearing established human rhabdomyosarcoma xenograft. Taken together, these findings validate IL-6/GP130 signaling as therapeutic target in rhabdomyosarcoma and provide first evidence that Bazedoxifene may serve as a novel promising drug targeting IL-6/GP130 for treatment of rhabdomyosarcoma.

Fig 1. Docking modeling of drug candidate Bazedoxifene binding to GP130.

Docking drug bazedoxifene (gray stick) to GP130 D1 domain shows that the indole moiety and seven-membered ring azepanyl of bazedoxifene effectively compete the native Trp157 and Leu57 binding sites (red sticks) of IL-6, respectively. Bazedoxifene also forms two hydrogen bonds with ASN92 and CYS6 of GP130 (rendered in green dot line). GP130 D1 domain (PDB code 1P9M) is represented as molecular surfaces. Picture is made using AutoDockTools (ADT).

Fig 2. Human rhabdomyosarcoma cell lines are sensitive to GP130 inhibition.

A, RH30 and RD cells were treated by neutralized anti-GP130 antibody (50 μg/ml) for 24 hours. STAT3 phosphorylation was detected by western blot. B, Cell viability was measured by MTT assay in RH30 or RD cells treated by neutralized anti-GP130 (100 μg/ml) antibody for 48 hours. The data represent mean ± SD, **, P < 0.01. C, GP130 and PSTAT3 (Y705) expression was evaluated by Western blot analysis in RH30 cells transfected with GP130 shRNA (C. shRNA: control shRNA). D, Cell viability was measured by MTT assay in RH30 cells transfected with GP130 shRNA (C. shRNA: control shRNA). Error bars indicate SD of mean, **, P < 0.01.

Fig 3. Bazedoxifene suppresses STAT3 phosphorylation, induces apoptosis, inhibits DNA binding, and decreases down-stream genes expression in human rhabdomyosarcoma cells.

A, RH30, RD, and RH28 cells were treated with Bazedoxifene at the indicated concentration overnight. The protein expression of interest was determined by Western blot analysis with GAPDH as loading control. B, STAT3 DNA binding activity was measured by DNA binding assay in RH30 cells treated with Bazedoxifene (10 and 20 μM) overnight. The data represent mean ± SD, *, P < 0.05; **, P < 0.01. C, CYCLIN D1, SURVIVIN, and BCL-XL gene expression were detected by RT-PCR in RH30, RD, or RH28 cells treated with Bazedoxifene overnight at the indicated concentration. D, miR21 and miR-181b gene expression were analyzed by real-time quantitative RT-PCR in RH30, or RH28 cells treated with Bazedoxifene overnight at the indicated concentration, **, P < 0.01; ***, P < 0.001.

Fig 4. Bazedoxifene-mediated inhibition is reversed by excess of IL-6 treatment or expression of constitutively active STAT3-C.

A, Colony formation assay was conducted in RH30 and RD cells as described in materials and methods. Cells were treated with Bazedoxifene (20 μM) for 6 hours, and then cultured with or without of IL-6, IFN-γ or IL-4 respectively for 30 minutes. Then, cells were reseeded and cultured for 2–3 weeks to grow clones. B, After treatment with Bazedoxifene (20 μM) for 6 hours, RH30 cells were stimulated with IL-6 (50 ng/ml) for 30 minutes. STAT3 phosphorylation was determined by Western blot analysis. C, RH30 cells were transfected with STAT3-C, and stable clones were selected. Selected clone cells were reseeded and treated with Bazedoxifene at indicated concentration for 48 hours. Cell viability was detected by MTT assay. Data are shown as means ±SD, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5. Bazedoxifene blocks angiogenesis and invasion in vitro.

A, Bazedoxifene inhibits tube formation. HUVECs were seeded on Matrigel-coated wells with VEGF (10 ng/ml) in the absence or in the presence of Bazedoxifene and incubated for 24 hours to form a capillary network. The total number of branched tubes was then counted and representative image of capillary network formation was taken. B, Bazedoxifene downregulates several angiogenic factors in vitro. The proteome profiler antibody angiogenesis array was performed. The relative level of selected angiogenesis-related proteins was determined in parallel between untreated (left) and treated (right) HUVEC cells. C, Bazedoxifene inhibits endothelial cellular invasion. HUVECs (1 × 10⁶ cells/ml) were added to transwell chamber coated with Matrigel and treated with VEGF (20 ng/ml) in the absence or in presence of Bazedoxifene. After 24 hours, the number of invaded cells was counted, and results are expressed as percentage of invasion (basal invasion with no treatment). D, Bazedoxifene blocks rhabdomyosarcoma cells invasion. Parental RH30 and RH28 cells were starved in 0% FBS medium for 24 hours. After that, cells were seeded (5 × 10⁴ cells/well) to the top chamber, and 10% FBS was added into the lower chamber. Cells were treated with Bazedoxifene for 24 hours, and then invasion cells were detected in the bottom chamber using a Cultrex BME Cell Invasion Assay. Statistical analysis of three independent experiments was shown as means, *, P < 0.05.

Fig 6. Bazedoxifene inhibits tumor growth of human rhabdomyosarcoma xenograft and STAT3 phosphorylation in vivo.

RH30 Rhabdomyosarcoma cells (5 × 10⁶) were injected subcutaneously into nude mice. After the tumor development, vehicle or Bazedoxifene (5 mg/kg) was administered daily by oral gavage. A, Tumor size and B, Body weight were measured on the indicated days. Data represents mean ± SD, *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, STAT3 phosphorylation from the harvested tumor tissue was determined using Western blot analysis (V: vehicle, T: tumor).