UCSF scientists are reporting key insights into the p53 tumor-suppressor gene that they say should help harness the gene to treat cancer.
The gene is disabled in most forms of the disease, and its loss is often associated with increased malignancy, resistance to treatment and decreased patient survival.
Scientists have long considered restoration of the p53 gene a possible therapeutic strategy. However, they have not known whether restoring the gene would lead to its activation or what impact, if any, such activation would have on established tumors - until now.
In a unique model of p53 signaling, UCSF scientists restored the gene in the tumor cells of mice with established lymphoma. The initial results were dramatic.
"The tumors were completely dead within hours," says senior author Gerard I. Evan, PhD, the Gerson and Barbara Bass Bakar Distinguished Professor of Cancer Research at the UCSF Comprehensive Cancer Center. The animals also survived significantly longer than mice in which p53 had not been activated.
"These results demonstrate that signals exist in established lymphomas that can engage and trigger p53," he says. "All the machinery is there - all the signals leading to p53 - and all the machinery downstream of p53 that can destroy cancer cells is in place. The only thing missing is p53 itself. Restoring p53 in these tumor cells is like engaging the clutch in your car - it reconnects the cancer-sensing 'engine' to the cell death 'transmission.'
"This result is very good news to the many of us who are thinking about trying to restore p53 function in established human cancers."
At the same time, he notes, the tumors eventually re-emerged in all of the mice. The team analyzed how this occurred, and identified tactics for optimizing the gene's therapeutic impact.
The study was considered significant enough by
Cell that it was published online on Dec. 21, in advance of its publication in the Dec. 29 issue of the journal. The study was led by Carla P. Martins, PhD, a postdoctoral scholar in the lab of senior author Evan.
The p53 tumor-suppressor gene has been of intense interest to biomedical scientists for a quarter-century. It encodes a protein that helps preserve the integrity of cells. When the protein detects insults - DNA damage, oxygen and nutrient deprivation or mutated genes - it triggers programs that arrest that cell's normal cycle and growth until it can repair itself or, if the damage is too severe, induces cell suicide.
In virtually all cancers, the p53 gene, or one of its upstream activators or downstream mediators, always is inactivated or deleted. Such loss of function allows mutated genes known as oncogenes, which hijack the cell cycle, to go unchecked. Once a critical ensemble of oncogenes has accumulated, the cell spirals into unbridled expansion, the hallmark of cancer.
Scientists have speculated that if the p53 gene could be restored in tumors, it might re-engage its protective functions and arrest or reverse the cancer. However, they have not known at what point in the evolution of a tumor that the loss of p53 has its effect, or why: Is inactivation of p53 required only transiently during early tumor evolution, allowing the cancer to escape a bottleneck in its progression? Or does inactivity of p53 remain a continuous requirement, even for established cancers?
Given these questions, scientists have not been certain that p53 would function when restored in established tumors. Would the upstream and downstream genes in its signaling pathway be present to support its function? Would other cellular factors prevent its restoration?
And if it were restored, what would its impact be? Would the tumor cells die, stop or ignore it? Would the tumor then respond, regress or grow back?
The scientists' novel model was designed to answer these questions. Using a technique previously reported by members of the team (
Nature Genetics online, May 29, 2005), the scientists genetically engineered mice so that one of the two normal p53 gene copies in each cell had been replaced with one that encoded a p53 protein with a built-in switch, allowing the scientists to toggle p53 function on and off at will in the genetically modified mouse. At the outset of the experiment, this switchable p53 was switched to the "off" position. The other copy of p53 remained normal and functional.
The team then expressed the myc oncogene, which drives the development of lymphoma and many other tumor types, into the lymphocytes (a type of white blood cell) of the mice. After a few weeks, the mice developed lymphomas and, as expected, the functional p53 gene copy had been inactivated in all the lymphoma cells.
The scientists then transplanted the tumors into normal mice, a technique that allows much more reproducible monitoring of how the cancers grow and greatly reduces the number of animals that need to be used. Then, using the on-off switch mechanism, they restored p53 function in the transplanted tumor cells and assessed the short- and long-term therapeutic impact.
After observing the immediate results - destruction of the tumors and increased life span of the animals - the team analyzed what led to the eventual relapse and re-emergence of the tumors, and what could be done to address the problem.
The recurring tumors, they noted, had either disabled their spare, switchable p53 or had inactivated a protein upstream of p53 called p19ARF. Tumors that deleted p53 retained p19ARF, while those that inactivated p19ARF retained p53. This indicates, says Evan, that both p19ARF and p53 are essential connectors in a sequential pathway. p19ARF is the conduit that links p53 to oncogenic mutations in the cell, triggering p53 when the cell cycle machinery becomes corrupted.
Interestingly, however, says Evan, inactivation of p19ARF does not prevent p53 from being activated by many other triggers, such as DNA damage. This appears to imply that activation of p53 by oncogenes is the only significant trigger of p53 function in established tumor cells. DNA damage and other forms of stress appear not to be significant activators of p53 in established tumors.
This, he says, was a "real surprise." In principle, then, those tumors that recur after p53 therapy because they kick out their newly restored version of p53 could be treated again merely by reinstating p53 yet again.
Those recurring tumors that lose p19ARF, however, would present a greater challenge because these cancers no longer have the upstream signal that is needed to engage p53. The team saw a possible work-around, though: The recurring tumors that lose p19ARF retain functional p53. Hence, it might be possible to activate this residual p53 to therapeutic effect via other p53-activating pathways.
To test this idea, the team restored p53 in the animals and then treated them with radiation - which causes DNA damage and activates p53 without the need for p19ARF. The result was a significant delay in the onset of tumors, compared with either treatment alone. In fact, says Evan, the impact was synergistic rather than merely additive. Thus, simultaneously engaging two pathways that each, independently, activates p53 is significantly better than engaging each one in turn, suggesting some interesting strategies for optimizing p53-based treatments in cancer patients.
As to why the tumors become resistant to p53 and p19ARF at all, Evan thinks the answer is that some cells within the original tumor populations are innately resistant, due to random mutation in either p53 or p19ARF, from the outset of tumor formation. However, it is only when all the other sensitive tumor cells die following restoration of p53 that the few resistant cells multiply and surge to the fore.
"The good news," says Evan, "is that those cells that lose p53 or p19ARF retain all the other signals to activate the pathway. So if there were a way to restore the genes continuously, the tumor could be contained."
The other co-author of the study was Lamorna Brown-Swigart, PhD, a specialist in the UCSF Comprehensive Cancer Center. The study was funded by the National Institutes of Health and the Leukemia & Lymphoma Society.
Modeling the therapeutic efficacy of p53 restoration in tumors
Carla P. Martins, Lamorna Brown-Swigart and Gerard I. Evan
Cell 127;7:1323-1334. December 29, 2006
Summary |
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Temporal Dissection of p53 function in vitro and in vivo
Maria A. Christophorou, Dionisio Martin-Zanca, Laura Soucek, Elizabeth R. Lawlor, Lamorna Brown-Swigart, Emmy W. Verschuren, Gerard I. Evan
Nature Genetics 37:718-726. May 29, 2005
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Related Links:
UCSF Researchers Challenge Paradigm of How a "Tumor Suppressor" Works
UCSF Today, September 6, 2006
Laboratory of Gerard Evan
UCSF Comprehensive Cancer Center
