Abstract 4133: Comparative gene expression and proteomic analysis of IGF-I and insulin signaling in a large panel of breast cancer cell lines

Abstract

Abstract IGF-I signaling is involved in tumor progression and drug resistance and many anti-IGF-IR therapeutic agents are currently in clinical trials. Moreover, growing evidence also suggests that insulin signaling may play an important role in cancer development and progression. However, little is known regarding similarities and difference in IGF-I and insulin signaling in cancer, and there are currently no biomarkers to predict patient response to IGF targeted therapy. To gain a better understanding of these signaling pathways in human breast cancer, we measured the levels of insulin receptor (IR) and IGF-IR, and the activity of insulin and IGF-I, in a large panel of human breast cancer cell lines. Comparative gene expression analysis was performed using publicly available gene expression data and IR and IGF-IR mRNA levels in 17 breast cancer cell lines were measured by Q-RT-PCR. IGF-IR and IR levels were found to be highly variable in breast cancer cell lines. MCF7 and MDA-MB-134 have high levels of IGF-IR expression and MDA-MB-468 and ZR-75-1 have high levels of IR expression, relative to other breast cancer cell lines. Reverse phase protein array (RPPA) analysis using 134 different antibodies was performed on MCF7 cells stimulated with increasing doses of insulin or IGF-I. One Way ANOVA was performed to identify proteins affected by insulin and/or IGF stimulation. Maximal response to insulin and IGF-I stimulation was observed at a 10nM concentration of both ligands. Therefore, we further performed RPPA on 21 breast cancer cell lines treated with 10nM of insulin and IGF-I for 6 time points (5min, 10min, 30 min, 6hrs, 24hrs, and 48hrs). Spearman rank correlation was performed on IR and IGF-IR protein levels to mRNA levels from Q-RT-PCR. There was a significant correlation between IGF-IR protein and mRNA levels (r=0.559, p=0.024), but not between IR protein and mRNA levels. Principle component analysis showed that MCF7, MDA-MB-134, T47D and ZR-75-1 are insulin and IGF-I responsive. Results from ANOVA with contrast in MCF7 cells showed that insulin and IGF-I affected similar set of proteins at 10 minute and 24 hours time points, however, there were several proteins affected only by IGF-I or insulin stimulation. For example, phospho-estrogen receptor (p-ER) was found to be increased by 10 minutes IGF-I stimulation, but not by insulin stimulation. Beta-catenin and c-Myc were significantly suppressed by IGF stimulation while PARP was significantly suppressed by insulin stimulation at the 24 hour time point. Moreover, IGF affected twice the number of proteins compared to insulin at the 48 hour time point. For instance, Bcl2 and SMAD3 were suppressed and Raf and FOXO3a were increased by IGF stimulation after 48 hours of stimulation. Further statistical and system biology analysis of the temporal activation of signaling by IGF-I and insulin in the 21 cell lines is ongoing and will be presented. 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 4133.