Researchers at Children’s Medical Research Institute have demonstrated why chemotherapy drugs work better with some types of cancers than with others.
Dr. Pragathi Masamsetti, the lead author of a new paper published in Nature Communications, has been studying the pathways of cell death caused by chemotherapy drugs for the past five years after a surprise initial discovery piqued her interest.
Now that her Ph.D. is finished, she is optimistic that her research will form the groundwork for other scientific or clinical developments in cancer treatment.
“This research could influence which class of chemotherapy is used for each cancer by giving doctors and scientists a better understanding of how the mutations in each type of cancer respond to various chemotherapy drugs.”
Chemotherapy drugs work by causing a lethal level of stress on the processes occurring when cells are rapidly dividing, a hallmark of cancerous tissue.
But these drugs, which attempt to stop rapid cell division, are a blunt solution and are not always effective.
“Chemotherapy can be very powerful in the right situation, but we are only beginning to understand the mechanism behind why some cancer cells die and why some proliferate when targeted by chemotherapy.
“Depending on the type of mutation in a patient, the cancer cells can avoid dying.
These cells may go on to proliferate and actually promote the cancer.”
Dr. Masamsetti’s research revealed that cancers avoid cell death via a range of cellular pathways.
The exact pathway the cancer cell uses depends on the particular mutations and other factors present in the cell.
Cells with certain cancer mutations, such as the common p53 mutation, are adept at avoiding their own death and go on to create further instability in the genome, making the cancer even more diverse and hard to eradicate.
Well-known P53 functions
P53 is often mutated in solid tumors, in fact, somatic changes involving the gene encoding for p53 (TP53) have been discovered in more than 50% of human malignancies. P53 is a transcription factor able to regulate several intracellular pathways involved in cell survival, DNA-repair, apoptosis and senescence.
In normal and un-stressd conditions, p53 is inactivated by its negative regulators, such as murine double minutes2 (MdM2) and phosphatase and Tensin homolog (PTEN).
MdM2 is an E3 ubiquitine ligase which favors the p53 degradation via proteasome.
In stressed conditions, such as, the presence of genotoxic stimuli, p53 is stabilized by several post-translational covalent modifications which are mediated by another important enzyme named PML (promyelocytic leukemia).
As result, it can accumulate and form tetramers with other p53 subunits which exert their function as transcription factors. P53 mainly promotes cell cycle arrest, DNA repair and, if the damage is anbnormally wide, apoptosis.
Cell cycle arrest and senescence are mainly mediated by a p53 downstream effector named p21 (encoded by the gene WAF).
P21 acts inhibiting the hetero-dimers formed by ciclins/Cdk, thus arresting cell-cycle progression and favoring the cell entrance in G0 phase (senescence).
Moreover, while cell cycle is arrested, p53 promotes DNA repair, mainly activating GADD45 (Growth Arrest and DNA Damage) and P53R2 (p53 inducible ribonucleotide reductase) genes.
As result, DNA damage is repaired, avoiding mutations accumulation and their perpetration to descendent cells during mitosis.
In response to severe or sustained stress signals causing irreparable DNA changes, p53 usually induces genes involved in apoptosis, namely PUMA (p53 upregulated modulator of apoptosis), Bax, Fas, PIG3 and Killer/DR5 (3,7–9).
Peculiar P53 functions
Recent studies have demonstrated that in addition to cell cycle arrest, senescence induction, DNA repair and apoptosis, p53 is able to exert additive functions, such as regulation of energy metabolism and anti-oxidant defense.
Metabolic changes are a hallmark of tumor cells, in fact, cells rapid growth and division requires energy and precursor for macromolecule byoshyntesis.
P53 has been discovered to regulate glycolysis, pentose phosphate pathway (PPP), mytochondrial oxidative phosphorylation, lipids and nucleotides metabolism and, importantly, the cell response to oxidative stress.
Unlike the majority of cells, which depend on oxidative phosphorylation to provide energy, tumor cells mainly utilize glycolysis even in presence of sufficient oxygenation.
This phenomenon (glycolysis in aerobic conditions) is named Warburg effect. Wild type P53 upregulates oxidative phosphorylation and down-regulates glycolysis inducing its target genes, such as SCO2 (synthesis of cytocrome oxidase 2) and GLS2 (mitochondrial glutaminase), which promote mitochondrial oxidative phosphorylation, and TIGAR (TP53 indicible glycolysis and apoptosis) which inhibit glycolysis (12).
