One Protein to Rule Them All

In the 1970s, scientists knew that some viruses and chemicals caused cancer, but they didn’t know how. Arnold Levine, a biologist currently at the Institute for Advanced Study researched DNA viruses at Princeton University at the time. “Just like the RNA virus virologists didn’t talk to the DNA virologists [about viruses] that cause tumors, the people who worked on chemical carcinogenesis, never spoke to the virologists,” he recalled. But in 1976, a study demonstrated that an oncogene from an RNA virus that caused tumors in avian cells originally came from the host cell.¹ This discovery indicated that many animals have genes that, if manipulated by a viral agent or other stressor, act as oncogenes to induce tumors. DNA virologists then turned their attention to searching for tumorigenic culprits among viruses.

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Ten years later, it changed from an oncogene to a tumor suppressor gene. And that just changed the field completely.
– Arnold Levine, Institute for Advanced Study

One DNA virus stood out. Simian Virus 40 (SV40), which infected human and mouse cells, induced tumorigenesis in both cell types and caused tumors in mice. By digging into its mechanisms, scientists identified a viral oncoprotein, large T antigen, which was necessary for tumorigenesis.^2,3 In the late 1970s, four teams working independently reported a host protein of about 53 kiloDaltons (kD) in size complexed to the large T antigen.

Michel Kress, then a graduate student in Pierre May’s lab at the Cancer Research Institute and today a biologist at Sorbonne University, found this new protein by infecting several different cell lines from multiple species with SV-40.⁴ David Lane, a postdoctoral fellow in Lionel Crawford’s lab at the Imperial Cancer Research Fund (now Cancer Research UK) and today an immunologist at the Karolinska Institute also identified it from SV40-transfected cells and demonstrated that the complex could be isolated consistently in the same ratio even with limiting amounts of antiserum, but that the 53kD protein was distinct from the large T antigen since serum specific for this viral protein did not bind to purified 53kD protein.⁵ Levine and his graduate student, Daniel Linzer, found the new protein in SV40 infected cells, but also identified it in noninfected embryonal carcinoma cells.⁶ Finally, Lloyd Old, at the time an immunologist at Memorial Sloan Kettering Cancer Center, and his group showed that this new protein could also be detected in chemically-induced tumor cells.⁷

All four groups independently published their findings in separate journals in 1979. Since they all used different names to describe this protein, it took some time for scientists to realize that all four papers described the same protein. Once everybody got on the same page, the community settled on a common name: p53.

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“The field looked at this and didn’t know what to make of it,” Levine said. “Because it wasn’t isolated like an oncogene, it was just isolated as an interacting protein with an oncogene.” The groups interested in studying p53 next wanted to investigate its interactions with T antigen.

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p53: A Cancer Promoter

Eventually, scientists cloned tp53 (the human gene; in mice, it is Trp53) from their cell cultures and studied it in the context of cancer.^8,9 When scientists inserted the p53 gene into normal cells, those cells became cancerous; its introduction could also make tumors more aggressive.^10,11 They predominantly detected p53 in cancer cells, but noted that it was nearly absent in healthy cells.¹² All of the evidence pointed to p53 promoting the progression of tumors under certain conditions.

Scientists made these findings as the theory of oncogenes, the idea that mutated host growth-promoting genes in cells caused cancer, was becoming popular.¹³  This new explanation to the cause of cancer united the observations of chemically-induced and virally-induced cancer transformation. Around this time, cancer researchers across different disciplines concluded that the gene encoding p53 was an oncogene.

Once they started searching, scientists found p53 in almost every cancer type. Groups studying p53 and its gene across different model organisms even found this protein in nonmammalian organisms like frogs and trout, which enabled them to piece together the domains of the protein.¹⁴

Except there was a problem. As researchers looked deeper into the data from different cancers, the amount of p53 that they observed wasn’t consistent across cell lines and tumors.¹⁵ While some researchers found rearrangements in the p53 gene in tumors that elevated its expression, others found that the gene was nearly entirely deleted in some cases.^16-18

Switching Sides: Stopping Cancer

In the excitement of discovery that sparked new explanations for cancer, scientists had quickly fit a label onto p53, but an oversight soon came to light. All the various tp53 and Trp53 clones used in cancer studies came from transformed cell lines; they all contained mutations.

Levine and his team demonstrated that cloning the gene for p53 from noncancerous cells didn’t induce transformation and showed that normal p53 could actually suppress tumor formation, which was confirmed by another group.^19,20 Around the same time, another team showed that for cancer to develop, both copies of p53 needed to be inactivated.²¹

This result echoed findings from research on retinoblastomas showing that most tumors developed only after two mutation events. ^22,23  This result laid the foundation of the concept for the “two-hit” hypothesis that tumor suppressor genes required both alleles to be mutated for cancer to develop; in contrast, oncogenes activated if just one gene was mutated. “Ten years later, it changed from an oncogene to a tumor suppressor gene,” Levine said. “That just changed the field completely.”

