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KEEPING CANCER IN CHECK WITH p53 Researchers worldwide work to unravel how this protein functions and devise drugs to boost its usefulness Rebecca L. Rawls C&EN Washington
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molecular weight of 53,000. The gene that carries the information to make this protein goes by the same name, though to lessen confusion, the gene name is italicized. The strongest proof that p53 is impor tant for preventing cancer is what hap pens when it's unable to function proper ly. Of the approximately 6.5 million can cer cases worldwide each year, about 2.4 million involve tumors containing p53 mu tations, estimates Curtis C. Harris, chief of the laboratory of human carcinogenesis at the National Cancer Institute. Although the estimate is necessarily crude, that amounts to nearly 40% of all cancers
omeday, when the committee of the Karolinska Institute decides the time isright,it's almost a sure bet that the discoverers of the p53 gene and its protein product will be making a trip to Sweden to receive Nobel Prizes. The gene and the protein it codes for stand at the center of the body's defense against cancer, directing the machinery of the cell to take notice and respond when the stability of the cell's DNA is threatened. The gene and its protein product stand, as well, at the center of one of the hottest areas in cancer research or, indeed, in all of molecular biology. More than 3,000 researchers worldwide Tetrameric in one way or another study p53, publishing papers at a current rate of several thousand per year. "It's a testimony to the complexity of this particular gene and its product that it's managed to keep so many people busy for so long," says David H. Beach, senior staff scientist and a Howard Hughes Medical Institute investigator at Cold Spring Harbor Laboratory in New York. He adds: "This protein is always throwing surprises up." It's of overwhelming interest, too, to drug companies because of the promise it offers of new approaches to fighting cancer. "Nearly every pharmaceutical company I know of has a p53 program of some sort now," says David P. Lane, profes^ sor of biochemistry at the University of Dundee, Scotland. Lane, along with Princeton University molecular biologist Arnold J. Levine, is codiscoverer of p53. Not bad for a protein that just barely has a name. In the jargon of molecular biology, the ρ of p53 merely identifies it as a protein, and the 53 indicates its approximate 38 FEBRUARY 17, 1997 C&EN
p53 binds DNA
worldwide. It's by far the most commonly mutated gene found in cancer cells. The frequency of p53 mutations var ies from one form of cancer to another, with even higher rates in some of the most common cancers in the U.S. Harris estimates about 55% of lung cancers in volve p53 mutations, about half of co lon and rectal cancers, and some 40% of lymphomas, pancreatic, and stomach cancers. In still other types of cancer, the p53 protein appears to be normal but inactivat ed. The human papilloma virus, for exam ple, which causes many cervical cancers, produces a protein that inactivates p53 in about 90% of these cancers. Roughly a third of soft-tissue sarcomas contain nor mal, but inactive p53, and so do many neuroblastomas. Altogether, lack of prop erly functioning p53 has been linked to some 50 to 60% of all cancers. Not only does p53's protective role reach to many different kinds of tumors, but protecting against cancer seems actu ally to be its job. "The sole purpose of the protein seems to be to suppress tumor for mation," Beach says. That's different from many genes that are mutated in human can cer cells, which probably serve their prima-
ry function during embryonic development. Such genes only become mutated after birth, in the process predisposing the cells that contain them to become cancerous. When mutated forms of these genes are inserted into the fertilized eggs of mice to replace the normal genes, the animals die as embryos because they are missing a key protein needed for their development. That's not what happens with mutated p53- Mice that completely lack function ing p53 develop to birth and are normal for the first several weeks after that. But nearly all of them develop tumors within the first six months of their lives. Even mice with one mutant p53 gene and one normal one are cancer prone, suggesting that they already have one strike against them in a multistep process that ultimately leads to cancer. Some peo ple inherit a mutated form of the p53 gene, too, in a condition known as LiFraumeni syndrome. They seem to be normal in all ways except they have an increased risk for many types of cancer including leukemia, breast cancer, softtissue sarcoma, brain tumors, and certain bone cancers. "The first thing to understand about p53 is that it's involved in diverse chemi
