The 4q12 amplicon in malignant peripheral nerve sheath tumors: consequences on gene expression and implications for sunitinib treatment

Abstract

Background: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive tumors which originate from Schwann cells and develop in about 10% of neurofibromatosis type 1 (NF1) patients. The five year survival rate is poor and more effective therapies are needed. Sunitinib is a drug targeting receptor tyrosine kinases (RTK) like PDGFRalpha, c-Kit and VEGFR-2. These genes are structurally related and cluster on chromosomal segment 4q12.

Methodology/principal findings: Here we characterize this region by multiplex ligation-dependent probe amplification (MLPA) in MPNST. Our probe set encompasses the 3 adjacent RTK genes (PDGFRA, KIT, KDR) and 6 flanking genes. We found amplification of several genes within this region in a subset of MPNST and MPNST cell lines. Transcript and protein expression of PDGFRA matched well with its increased copy number suggesting a central role of PDGFRA within the amplicon. Studying the effect of sunitinib on 5 MPNST cell lines revealed that cell line S462 harboring the 4q12 amplicon was extremely sensitive to the drug with an IC50 below 1.0 microM. Moreover, sunitinib induced apoptosis and prevented PDGF-AA induced signaling via PDGFRalpha as determined by western blotting. Co-expression of VEGF and its receptor VEGFR-2 (KDR) was present in MPNST cell lines suggesting an autocrine loop. We show that VEGF triggered signal transduction via the MAPK pathway, which could be blocked by sunitinib.

Conclusions/significance: Since multiple receptors targeted by sunitinib are expressed or over-expressed by MPNST cells sunitinib appears as an attractive drug for treatment of MPNST patients. Presence of the 4q12 amplicon and subsequent over-expression of PDGFRA might serve as predictive markers for efficacy of sunitinib.

Figure 1. Amplification and expression of genes located on chromosomal segment 4q12.

A) Amplification pattern of 9 genes in an MPNST and its corresponding cell line S462. Sunitinib targets are highlighted in grey. B) Transcript and protein levels in 5 MPNST cell lines, dermal fibroblasts and neurofibroma derived Schwann cells (SC).

Figure 2. PDGFRA and CHIC2 transcript levels in MPNST, plexiform neurofibromas (pNF) and dermal neurofibromas (dNF) as determined by real time PCR.

Normal nervous tissue and brain served as control. PCR was performed in triplicates and accepted when Ct value variation was less than 0.5 cycles. MPNST with gene amplification are marked with *.

Figure 3. Effect of sunitinib on MPNST cell lines and fibroblasts.

A) Dose dependent inhibition of MPNST cell lines after 4 days of treatment. B) For better comparability of cell lines treatment with 1.0 µM sunitinib is depicted in the bar chart. The dashed line marks IC₅₀. C) Detection of apoptosis in S462 cells treated for 3 days with 10µM sunitinib. Note formation of apoptotic bodies by DAPI staining (upper panel) and DNA fragmentation as detected by the Apoptag assay (lower panel).

Figure 4. Expression of VEGF and VEGFR-2 in MPNST cell lines and inhibition of growth factor induced signaling by sunitinib.

A) Transcript levels of KDR (VEGFR-2) and VEGF. Corresponding concentrations of VEGF in cell lysates is shown below the bar chart (nt: not tested; ND: not detected). On the right side cytochemistry of VEGF and VEGFR-2 in S462 cells is shown. B) Serum starved MPNST cell lines ST88-14 and S462 were stimulated with PDGF-AA (50ng/ml). Where indicated cells were pre-incubated with 5µM sunitinib for 1h to block signaling. C) Serum starved HUVECs were stimulated with VEGF-165 (100ng/ml) or HUVEC medium (HM). D) VEGF-165 (100ng/ml) was used to stimulate serum starved NSF1 cells. Sunitinib blocked VEGF induced signaling. E) VEGF-165 and conditioned S462 medium (CM) was used to stimulate serum starved S462 cells. EGF (100ng/ml) served as positive control. Cells were pre-incubated with 5µM sunitinib where indicated. Densidometric analysis of pMAPK/total MAPK is shown in the bar chart.