Profiling Enzyme Activities in Models of Human Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Benjamin Cravatt, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8WB-0016 Award: $369,508
Award Type: STEP Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2002)
The key molecular features of metastatic breast carcinomas that support their motile and invasive behavior are complex and poorly defined. It is thought that proteins, like proteases and catabolic enzymes, play a central role in allowing cell migration leading to metastasis to distant sites in the body. Conventional genomics and proteomics methods measure changes in transcript (RNA) and protein abundance, respectively, and therefore provide only an indirect assessment of protein function. For example, posttranslational forms of enzymes would remain undetected by these approaches. In our research group, we are addressing the need for higher-order functional proteomics technologies by developing and applying chemical probes that measure protein activity rather than abundance. We refer to this method as "activity-based protein profiling" (ABPP), and propose in this application, to apply ABPP to the analysis of enzyme activities expressed by human breast cancer line xenografts, in particular those protein enzmes related to cell migration.

Our aim is to provide a dynamic view that assigns "activity" (i.e, catalytic power) to specific changes during tumorigenesis.

We have developed and applied ABPP to whole cell, tissue, and fluid samples. In our initial research efforts, supported by a 2000-2002 CBCRP award, we successfully applied ABPP probes to the proteomic analysis of several enzyme classes across a panel of human breast cancer lines. These studies resulted in the identification of multiple enzyme activities, including several serine proteases, lipases, and glutathione S-transferases, that were differentially expressed in estrogen receptor (ER)(+) and ER(-) breast cancer lines.

A critical next step in validating the association of these enzymes with breast cancer is to demonstrate that their expression pattern is maintained in breast tumors in vivo, a setting more relevant to the actual disease. Accordingly, we now propose to determine the enzyme activity profiles of tumors and metastases derived from human breast cancer line xenografts grown in immunodeficient mice. We intend to take advantage of the mixed species nature of this breast cancer xenograft model to distinguish tumor enzyme activities that are expressed by cancer cells (human) and by infiltrating stroma (mouse). Finally, we will determine the enzyme activity profiles of metastasized variants of human breast cancer lines (e.g., bone, lung, and lymph nodes) and compare these ABPP profiles to those of the parent lines.

By extending our studies to more relevant models of the disease, adding the stromal component, and studying metastatic variants from primary tumors we hope to better place specific proteases as promising targets for diagnosis and treatment of breast cancer.

Final Report (2004)
Note: The abstract below was derived from the PNAS paper conclusions section from the PIís lab and collaborators that summarize the project. In addition, the PI used CBCRP funding to develop the basic methodology of the ABPP proteomic method, which is summarized in a recent paper (Chemistry and Biology,11:535-46, 2004).

Using a suite of chemical probes for our proteomic approach, called ABPP, we have comparatively profiled human breast cancer cells grown in culture and during and after in vivo passage as orthotopic xenograft tumors in the mfp (mammary foot pad) of immunodeficient mice. These functional proteomic studies identified at least seven types of enzyme activities with distinct expression patterns: (i) tumor-associated activities derived from parental MDA-MB-231 cells (e.g., hCathepsin A); (ii) tumor-specific activities (absent in both parental cells and mfp; e.g., human monoacylglycerol lipase); (iii) stromal activities excluded from tumors (e.g., mMGC18894); (iv) stromal activities present in tumors (e.g., mCE-1); (v) carcinoma activities expressed selectively in the parental line (e.g., hPFK); (vi) carcinoma activities expressed selectively after in vivo passaging (e.g., huPA and htPA); and (vii) carcinoma activities reconstituted after in vivo passaging (e.g., hGST ). Together, the myriad changes observed in the MDA-MB-231 proteome after cultivation in the mouse mfp point to the existence of a subpopulation of breast cancer cells (231mfp cells) that exhibits increased tumor growth rates and metastasis in vivo. Although it remains to be determined whether the differentially expressed enzyme activities identified by ABPP contribute to the enhanced tumorigenic properties of 231mfp cells, it is nonetheless significant that many of the most dramatic alterations in enzyme activities occurred as a result of posttranscriptional events (e.g., the up-regulation of tPA and uPA activity in 231mfp cells). These findings indicate that large-magnitude changes in cancer cell proteomes may occur without corresponding alterations in transcript expression and thus underscore the value of ABPP for measuring the functional outcome of posttranscriptional and posttranslational events that ultimately govern enzyme activity in vivo.

