ACTIVITY-BASED PROTEIN PROFILING OF HUMAN BREAST CANCER

INTRODUCTION. Considering that proteins mediate nearly all biochemical events underlying pathophysiological processes such as cancer, the need to develop general methods to measure levels and activities of these biomolecules is apparent. To this end, a chemical proteomics method, "activity-based protein profiling" (ABPP), was developed to compliment conventional genomic and proteomic methods that focus on measuring abundance rather than activity (1-3). We have previously demonstrated the utility of ABPP for the functional analysis of human cancer (4, 5), and demonstrated the dramatic functional differences that exist between cancer cells grown in vitro (cultured cancer cell lines) and in vivo (xenografts), both of which serve as important, widely used, research models of human cancer. Here, we describe further results from ABPP analysis of orthotopic xenograft tumors formed by the invasive breast cancer line MDA-MB- 231 that suggest that the in vivo microenvironment of the mouse mammary fat pad (mfp) cultivates the growth of human breast cancer cells with elevated tumorigenic properties. METHOD. Orthotopic xenograft tumors were established with the invasive human breast cancer line MDA-MB-231 in the mouse mfp of immunodeficient SCID mice. Isolated tumors were then compared to cultured preparations of MDA-MB-231 cells by treatment with ABPP probes, and subsequent in-gel fluorescent analysis. Molecular identification of tumor enzymes was carried out by mass-spectrometry analysis, as previously described (1, 2). RESULTS AND DISCUSSION. Cancer cells were isolated from MDA-MB- 231 tumors and recultured to provide a propagatable subpopulation referred to as 231mfp cells, a name intended to signify the in vivo passaging of these cells as tumors in the mouse mfp prior to culturing. Comparative ABPP analysis of 231mfp and parental MDA-MB-231 cells revealed dramatic differences in the levels of specific enzyme activities, including upregulation of the serine proteases tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the 231mfp line. The distinct enzyme activity profiles of MDA-MB-231 cells grown in vitro (parental) and in vivo (231mfp) indicate that these lines most likely represent two fundamentally different populations of cells, with the 231mfp line