Sorafenib inhibits tumor growth and vascularization of rhabdomyosarcoma cells by blocking IGF-1R-mediated signaling

Abstract

The growth of many soft tissue sarcomas is dependent on aberrant growth factor signaling, which promotes their proliferation and motility. With this in mind, we evaluated the effect of sorafenib, a receptor tyrosine kinase inhibitor, on cell growth and apoptosis in sarcoma cell lines of various histological subtypes. We found that sorafenib effectively inhibited cell proliferation in rhabdomyosarcoma, synovial sarcoma and Ewing's sarcoma with IC(50) values <5 μM. Sorafenib effectively induced growth arrest in rhabdomyosarcoma cells, which was concurrent with inhibition of Akt and Erk signaling. Studies of ligand-induced phosphorylation of Erk and Akt in rhabdomyosarcoma cells showed that insulin-like growth factor-1 is a potent activator, which can be blocked by treatment with sorafenib. In vivo sorafenib treatment of rhabdomyosarcoma xenografts had a significant inhibitory effect on tumor growth, which was associated with inhibited vascularization and enhanced necrosis in the adjacent tumor stroma. Our results demonstrate that in vitro and in vivo growth of rhabdomyosarcoma can be suppressed by treatment with sorafenib, and suggests the possibilities of using sorafenib as a potential adjuvant therapy for the treatment of rhabdomyosarcoma.

Keywords: kinase inhibitors; soft tissue sarcoma; targeted therapy; vascularization.

Figure 1

Effect of sorafenib on cell cycle in alveolar rhabdomyosarcoma (RMS) cells. A) Sorafenib inhibits proliferation of RMS in a dose dependent manner. Rhabdomyosarcoma cells were cultured and treated with 0–5 μM sorafenib for 48 hrs and visualized using light microscopy. B) Estimation of sub-G1 content. Cells were treated as above and sub-G1 fraction of cells was determined by PI staining and FACS analysis. C) Annexin V staining of sorafenib-treated RMS cells. Cells were treated with sorafenib as above and the level of Annexin V staining was determined by FACS analysis. Abbreviation: PI, propidium iodide.

Figure 2

Sorafenib inhibits Erk and Akt activation in alveolar rhabdomyosarcoma (RMS) cells. A) RMS cells were starved for 24 hrs, treated with 0–5 μM sorafenib for 4 hrs and simulated with serum for 10 minutes. Cells were harvested for western blotting by direct lysis in loading buffer. Levels of phosphorylated Erk and Akt were detected using phosphor specific antibodies. Levels of total Erk and Akt were used as a loading control. B) RMS cells were serum starved for 24 hrs, pre-treated with 5 μM sorafenib and stimulated with serum for the indicated time points. Levels of phosphor Erk and Akt were detected as above.

Figure 3

Expression of receptor tyrosine kinases in rhabdomyosarcoma cells. Rhabdomyosarcoma cells (RMS, RH30, and RD) were cultured in chamber slides, fixed and incubated with antibodies against the indicated receptor tyrosine kinase. Receptor tyrosine expression was visualized using the appropriate fluorescent conjugated secondary antibody.

Figure 4

Sorafenib inhibits activation of Erk and Akt through IGF-1 signaling in rhabdomyosarcoma cells. Rhabdomyosarcoma cells were starved for 24 hrs, treated with 5 μM sorafenib and stimulated with 50 ng/ml of the appropriate ligand (SCF, VEGF, PDGF, FLT-3 or IGF-1) or serum for 30 minutes. Cells were lysed directly in loading buffer and immunoblotting of Erk and Akt phosphorylation was performed for A) RMS, B) RH30, and C) RD cells using phosphor-specific Erk and Akt antibodies. Levels of total Erk and Akt were used as loading controls. Abbreviations: IGF, insulin-like growth factor; SCF, stem cell factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; FLT3, FMS-like tyrosine kinase 3.

Figure 5

Sorafenib inhibits IGF-1-induced cell growth in rhabdomyosarcoma cell lines. Rhabdomyosarcoma cells (RMS, RH30, and RD) were grown in the presence or absence of serum or IGF, followed by treatment with 5 μM sorafenib for 48 hrs. Cell proliferation was estimated using WST-1 proliferation reagent as previously described. Abbreviation: IGF, insulin-like growth factor.

Figure 6

Effect of sorafenib on alveolar rhabdomyosarcoma (RMS) tumor xenografts. BALB/c SCID mice were inoculated subcutaneously with RMS cells. When the tumors were palpable, the mice were treated with 40, 60, 80 mg/kg body weight sorafenib or placebo per orally for 7 days. At the end point of the experiment, mice were euthanized, tumors were excised and fixed in formalin and stained immunohistochemically with HTX, Ki-67 and CD31. A) Histogram showing the mean tumor mass at end point of experiment of rhabdomyosarcoma xenograft tumors treated with placebo or sorafenib. B) Hematoxilin (HX) and Ki-67 stained formalin-fixed paraffin embedded sections of RMS tumors. (N: necrosis, S: septa/fibrovascular tissue). C) Effect of sorafenib on tumor vascularization of formalin-fixed paraffin-embedded sections of RMS tumors were stained with CD31. Abbreviation: E, endothelial cells.