Trypsin-like Proteases as Metastatic Agents in Breast Cancer

Institution: University of California, Riverside
Investigator(s): Kathryn DeFea, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7KB-0093 Award: $295,893
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2001)
The ability of tumors to metastasize from their original location to the brain, bone and lymphatic tissue, is not well addressed by current therapies. Normal cells exist in contact with a complex protein network, called the extracellular matrix (ECM). The ECM is a barrier to cell movement and cancer cells must devise ways to break their attachments, degrade, and move through the ECM in order to metastasize. Proteases are enzymes that degrade other proteins and have long been thought to aid in freeing the tumor cells from their original location by chewing up the ECM. Recent studies have suggested that they may promote cell shape changes and motility through the activation of a protein in the tumor cell membrane called Protease-Activated Receptor-2 (PAR2). This leads to a cascade of intracellular reactions that activates the motility apparatus of the cell. Thus, the ECM-degrading proteases serve two functions by, (1) reducing the extracellular resistance to cell movement, and (2) activating the motility processes inside of cells though specific receptors. Because a number of inhibitors of the proteases that activate PAR2 are already commercially available, understanding the precise mechanisms by which PAR2 leads to metastatic changes in tumor cells could lead to the development of a new class of anti-metastatic agents.

In this project we first aim to investigate whether PAR2 can: (a) make cells form extensions, called lamelipods, (b) induce net cell motility, and (c) activate key cell surface receptors, called integrins in breast cancer cells. Because lamelipods are composed primarily of a protein called actin, we will watch these structures form by observing the movement of fluorescently-tagged actin in the presence and absence of proteases and protease inhibitors. Changes in the structure and shape of cells mediated by PAR2 will be determined using a video microscope. Cell motility will be measured by growing cells on a grid and monitoring their movement across the grid in response to proteases and protease inhibitors over time, either in real time using a video microscope or at fixed intervals by counting the cells that have moved a specific distance. Second, we will identify other proteins involved in the PAR2 cascade using a radioactive label to tag proteins modified in response to PAR2 activation and mass spectrometry to analyze these modified proteins. We will then test these proteins for their role in cell shape change, motility and integrin activation. Finally, we will assess the efficiency of inhibiting PAR2, either using protease inhibitors or other chemicals that block PAR2 action, on the metastasis of tumors in a mouse model of breast cancer. With an increased understanding of the biology of PAR2, we will be able to assay inhibitors of different steps of the PAR2 pathway to see which ones are most effective at blocking metastasis.

These studies have the potential to provide a host of new anti-metastatic agents that could prevent tumor cell migration at early stages. Additionally, identification of other proteins involved in this pathway may provide a new diagnostic tool for identifying tumors that are likely to metastasize or provide targets for other anti-metastatic drugs. Such therapies, when used with anti-growth therapies, could greatly reduce the lethality of breast cancer.

Final Report (2004)
Introduction: Cell migration during wound healing and inflammation depends on directed movements that result from a cell’s ability to “sense” a chemical gradient. Our work involves studying the cell surface-signaling-cytoskeletal connections in cancer cells. When a cell, such as a tumor cell, receives a signal to migrate, it organizes itself so that the edge nearest the signal (the leading edge) is actively extending while the back of the cell (the trailing edge) is disassembling its contacts with the surrounding tissue. Recent studies in our lab suggest such polarized cell shape changes and motility might occur through the activation of a protein called Protease-Activated-Receptor-2 (PAR2) leading to a cascade of intracellular reactions. Because a number of inhibitors of the proteases that activate PAR2 are already commercially available, understanding the precise mechanisms by which PAR2 leads to metastatic changes in tumor cells could lead to the development of a new class of anti-metastatic agents. The studies proposed in this project were aimed at understanding the potential role PAR2 in promoting tumor metastasis by: (1) examining PAR2-stimulated cell shape change and motility and the effect of protease inhibitors on these events; (2) identifying cellular targets of PAR2 action involved in promoting cell shape change and motility; and (3) investigating the ability of protease inhibitors to reduce metastasis in a mouse model of breast cancer. Drugs that target the early steps in cell migration could potentially be far more powerful in preventing metastasis than those that prevent tumor growth and reattachment in a new location.

