S and development. Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes which are activated through phosphorylation or over-expression in the development and progression of many types of cancer. They have emerged as promising drug targets for cancer therapy. In the current study, we identified thirteen phosphorylated RTKs that were over-expressed in three RMS cell lines RD (ERMS), RW9019 (ARMS) and RH30 (ARMS) using the phospho-RTK array (Figure 1). Importantly, other groups have also reported the over-expression of several RTKs in these cell lines. For instance, it has been reported that IGF-IR is highly expressed in RD and RH30 cell lines and could be a potential therapeutic target for RMS both in vitro and in vivo [19]. In addition, EGFR is highly expressed in ERMS tumor tissue [20-22]. Expression of ErbB2 is more prevalent in ARMS tumor tissue where it is found in the majority of RMS tumors of the head and neck [23]. ErbB3 is overexpressed in RMS cells and may play a role in regulating differentiation [24]. We have focused on another RTK, HGFR/c-Met, mainly due to its over-expression in all three RMS cell lines, which was consistent with previous reports [25,26]. We found that 14 of 24 (58.3 ) RMS tumor tissues showed high expression level of phosphorylated cMet (Figure 2). Over-expression of HGF and c-Met has been reported to correlate with increased aggressiveness of tumors and a poor prognosis in cancer patients [27]. In tumor cells, c-Met activation triggers a diverse series of signaling cascades resulting in cell growth, proliferation, invasion, and protection from apoptosis [28,29]. Data from cellular and animal tumor models suggest that the underlying biological mechanisms for tumorgenicity of c-Met are achieved in three different ways: (i) with the establishment of HGF/c-Met autocrine loops; (ii) via c-Met or HGF over-expression; and (iii) in the presence of kinase-activating mutations in the c-Met PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28878015 receptor coding sequence [28,30-32]. There were no activating mutations in the tyrosine kinase region of the c-Met receptor in the RMS cell lines used in our experiments (RD, CW9019 and RH30), and no functional autocrine regulatory loops were present. One explanation for higher expression of c-Met in these RMS cells is modulation by the PAX3/7-FOXO1 fusion gene [33]. In this study, both ARMS cells lines have the PAX3FOXO1 (RH30) and PAX7-FOXO1 (CW9019) translocations and express more phosphorylated c-Met than the PAX-FOXO1-negative ERMS cell lines (RD). It has been proposed that targeting c-Met by novel biological agents will inhibit tumor progression at the molecular level. Recently, cell proliferation in vitro and tumor burden in mouse xenograft models weredecreased by targeted knockdown of c-Met using siRNA in human RMS cell lines [34,35]. In order to improve clinical application of this concept, several different strategies are being explored, including the development of competitors of c-Met/HGF, monoclonal antibodies directed against HGF and c-Met, and small-molecule tyrosine kinase inhibitors directed against c-Met [8]. We hypothesized that inhibition of phosphorylation on c-Met with the specific, small-molecular inhibitor Lixisenatide biological activity SU11274 might induce anti-tumor effects. We examined the cytotoxicity of SU11274 without and with HGF treatment on three RMS cell lines and one normal muscle cell line (Figure 3A and 3B). SU11274 inhibited the proliferation of RMS cells that exhibited high levels of phosphory.
