Abstract 710: Kinetic of phosphoproteins in the PI3K and MAPK pathway reflect responsiveness of Ewing's Sarcoma cells to R1507, a human IgG1 Mab that binds IGF-IR
Authors: Hofmann Marco H. , Kuenkele Klaus-Peter 
Published: February 2011

Abstract
The Ewing Sarcoma (ES) family represents 3% of all pediatric cancers (Ludwig J.A. 2008). Following a multimodal treatment scheme ES Patients with metastases have a 5 year survival rate of less than 25% (Subbiah V. et al. 2009). For these patients, new therapy options are imperative. Specific chromosomal translocations account for 95% of all ES and lead to alteration of gene expression levels including genes up regulating IGF-IR signaling (Benini S. et al. 2004). Thus, targeting IGF-IR may be useful to interfere with tumor growth in ES.
R1507 is a human IgG1 Mab that binds IGF-IR, inhibits IGF-I and II binding, leads to down modulation of the IGF-IR receptor and inhibits proliferation of cancer cells. Responsiveness to R1507 was studied in 5 ES-cell lines. RD-ES and TC71 cell lines showed maximal inhibition of tumor cell proliferation (&gt;68%) in a 3-D, 7-days proliferation assay in vitro. MHH-ES and RH1 showed medium responsiveness (30%) but no responsiveness was observed in CADO-ES1 cells. Compared to RD-ES, CADO-ES1 harbors 5 times more insulin receptor (IR) but treatment with a combination of R1507 and IR antibody 83-14 reduced proliferation by only 11%. Thus the high expression of IR does not explain the resistance of CADO-ES1 versus R1507 treatment. Phosphorylation kinetics of 8 proteins within the Phosphoinositide 3-kinase (PI3K)-pathway and the mitogen activated protein-kinase pathway were determined with Luminex bead-based assays 0,5-50 hours after treatment with R1507.
Responsive RD-ES and MHH-ES1 cell lines showed a sustained down regulation of phospho-IGF-IR (Tyr1146) by more than 80% after treatment with R1507. The non responsive CADO-ES1 cell line showed only temporary decrease of phospho-IGF-IR by 40% after 30 minutes. IRS-1 has multiple phosphorylation sites which differentially regulate the PI3K pathway. Tyrosine phosphorylation of IRS-1 initiates the PI3K signaling. Serin phosphorylation inhibits IRS-1 signaling by uncoupling IR or IGF-IR/IRS-1 interaction and inhibiting signal transduction via tyrosine phosphorylation. After treatment with R1507, tyrosine phosphorylation of IRS-1 was decreased in responsive cell lines by 60% but phosphorylation of Ser636/Ser639 was increased 2,5-fold. To analyze this effect in more detail, a panel of 13 human tumor cell lines was analyzed. Correlation of increased Ser636/Ser639 IRS-1 phosphorylation and responsiveness towards R1507 was observed in most cell lines. Furthermore in responsive cell lines a 60% reduction of phospho-Akt (Ser473) and phospho-p70S6kinase (Thr421/Ser424) can be observed. In contrast no effect on phosphorylation status for IRS-1, Akt and p70S6 kinase was observed in non responsive cell line CADO-ES1.
In conclusion the kinetic of phosphoproteins and especially the phosphorylation status of IRS-1 might be indicative for sensitivity of cancer cells to IGF-IR targeting reagents.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 710.