Moreover, P53 blocks the intracellular glucose uptake inhibiting the expression og GLUT1 and GLUT4 (glucose transporter) and also reduces the activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD), which has an important role in initiate the PPP. The PPP is an alternative pathway to glycolysis and is often employed by tumor cells (13).
Tumor cells strongly needs of fatty acids (FAs) synthesis with the aim to perform rapid cell membrane production and intracellular signaling transduction.
Wild type P53 has been reported to stimulate β-oxidation of FAs in mitochondria and to arrest FAs biosynthesis acting on FASN (fatty acids syntase) and ACLY (ATP citrate lyase), thus blocking the formation of new phosphor-lipid membrane to support the rapid growth and division of tumor cells (14).
Oxidative stress often leads to DNA-damage and apoptosis through the ROS (reactive oxygen species) production, and wyld type p53 is the main controller of intracellular ROS level. Specifically, p53 regulates the level of NRF2 (nuclear factor erythroid-related factor 2) through p21 up-regulation. NRF2 is linked by p21 and translocated into nucleus allowing its transactivator function upon a number of genes which encodes for anti-oxidant enzymes, such as NQOI (NADH-quinone oxidoreductase 1). In response to mild stress, p53 up-regulates NRF2 through p21, and NRF2, once translocated into nucleus, induces the synthesys of anti-oxidant enzymes. When the stress is severe, p53 inhibits NRF2 allowing the ROS accumulation and apoptosis induction (15).
TP53 mutations and cancer
Majority of tumor suppressor genes, such as APC (adenomatous polyposis coli), RB (retinoblastoma-associated protein) and VHL (Von-Hippel-Lindau) are inactivated by deletion or early truncation mutations in tumors, resulting in the decreased or loss of expression of their proteins.
On the other hand, most p53 mutations in human cancer are missense mutations, which result in the production of full-length mutant p53 proteins.
In fact, only 10-15% of TP53 mutations are defined as “disruptive mutations”, namely those leading to a inactive or truncated protein, while the remaining 85-90% often lead to the synthesis of functioning proteins.
Missence mutations are often clustered in the 4–9 exones of the TP53 gene, which correspond to a particular sequence of the gene, representing the p53 DNA- binding-domain.
As a consequence, missense p53 mutations are able to modify its ordinary function of transcriptional factor (16).
Interestingly, several studies have demonstrated that many mutant p53 proteins, not only lose their tumor suppression functions, but also gain new oncogenic functions.
This phenomenon is termed “the gain of function of mutant p53”.
More specifically, mutant p53 interacts with proteins that normally partner with wild type p53.
This new association deprive them of their anticancer activities and in place, they are corrupted to act as cancerogenesis promoters (17).
An example is given by the p53-PML interaction. PML stabilizes and activates wild type p53 only in presence of stressed conditions, while, when p53 is mutated its association with PML is constitutive.
Mutant p53-PML stabilized interaction leads p53 to aberrantly transcribe its targets. The major consequences of gain of functions mutant p53 affect the metabolism and the response to oxidative stress of cancer cells.
Rapid cell proliferation strongly needs of a ready supply of energy and basic macromolecules, so mutant p53 proteins have been reported to facilitate the supply of cancer cells.
Proper repair of damaged DNA, not only requires DNA-repair enzymes, but also the availability of nucleotides, and, mutant p53 constitutively activates the ribonucleotide reductase (RRM2) to facilitate the conversion of ribonucleoside diphosphate to deoxy-ribonucleoside diphosphate, which is an essential step for DNA synthesis.
In a similar manner, mutant p53 is able to modify the lipids metabolism in a way that encourages rapid cell proliferation and intracellular signaling.
In fact, mutant p53 engages the SREBPs (sterol regulatory element-binding protein) and leads to FAs synthesis, β-oxidation arrest and cholesterol synthesis, favoring so, the generation of a robust supply of lipids for cell membranes formation (18).
The strongest effect of mutant p53 on the metabolism regards the glucose. Mutant p53 constitutively stimulates glycolysis acting in various ways.
First of all, it up-regulates GLUT1 and GLUT 4 favoring the rapid uptake of glucose in tumor cells, moreover, mutant p53 stimulated genes involved in glycolysis activation such as TIGAR, and finally it stimulates the PPP acting on G6PD (13).
The final result is a wide supply of glucose which can be consumed in order to produce energy also in aerobic conditions (Warburg effect).
