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The FAKs of Metastasis
A psychologist who specializes in the dynamics of small groups of people might say that within any such small group, one of the most important traits for building and maintaining good interpersonal relationships is empathy—understanding, being aware of, having sensitivity to, and otherwise fully communicating with one another. There are parallels in the way cells interact. While biological molecules don't use empathy, they do communicate among themselves during cell signaling, using their own set of subtle and intricate signals. David Schlaepfer, associate professor in the Department of Immunology at The Scripps Research Institute (TSRI), has an appreciation for these interactions are their complex nature. He studies an intricate group of signaling molecules involved in cell motility and cancer. In particular, he investigates how certain cues can cause epithelial cells, which cover most inner and outer “surfaces” in the body, to move when they are cancerous. “All the players [responsible for this] are not known,” says Schlaepfer. Just the FAKs However, it is known that a protein called focal adhesion kinase (FAK), a large, important signaling molecule of about 120,000 daltons, is involved in cancer as well as a variety of other roles in the body. FAK is an enzyme that attaches phosphate groups to tyrosine residues on other target proteins inside cells, thereby modulating their function. As its name suggests, FAK is involved in the regulation of focal adhesion formation—the physiological phenomenon whereby cells attach to a scaffold-like arrangement of sugars, proteins, collagens, and other molecules forming the extracellular matrix. The extracellular matrix is a cementing agent that holds cells together and maintains tissue architecture. “In addition,” says Satyajit Mitra, a postdoctoral fellow in Schlaepfer’s laboratory, “FAK is important for turning over focal adhesions.” FAK does not form focal adhesions, Mitra adds, but what it does is turn them over rapidly. In the absence of FAK, cells get stuck because they have more focal adhesions with the extracellular matrix and stronger ones, too. Cells attach to the extracellular matrix through proteins on their surface called integrins. Integrins are cell surface receptors that transduce survival, angiogenesis, motility, and positional cues. To deliver these signals, integrins recruit a large assembly of signaling proteins, with FAK as a key and central component. However, FAK is not merely an interesting protein that one might encounter occasionally in the scientific literature. FAK, because of its role as a regulator of focal adhesion turnover and cell motility, is also highly important in cancer and the worst kind of tumor cell growth. “In clinical settings,” says Schlaepfer, “the expression of FAK is tightly correlated with increased human tumor cell metastasis.” FAK and Cancer Cancer is the second leading cause of death in the United States, claiming over 500,000 lives and costing over $100 billion in healthcare and related costs every year, according to the Centers for Disease Control and Prevention. The role of basic science in this context is to understand cancer—to understand how normal, healthy cells can mutate into dangerous changelings. These transformations often lead the cells to acquire abilities that they did not have before. The ability to stay alive and divide over and over, forming a tumor, for example, is not something normal cells do. Nor is the ability of a cell that is normally fixed in one place in one tissue of the body to get up and move through the bloodstream, anchor down in a distant tissue, and begin a new cancer—in other words, to metastasize. Metastasis is a dangerous phenomenon through which many cancers are able to claim lives by spreading to multiple tissues and organs and compromising their function. While surgeons can remove cancerous tissue, such procedures are greatly complicated if a tumor establishes new tumors in other tissues, a process known as metastasis. One of the aims of basic science is to elucidate the mechanism of and find ways to deal with cancer metastasis. Metastasis depends on the cancerous cells acquiring two separate abilities—increased motility and invasiveness. Motility, the ability of a cell to get up and go, is an obvious requirement for metastasis. The word “metastasis” comes from the Latin construction meaning to change position. Perhaps not so obvious is the need for cancerous cells to invade tissue, to burrow in and set up shop somewhere else. Invasion is the completion and therefore a requirement for metastasis. These “phenotypic” abilities do not arise out of thin air. Cancerous cells acquire them in part through mutations to their DNA that result in changes of expression of one or more genes. Often as these expression levels change, so do the levels of other, related genes. Schlaepfer and his colleagues are specifically interested in the role that FAK plays in all of this and they are trying to get at these mechanisms in tumor cells to see if they can intervene in that process. Methods for