PDGFRA, PDGFRB, EGFR, and downstream signaling activation in malignant peripheral nerve sheath tumor

Abstract

We investigated the activation of platelet-derived growth factor (PDGF) receptor A (PDGFRA), PDGF receptor B (PDGFRB), epidermal growth factor receptor (EGFR), and their downstream pathways in malignant peripheral nerve sheath tumors (MPNSTs). PDGFRA, PDGFRB, and EGFR were immunohistochemically, biochemically, cytogenetically, and mutationally analyzed along with the detection of their cognate ligands in 16 neurofibromatosis type 1 (NF1)-related and 11 sporadic MPNSTs. The activation of the downstream receptor pathways was also studied by means of v-akt murine thymoma viral oncogene homolog (AKT), extracellular signal-regulated kinase (ERK), and mammalian target of rapamycin (mTOR) Western blotting experiments, as well as rat sarcoma viral oncogene homolog (RAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), phosphoinositide-3-kinase, catalytic, alpha polypeptide (PI3KCA), and phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutational analysis and fluorescence in situ hybridization. PDGFRA, PDGFRB, and EGFR were expressed/activated, with higher levels of EGFR expression/phosphorylation paralleling increasing EGFR gene copy numbers in the NF1-related cases (71%). Autocrine loop activation of these receptors along with their coactivation were suggested by the expression of the cognate ligands in the absence of mutations and the presence of receptor tyrosine kinase (RTK) heterodimers, respectively. Both MPNST groups showed AKT, ERK, and mTOR expression/phosphorylation. No BRAF, PI3KCA, or PTEN mutations were found in either group of MPNSTs, but 18% of the sporadic MPNSTs showed RAS mutations. PTEN monosomy segregated with the NF1-related cases (50%, p = 0.018), but PTEN protein was expressed in all but two cases. In conclusion, PDGFRA, PDGFRB, and EGFR seem to be promising molecular targets for tailored treatments in MPNST. In particular, the ligand- and heterodimerization-dependent RTK activation/expression coupled with a downstream signaling phosphorylation, mediated by the upstream receptors or RAS activation, may provide a rationale to apply combined RTK and mTOR inhibitor treatments both to sporadic and NF1-related cases.

Fig. 1

(A–C) Receptor tyrosine kinase (RTK) biochemical analyses. (A) Platelet-derived growth factor (PDGF) receptor A (PDGFRA) immunoprecipitation (IP) and Western blotting (WB) experiments revealed a 170-kDa band corresponding to the activated (upper arrow) and expressed (lower arrow) receptor in the tumor samples. PDGFRA phosphorylation levels were similar to (cases 10 neurofibromatosis type 1 [NF1], 7 NF1, and 2s) or less intense than (cases 11s and 5s) those found in the positive control (NIH3T3 cell line). pTyr, phosphorylated tyrosine. (B) PDGF receptor B (PDGFRB) IP and WB revealed a 180-kDa band corresponding to activated (upper arrow) and expressed (lower arrow) PDGFRB in all analyzed cases. The samples showed different phosphorylation levels: similar to (NF1-related cases 10 [10NF1] and 7NF1), more intense than (case 2NF1), or less intense than (cases 6s and 2s) those observed in the 2N5A cells used as positive controls. (C) Epidermal growth factor receptor (EGFR) analysis revealed a band of 170 kDa in all of the analyzed samples, thus indicating the presence of activated (upper arrow) and expressed (lower arrow) EGFR. The level of phosphorylation was similar to (cases 12s and 2s) or more intense than (cases 5NF1 and 8NF1) those observed in the positive controls (Cal27 cell line). C+, positive control; s, sporadic case. (D–F) RTK fluorescence in situ hybridization analyses. (D) The nuclei in sporadic case 2 showed PDGFRA gene amplification (green cluster) in a high-polysomy chromosome 4 (centromere 4, red signals) where PDGFRA is located (green signals). (E) The nuclei in NF1-related case 2 showed a high-polysomy chromosome 5 (centromere 5, red signals) where PDGFRB is located (green signals). (F) The nuclei in NF1-related case 1a showed EGFR gene amplification (red cluster) in disomy represented by two centromere 7 signals (green signals).

Fig. 2

Epidermal growth factor receptor (EGFR)–platelet-derived growth factor (PDGF) receptor A (PDGFRA) and EGFR–PDGF receptor B (PDGFRB) heterodimerization. (A) EGFR immunoprecipitation (IP) and PDGFRA Western blotting (WB) experiments revealed a 170-kDa band corresponding to PDGFRA showing similar expression in all three analyzed cases (sporadic cases 2 [2s] and 12s and NF1-related case 10 [10NF1]). NIH3T3 cell lines were used as positive control for PDGFRA. Afterward, the filter was stripped and incubated with EGFR antibody, which revealed a band of 170 kDa in all of the analyzed samples, thus indicating less intense expression of EGFR than observed in the positive control A431 cells. (B) EGFR IP and PDGFRB WB experiments revealed a 180-kDa band corresponding to PDGFRB showing similar expression in all three analyzed cases (cases 2s, 10NF1, and 12s). 2N5A cell lines were used as positive control for PDGFRB. Afterward, the filter was stripped and incubated with EGFR antibody, which revealed a band of 170 kDa in all of the analyzed samples, thus indicating the expression of EGFR.

Fig. 3

PTEN, AKT, ERK, and mTOR biochemical analysis. (A) Membrane incubation with phosphorylated Akt Ser 473 and anti-Akt polyclonal antibodies revealed a 60-kDa band corresponding to the activated (upper arrow) and expressed (lower arrow) Akt form. The samples showed phosphorylation levels similar to (NF1-related case 10 [10NF1]) or more (case 8NF1) or less (sporadic cases 7s and 1s) intense than 2N5A cells were used as positive controls. (B) Membrane incubation with both phosphorylated ERK and anti-ERK polyclonal antibodies revealed two bands of 42 and 44 kDa, corresponding to the ERK1 and ERK2 activated (upper arrow) and expressed (lower arrow) forms, respectively. ERK1 and ERK2 phosphorylation levels were similar to (cases 2s, 7s, and 10NF1) or less intense (case 7NF1) than those observed in the 2N5A cells used as positive controls. (C) Membrane incubation with phosphorylated mTOR and anti-mTOR polyclonal antibodies revealed a 289-kDa band corresponding to the activated (upper arrow) and expressed (lower arrow) mTOR form. Phosphorylation levels were similar to (cases 2s and 5s) or less intense (cases 7NF1 and 8NF1) than those observed in A431 cells used as positive control. (D) Membrane incubation with PTEN antibody revealed a 54-kDa band in all cases (cases 1s, 2s, 7NF1, and 11NF1) except in one case (case 5NF1) that showed reduced PTEN expression compared to A431 cell line used as positive control. C+, positive control.