Inhibition of STAT3 blocks protein synthesis and tumor metastasis in osteosarcoma cells

Abstract

Background: Osteosarcoma is the most common bone cancer. Despite advances, molecular mechanisms associated with osteosarcoma have not been fully understood. Hence, an effective treatment for osteosarcoma has yet to be developed. Even though signal transducer and activator of transcription3 (STAT3) has been implicated, its role in pathogenesis of osteosarcoma is not fully determined. In this study, we investigated the antitumor effect of napabucasin (NP) (BBI608), an inhibitor of STAT3 on osteosarcoma in vitro and in vivo and studied the underlying molecular mechanism.

Methods: Cell viability, colony formation, apoptosis, tumor growth and metastasis assays were performed to examine the effect of NP on osteosarcoma in vitro and in vivo. Real-time RT-PCR, western analysis, immunofluorescence and reporter assays were used to monitor the expression and activity of proteins and underlying molecular pathways. Protein synthesis, co-immunoprecipitation and CAP binding assays were carried out to understand NP-mediated mechanism of actions in osteosarcoma cells.

Results: Our results show that NP treatment decreases cell viability and induces apoptosis in several osteosarcoma cell lines. NP treatment suppresses both expression and phosphorylation of STAT3 in addition to blocking STAT3-mediated transcription and downstream target proteins in osteosarcoma cells. Furthermore, NP inhibits protein synthesis through regulation of the eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1). NP also inhibits the progression of osteosarcoma tumors and metastasis in vivo in an orthotopic tibial model of osteosarcoma.

Conclusions: Taken together, our investigation reveals that NP acts through a novel mechanism and inhibits osteosarcoma growth and metastasis, and could be investigated clinically for treating osteosarcoma patients alone or in combination with other drugs.

Keywords: 4EBP-1; Apoptosis; Napabucasin; Osteosarcoma; Protein synthesis; eIF4E.

Fig. 1

NP decreases cell viability and proliferation of human osteosarcoma cells. a, Human osteosarcoma cells (143B, MG63, U2OS, KHOS) were treated with vehicle (Veh) (0.1% DMSO) or NP at various concentrations for 24, 48, and 72 h, and cell viability was measured by MTS assay as described in the Methods section of the text. b and c, The cell colony formation assay was carried out in 143B and MG63 treated with Veh or NP at indicated concentrations. d, 143B cells were treated with Veh or NP for 24 h and analyzed by immunofluorescence using anti–Ki-67 antibodies. The data are representative of 3 independent experiments. *P < 0.05 versus vehicle control; ** P < 0.01 versus vehicle control

Fig. 2

NP induces apoptosis in osteosarcoma cells in a dose-dependent manner. a, 143B and MG63 cells were treated with vehicle (Veh) or indicated NP (3.0 μM) for 24 h, and apoptotic morphologic changes were evaluated by fluorescence microscopy using Hoechst 33342. b-d, 143B and MG63 osteosarcoma cells were treated with Veh or indicated concentrations of NP for 24 h and evaluated by flow cytometry with Annexin V-FITC and PI staining. The number of apoptotic, necrotic, and live cells was quantitated. e and f, 143B and MG63 cells were treated with NP at the indicated concentrations and time. The expressions of cleaved PARP, caspase-3 were determined by Western blot using specific antibodies. The data are representative of 3 independent experiments. *P < 0.05 versus vehicle control; ** P < 0.01 versus vehicle control

Fig. 3

NP blocks STAT3 activation and STAT3-dependent gene expression. a, MG63 cells treated with vehicle or 3 μM NP for 24 h were analyzed by immunofluorescence using anti-phospho-STAT3^Tyr705 antibodies. b, Cytoplasmic extracts from osteosarcoma cells treated with NP were analyzed by Western blot using anti-STAT3, anti-phospho-STAT3^Tyr705, anti-phospho-STAT3^Ser727, and anti-GAPDH antibodies as described in the Methods section. c, Cytoplasmic extracts prepared from143B and MG63 cells treated with vehicle, NP, and IFN-γ for 24 h were analyzed by Western blot analysis. d, 143B and MG63 cells transiently transfected with GAS-luciferase reporter plasmids were treated with vehicle and NP (3.0 μM) for 24 h, and luciferase activity was analyzed. e, 143B and MG63 cells were treated with vehicle or indicated concentrations of NP for 24 h. The cytoplasmic extracts prepared following the treatment were analyzed by Western blot using anti–VEGF-A, anti–c-Myc, Bcl-2, anti–Bcl-xl, anti-survivin, and anti-GAPDH antibodies. f, Quantitation of Western blot using densitometry. *P < 0.05 versus vehicle control; **P < 0.01 versus vehicle control

Fig. 4

NP treatment inhibits protein synthesis in osteosarcoma cells. a, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) for 24 h, and STAT3 mRNA expression was determined by RT-PCR. b, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) with or without protein synthesis inhibitor cycloheximide (CHX) for 0, 12, and 24 h. Cytoplasmic extracts were prepared and analyzed by Western blot using anti-STAT3 antibodies. c and d, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) in the presence and absence of proteasome inhibitor MG132 (5.0 μM) for 24 h. Cytoplasmic extracts were prepared and analyzed by Western blot using anti-STAT3 antibodies. e and f, 143B and MG63 cells were treated with vehicle or NP (3.0 μM) with or without lysosome protein degradation inhibitor Baf-A1 (200 nM) for 24 h, and cytoplasmic extracts were prepared and analyzed by Western blot using anti-STAT3 antibodies. g, ¹⁴C-valine–labeled MG63 cells were treated with vehicle and NP (3.0 μM) in the presence and absence of MG132 (5.0 μM) for 4, 12, and 16 h, and protein degradation was analyzed as described in the Methods. *P < 0.05 versus vehicle control; ** P < 0.01 versus vehicle control

Fig. 5

NP regulates eIF4E and 4E-BP1 functions in osteosarcoma cells. a, Protein synthesis was measured following vehicle and NP treatment in 143B and MG63 cells at 24 h through pulse labeling with ³H-leucine. b, Cytoplasmic extracts were prepared from 143B and MG63 osteosarcoma cells at 16 and 24 h following Veh and NP (3.0 μM) treatment. The extracts were subjected to immunoprecipitation (IP) with anti-4E-BP1 antibodies (b) or cap-binding (c) and analyzed by Western blot analysis using anti-eIF4E antibodies. d, Cytoplasmic extracts were analyzed by Western blot using anti–4E-BP1, anti–phosph-4EBP1Thr37/46, anti–non-phospho-4E-BP1, anti-eIF4E, and anti-GAPDH antibodies. Arrows represent phosphorylated proteins and dotted lines represent non-phosphorylated proteins

Fig. 6

NP inhibits osteosarcoma growth in vivo. Measurement of tumor volume (a) and body weight (b) following Veh and NP treatment in osteosarcoma animals. c and d, Quantitation of lung metastases (shown by black arrows) and hematoxylin and eosin staining of lung tissues following Veh and NP treatments in mice with osteosarcoma. e, Representative micro-CT images of tibia osteosarcoma tumors in mice after treatment with Veh and NP. f, Bone volume measurements. *P < 0.05 versus vehicle control; **P < 0.01 versus vehicle control