IGF1R Endocytosis in Ewing Sarcoma Figure 5. Effects of IGF1R tyrosine kinase inhibition combined with CAV1 or clathrin inhibition on ES cell proliferation and apoptosis. A) Isobolograms of AEW combined with MCD/CPMZ. B) Combination index (CI) of AEW combined with MCD/CPMZ. AEW combined with MCD shows additive effects in the inhibition of ES cell line proliferation (CI around 1). In contrast, regarding AEW combined with CPMZ the effects were synergistic (CI between 0.73–0.86) in the A4573 and TC71 cell lines, and only additive in the A673 cell line. C) AEW combined with MCD/CPMZ had additive effects on the induction of apoptosis in ES cell lines, mainly inducing late apoptosis, with few cells undergoing necrosis. A: AEW, C: CPMZ and M: MCD. Data presented as the average of 4–6 independent replicates. doi:10.1371/journal.pone.0019846.g005 endocytosis mainly regulates the RAS/RAF/MEK/ERK path- way in ES. Moreover, we believe that endocytosis further improves the IRS1/PI3K/AKT signaling pathway, since CPMZ treatment also reduced AKT, TOR and p70S6K phosphorylation. None- theless, we assume that the PI3K/AKT pathway undergoes its strongest activation either directly from the membrane, with IRS1 activation independent of IGF1R internalization (like the one reported by Chow et al.,[36]) or with an extra regulation, through caveolin-dependent internalization [13]. The data supporting this assumption show that the phosphorylation of AKT is already reduced by about 50% with AEW treatment (confirming that IGF1R signaling pathway activation is not totally dependent upon internalization), even though it showed a large improvement with the triple treatment, 80% of inhibition being reached. More importantly, the residues whose phosphorylation is reduced in the AKT, TOR, AMPK and p70S6K proteins are different, depending on the treatment used (CPMZ vs MCD vs combina- tion). This constitutes additional evidence that endocytosis not only provides additional control over the ‘‘quantity’’ of RTKs signaling, but also over the ‘‘quality and selectivity’’ of such signaling. The model suggested here (see figure 7) provides new insight into the selectivity and specificity of IGF1R signaling driven from different cellular compartments. With the studies on the effects of AEW541 and the CPMZ/ MCD combination on ES cells, here we demonstrate that IGF1R tyrosine kinase inhibition is improved when combined with CAV1 or clathrin inhibition, reducing the levels of proliferation and the induction of apoptosis in ES cell lines. The effects of the drug combination had an impact on the phosphorylation status of several proteins, namely AKT, SRC p38, TOR, MSK1/2, MEK1/2, among others, confirming once again that IGF1R signaling depends on its internalization. Also, proteins such as HSP27, AKT, b-catenin, AMPKa and STAT2/4/3 exhibited the highest reduction in phosphorylation with the triple treatment, supporting the idea of combining TK inhibitors with internaliza- tion blockers in order to decrease signaling. It should be noted that RTK-blocking antibodies would, in theory, only block receptors located in the cellular membrane, not affecting the receptors already internalized. In this case, the small-molecule tyrosine kinase inhibitors would be a better choice since they would diffuse to the cytoplasm and also inhibit some of the receptors within the endocytic vesicles. PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19846