(283b) Profiling Signaling Pathways Using Comparative Proteomics: An Illustrative Example From Breast Cancer

Genetic rearrangement in cancer alters the relative contributions of different cellular signaling pathways. The objective of this study was to identify the predominant protein signaling pathways associated with two breast cancer cell lines using proteomic profiling.

A comparative proteomic analysis of total cell lysates was obtained from two cellular models of breast cancer: BT474 (HER2+/ER+) and SKBR3 (HER2+/ER-). 2-DE was used to analyze the proteome. Differentially expressed proteins were detected using SameSpots and identified using MALDI-TOF-MS. Protein networks and canonical pathways were identified from the Ingenuity Pathway Knowledgebase (IPK) based on their significance to the differentially expressed proteins.

Of the 803 common protein spots between the two cell lines, 304 were picked and 167 identified by peptide mass fingerprinting. A threshold of 1.5-fold was used to select 62 proteins used in the analysis. IPK analysis suggested that metabolic pathways were highly associated with protein expression in SKBR3 while cell motility pathways were highly associated with BT474. Inferred protein networks were confirmed by observing an up-regulation of IGF-1R and profilin in BT474 and up-regulation of Ras and enolase in SKBR3 using western blot.

We have performed a proteomic profiling study of these human breast cancer cell lines and identified proteins that are up-regulated and pathways that are altered. Future efforts will focus on understanding the underlying mechanisms by which these pathways become altered upon response to molecularly targeted therapies.