Profiling Serine Protease Activities in Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Benjamin Cravatt, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6JB-0091 Award: $265,950
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
The spread of breast cancer cells in the body occurs by a variety of processes. The most localized phases of tumor spread appear to be governed by protein-digesting proteases that are secreted by cancer cells and create a path by which the cells can move through their surrounding extracellular matrix. However, the actual functions played by individual proteases in breast cancer motility and invasiveness remain elusive. While given serine proteases and their endogenous inhibitors clearly show altered expression patterns during breast tumorigenesis, the overall functional outcome of such changes remain unclear. The reason for this is simple. Protein analysis is usually based on measuring quantity and not functional activity. Indeed, when data relating serine proteases and their endogenous inhibitors to breast cancer are evaluated, seemingly paradoxical trends emerge. For example, while the advancement of breast carcinomas is often linked to a lower amount of some protease inhibitors (e.g., maspin and MEPI), the enhanced expression of other serine protease inhibitors (e.g. PAI-1) increases with late stages of breast tumorigenesis. Likewise, some serine proteases increase, while other decrease, as breast carcinomas become more aggressive. The resolution of this paradox requires the ability to measure protease activities in a complex mixture of both proteases and their endogenous inhibitors. Simple static measures of protease and inhibitor quantity are insufficient to explain the dynamic nature of their interactions that occurs in the breast tumor environment.

To better understand how serine protease activities impact breast cancer, we will utilize a novel chemical strategy developed in our laboratory termed Activity-Based Protein Profiling (ABPP). This technique allows us to simultaneously monitor the catalytic activities of numerous serine proteases from whole cell, tissue, and fluid samples. Using the CBCRP funding, we will use ABPP to analyze both estrogen receptor (ER) positive and negative human breast cancer cell lines. In breast carcinomas, a strong inverse relationship exists between ER expression and the resulting cellular metastatic properties. Our goal is to take the observations on cell invasiveness and ER status to a specific understanding of serine protease activity. Using ABPP, we will not only detect the serine protease activities found in human breast cancer cells, but also isolate and characterize these enzymes at the molecular level.

We anticipate that these studies will identify functional changes in key serine proteases involved in promoting or retarding breast tumorigenesis. These enzymes should in turn serve as both markers for breast cancer progression and targets for pharmaceutical efforts aimed at treating this disease.

Final Report (2002)
Note: Dr. Cravatt was funded through a new two-year grant from the CBCRP in 2002 to continue this research.

Introduction: 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, called proteases, in a central role in promoting the aggressive behavior of cancer cells. Thus, proteases 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.

Topic addressed: 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 activities actually change during the progression of this disease. To better understand how proteases impact 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 this grant, we have applied ABPP methods to discover new enzymes implicated in breast cancer that may serve as both diagnostic markers and therapeutic targets for this disease.

Progress towards specific aims: Over the period of this grant, we have completed both specific aims put forth in our application. First, we have succeeded in detecting and identifying several serine protease activities that are differentially expressed in human breast cancer cell lines. These enzymes include both established markers of breast cancer progression, like urokinase, and completely novel proteins like the uncharacterized enzyme KIAA1363, which we found to be upregulated in invasive, estrogen-receptor negative breast cancer lines. Secondly, we have used several new ABPP probes developed by our research group to identify many other types of enzymes that are upregulated in invasive breast cancer cells. These may include detoxification enzymes of the glutathione S-transferase and transglutaminase families. We think that these enzymes may support the drug resistant behavior of aggressive breast cancer cells. This research has served to validate our ABPP method as a valuable approach to identify new candidate markers and targets for the diagnosis and treatment of breast cancer.

Future directions and impact: Progress towards better diagnostic and therapeutic agents to treat breast cancer relies heavily on our ability to identify new proteins that are both: (1) upregulated in breast cancer samples, and (2) of functional relevance to the progression of this disease. We anticipate that the enzymes identified by our ABPP methods may represent valuable markers and targets for future studies aimed at understanding, diagnosing, and ultimately treating breast cancer. To follow up on this notion, we are currently using ABPP methods to determine whether these enzyme activities are also upregulated in primary animal and human breast tumors. We are also developing chemical inhibitors of these enzymes which can be used to the function that they play in supporting breast cancer progression.

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: 16 Abs: Pg:10335-40

Chemical strategies for functional proteomics
Periodical:Molecular and Cellular Proteomics
Index Medicus: Mol Cell Proteomics
Authors: Adam GC, Sorense EJ, Cravatt BF
Yr: 2002 Vol: 1 Nbr: 10 Abs: Pg:781-90

Trifunctional chemical probes for the consolidated detection and identification of enzyme activities from complex proteomes
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:828-35

Profiling serine hydrolase activities in complex proteomes.
Index Medicus: Biochemistry
Authors: Kidd D, Liu Y, and Cravatt BF
Yr: 2001 Vol: 40 Nbr: Abs: Pg:4005-4015