Studying FAK Schlaepfer and his laboratory have been asking several questions related to FAK, including, on the most basic level, what role it plays in tumors. Tumors over-expressing FAK are larger, have a significant advantage for growth, and have a cunning ability to invade new tissue. “We have found that tumor cells lacking FAK don’t metatasize and we can re-initiate this process by genetically re-expressing FAK in these cells,” says Schlaepfer. Schlaepfer and his colleagues are using a set of cell culture assays and in vivo models to tease apart the role of FAK in different cells and under different conditions. They compared the gene expression profile in tumor cells that do not express FAK, those that do, and those that have been “reconstituted” by putting FAK expression back in to discover its properties. By doing these experiments, they were able to go beyond simply asking which genes are upregulated and which are downregulated in the tumor cell. Instead, they are determining which genes are regulated as a direct result of FAK expression. Furthermore, Schlaepfer and his colleagues established in vivo models in which they can effectively take away the ability of FAK to invade tissues. They used an inhibitor of FAK activity to selectively disrupt the invasion component alone. The inhibitor is actually just a fragment of the FAK gene itself that competes with endogenous FAK for binding to integrins. “We’re throwing a wrench into the FAK signaling system to answer the question, if we stop its function, what happens?” says Schlaepfer. Interestingly, they found that stopping FAK takes away, from tumor cells, the ability to metastasize but does not affect their motility. This enabled them to dissociate the role of FAK in motility versus its role in invasion. It also led to an interesting direction for the research. FAK in Motility and Invasion FAK has a role to play in motility and invasion because it is present in the projections that cells form when they are invading new tissue. In the parlance of cell biologists, these feet are referred to as “invadopodia” or “pseudopodia” Podia, in Latin, means feet. Pseudopodia are foot-like extensions that cells use for probing an area and crawling. And within these pseudopodia, FAK is highly expressed. Staining cells growing in culture for phosphotyrosine, a sure sign of FAK activity, will show hotspots at the ends of actin filaments, where the FAK signaling is taking place. “During invasion, these same feet squeeze between cells,” says Mitra. “We’ve seen FAK specifically enriched [in invading cell extensions].” Another important cancer enzyme that is often overexpressed in cancer cells and is localized to pseudopodia are enzymes known as matrix metalloproteinases (MMPs). MMPs are secreted enzymes that play a number of important biological roles in both the early development of organ structures and in tissue remodeling. Their physiological function is to remodel the extracellular matrix, and because of the potential damage that this could do to tissues, MMPs are one of the most highly regulated enzymes in the body. “If they weren’t regulated,” says Schlaepfer, “our bodies would dissolve, basically.” Unfortunately, this sophisticated regulation does not prevent cancer cells from subverting MMPs for their own purposes—cancer cells secrete these enzymes in order to break free of the extracellular matrix and tissue stroma, allowing them to move. It also allows them to dissolve barriers that come in their way to the bloodstream or to distant tissues during metastasis. Significantly, when FAK is upregulated in a tumor cell, that cell will correspondingly upregulate MMP expression and activity as well. This leads to the tantalizing possibility that FAK is one of the signaling proteins that cancer cells use to activate MMPs and achieve metastasis. Schlaepfer and colleagues are testing the connections between FAK, MMPs, and metastasis. “If we can figure out how FAK is functioning, and if we can get a good inhibitor then we might be able to stop cells from metastasizing,” says Schlaepfer. “These drugs might contain a tumor, preventing it from spreading if it is found early enough.” In addition to the regulation of MMPs, Schlaepfer is also looking at the effect of FAK inhibition on certain other genes within the cells. Looking at these “peripheral” markers that are up- or down-regulated by FAK expression, might be the easiest way to gauge the effectiveness of a future FAK inhibitor in vivo and could be a useful application for testing whether any given FAK inhibitor works in a clinical setting.
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Associate Professor David Schlaepfer (center) and his laboratory have been asking several questions related to the signaling molecule FAK, including what role it plays in tumors. Other lab members include (left to right) Satyajit K. Mitra, John E. Molina, and Datsun A. Hsia.
This FAK-null tumor cell has been reconstituted with FAK and stained to show where the protein localizes. The staining shows FAK concentrated in areas of focal contact (top arrowheads) and of pseudopodia formation (lower arrow). | |