Lately, an additive and very important mutant p53 function has been documented. Rapid cell proliferation produces high levels of ROS, which may dampen cell membranes, proteic structures and DNA inducing apoptosis. Mutant p53, as previously seen, can constitutively stimulate genes involved in antioxidant cell response.
The greater response to the activation of these genes is the synthesis of molecules able to neutralize ROS, such as glutathione (19). Yogev et al. demonstrated that in MYC-driven neuroblastoma, the acquisition of p53 mutations can greatly worsen the prognosis of patients affected, and this feature can be mainly due to metabolic changes in tumor cells.
In fact, Synergistic effect of Myc protein and mutant p53 strongly up-regulates the GSH (Glutathione S transferase) family, such as Gstz1 and Gstp1, resulting in an increased intracellular pool of glutathione.
This characteristic not only preserves tumor cells from oxidative-stress-induced damage, but also favors the resistance to ionising radiations. Up-regulation of GSH strongly predisposes to chemo and radioresistance and not only in Myc-driven neuroblastoma, but also in other solid tumors and it can be due to p53 missense mutations (20).
This protein is mutated in half of all cancers. New drugs aim to fix it before it’s too late
By Robert F. ServiceOct. 5, 2016 , 12:00 PM
It has been nearly impossible to get a good look at Rommie Amaro’s favorite protein in action. Called p53, the protein sounds the alarm to kill cells with DNA damage and prevent them from becoming cancerous—one reason why it has been called the “guardian of the genome.” But it is big and floppy, a molecular shapeshifter that is hard to follow with standard imaging tools. So Amaro, a computational biologist at the University of California (UC), San Diego, turned to supercomputers. She plugged in new x-ray snapshots of p53 fragments and beefed up her program to make a movie of the quivering activity of each of the protein’s 1.6 million atoms over a full microsecond, an eternity on the atomic scale that required about a month of supercomputer time. She watched as four copies of p53 linked up and wrapped themselves around a DNA strand, an essential dance the protein performs before it sends off messages for cellular self-destruction.
Amaro wasn’t just interested in the behavior of healthy p53: She wanted to understand the effects of mutations that the gene for p53 is prone to. In dozens of simulations, she and her colleagues tracked how common p53 mutations further destabilize the already floppy protein, distorting it and preventing it from binding to DNA. Some simulations also revealed something else: a fingerhold for a potential drug. Once in a while, a small cleft forms in the mutated protein’s core. When Amaro added virtual drug molecules into her models, the compounds lodged in that cleft, stabilizing p53 just enough to allow it to resume its normal functions.
For Amaro and a few other researchers, those computer simulations are an inspiration. “A long-standing dream of cancer biology is to find small molecule drug compounds to restore the activity of p53,” Amaro says. “We’re very excited about this.”
The beauty of these [drugs] is that they are broadly applicable.Alan Fersht, University of Cambridge
Of the nearly 1.7 million people diagnosed with cancer each year in the United States alone, about half have mutated versions of p53—a sign of how important the normal protein is in preventing the disease. It is one of the most intensely studied proteins in science, and a highly sought-after target for drugmakers. But of the dozens of p53 drugs in development, the vast majority simply try to boost levels of healthy p53. And despite decades of effort, none has made it to market.
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Amaro’s work illustrates how a handful of academic labs and small companies are making progress with a fresh approach to targeting p53: rescuing it when it’s sick. They’re finding drugs that bind to and prop up copies of mutated p53, restoring its shape and ability to carry out its job. One such drug has already passed an early stage safety trial in humans, and a more advanced clinical trial is now underway in Europe. Other would-be medicines are nearing human tests. If any succeed in the clinic, they could dramatically change the landscape for cancer treatment—and for other diseases that involve misfolded proteins, perhaps even Alzheimer’s.
It won’t be easy. Restoring normal function to a mutated protein is more difficult than simply blocking a protein, the strategy used by most medical therapies, says Klas Wiman, a tumor cell biologist at the Karolinska Institute in Stockholm. As a result, large drug companies have shied away from the rescue approach and progress has been slow, he says. “It’s a little out of the mainstream for big pharma.”
The payoff could be big, however. Not only could the strategy treat many kinds of cancer, but just a handful of drugs might be enough, particularly when coupled with chemotherapy drugs that induce the tumor cell damage to which p53 responds. P53 mutations tend to be clustered in the core of the protein, where it binds to DNA, and they have similar effects on its shape. Cell assays and animal studies suggest that drugs that restore p53’s activity work with not just one mutant form of the protein, but many, says Alan Fersht, a chemist at the University of Cambridge in the United Kingdom. “The beauty of these things is that they are broadly applicable.”
