Substrate Profiling of Breast Cancer Related Proteases

Institution: Scripps Research Institute
Investigator(s): Melissa Dix, B.S. -
Award Cycle: 2009 (Cycle 15) Grant #: 15GB-0017 Award: $76,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2009)

The discovery and evaluation of critical protein mediators of breast cancer development and metastasis remains an important challenge for breast cancer researchers. Serine proteases have been shown to be important regulators of breast cancer growth and metastasis. This fact is exemplified by urokinase-type plasminogen activator (uPA), an extracellular serine protease which has proven to be a useful marker for the clinical diagnosis of breast cancer. Despite the fundamental importance of proteases in breast cancer, an endogenous substrate profile for most human proteases is lacking. As such, there is an urgent need for further understanding of the roles that serine proteases play in breast cancer tumorigenesis and metastasis, as these enzymes, and the cellular pathways they control, offer promising new therapeutic targets and biomarkers.

In my dissertation project funded by the CBCRP, the first aim is to identify and catalog the substrates of plasminogen activation system proteases and determine how the increased activity of these proteases is associated with increases in aggressiveness (i.e., migration potential) of breast cancer cells. Secondly, we aim to address the underlying biological mechanisms responsible for the increase in protease activity. Finally, we plan to map and functionally characterize the breast cancer cell signaling pathways “upstream” (i.e, regulatory factors) for the plasminogen proteolytic system. A key technical element for these studies is the use of PROTOMAP (PROtein Topography and Migration Analysis Platform) technology, a novel proteomic method for protease substrate identification that combines electrophoresis with mass spectrometry. In addition, we will employ shRNA technology (a method to silence gene expression by RNA inhibition), protease mutants, and small molecule inhibitors to diminish individual protease activities. We have constructed breast cancer-based cell models to explore biological mechanisms of plasminogen protease substrate and feedback regulation. Hopefully, we can progress to performing tumor xenograft studies in plasminogen knockout mice.

Successful completion of this proposal may identify and validate novel biomarkers which could then be integrated into a screening platform that would allow better detection of breast cancer and measure potentially aggressive disease. Also, increased knowledge on the normal protease pathways commonly “hijacked” by cancer cells to increase aggressiveness will illuminate novel therapeutic targets.

Final Report (2011)

Despite catalyzing one of the most ubiquitous post-translational regulatory processes in biology, a complete endogenous substrate profile for most human proteases is lacking. Upregulation of a variety of proteases has been strongly linked to tumorigenesis, although the precise role played by these enzymes in cancer remains unknown. Three such proteases, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasmin (pin), have been linked to increased breast cancer aggressiveness. The complete in vivo substrate profile for these proteases remains uncharacterized, which has hindered researchers efforts to understand the functional significance of the plasminogen system in breast cancer.

We have previously isolated an in vivo-derived hyper-aggressive variant of the MDA-MB-231 breast cancer cell line, termed 231 MFP. The 231 MFP cell line secretes much higher levels of active uPA and tPA, making it an excellent experimental system to study this proteolytic cascade . We have developed an unbiased protease substrate discovery platform termed PROTOMAP (PROtein TOpography and Migration Analysis Platform), that combines lDSDS-PAGE fractionation with mass spectrometry to identify shifts in protein migration indicative of a proteolytic event.

My dissertation project funded by the CBCRP focuses on application of this method to identify the endogenous substrate profile for uPA, tPA, and plasmin, and how cleavage of these substrates can affect cancer aggressiveness. First, we completed a PROTOMAP analysis of plasminogen-treated 231 MFP conditioned media and identified 175 putative substrates of plasmin. Further in vitro verification of these cleavage events show that while the majority of proteins identified appear to be plasmin-specific substrates, a few proteins can also be cleaved to some extent by uPA or tPA. A cleavage event on the most basic level can either be an activating or inactivating event. We found that overexpression of this protein alters intracellular downstream signaling pathways. This led us to develop a new technology that could simultaneously evaluate the relationship between phosphorylation and proteolysis.

Next, a major accomplishment this past year was the development and application of quantitative-phosphoPROTOMAP. As proof-of-concept for the method, we chose to analyze a T-cell lymphoma line, Jurkat cells, treated with staurosporine, a potent activator of the intrinsic apoptotic cascade. We identified extensive cross-talk between protein phosphorylation and subsequent cleavage. With this method in hand, we can now begin to access the functional consequences of plasmin-mediated cleavage events and the intracellular signaling pathways these events could control.

Activity-based protein profiling for biochemical pathway discovery in cancer.
Periodical:Nature Reviews. Cancer
Index Medicus: Nat Rev Cancer
Authors: Nomura DK, Dix MM, Cravatt BF.
Yr: 2010 Vol: 10 Nbr: 9 Abs: Pg:630-8