Activity Based Profiling of Breast Cancer Xenografts

Institution: Scripps Research Institute
Investigator(s): Nadim Jessani, B.S. -
Award Cycle: 2003 (Cycle IX) Grant #: 9GB-0065 Award: $30,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2003)
The molecular features special to metastatic breast carcinomas that support their motile and invasive behavior are complex and ill-defined. It is thought that extracellular enzymes, called proteases, play a central role in promoting their aggressive properties. However, studies that measure the abundance of cancer-associated proteases may not provide of clear picture of their function. For example, the coincidental or coordinated upregulation of both a protease and its endogenous inhibitor (as occurs in breast cancer for uPA and its inhibitor PAI-1) might result in little net difference in enzyme activity, thus producing a protease whose quantity, rather than catalytic power, changes during tumorigenesis.

Conventional genomics (DNA/RNA-based) and proteomics (protein/peptide-based) methods measure changes in transcript and protein abundance, respectively, and therefore provide only an indirect assessment of protein function. 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).

Our laboratory’s initial research efforts, supported by previous CBCRP awards to Dr. Cravatt, allowed us to successfully apply ABPP probes to the proteomic analysis of several enzyme classes across a panel of human breast cancer cell lines. These studies resulted in the identification of multiple enzyme activities, including several serine hydrolases that were differentially expressed in estrogen receptor (ER)-positive and ER-negative 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 human tumors grown in animal models (i.e., xenografts). Taking advantage of the mixed species nature of these breast cancer xenograft models, we will also attempt to distinguish tumor enzyme activities that are expressed by cancer cells (human) and by infiltrating stroma (mouse). Next, we also plan to determine the enzyme activity profiles of circulating tumor cells. Finally, we will determine the enzyme activity profiles of metastasized variants of human breast cancer lines and compare these profiles to those of the parent lines.

Progress towards better diagnostic and therapeutic agents to treat breast cancer relies heavily on the 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 our chemical proteomics studies will identify novel key enzymes that support the growth and metastatic properties of breast cancer in vivo. These enzymes will in turn represent valuable markers and targets for future studies aimed at understanding, diagnosing, and ultimately treating breast cancer.

Final Report (2004)
Note: The grant was resigned after one year, because the PI graduated from his doctoral program. The abstract below was derived from the PNAS paper conclusions section authored by the PI, his mentor, and collaborators that summarize the project.

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.

Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo. (TrackII) Proc. Natl. Acad. Sci USA 2004, 101, 13756-1361. relevant
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, et al., Yates JR, Mueller BM, Cravatt BF., Cravatt, BF, and Stromal Enzyme
Yr: 2004 Vol: 101 Nbr: 38 Abs: Pg:13756-61