An understanding of p53’s seemingly magical powers to suppress tumors didn’t emerge until well after the protein’s discovery in 1979. Early on it was thought to be an oncogene capable of turning a cell cancerous under some circumstances. Only a decade later was it confirmed to bind to DNA and turn on the expression of other genes aimed at healing cell damage. If that damage is deemed too extensive by other cellular actors that interact with p53, it triggers p53 to launch the call for the cell to commit suicide.
The protein is now known to interact with and control dozens of different genes and proteins, and it helps regulate the cycle of molecular events by which cells grow and reproduce. Because of its outsize importance, its presence in cells is tightly controlled. Another protein, MDM2, latches onto p53 molecules and destroys them, keeping their numbers in check.
But this control mechanism can fail in multiple ways. For starters, when p53 itself is mutated, MDM2 cannot attack it. As a result, the malfunctioning protein builds up in cells unchecked and keeps the remaining healthy p53 from doing its job. Without the genome’s guardian on patrol, precancerous cells survive and reproduce. This gives them the opportunity to build up the additional mutations they need to become fully malignant.
Her research paints a picture of what must be present or absent in the cancer cell in order for a cell to avoid its own death via one pathway or another.
“I feel like this will give a lot of scope for future study into what is happening when chemotherapy is effective and when it’s not.
Building on previous knowledge with new techniques has allowed a new level of detail, so now hopefully other labs can better understand the relationship between cancer cells, chemotherapy and genetic mutations.
This will be a big bridge to connect all the stories.”‘
The research made extensive use of live cell imaging, adding chemotherapy drugs to cells in real time in the ATAC facility at Children’s Medical Research Institute. While this study was fundamental scientific research, Dr. Masamsetti sees her work as assisting clinics on the path toward personalized medicine.
“By knowing the ways in which cancer cells avoid cell death, it will help us to identify cancer treatments in the future.
Once we can identify the mutations involved in each cancer then we can tailor our treatment approach as specifically to the instance of cancer as possible.
“Ultimately this is a step towards more personalized medicine, but we have to do more fundamental scientific research in order to understand how cancer works.”
How chemotherapy kills cancer cells
Chemotherapy circulates throughout your body in the bloodstream. So it can treat cancer cells almost anywhere in the body. This is known as systemic treatment.
Chemotherapy kills cells that are in the process of splitting into 2 new cells.
Body tissues are made of billions of individual cells. Once we are fully grown, most of the body’s cells don’t divide and multiply much. They only divide if they need to repair damage.
When cells divide, they split into 2 identical new cells. So where there was 1 cell, there are now 2. Then these divide to make 4, then 8 and so on.
In cancer, the cells keep on dividing until there is a mass of cells. This mass of cells becomes a lump, called a tumour.
Because cancer cells divide much more often than most normal cells, chemotherapy is much more likely to kill them.
Some drugs kill dividing cells by damaging the part of the cell’s control centre that makes it divide. Other drugs interrupt the chemical processes involved in cell division.
The effects on dividing cells
Chemotherapy damages cells as they divide.
In the centre of each living cell is a dark blob, called the nucleus. The nucleus is the control centre of the cell. It contains chromosomes, which are made up of genes.
These genes have to be copied exactly each time a cell divides into 2 to make new cells.
Chemotherapy damages the genes inside the nucleus of cells.
Some drugs damage cells at the point of splitting. Some damage the cells while they’re making copies of all their genes before they split. Chemotherapy is much less likely to damage cells that are at rest, such as most normal cells.
You might have a combination of different chemotherapy drugs. This will include drugs that damage cells at different stages in the process of cell division. This means there’s more chance of killing more cells.
Why chemotherapy causes side effects
The fact that chemotherapy drugs kill dividing cells helps to explain why chemotherapy causes side effects. It affects healthy body tissues where the cells are constantly growing and dividing, such as:
- your hair, which is always growing
- your bone marrow, which is constantly producing blood cells
- your skin and the lining of your digestive system, which are constantly renewing themselves
Because these tissues have dividing cells, chemotherapy can damage them. But normal cells can replace or repair the healthy cells that are damaged by chemotherapy.
So the damage to healthy cells doesn’t usually last. Most side effects disappear once your treatment is over. Some side effects such as sickness or diarrhoea might only happen during the days you are actually having the drugs.