The Guardian of the Genome and Beyond

 Once researchers redefined p53 as a tumor suppressor gene and generated nonmutated clones, they began piecing together the puzzle of its role in the cell. One group showed that p53 controlled the progression of the cell cycle, but it wasn’t clear how.²⁴

Guillermina Lozano entered the p53 field as a postdoctoral fellow in Levine’s lab in 1985 and continued researching its functions later as a geneticist at University of Texas MD Anderson Cancer Center. When analyzing the p53 sequence, she noticed that it looked similar to many other known transcription factors. “At the time, my first hypothesis was it was a transcriptional repressor,” Lozano recalled.

It’s amazing that an individual point mutation could cause an entire organism to develop cancer.
– Deborah Kelly, Pennsylvania State University

They didn’t know what genes p53 regulated, so she and her team replaced tp53’s DNA binding domain with the one from GAL4, a galactose-responsive transcription factor in yeast, for which the researchers knew the target binding sequence. The scientists fused the target binding sequence to a reporter with strong expression to assess if p53 could repress its transcription. When the results didn’t show any reduction in gene expression, Lozano noticed a small increase in the already highly expressed reporter gene that they were trying to repress with their fused p53 gene construct. “All of a sudden, I looked at the data, I said ‘we’re doing it all wrong,’” Lozano said.

Her team created a new reporter fused with chloramphenicol acetyltransferase, which is a lowly expressed gene, to steer p53 to activate its expression. “It went through the roof,” Lozano remembered. She had successfully shown that p53 was a transcriptional activator.²⁵

Shortly after this, researchers solved another piece of the p53 puzzle when they identified the gene murine double minute 2 (mdm2), encoding MDM2, which bound Trp53 and targeted it for degradation.^26,27 Another group showed that p53 drove the expression of mdm2, suggesting that p53 operated in a negative feedback loop. ²⁸ Because of this regulatory mechanism, when Lozano and her group turned to mouse models to explore more of p53’s functions, they discovered that deleting mdm2 alone in mice was lethal during development, and she and her team had to delete Trp53 in addition to mdm2 to prevent excessive cell death.²⁹

Through this feedback loop, p53 remained inactive and at low levels in the cell under homeostatic conditions. However, if something like a virus or a stressor like ultraviolet light induced DNA damage, p53 sprang into action, stopping the cell cycle by activating p21 and halting DNA replication.^30,31 The protein could promote DNA repair, or if the damage was too extensive, initiate apoptosis.³² Later, Lozano showed that p53 stopped the cell cycle independently of inducing apoptosis when preventing tumors, underscoring this protein’s extensive control over cell growth.³³

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p53 wasn’t just an anticancer protein though. Animal studies demonstrated that p53 was expressed at varying levels in mouse and frog embryos throughout their development.^34-36 Although researchers successfully raised mouse colonies with the p53 gene deleted, further investigation demonstrated that loss of p53 not only increased the risk of cancer in offspring, but also led to a variety of developmental defects including embryonic or postpartum demise, particularly in female mice.^37-40

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“It turns out that p53 regulates a whole different set of genes in the embryonic stem cells,” said Jing Huang, a senior investigator at the National Cancer Institute who previously studied p53’s role in embryonic development and currently investigates its gain-of-function activity in cancer. Huang’s team demonstrated novel p53 targets and regulatory mechanisms in mouse embryonic stem cells.^41,42

Scientists also found that p53 played a role in inflammation, metabolism, migration, aging, and cell differentiation.^43-47 Researchers are still investigating how p53 determines which role it will play and which decisions it makes within cells. Previous studies showed that p53 executes most of its functions as a dimer of dimers to create a DNA-binding tetramer unit, though others indicated that p53 dimers, splice variants, or interactions with the homologues p63 and p73, could control p53’s diverse and tissue-specific actions.^48-51 While p53 is known as the guardian of the genome, it’s apparent that this protein more accurately acts as the ultimate cellular maestro.

Making Molecular Models

As far reaching as p53’s cellular functions appear to be, whether it is halting the cell cycle for repair, promoting differentiation toward a defined function, quieting cells through senescence, or outright killing cells, Lozano sees a common thread. “All those are dead ends,” she said. “You arrest, you senesce, you die, or you differentiate.”

However, when a mutation disrupts this great conductor, mutated cells multiply and become cancerous. Whereas most other tumor suppressor genes become inactivated through deletions or truncations, the majority of p53 mutations gives rise to a functional protein with altered activity. While some mutations merely prevent p53’s induction of cell cycle arrest and apoptosis, others, termed “gain-of-function” mutations, cause p53 to bind to atypical proteins that promote DNA replication and dampen genome repair mechanisms, as well as repress a nonmutated allele of p53 if one is present, leading to more aggressive tumors.^52-55

Huang and his group study p53 gain-of-function in breast cancer models to identify mutant p53 binding partners to understand how these mutations alter p53’s transcriptional activity. “If we can find this unifying gain-of-function mechanism, then potentially, we can design a therapeutic strategy,” he said.

