The word cancer applies to more than a hundred diseases—some evolve rapidly, others only appear after decades, some are highly curable and others are unavoidably fatal—but all have one thing in common: the uncontrolled proliferation of cells. Based on international studies and data obtained from his own laboratory at the Federal University of Rio de Janeiro (UFRJ), biochemist Jerson Lima da Silva believes that at least some cancers are triggered by the same molecular mechanism as Creutzfeldt-Jakob disease, the human version of mad cow disease, which causes premature cell death and leaves the brain as porous as a sponge.
According to this view, advanced by Silva and his colleagues in a paper published June 2013 in Bioscience Reports, both in cancers characterized by the perpetuation of cell life, and in Creutzfeldt-Jakob disease, in which cell death is premature, the source of the problem is the same: an abnormal protein fold. It may seem too subtle a cause for damage so great. But researchers believe it makes sense. After all, it is the three-dimensional structure of these large, complex molecules that is crucial to defining the structure and functioning of the cells and determining the role they will play. When the folds are incorrect, proteins generally no longer function as they should and even take on additional functions. The difference between cancer and Creutzfeldt-Jakob disease lies in the affected protein.
In the forms of cancer analyzed by the UFRJ group, the deformation occurs in the p53, the protein that has been called the guardian of the genome, because it coordinates DNA repair in the case of minor damage and is responsible for the death of cells whose defects cannot be repaired. Yet in Creutzfeldt-Jakob disease the altered protein is the cellular prion, the molecule anchored on the outer surface of the cell that controls the movement of information from the external environment to the internal environment (see Pesquisa FAPESP Issue nº 148). In both cases, the defective fold appears to confer on the altered protein a characteristic typical of traditional infectious agents such as viruses and bacteria: the ability to autopropagate and infect other cells.
The idea that deformed versions of a protein can cause disease is not new. It was proposed in the 1980s by American researcher Stanley Prusiner to explain the origin of a group of neurodegenerative diseases that include Creutzfeldt-Jakob disease and mad cow disease—the spongiform encephalopathies. While investigating the causative agent of an encephalopathy that affects sheep, Prusiner did not find the viruses that he anticipated. Instead, he was only able to identify a defective protein, which he called a prion (short for proteinaceous infectious particle) and formulated an explanation of how prions deform healthy proteins. According to this hypothesis, for which Prusiner won a Nobel Prize in 1997, the mere contact of the deformed molecule with normal proteins is enough to alter their three-dimensional structures. It is a chain of events that once started cannot be stopped, a kind of domino effect. It is also an effect that is difficult to reverse. The defective proteins have a more stable structure than the healthy ones and adhere to each other, giving rise to long fibers that are toxic to neurons.
“We believe the same process occurs in most cancers in which p53 is deformed,” said Silva in his laboratory in early August, a few days after an event in which he compared his results with Prusiner’s. According to Silva, recent international studies also suggest that some deformed versions of p53 could travel from one cell to another. This does not mean, however, that they are infectious and could be transmitted between individuals of the same species. “These proteins can be transmitted from cell to cell, but are not infectious,” explained Silva, who coordinates the National Institute of Science and Technology for Structural Biology and Bioimaging and is the scientific director of the Rio de Janeiro Research Foundation (Faperj).
The strongest evidence that defective versions of p53 can act as a prion—researchers call them prionides as their actions are prion-like—and induce remodeling of healthy proteins, causing them to lose their original function, has emerged in the past two years. In partnership with a team of geneticists led by Cláudia Moura-Gallo of Rio de Janeiro State University (UERJ), Silva ‘s group analyzed breast tumor samples from 88 women and found that in most cases aggregates were formed by defective p53 molecules, similar to amyloid aggregates of diseases caused by prions—such fibers and aggregates also appear in other neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. The deformed proteins were almost always generated as a result of minor alterations of TP53, which contains the recipe for production of this protein.
