Tyrosines in the MUC1 cytoplasmic tail modulate oncogenic signaling pathways

946 MUC1 is an oncogene and tumor marker, which is overexpressed in numerous adenocarcinomas. Structurally, it is a transmembrane heterodimer with an extracellular domain composed primarily of heavily glycosylated twenty amino acid tandem repeats and a 72 amino acid cytoplasmic tail (CT). Several protein-protein interaction motifs have been identified in the CT that contain tyrosines, phosphorylation of which leads to the recruitment of receptor and non-receptor tyrosine kinases. The impact of subsequent signaling events is poorly understood. We have developed a cell line based model system to study the significance of tyrosine based signaling of the MUC1 CT within the context of full-length MUC1. COS-7 cells, a MUC1 non-expresser, were utilized to prevent the effects of signaling due to endogenous expression. Critical function(s) of the MUC1 CT tyrosines was addressed by direct comparison of cells expressing wildtype MUC1 (MUC1WT) and MUC1 lacking tyrosines in the CT (MUC1Y0). The broad implications of signaling via the MUC1 CT were determined by studying pathways commonly dysregulated in cancers. To this end, we first addressed the impact of MUC1-dependent signals on proliferative index (PI). Surprisingly, the presence of MUC1WT did not alter the PI, although MUC1Y0 resulted in an approximately 30-fold increase in PI as measured by 3 H-thymidine incorporation, which correlated with a robust increase in Erk1/2 phosphorylation. A downstream effector of Erk1/2 activity is AP-1, which was assayed using a luciferase reporter. Cells expressing MUC1Y0 exhibited 2.5 and 5-fold increases in AP-1 activity compared to cells expressing MUC1WT and the neo control, respectively. Secondly, we examined the invasive potential of the cells expressing MUC1WT or MUC1Y0 and found that MUC1Y0 confers a 2-3-fold increase in invasion. In line with these results, we observed a 40% decrease in basal cell death and 33% increased sensitivity to Taxol-induced cell death in the MUC1Y0 cells as compared to MUC1WT or neo control. The relevance of MUC1 CT tyrosine-mediated interactions in modulating cell death via transcriptional regulation of NF-κB complexes was analyzed using a luciferase reporter, which showed 2-fold increased activity for MUC1WT compared to the neo control, while the MUC1Y0 displayed a fifth of the neo control activity. This study demonstrates the importance of the CT tyrosines, the lack of which induced substantial changes in various signaling pathways, and demonstrates for the first time that MUC1 signals can modulate the transcriptional activity of AP-1 and NF-κB. Experiments that delineate this effect of the MUC1 CT via other interactions, which impinge on both cytoplasmic signaling and modulation of transcription of various complexes, are underway. Supported by NIH CA94389 & ACS PF-04-256-01-M601