Understanding a protein’s structure and how it changes during mutations can also help scientists develop therapeutics against it. “When there are mutations in these proteins, or when there are changes that affect the way that they look and act inside cells, they no longer do their productive duties as efficiently in terms of preventing cancer as tumor suppressors,” said Deborah Kelly, a biophysicist at Pennsylvania State University who uses cryogenic electron microscopy (cryo-EM) and other biophysical methods to study tumor suppressor protein structures.

However, imaging the p53 structure is not easy. “It’s a really hard protein to work on because most of it is flexible. There’s a lot of disorder,” Kelly explained.

Scientists determined that p53 has five distinct domains: an N-terminal domain with two transactivation regions, a proline-rich regulatory domain, a tetramerization domain, a DNA-binding core domain, and a C-terminal domain, which also interacts with DNA nonspecifically.⁵⁶ Unstable domains complicate imaging efforts, so many early structural analyses focused on the more stable DNA-binding domain.^57,58 Subsequent studies used cryo-EM, single-molecule fluorescence resonance energy transfer, and mass spectrometry to create models of recombinantly expressed protein with the help of computer modeling software to improve the picture of p53.^59-62

Although a high-resolution, full-length model of p53 has yet to be solved, Kelly and her team are pushing the view of p53 forward as technology advances. Using cryo-EM, the team developed the first full-length model of normal p53 from human cells as opposed to recombinantly expressed protein.⁶³ With this model as a reference, they compared the effects of frequent p53 mutations on the protein’s structural properties through molecular simulations in an effort to better understand how these errors contribute to specific changes in the protein’s overall conformation or even environmental charge.⁶⁴

“It’s amazing that an individual point mutation could cause an entire organism to develop cancer,” Kelly said. However, if these mutations affect local electronic charges or correct folding, it could prevent proper post-translational modifications, which p53 relies on heavily for proper functioning. While the field is still working on a high-resolution, full-length structure of p53, current models inform scientists about how mutations alter the function of p53, which in turn, can improve the development of novel therapies.

Inspiration From the Wild Side

Another route to understand how p53 balances cell growth with cell survival is to look beyond humans for answers. Comparative oncology investigates the reasons behind cancer susceptibility and resistance in animals to help answer questions about how human cancers may form and to potentially offer therapeutic solutions.^65,66 When exploring cancer resistance and p53, one animal that captured the interest of many scientists, including Joshua Schiffman, a pediatric oncologist at the University of Utah, was the elephant. Despite some species of elephants living as long as humans, and the animals outweighing the average person by a couple orders of magnitude, these creatures have remarkably low cancer rates.⁶⁷ The answer could in part be due to p53.

Elephants have twenty copies of p53, thus in a single cell, they have 40 p53 alleles.⁶⁸ Although many of these are retrogenes that don’t give rise to functional proteins, at least some of these transcripts seem to provide other procellular growth control effects independent of p53’s normal transcriptional role, such as alternative binding affinity to MDM2.⁶⁹ Schiffman’s group showed that elephant p53 tolerates DNA damage less than the human version, with the elephant protein triggering apoptosis preferentially over cell cycle arrest.

However, the biggest question remained whether this had any potential as a future therapeutic for cancer in humans. To begin to answer this question, Schiffman and his team collaborated with their local zoo, Hogle Zoo, and animal conservatories to obtain blood and tissue samples from these animals during routine procedures. Once they had elephant cell lines in their incubator, they replaced the human p53 in a cancer cell line with that of elephant p53 and its retrogenes.⁷⁰ “Whatever type of cancer we tested, when we inserted elephant p53 and it was overexpressed, these cancer cells shattered,” Schiffman said.

Schiffman’s team also studies cancer resistance and p53 function from many other animal species through their collaboration with zoos and wildlife preserves. “We really decided to focus on how nature, over hundreds of millions of years of evolution, decided to control this master regulator of cancer,” Schiffman said.

Schiffman cofounded and currently serves as the chief executive officer for the biotechnology company Peel Therapeutics, which incorporates evolutionary biology in their search for treatments. While he acknowledged that these results were far from clinical trials, Schiffman and his group are optimistic about the potential this finding has in treating patients with p53 mutations and cancer.

“The next step is really to identify the mechanisms of cancer defense across species and then validate those,” said Lisa Abegglen, a comparative cell biologist in Schiffman’s lab. These findings may help scientists better understand the unique functions of human p53 isoforms and identify new potential therapeutic ideas, such as restoring p53 function with the help of small molecules, or even targeting specific downstream proteins of p53 to activate cell death pathways. However, Abegglen noted that some of these mechanisms may be independent of p53. “Throughout evolution, p53 has evolved enhanced function. And if we can understand how that happened, and what changes in p53 can confer enhanced function, then we can potentially develop a better version of p53 to treat human cancers,” said Abegglen.

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