cal and cellular pathways," Beach says. One of its best understood roles is to pre vent cells from continuing to grow and di vide if they have been damaged. If a cell's DNA is damaged—by exposure to ionizing radiation, for example, or by certain chemotherapeutic agents—p53 levels within the cell go up. Those extra p53 molecules, among other things, trigger a process that arrests the cell's cycle of growth and divi sion until the DNA damage has been re paired. Without this brake on the cell cy cle, subsequent cell divisions would ampli fy the mutations, produce chromosomal segregation abnormalities, and lead to a very genetically unstable and potentially tumorigenic cell. But sometimes higher levels of p53 cause a different effect: Instead of halting cell division, they trigger a process of cel lular self-destruction called programmed cell death or apoptosis. "It's slightly myste rious in certain cell types why p53 some times does one and sometimes the other," Beach says. Both, however, can be viewed as protective mechanisms. In one case, p53 gets rid of a cell that's so damaged the body doesn't want it around anymore. But if the damage is less, it seems that p53 merely halts cell division until the damage can be repaired. At the molecular level, p53 is a transcription factor. It can bind to the regulatory regions of a specific set of genes and by doing so instruct the cell to make more of these genes' pro teins. At least 10 genes have been identified that are regulat ed by p53. Puzzling out exacdy what these genes do is one of the forefront areas of current p53 research. One of them, called p21, seems clearly to trigger p53's ability to arrest the cell cycle. Another, MDM2, produces a protein that inhibits p53, probably as part of a regu latory feedback mechanism. Still another, ΒΑΧ, looked in early studies to be the key play er in p53's ability to induce ap optosis. Now that picture is less clear, with more recent work suggesting that, although ΒΑΧ likely plays some role, the pathway by which p53 induces cell suicide is very complex. Beach's lab, which discov ered the p21 gene, has been Beach: p53 protein Is always throwing surprises up
heavily involved in piecing together how that gene's product stops the cell cycle. From work there and in many other labs, it now seems clear that the p21 protein serves as a checkpoint in the cycle. It binds to en zymes called cyclin kinases to inhibit their catalytic activity. Without active cyclin ki nases to interact with various proteins called cyclins, cells can't enter the different stages of the cell cycle. But mice that lack a functioning p21 gene don't develop tumors. And virtually no human tumors contain mutated p21 genes. "That begins to lead to a suspicion that it's the apoptosis pathway, or possibly a mix ture of the two, that's needed for the tumor suppressive activity of p53" Beach says. With such a central role in protecting cells from cancer, it's hardly surprising that p53 has become a major target for drug development. Perhaps the most straightforward ap proach—though far from simple—is to use gene therapy techniques to put an unmutated p53 gene into tumor cells that lack it. A few such attempts are currently in the first phase of clinical trials. At the University of Texas' M. D. Ander son Cancer Center in Houston, for example, a group led by Jack A. Roth, professor of thoracic and cardiovascular surgery, has used a retrovirus to carry a normal p53 gene into tumor cells in lung cancer patients whose tumors carry mutant p53 genes. The researchers injected the retrovirus di rectly into the tumor cells. Nobody has been cured in these early trials, but in the first round of tests the treatment triggered apoptosis in six of the seven patients. The tumors shrank for three patients and stopped growing in the other three. "It's a kind of simple-minded ap proach," Roth says. 'On the other hand, it seems to work, which is a big advantage." Cancer cells are very good taigets for gene therapy, he points out. One advan tage they have is that the gene only needs to express its protein for a relatively short period of time to do its job. For p53, ex pression for 24 hours is long enough to trigger programmed cell death in many cancer cells. And p53 seems to trigger cell death only in cancer cells. Elevating the p53 level in a normal cell doesn't seem to do it any harm, possibly because apoptosis may require extensive DNA damage as well as elevated levels of p53 protein. The Roth group is continuing these stud ies, trying to lower the dose of retrovirus needed. They've begun clinical trials, as well, using an adenovirus as the vector to carry ihtp53 gene into tumor cells. Both vi ruses have been modified so that they can't FEBRUARY 17, 1997 C&EN
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scïenc#|ti|^^^^^^H
ffi Short segment ofp53 (yellow) fits snugly into enzymelike binding pocket of regulatory protein MDM2. Key to the binding are a triad of hydrophobic amino acids from p53: (second from left) leucine, tryptophan, and phenylalanine. Also shown, but less Important to binding, are proline (far left) and leucine (upper right). Drug molecules that could block this binding site might boost p53 activity.
