*2.5. Detailed Analysis of Signaling Pathways Revealed Constitutive Phosphorylation of MEK1/2 and ERK1/2 Proteins*

In the next step, we analyzed the phosphorylation of PDGFR-beta, EGFR and downstream kinases, which can be activated by these RTKs after treatment with kinase inhibitors. In all experiments, cells were cultivated for 24 h in medium containing an inhibitor but not FCS. After 24 h, some cells were stimulated with PDGF-BB or/and EGF for 15 min to observe the effects of inhibitors on ligand-stimulated cells. Cells that were serum starved for only 24 h, and cells that were cultivated with FCS were used as negative controls.

Sunitinib alone decreased the phosphorylation of PDGFR-beta (Figure 4C). Akt phosphorylation was also decreased after sunitinib treatment, but a substantial decrease in MEK1/2 and ERK1/2 phosphorylation was not observed. Erlotinib decreased the phosphorylation of EGFR, but only at higher concentrations, and Akt phosphorylation was also slightly decreased (Figure 4D). No effect of erlotinib on MEK1/2 and ERK1/2 phosphorylation was observed. Surprisingly, U0126 did not decrease the phosphorylation of MEK1/2 (Figure 4E). Similarly, FR180204 treatment had no effect on ERK1/2 phosphorylation (Figure 4F). As expected, the combination of sunitinib and erlotinib markedly decreased the phosphorylation of PDGFR-beta, EGFR and Akt, but no effect was observed on MEK1/2 and ERK1/2 phosphorylation (Figure 4G).

Altogether, sunitinib and erlotinib showed inhibitory effects on RTKs and Akt. Interestingly, no substantial changes in MEK1/2 and ERK1/2 phosphorylation were observed after treatment with any inhibitor.

#### *2.6. Serum Starvation of NSTS-47 Cells Induces an Increase in PDGFA Expression*

In some cases, our data indicated higher phosphorylation of PDGFR-beta and EGFR in serum-starved cells than in cells cultivated in DMEM supplemented with FCS (Figure 4A,B). Therefore, the expression of selected EGFR and PDGFR-beta ligands was measured to investigate whether there is a possible autocrine PDGF/PDGFR or EGF (TGF-alpha)/EGFR signaling loop that could contribute to the higher phosphorylation of RTKs. Expression of *EGF*, *PDGFA*, *PDGFB* and *TGFA* was analyzed under normal serum conditions (DMEM supplemented with 20% FCS) and under serum starvation conditions using qPCR. Substantial differences were observed in the transcriptional response of serum-starved cells (Figure 5). qPCR analyses also showed increased levels of *PDGFA* expression, while *EGF* and *PDGFB* mRNA levels were not significantly influenced by serum starvation, and *TGFA* expression was considerably decreased.

**Figure 5.** Effect of serum starvation on *EGF*, *PDGFA*, *PDGFB* and *TGFA* expression in the NSTS-47 cell line. Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% FCS or in DMEM without FCS. After 24 h, cells were harvested, and the expression of selected genes was analyzed using qPCR. The results represent the mean ± SD of nine (six in case of *PDGFB*) independent experiments. \* *p* < 0.05 indicates statistically significant differences.

#### **3. Discussion**

IM is a rare disorder of mesenchymal proliferation that is characterized by the development of nonmetastatic tumors [1]. Several studies have confirmed that specific point mutations in the *PDGFRB* gene are involved in the pathogenesis of IM [1,7,11]. However, mutations in the *PDGFRB* gene presumably show incomplete penetrance and variable expressivity, and other genes may be involved in the pathogenesis of IM [1,18,19].

The main goal of this study was to analyze the effects of various protein kinase inhibitors (PKIs) on the NSTS-47 cell line, which harbors the IM-associated c.1681C>T (p.R561C) mutation in *PDGFRB*. The results showed that sunitinib, a potent inhibitor of PDGFR-beta phosphorylation, can significantly decrease the proliferation of NSTS-47 cells.

