[Show abstract][Hide abstract] ABSTRACT: Achieving information content of satisfactory breadth and depth remains a formidable challenge for proteomics. This problem is particularly relevant to the study of primary human specimens, such as tumor biopsies, which are heterogeneous and of finite quantity. Here we present a functional proteomics strategy that unites the activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT) for the streamlined analysis of human samples. This convergent platform involves a rapid initial phase, in which enzyme activity signatures are generated for functional classification of samples, followed by in-depth analysis of representative members from each class. Using this two-tiered approach, we identified more than 50 enzyme activities in human breast tumors, nearly a third of which represent previously uncharacterized proteins. Comparison with cDNA microarrays revealed enzymes whose activity, but not mRNA expression, depicted tumor class, underscoring the power of ABPP-MudPIT for the discovery of new markers of human disease that may evade detection by other molecular profiling methods.
[Show abstract][Hide abstract] ABSTRACT: Human cell lines constitute powerful model systems for the in vitro and in vivo analysis of cancer. Cancer lines that are invasive in culture often form tumors and metastases in immune deficient mice. It is generally assumed that, in such cases, the principal population of cancer cells in culture corresponds to the tumor-forming cells in vivo. Here, we high-light a recent functional proteomics investigation that suggests the contrary. In this study, cells derived from orthotopic xenograft tumors formed by the invasive breast cancer line MDA-MB-231 were found to exhibit profound differences in their enzyme activity profiles and increased tumor growth rates and metastasis when compared to the parental line. These findings suggest that the in vivo microenvironment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties. Characterization of the unique molecular properties of these tumor-forming cells may reveal new strategies for the diagnosis and treatment of breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Cancer research depends on the use of human cell lines for both the in vitro (culture) and in vivo (xenograft) analysis of tumor progression and treatment. However, the extent to which cultured preparations of human cancer lines display similar properties in vivo, where important host factors may influence tumor biology, remains unclear. Here, we address this question by conducting a functional proteomic analysis of the human breast cancer line MDA-MB-231 grown in culture and as orthotopic xenograft tumors in the mammary fad pad of immunodeficient mice. Using a suite of activity-based chemical probes, we identified carcinoma (human) enzyme activities that were expressed selectively in culture or in xenograft tumors. Likewise, distinct groups of stromal (mouse) enzyme activities were found that either infiltrated or were excluded from xenograft tumors, indicating a contribution by specific host components to breast cancer development. MDA-MB-231 cells isolated from tumors exhibited profound differences in their enzyme activity profiles compared with the parent cell line, including the dramatic posttranscriptional up-regulation of the serine proteases urokinase plasminogen activator and tissue plasminogen activator and down-regulation of the glycolytic enzyme phosphofructokinase. These altered enzyme activity profiles correlated with significantly greater tumor growth rates and metastases for xenograft-derived MDA-MB-231 cells upon reintroduction into mice. Collectively, these data indicate that the in vivo environment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties and highlight the value of activity-based protein profiling for identifying proteomic signatures that depict such changes in cancer cell biology.
Proceedings of the National Academy of Sciences 10/2004; 101(38):13756-61. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.
Proceedings of the National Academy of Sciences 08/2004; 101(27):10000-5. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.
Current Opinion in Chemical Biology 03/2004; 8(1):54-9. · 7.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: By primarily measuring changes in transcript and protein abundance, conventional genomics and proteomics methods may fail to detect significant posttranslational events that regulate protein activity and, ultimately, cell behavior. To address these limitations, activity-based proteomic technologies that measure dynamics in protein function on a global scale would be of particular value. Here, we describe the application of a chemical proteomics strategy to quantitatively compare enzyme activities across a panel of human breast and melanoma cancer cell lines. A global analysis of the activity, subcellular distribution, and glycosylation state for the serine hydrolase superfamily resulted in the identification of a cluster of proteases, lipases, and esterases that distinguished cancer lines based on tissue of origin. Strikingly, nearly all of these enzyme activities were down-regulated in the most invasive cancer lines examined, which instead up-regulated a distinct set of secreted and membrane-associated enzyme activities. These invasiveness-associated enzymes included urokinase, a secreted serine protease with a recognized role in tumor progression, and a membrane-associated hydrolase KIAA1363, for which no previous link to cancer had been made. Collectively, these results suggest that invasive cancer cells share discrete proteomic signatures that are more reflective of their biological phenotype than cellular heritage, highlighting that a common set of enzymes may support the progression of tumors from a variety of origins and thus represent attractive targets for the diagnosis and treatment of cancer.
Proceedings of the National Academy of Sciences 09/2002; 99(16):10335-40. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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