replicate themselves, and both are taken up well by tumor cells, Roth says. If they work, adenoviruses may be better vectors forp53 gene therapy, he notes, because they can be produced in much larger quantities than retroviruses. And, unlike retroviruses, they can infect cells even when they are not dividing. That may mean a single injection will be all that's needed, compared with five injections on consecutive days using a retrovirus. Altogether the group has treated about 30 patients with one or the other of their /?53-carrying vectors. Neither vector shows significant toxicity, Roth says. Frank McCormack, professor of cancer biology and director of the Cancer Research Institute at the University of California, San Francisco, and colleagues at Onyx Pharmaceuticals, Richmond, Calif., have a different way to use genetically engineered viruses to kill p53-deficient cancer cells. They have engineered a virus that replicates and kills cells that don't have functioning p53 but leaves other cells unaffected. Normally when a virus infects a cell, McCormack explains, one of the steps in the process of tricking the cell into replicating the virus is to turn off p53. Adeno-
viruses do this by producing a protein, called the early region IB (E1B) protein, that binds to p53 and inactivates it. McCormack and his colleagues have made an adenovirus that's missing the gene for the E1B protein. Without E1B, their virus can't inactivate p53, and so doesn't survive in cells that contain this protein. In tumor cells that lack functioning p53, however, the virus replicates, takes over the cells, and kills them. The engineered virus is in clinical trials designed to determine the appropriate dose at the University of Texas, San Antonio, and the University of Glasgow in Scotland. "We're getting up to high doses of
X-ray crystal data from three regions ofp53 protein important to its activity come together in this ribbon diagram of the structure from Pavletich's lab at Memorial Sloan-Kettering Cancer Center. The protein's four identical subunits are shown in red, green, yellow, and purple. At left is the protein's carboxy terminus, which controls tetramer formation. In the center is the DNA-binding domain, with DNA itself shown in gray. At right, the regulatory region at the amino terminus of each tetramer binds in the cleft of Its own MDM2 molecule, blue. Flexible links, shown as dotted lines, connect the different regions of the peptide. A different view of the DNA-binding domain of the tetramer is shown on page 38.
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FEBRUARY 17, 1997 C&EN
virus now, and we see no side effects of the virus so far," McCormack says. "The beauty of this approach, if it works, would be that you only have to infect a small number of cancer cells," he points out. "The virus will spread through the cancer, killing the tumor cells as it goes along, and stopping when it reaches normal cells." McCormack and his colleagues have produced a number of second-generation viruses that they hope may be improvements over the one now in clinical trials. "But until we analyze the results of the first trial, we don't know exacdy what we need to change," he says. At the other end of the therapeutic spectrum are efforts by many research groups and pharmaceutical companies to find small molecules that can stimulate p53's normal function or keep it from being inactivated. These studies are in the conceptual, exploratory stage, with
mutants that cause the protein to fall apart we can perhaps find a small molecule—a drug compound, if you want to call it braries and high-throughput screen- that—that will bind somewhere to the suring techniques, Lane and his col- face and by binding to the folded state, leagues have identified peptides stabilize it a little bit." It's something of a leap of faith to try to seven or eight amino acids in length that have a very high affinity for the find small molecules that will bind to muMDM2 binding site. Some of them tant p53 and stabilize its folding enough can displace MDM2 from p53, leav- that it will function like the unmutated protein, Pavletich admits. People have ing active p53 behind. "We're at the stage of very in- shown in some model systems that small tense chemical effort to try and molecules can bind in a surface crevice of improve leads," Lane says. "It's go- the protein and stabilize its folding, he says, but whether such compounds can be ing very well." The Lane group has also been found for p53 and whether they will stabitrying to understand how p53 is lize it enough to restore its function reactivated. It can be controlled mains to be seen. And earlier attempts to through protein phosphorylation, use this approach with other proteins which, in turn, is regulated by a have been notoriously