Previously published results [21] show that PDGFR-beta p.R561C mutant cells have constitutively phosphorylated PDGFR-beta and are able to induce the phosphorylation of ERK1/2, PLC-gamma, STAT3, STAT5 and Akt in the absence of PDGF. These results are in accordance with our observations. We found that PDGFR-beta and ERK1/2 kinases were highly phosphorylated in both s and even in NSTS-47 cells that were serum starved for 24 h. We also detected increased phosphorylation of Akt2 in Tumor Sample 1.

The same study that revealed a role for the p.R561C mutation in PDGFR-beta [21] showed that imatinib, nilotinib, and ponatinib can decrease PDGFR-beta phosphorylation and inhibit cell proliferation. We studied the effects of sunitinib, a multi-tyrosine kinase inhibitor that is able to target PDGFR-beta. Sunitinib was chosen because siblings from whom tumor tissue samples were obtained responded very well to treatment with this inhibitor [8]. Sunitinib alone significantly decreased the proliferative activity of the NSTS-47 cell line, and this finding could explain the response of the siblings to the targeted therapy.

Western blot analyses showed that sunitinib is able to decrease the phosphorylation of mutant PDGFR-beta even in the presence of high PDGF-BB levels and can also decrease the phosphorylation of Akt. Because activated Akt is a well-established survival factor [28], these effects of sunitinib on PDGFR-beta and Akt phosphorylation can explain why sunitinib reduced the proliferative activity of NSTS-47 cells.

A similar inhibitory effect was observed for EGFR and erlotinib (the inhibitor of EGFR phosphorylation). Erlotinib also decreased NSTS-47 cell proliferation, and Western blot analysis showed that it was able to decrease EGFR and Akt phosphorylation. However, neither sunitinib nor erlotinib inhibited the phosphorylation of the corresponding receptor completely, and some receptor molecules remained phosphorylated even when high doses of those inhibitors were used.

Surprisingly, phosphorylation of MEK1/2 and ERK1/2 proteins was not significantly influenced by any inhibitor. This observation could explain why sunitinib and erlotinib incompletely decreased proliferative activity and why U0126 and FR180204 did not influence proliferative activity. MEK1/2 and ERK1/2 belong to the Ras/MAPK signaling cascade, which transmits signals from receptors and participate in regulating the cell cycle, apoptosis and differentiation [29]. All tyrosine kinase inhibitors have been previously shown to be able to simultaneously decrease PDGFR-beta and ERK1/2 phosphorylation, which resulted in the inhibition of proliferative activity [21]. In NSTS-47 cells, sunitinib and erlotinib decreased the phosphorylation of PDGFR-beta, EGFR and Akt, but for yet unknown reasons, MEK1/2 and ERK1/2 kinases remained phosphorylated at levels that were comparable with those detected in untreated cells.

Interestingly, incomplete penetrance of the c.1681C>T (p.R561C) mutation was found in a family with two children suffering from IM [19]. Genetic analyses revealed a c.1681C>T (p.R561C) mutation in *PDGFRB* in both siblings and, surprisingly, also in their healthy mother. However, both siblings had inherited a heterozygous c.1276G>A (p.V426M) mutation in *PTPRG* from their healthy father. The *PTPRG* gene encodes a protein called receptor-type tyrosine-protein phosphatase gamma that can dephosphorylate PDGFR-beta [19,20]. Therefore, the mutation in *PTPRG* could probably decrease the efficiency of the phosphatase to dephosphorylate its substrates and thus positively influence the phosphorylation of PDGFR-beta and the penetrance of mutant *PDGFRB* [19].

Finally, our analyses of gene expression showed that the phosphorylation status of PDGFRs in NSTS-47 cells was not influenced by only mutations in PDGFR-beta. We analyzed the gene expression levels of *EGF*, *PDGFA*, *PDGFB* and *TGFA* in NSTS-47 cells that were serum starved for 24 h. The expression of *TGFA* decreased, but no difference was observed in the expression of *EGF* and *PDGFB*; however, *PDGFA* gene expression was significantly increased. The increase in *PDGFA* expression was unexpected and could result in the stimulation of cells via an autocrine mechanism, an increase in PDGFR-alpha phosphorylation and improved survival of NSTS-47 cells in the absence of serum.
