Sorafenib inhibits growth and mitogen-activated protein kinase signaling in malignant peripheral nerve sheath cells

Abstract

Malignant peripheral nerve sheath tumors (MPNST) are soft-tissue tumors with a very poor prognosis and largely resistant to chemotherapy. MPNSTs are characterized by activation of the Ras pathway by loss of tumor suppressor neurofibromatosis type 1. In view of this, MPNST may be susceptible to inhibition of the activated Ras/Raf/mitogen-activated protein kinase pathway by the B-Raf inhibitor sorafenib. MPNST (MPNST and ST8814) and dedifferentiated liposarcoma (LS141 and DDLS) human tumor cell lines were characterized for Ras activation and B-Raf expression. Tumor cells were treated with sorafenib and examined for growth inhibition, inhibition of phospho-MEK, phospho-ERK, cell cycle arrest, and changes in cyclin D1 and pRb expression. MPNSTs were sensitive to sorafenib at nanomolar concentrations. This appeared to be due to inhibition of phospho-MEK, phospho-ERK, suppression of cyclin D1, and hypophosphorylation of pRb at the CDK4-specific sites, resulting in a G(1) cell cycle arrest. These effects were not seen in the liposarcoma cells, which either did not express B-Raf or showed decreased Ras activation. Small interfering RNA-mediated depletion of B-Raf in MPNSTs also induced a G(1) cell cycle arrest in these cells, with a marked inhibition of cyclin D1 expression and Rb phosphorylation, whereas depletion of C-Raf did not affect either. With growth inhibition at the low nanomolar range, sorafenib, by inhibiting the mitogen-activated protein kinase pathway, may prove to be a novel therapy for patients with MPNST.

Figure 1. Detection of active Ras, and B-Raf and C-Raf expression in STS cell lines

A. In vitro Ras activity in cells lysates (500μg) from each cell line were incubated with GST-Raf1-RBD and a SwellGel® Immobilized Glutathione Disc to pull down active Ras. The eluted samples and 25 μg of whole cell lysates were analyzed by Western blotting using an anti-Ras mouse monoclonal antibody. B. Cell extracts from STS cells were prepared and examined by Western blotting for B-Raf and C-Raf. Membranes were stripped and re-probed with an anti-KU-70 antibody to confirm equal protein loading.

Figure 2. Effect of sorafenib therapy on STS growth inhibition

STS cell lines in log phase growth were incubated with sorafenib at the indicated concentrations for 4 days. Cell viability was determined using the Cell Counting Kit 8 in 96 well plates. Points, mean percent absorbance of three replicate wells relative to untreated controls. The experiments were repeated on three separate occasions with similar results.

Figure 3. Inhibition of the MAPK pathway in sorafenib-sensitive MPNST cells

A. STS cell lines were treated with 100 nM sorafenib (+) for 24 hours. Cell extracts were analyzed by Western blotting for a p-MEK, total MEK, p-Erk, and total Erk. B. Time-dependent expression of Erk and MEK in MPNST and ST8814 cells treated with 100 nM sorafenib for 1 to 24 hours. KU-70 antibody was used to confirm equal protein loading. C. Effect of 100 nM sorafenib treatment for 24 hours on Mcl-1 protein levels in STS cell lines. KU-70 antibody was used to confirm equal protein loading.

Figure 4. Effect of sorafenib on cell distribution, pRb and cyclin D1 expression in MPNST treated cells

A. Cells were treated with 100nM of sorafenib for 24 hours, labeled with MPM2, stained with propidium iodide and analyzed by flow cytometry. B. Expression of phospho-Rb (pRb) at CDK-4 specific sites S⁸⁰⁷/S⁸¹¹, total Rb and cyclin D1 in cells treated with 100 nM sorafenib (+) for 24 hours. The expression of KU-70 was used to confirm equal protein loading.

Figure 5. Effect of B-Raf and C-Raf siRNA suppression on the cell cycle distribution, pRb and cyclin D1 expression in MPNST cells

MPNST and ST8814 cells were transfected with B-Raf, C-Raf-specific siRNAs, or control siRNA. A. The expression of B-Raf, C-Raf, pRb, and cyclin D1 were analyzed by Western blotting. The expression of α-tubulin was used to confirm equal protein loading. B. Cell cycle analysis was carried out 3 days after transfection, by flow cytometry. Cells transfected with B-Raf siRNA were also co-treated with 100 nM sorafenib. A representative experiment is shown.