unsuccessful. The region at the canboxy terminus of best known of these is probably work in the molecule, Lane says. "We've the 1970s to use small-molecule binding to been able to mimic that interac- get mutant hemoglobin to fold correctly in tion with small-peptide molecules, sickle-cell disease. The idea that this approach might so that we can turn p53 on and off. Again, the plan is to try to develop this work for p53, though controversial, has many supporters. Chief among them into more druglike molecules." But many kinds of cancer cells don't may be Lane. In his view, the mutant p53 have functioning copies of p53, and devis- proteins found in many human tumors ing drug strategies to attack these tumors are not completely unfolded, they are is a much more difficult problem. "A gene simply "breathing" at a faster than norwhose function is lost by mutation is not mal rate. "Proteins breathe," he explains. "When the most obvious drug target," as Beach puts it. "But p53 has such a central role in you see a crystal structure, it looks rigid, cancer that people have been thinking but in fact, these are extremely dynamic along a lot of creative approaches to tack- molecules that are always moving between different conformational states. And le the problem." Not surprisingly, considering that p53 does this particularly easily. The molep53's function is to bind to DNA, most of cule is poised to become active or inacthe mutations that inactivate it occur in tive; it's very easily denatured." Lots of people were skeptical, Lane the portion of the protein where DNA binding takes place. Pavletich and his says, when he first began looking for colleagues have determined the crystal small molecules to stabilize p53 folding. structure of this region bound to DNA "People told me you can't renature both for the unmutated form of p53 and boiled milk and other discouraging for several mutant forms. Broadly speak- things like that." The lab has had some ing, the mutations fall into two classes. encouraging results, both with antibody One class eliminates amino acids that stabilizers and with small peptides, he would bind to DNA. There's not much adds. that can be done to fix those, Pavletich "It is a very difficult target, compared notes. But a second set of mutations af- to work we've done on protein-protein fect the stability of the protein in its fold- interaction, for example, or on trying to ed state. "Tumors with this type of muta- inhibit an enzyme," Lane notes. "It's tion make p53, but the p53 can never hard to devise the screen. And it's hard fold," Paveltich says. to produce the protein poised to be able In recent work, the researchers have to sensitively detect changes that might used calorimetry techniques to determine occur to it in the presence of a small the stability of these mutant proteins. "We molecule. But these are all technical can show that many of these mutants fall things, and I think people are making apart at temperatures below 37 °C, which progress on them." Given the importance of the target, means that in our bodies, they don't have a chance of staying folded," Pavletich says. there's no question a great many people "Our hope is that for this second family of will continue to try.^ Pavletich: crystal structures reveal p53 binding interactions
even the most promising candidate compounds far from clinical trials. One of the more promising targets is the interaction of the p53 protein with its natural inhibitor, MDM2. Some tumors—certain soft-tissue sarcomas, for example—make too much MDM2. Although their p53 is normal, the cells nevertheless become cancerous, presumably because the MDM2 ties up all the p53 so that it can't bind to DNA. In such tumors, an agent that could block MDM2 activity might very well allow p53 to get on with its tumor suppressing activity. At least that's the theory. X-ray crystallographer Nikola P. Pavletich, associate member in the cellular biochemistry and biophysics program at Memorial SloanKettering Cancer Center in New York City, and his colleagues are one of the groups investigating this approach. Last fall, they reported the crystal structure of a portion ofp53 bound to MDM2. "To our surprise, the structure shows a nice, deep pocket on MDM2 into which p53 inserts a few amino acids," Pavletich says. "We like nice, deep pockets because they are reminiscent of enzyme-active sites, and scientists have traditionally been very good at inhibiting enzymes with drugs. This protein looks to be almost as good a drug-binding site as an enzyme would be." At the University of Dundee, Lane has also been studying the interactions of p53 with the MDM2 protein. The work is in conjunction with the pharmaceutical company Novartis. Using synthetic peptide li-
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