It was the first time that p53 fibers had been found in tumor cells. But just the identification of these fibers was not enough to demonstrate that the altered protein could trigger the deformation of healthy proteins—a phenomenon called seeding, which is a characteristic property of prions. In Silva’s laboratory, biomedical researcher Ana Paula Ano Bom, pharmacist Luciana Rangel, and biochemist Danielly Costa then began tests to identify the conditions under which p53—both the healthy and deformed versions—generated aggregates. In order to do this, they measured the amount of time it took for the fibers to emerge and what form they assumed under different chemical (pH) and physical (temperature and pressure) conditions.
They found that the two forms of p53 spontaneously generated aggregates under conditions similar to those found by cells in the human body—a temperature of 37oC and a neutral or slightly acidic pH, which are characteristic of tumors. These fibers formed even when only the central segment, the part of p53 that interacts with DNA, was used in the experiments. The difference was that the toxic aggregates formed more rapidly from the deformed p53, as the researchers reported in August 2012 in the Journal of Biological Chemistry (see infographic above).
The most important result appeared when the researchers added tiny concentrations of the defective protein to containers with healthy p53. Like a drain draws off liquid, the deformed protein attracted normal molecules and induced their conversion into altered forms—confirming its seeding property. The ability to induce the deformation was greater when the altered version of p53 resulting from a mutation known as R248Q was used, one of the seven most frequent cancer mutations. “We found aggregates of this mutant form in breast cancer samples and in cell lines of this tumor grown in the laboratory,” says Costa.
Small changes, big effects
The formation of p53 clusters is not unique to breast tumors. Earlier this year a team led by Rakez Kayed of the University of Texas at Galveston, described these fibers in a very common type of skin tumor, basal cell carcinoma. More recently Silva’s group also identified them in samples of human glioblastoma, one of the most aggressive brain tumors known.
The tendency to aggregate does not occur as a result of the R248Q mutation alone. As noted by the UFRJ researchers, other specific changes in the gene generate versions of the protein that are prone to aggregation. In addition to modifying the structure of the healthy p53, the deformed versions of this molecule sequester and damage two other proteins of the same family: p63, which controls cell proliferation, and p73, which independently directs defective cells to apoptosis, a type of programmed cell death. These two proteins also play an important antitumor role,” says Costa.
In a study published in July 2013 in PLoS One, Xavier Roucou and his team at the University of Sherbrook, Quebec, Canada, demonstrated that a defective version of p53 is indeed capable of infecting healthy cells. The researchers added deformed molecules to cell cultures and found that the altered proteins were absorbed by sacs formed in the membrane. Once inside the cells, the misfolded p53 triggered the formation of fibers.
Like any new idea, there is no consensus surrounding the hypothesis that mutated versions of p53 can function as a prion. “The UFRJ team’s work is quite consistent, but more evidence is needed,” says Vilma Martins, a biochemical expert on prion-related diseases from the International Center for Research at the A.C. Camargo Cancer Center in São Paulo. One of the steps we need to demonstrate the prion-like action of p53 is to evaluate if the deformed protein in the laboratory gives rise to tumors in animal models. Despite the cautious approach, Martins believes that this mechanism may explain the origin of some cancers linked to mutations in the TP53 gene.
If the idea of the UFRJ group is correct, it will help in understanding the development of the so-called spontaneous tumors, which are not transmitted from one generation to another and arise as a result of genetic alterations in the already formed embryo or in adults. The prion-like action of p53 would provide a good explanation for spontaneous tumors, which occur in most cancers, especially when there is the so-named negative dominance. “In this biological phenomenon, changing only one of the two copies of a gene is enough to lead to the development of a disease,” says Silva, who raised the possibility in the case of p53 as early as 2003, when he began to study protein folding. He believes it may be possible to use p53 fibers in a test, as a molecular marker of cancer’s severity or its prognosis. And also that in the future it may become possible to interfere with this mechanism and try to slow the development of some tumors. “Maybe,” he says, “we can find a way to block the aggregation process.”
SILVA, J. L. et al. Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumor suppressor. Bioscience Reports. June 27, 2013.
SILVA, J. L. et al. Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins. Accounts of Chemical Research. v. 43, no. 2, p. 271-9. 2010.
ANO BOM, A. P. et al. Mutant p53 aggregates into prion-like, amyloid oligomers and fibrils: implications for cancer. Journal of Biological Chemistry, v. 287 No. 33, p. 28,152-62. August 10, 2012.