Progress: During the three years that these studies were funded by the CBCRP, we have nearly completed the first and second specific aims and made significant progress on the third. We have demonstrated that PAR2 activation can lead to changes in cell shape through the activation of two enzymes known as Intracellular-Regulated Kinases-1 and -2 (Erk 1,2), which in turn modify and possibly activate a set of proteins involved in involved organization of motile elements at the migrating edge of a cell. The mechanism by which PAR2 affects this protein is novel, as it involves proteins traditionally associated with signal termination acting to regulate distinct events at the leading and trailing edge of the cell. We have also characterized and compared various aspects of PAR2 function in cell lines of varying metastatic potential and found that components crucial for the unique mechanism by which PAR-2 promotes cell migration are constitutively activated (i.e., always “turned on’) in a highly aggressive model of breast cancer cell lines, but are dormant in a non-metastatic cell lines. We have preliminary data to suggest that serine protease inhibitors may block the migratory behavior of the highly metastatic cell line, which bodes well for the success of animal studies.

Future Directions/Impact: We have established a chick embryo model, and we will test both protease inhibitors and PAR-2 peptide inhibitors for their ability to block metastasis. The localization of PAR2 to the motile edge of more metastatic tumor cells, along with its ability to modify components of the cell’s motile machinery suggest that it plays an important role in tumor metastasis. Thus, inhibiting PAR2 activation with inhibitors of trypsin may prove to have a strong anti-metastatic effect. Furthermore, characterization of the other downstream targets of PAR2 action may lead to the development of other metastatic inhibitors.

Symposium Abstract (2003)
Both inflammation and tumor metastasis require extensive remodeling of the actin cytoskeleton and the formation of pseudopodia for directed cell motility. We have previously reported that Protease-activated receptor 2 (PAR-2), a proinflammatory receptor that is highly expressed in motile cells such as neutrophils, macrophages and tumor cells, is one of a growing family of receptors that utilizes a b-arrestin-dependent mechanism for activation of the 42-44 kD members of the MAPK family (extracellular signal regulated kinases 1 and 2 or ERK1/2). b-arrestin-bound PAR2 serves as a scaffold to sequester a pool of activated ERK1/2 in the cytosol; however, a specific role for the sequestered kinase activity has not been established. We now show that PAR2 activation promotes ERK1/2- and b-arrestin-dependent reorganization of the actin-cytoskeleton, polarized pseudopodia extension and chemotaxis. Using subcellular fractionation, confocal microscopy and physical isolation of pseudopodial proteins, we demonstrate that the previously identified PAR2/b-arrestin/ERK1/2-scaffolding complex is enriched in the pseudopodia along with RhoA-GTPases, where it prolongs ERK1/2 activation, leading to cell motility, potentially by modifying actin machinery at the leading edge. These studies provide evidence that PAR-2-stimulated chemotaxis requires the formation of a b-arrestin/ERK1/2 signaling complex and are the first example for a distinct cellular consequence of b-arrestin-sequestered ERK1/2 activity.

Rho-ROCK-LIMK-cofilin pathway regulates shear stress activation of sterol regulatory element binding proteins.
Periodical:Circulation Research
Index Medicus: Circ Res
Authors: Lin T, Zeng L, Liu Y, DeFea K, Schwartz MA, Chien S, Shyy JY.
Yr: 2003 Vol: 92 Nbr: 12 Abs: Pg:1296-304

Constitutive protease-activated-receptor-2 mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and 2.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Ge L, Shenoy SK, Lefkowitz RJ, Defea KA.
Yr: 2004 Vol: Nbr: Abs: Pg: