In the early 1970s, still a few years before molecular biology and genetics would revolutionize cancer research, scientists assumed that some general change in surface chemistry must occur on tumor cells to distinguish them from normal cells. Find that one difference, they reasoned, and oncologists would gain a foolproof marker to diagnose cancer.
Thirty years ago this past November in the Proceedings of the National Academy of Sciences, a then 28-year-old English postdoc named Richard Hynes, Ph.D., reported that he may have found that foolproof marker. It was a hitherto unknown structural protein positioned on the surface of normal cells, but which was rare or conspicuously absent on tumor cells. Although Hynes would not directly speculate in his paper, it was tempting to imagine that tumor cells might cleave this seemingly growth-promoting surface protein as an essential step in metastasis.
Hynes discovery and work in other laboratories set in motion a rush to characterize this mysterious "cancer" protein. Although the cancer breakthrough soon proved to be a false alarm, research on this new structural protein, called fibronectin, would catalyze a string of seminal studies, including the first molecular investigations of cellular adhesion proteins and their receptors, today two hot areas in cancer research.
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In the Beginning
During a recent interview, Hynes recalled the experiments that led to his single-author PNAS article while he was a fellow at the Imperial Cancer Research Fund in London. "Id been interested in cell surface proteins from my thesis, and there was a body of literature in the early 1970s suggesting that cell surface changes occur [during oncogenic transformation]," said Hynes, now a scientist at the Massachusetts Institute of Technology in Cambridge. "The question was: Could one find out which molecules were on the surface of cells and whether they changed?"
Hynes tried unsuccessfully to label and compare surface proteins in a number of ways before he finally had success about a year later using a then-new iodination technique.
"I realized that all of the gels from the normal cells had a big plug of iodine at the top, and the ones from the transformed cells didnt," Hynes said, a suggestion that the tumor cells had somehow eliminated the protein. "So, there was something different there. I thought, Oh, my goodness, this is fantastic. This is a big difference."
Hynes now laughs, but he and others did not know what to make of this abnormally large protein at the top of the gel. "One of the questions that came up was: Could proteins be that big?" said Hynes. "It seems silly now 30 years later, but a lot of people didnt believe proteins could be that big. I even had to convince some people that this wasnt some sort of big aggregate."
Hynes named his big find with the descriptive, "large external transformation sensitive" protein, or LETS. "The protein was large and external," he said. "It also was transformation sensitive, so that was the name, not to make any assumptions beyond what we knew."
Interestingly, Hynes was not alone in discovering the protein. Senitiroh Hakomori, M.D., Ph.D., and colleagues at the University of Washington in Seattle independently published a few weeks later their discovery of the same molecule, calling it "galactoprotein a." The following year, Antti Vaheri, M.D., Ph.D., and colleagues at the University of Helsinki weighed in with what they dubbed "surface fibroblast antigen."
But enthusiasm for the major breakthrough soon waned. At the time, dogma dictated that any cancer-initiating change must be true for all tumor types, and several groups found the change was not universal. But, more problematic, other laboratories could not consistently show that restoring the protein to tumor cells controlled their abnormal growth, as Hynes paper suggested. "It turned out not to be the central moleculethe switch," added Hynes. "Actually, we now know there is no one central molecule. But, back then, we thought there must be a transforming switch. Everybody in the field had such simple-minded views."
What Is It?
If LETS was not the switch, what was it? The protein clearly influenced tumor cells in culture, altering their shape and behavior. Follow-up work also would determine that it connects normal cells to the supportive, lattice-like extracellular matrix, much like a stitch joins fabric in a seam. It made sense that tumor cells either must snip the protein or possibly downregulate its production to break free of the extracellular matrix and metastasize. Even if fibronectin was not the central molecule, perhaps it still could point scientists in the right direction to find the elusive switch. Or, as turned out to be the case, the protein might lead scientists to more regulatory molecules and important cellular pathways often involved in the disease.
"There were lots of unanswered questions and work to do," said Hynes. "This protein was the first clean molecular difference between normal and tumor cells, and people were trying to do the same sorts of experiments at the same time. So, the field was just ripe for an explosion. Over the next few years, everybody purified and fragmented fibronectin and did a sort of anatomy on the molecule to find out which bits did what."
All of the slicing and dicing led to a Babel of names for the protein. Adding to the confusion was the Finnish groups then-stunning discovery that this cell-surface protein was the same as "cold-insoluble globulin," a protein that had been identified after World War II in plasma. Although researchers later determined that the proteins were slightly different structurally, the surprises continued when it was determined that the proteins were encoded by the same gene, marking one of the first examples of alternative splicing, now an important issue in biology.