Breast cancer remains one of the most prevalent and life-threatening forms of cancer for which effective treatments and cures are lacking. Several properties can define a cell type as cancerous, foremost of which being: (1) indefinite growth (immortalization), (2) uncontrolled proliferation (transformation), and (3) invasion and colonization of surrounding (malignancy) and distant (metastasis) tissues. It is this third phenotype that marks the most aggressive and debilitating forms of cancer. The molecular features special to metastatic breast carcinomas that support their invasive behavior are complex and ill-defined. One generally accepted hypothesis assigns proteins like proteases and catabolic enzymes a central role in promoting the aggressive behavior of cancer cells. The normal job of proteases is to degrade other proteins in a manner that is tightly regulated by our cells and tissues. Proteases participate in many important developmental and physiological events, including tissue remodeling and blood coagulation cascades. However, excessive or unregulated protease activity appears to be one of the main factors that permits tumor cells to invade and eventually take over normal tissues. Similarly, intracellular catabolic enzymes, which also serve an important function in normal cells (to detoxify foreign chemicals), may exhibit increased activity in cancer cells that lead to their resistance to conventional chemotherapeutic drugs. Thus, proteases and catabolic enzymes are considered to be not only important markers for breast tumorigenesis, but also candidate drug targets for breast cancer treatment. Nonetheless, the identities of the individual proteases and catabolic enzymes involved in breast cancer remain elusive. We hypothesize that much of the current confusion over the role that individual enzymes play in breast cancer arises from an inability to those enzymes whose catalytic activities actually change during the progression of this disease. To identify proteases and other enzymes involved in breast cancer, we have developed a novel chemical technique termed activity-based protein profiling (ABPP) that allows large enzyme families to be analyzed for changes in their activity directly in whole cell, tissue, and fluid samples. In the second year of this STEP award, we have completed a broad ABPP analysis to profile the progression of human breast tumors grown as xenografts in mice. Using ABPP methods, we detected dramatic functional differences that exist between cancer cells grown in culture or as tumors in vivo, both of which serve as important, widely used, research models of human breast cancer. Furthermore the altered enzyme activity profiles we observed were found to correlate with significantly greater tumor growth rates and metastases upon reintroduction of cells derived from tumors back into mice indicating 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 ABPP for identifying enzyme activity signatures that depict such changes in cancer cell biology.

Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Jessani N, Liu Y, Humphrey M, Cravatt BF
Yr: 2002 Vol: 99 Nbr: Abs: Pg:10335-10340

Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype
Periodical:Nature Biotechnology
Index Medicus: Nat Biotechnol
Authors: Adam GC, Sorensen EJ, Cravatt BF
Yr: 2002 Vol: 20 Nbr: 8 Abs: Pg:805-809

Trifunctional chemical probes for the consolidated detection and identification of enzyme activities in complex proteomes
Periodical:Molecular and Cellular Proteomics
Index Medicus: Mol Cell Proteomics
Authors: Adam GC, Sorensen EJ, Cravatt BF
Yr: 2002 Vol: 1 Nbr: Abs: Pg:828-835

Chemical strategies for functional proteomics
Periodical:Molecular and Cellular Proteomics
Index Medicus: Mol Cell Proteomics
Authors: Adam GC, Sorensen EJ, Cravatt BF
Yr: 2002 Vol: 1 Nbr: 10 Abs: Pg:781-790

Activity-based protein profiling in vivo using a copper(l)-catalyzed [3+2] azide-alkyne cycloaddition
Periodical:Journal of the American Chemistry Society
Index Medicus: J Am Chem Soc
Authors: Speers AE, Adam GC, Cravatt BF
Yr: 2003 Vol: 125 Nbr: Abs: Pg:4686-4687

Profiling enzyme activities in vivo using click chemistry methods.
Periodical:Chemistry and Biology
Index Medicus: Chem Biol
Authors: Speers AE, Cravatt BF.
Yr: 2004 Vol: 11 Nbr: Abs: Pg:535-546

Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo.
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Jessani N, Humphrey M, McDonald WH, Niessen S, Masuda K, Gangadharan B, Yates JR 3rd, Muel
Yr: 2004 Vol: 101 Nbr: 38 Abs: Pg:13756-13761