Identifying Breast Cancer Targets for Protease Inhibitors

Institution: Scripps Research Institute
Investigator(s): Yongsheng Liu, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6FB-0066 Award: $42,120
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
Breast cancer cells become life threatening when they acquire the ability to migrate both in the immediate micro-environment of the tumor and via the circulatory system to other organs. Our interest is in the molecular processes that allow cancer cells to move locally in the tumor region. One generally accepted notion assigns extracellular proteolytic enzymes a central role in promoting the invasive properties. However, endogenous inhibitors regulate the activity of these enzymes, such that there is a fine tuned balance between invasion-promoting proteases and their regulatory inhibitors. Consistent with this thinking, several metastatic breast cancer cell lines suppress the serpin family‚ of serine-type protease inhibitors, which suggests that serine proteases support the aggressive behavior of these cells. The invasion activity of the serine proteases in breast cancer is also indicated by studies in which the application of serpins, such as maspin and MEPI, to metastatic breast carcinomas reduces their migratory and invasive potential. But, the serine proteases that are targeted by serpins like maspin and MEPI and their net activity in the cellular or tumor mico-environment have not been determined.

We plan to use the BCRP funding to apply a novel technique to analyze the relationship between serpins, their endogenous serine protease targets, and invasion properties of breast cancer cells. A novel experimental strategy has been developed by our laboratory, called activity based protein profiling (ABPP), to identify specific, and perhaps unknown, breast cancer-derived serine protease targets for maspin and MEPI. Using ABPP, we will compare the serine protease activity profiles of human breast cancer cell lines cultured in the presence and absence of these serpins. We will then isolate and molecularly characterize those serine proteases inhibited by maspin and/or MEPI. We anticipate that breast carcinomas express high activity serine protease targets for serpins like maspin and MEPI, and that these proteases are critically involved in promoting breast cancer metastasis.

Accordingly, the serine proteases identified in this study should represent valuable subjects for future pharmaceutical investigations aimed at inhibiting breast cancer metastasis.

Final Report (2001)
Note: Dr. Liu resigned the project in June 2001 to accept another research position.

We used the method of ABPP (Activity-Based Protein Profiling) to analyze several ER-negative breast cancer cell lines and validate the method. We were able to use urokinase activity and its inhibition by PA-1 as proof-of-principle. The next phase was to expand this and to recombinantly produce the protease inhibitors, MEPI and serpin, in breast cancer cells to directly assay their secreted proteomes. However, it was difficult to recombinantly express the serpin protease inhibitors, so we shifted to expressing them in the COS-7 (non breast cancer) cell line. Although the breast cancer cell detection studies were not completed, there remains a strong rationale for thinking that this approach will be successful. It is already known that serpins will inhibit breast cancer invasiveness, so the combination of APBB and recombinant serpins will allow us to identify a group of 'active' proteases in ER-negative breast cancer cells that are most likely involved in cell invasion. The inhibited proteases should stand as attractive targets for pharmaceutical intervention.

Profiling serine hydrolase activities in compex proteomes
Index Medicus: Biochemistry
Authors: Kidd D. Liu Y, and Cravatt BF
Yr: 2001 Vol: 40 Nbr: 13 Abs: Pg:4005-4015