How you have chemotherapy
You can have chemotherapy as:
- an injection into the bloodstream (usually through a vein)
- a drip (intravenous infusion) into the bloodstream through a vein
Chemotherapy drugs that you have in these ways circulate all round the body in the bloodstream. They can reach cancer cells almost anywhere in the body. This is known as systemic treatment.
How well chemotherapy works
The chance of the chemotherapy curing your cancer depends on the type of cancer you have.
- With some types of cancer, most people are cured by chemotherapy
- With other types of cancer, fewer people are completely cured
Examples of cancers where chemotherapy works very well are testicular cancer and Hodgkin lymphoma.
With some cancers, chemotherapy can’t cure the cancer on its own. But it can help in combination with other types of treatment.
For example, many people with breast or bowel cancer have chemotherapy after surgery to help lower the risk of the cancer coming back.
With some cancers, if a cure is unlikely, your doctor may still suggest chemotherapy to:
- shrink the cancer
- relieve your symptoms
- give you a longer life by controlling the cancer or putting it into remission
What remission means
Remission is a word doctors often use when talking about cancer. It means that after treatment there is no sign of the cancer.
You might hear your doctor talk about complete remission and partial remission.
This means that the cancer can’t be detected on scans, x-rays, or blood tests, etc. Doctors sometimes call this a complete response.
This means the treatment has killed some of the cells, but not all. The cancer has shrunk, but can still be seen on scans and doesn’t appear to be growing.
The treatment might have stopped the cancer from growing. Or the treatment could have made the cancer smaller so that other treatments are more likely to help, such as surgery or radiotherapy. This is sometimes called a partial response.
Another term doctors use is stable disease. This can mean that the cancer has stayed the same size or it might even have grown by a small amount.
Common Chemotherapy Drugs
There are dozens of chemotherapy drugs that doctors can prescribe. They’re often divided into groups based on how they work and what they’re made of. Each group of drugs destroys or shrinks cancer cells in a different way.
- Some drugs damage the DNA of cancer cells to keep them from making more copies of themselves. They are called alkylating agents, the oldest type of chemotherapy. They treat many different types of cancer, such as leukemia, lymphoma, Hodgkin’s disease, multiple myeloma, and sarcoma, as well as breast, lung, and ovarian cancers. Some examples of alkylating agents are cyclophosphamide, melphalan, and temozolomide. As they kill bad cells, though, they can also destroy your bone marrow in the process, which can cause leukemia years later. To lower this risk, you can take the drugs in small doses. One type of alkylating agent, platinum drugs like carboplatin, cisplatin, or oxaliplatin, has a lower risk of causing leukemia.
- One type of chemo drug interferes with the normal metabolism of cells, which makes them stop growing. These drugs are called antimetabolites. Doctors often use them to treat leukemia and cancer in the breasts, ovaries, and intestines. Drugs in this group include 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, and methotrexate, among many others.
- Anthracycline chemotherapy attacks the enzymes inside cancer cells’ DNA that help them divide and grow. They work for many types of cancer. Some of these drugs are actinomycin-D, bleomycin, daunorubicin, and doxorubicin, among others. High doses of anti-tumor antibiotics can damage your heart or lungs. So your doctor will have you take them for a short time.
- Drugs called mitotic inhibitors stop cancer cells from making more copies of themselves. They can also stop your body from making the proteins that cancer cells need to grow. Doctors might prescribe them for breast and lung cancers and types of myeloma, leukemia, and lymphoma. Mitotic inhibitors include docetaxel, estramustine, paclitaxel, and vinblastine.
- Another type of medicine, called topoisomerase inhibitors, also attacks enzymes that help cancer cells divide and grow. They treat some types of leukemia and cancer of the lung, ovaries, and intestines, among other types. This group of medicine includes etoposide, irinotecan, teniposide, and topotecan. Some of them, though, may raise your odds of getting a second cancer a few years later.
- Steroids are drugs that act like your body’s own hormones. They are useful in treating many types of cancer, and they can keep you from having nausea and vomiting after a round of chemo. They can also prevent allergic reactions to some of the drugs. Some of the steroids your doctor might prescribe are prednisone, methylprednisolone, and dexamethasone.
More information: V. Pragathi Masamsetti et al. Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection, Nature Communications (2019). DOI: 10.1038/s41467-019-12255-w
Journal information: Nature Communications
Provided by Children’s Medical Research Institute