By the mid 1970s, Vaheri and colleagues banded together and settled on a common namefibronectin, joining the Latin fibra, meaning fiber, and nectere, meaning to bind or connect. "Theres a joke about scientists that theyd rather use each others toothbrushes than each others nomenclature," Hynes said, laughing. "And so, those of us who had other names were resistant at first, but, by 1978, we just switched to fibronectin."
Seminal Studies
The discovery of fibronectin prompted a reevaluation of the structural glycoproteins and extracellular matrix. Until then, most biologists had written off the extracellular matrix as a boring conglomeration of inert, uninformative molecules. But fibronectin suggested a different story, in which structural glycoproteins linked cells to the extracellular matrix but also participated in orchestrating various aspects of cell behavior.
"Fibronectin is particularly important because it became the paradigm for a molecular understanding of cell adhesion molecules," noted Kenneth Yamada, M.D., Ph.D., who performed the first fibronectin reconstitution experiments on tumor cells and is now a scientist at the National Institute of Dental and Craniofacial Research at the National Institutes of Health. "Our field has established in a stepwise fashionusing analyses of the effects of cells in culturethat a single protein can regulate key cellular functions such as adhesion, shape, migration, and cell surface architecture."
A decade after the discovery of fibronectin, Erkki Ruoslahti, M.D., Ph.D., a distinguished professor at the Burnham Institute in La Jolla, Calif., and colleagues identified the three-amino-acid consensus sequence that is necessary for fibronectin to attach to cells. The tripeptide, called RGD, would lead the way to another major biological prizethe integrins, a family of protein receptors that form the transmembrane link between structural glycoproteins and the cytoskeleton. "One thing about RGD is we found it in fibronectin, but we also found it in vitronectin [an adhesive protein in plasma and serum], collagen, and many different proteins," said Ruoslahti. "So, its fundamental to the recognition of proteins by integrins."
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"But fibronectin has been a cool molecule to work on," he added. "In fact, the reason that it has kept going for 30 years is matrix and integrins are so important in a lot of different bits of biology. Fibronectin plays a role in thrombosis, development, cancer, inflammation, and other areas. So, you can work on it in a lot of situations and never get bored."
Fibronectin and Cancer
In the mid 1980s, Hynes wrote, "The study of fibronectin, initiated by work with tumor cells, may eventually come full circle to shed new light on the bases of malignant behavior." Almost 20 years later, is fibronectin starting to come full circle?
"It isnt there yet, but it is coming full circle," said Hynes. "I think we need to understand the role it plays in cancer progression better than we do. And, we need a better handle on its role in angiogenesis."
Some say fibronectin might be more than just a component feeding into a larger cellular system. As Jacqueline Labat-Robert, M.D., a scientist at the University of Paris who has a longstanding interest in structural glycoproteins, summarized in a recent review article, studies suggest our bodies produce more fibronectin as we age, and, as proposed 30 years ago, tumor cells secrete enzymes that snip the protein. According to Labat-Robert, this creates "a vicious circle" of steady fibronectin production that ends up scissored into smaller, free-floating fragments that possess altered chemical qualities and which are "a frequent phenomenon" of a number of pathological states, including potentially cancer.
"I am sure that this topic will remain of crucial importance for the next decades," noted Labat-Robert, adding that the fragment anastellin, which is derived from fibronectin, inhibits angiogenesis, tumor growth, and metastasis formation in laboratory studies. "I do agree that fibronectin fragments may have unique activities," said Yamada. "However, we feel that fibronectin itself is a novel developmental switch that is transcriptionally regulated and functions to modulate integrin and cadherin function at specific sites. That is, it is not just a ubiquitous adhesion protein, but can serve as a regulatory protein."
Ruoslahti said he now views fibronectin more broadly. "While fibronectin is the prototype adhesion protein, there is a lot of redundancy in the adhesion mechanisms," he said. "So, I dont think one can look at fibronectin in isolation. However, it is true that, in the anchorage dependence of tumor cells, its more important for their survival to be able to attach to fibronectin than to some other protein."
"I think that we all feel that fibronectin has been an incredible conceptual gold mine," continued Yamada. "The ideas and concepts that it has generated should have considerable relevance for our understanding of the metastatic process even if fibronectin turns out to play only an occasional role. More likely, though, it may well have roles, but often only in concert with the large number of other extracellular matrix proteins and proteoglycans that act through analogous molecular mechanisms."
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