Exploring Strategies for TP53 Mutated Gliomas

TP53 (tumor protein p53) is a gene encoding for the p53 protein. p53 is a trancription factor responsible for the cellular response to DNA damage. p53 can initiate cell cycle arrest or apoptosis (a form of cell death), depending on the severity of the damage, and is therefore known as a tumour suppressor. Notably, TP53 is the most commonly altered gene in human cancers, with approximately 50% of human tumours carrying one or more mutations in the TP53 gene.

Point mutations in the TP53 gene lead to the formation of altered p53 proteins, which not only inhibit the normal transcriptional activity of p53, but also cause the protein to gain novel, pro-tumorigenic functions. TP53 mutations are therefore now considered to be gain-of-function mutations in which the tumour suppressor function is lost, while tumour-promoting functions are gained.

Nearly all cases of IDH-mutant astrocytoma also harbour TP53 mutations [1], while about 34% of glioblastomas contain a TP53 mutation [2]. 95% of TP53 point mutations occur in the DNA-binding domain [3], including the most common mutations found in astrocytomas and glioblastomas, R273C (most common in IDH-mutant astrocytoma), R273H (more common in IDH wild-type astrocytoma and glioblastoma), and R175H. Oligodendrogliomas do not contain TP53 mutations, except in rare cases.

p53 and zinc

The p53 protein includes a single zinc ion in the DNA-binding domain, and this zinc is essential for the proper conformation and functioning of p53. Many mutant p53 proteins are prone to the loss of the zinc ion, disrupting the folding and function of the protein [3]. The effect of this loss of zinc from the p53 protein has been shown experimentally, as the removal of zinc from the protein by chelating agents leads to a loss of normal p53 function which can be restored by adding zinc. Many mutations in the TP53 gene at the DNA-binding domain can change the conformation of the protein as well as affecting the binding of zinc to the protein. These observations prompted a study [3] to determine if supplemental zinc could affect the function of p53 in p53-mutant cancer cell lines.

In this study 100 micromolar (uM) concentrations of zinc had restorative effects on mutant-p53 cancer cells. Zinc supplementation to breast cancer cells bearing the TP53 R175H mutation, and to U373 glioblastoma cells (TP53 R273H mutation), caused increased cell death when exposed to cisplatin and Adriamycin (doxorubicin) chemotherapy. In these same cell lines, zinc increased immunohistochemical positivity for active (folded) p53 and reduced positivity for denatured (unfolded) p53. The addition of zinc also restored normal transcriptional activity to mutant p53, such as the binding of p53 to the p21 and BAX promoters which leads to cell cycle arrest and apoptosis. Furthermore, zinc inhibited oncogenic functions of mutant p53, such as formation of p53/p73 complexes which interfere with the apoptotic function of p73.

To determine if these p53-restorative properties of zinc would translate in vivo, U373 glioblastoma cells (TP53 R273H mutation) were injected into the flanks of athymic mice. After tumour formation, the mice were treated orally with 10 mg zinc per kg body weight daily for two weeks, or cisplatin chemotherapy, or both agents combined. After 14 days, tumour volumes in mice treated with cisplatin plus zinc were 30% smaller than in mice treated with cisplatin alone. Zinc treatment alone did not slow tumour growth rates. Most significantly, when tumours were harvested and analyzed, zinc supplementation increased positivity for functional p53, while positivity for denatured p53 was decreased, showing that in zinc-treated animals, proper p53 conformation and function was restored to a degree.

Autophagy-dependent destruction of mutant p53 following zinc supplementation

A follow-up study by the same group (corresponding author Gabriella D’Orazi, University “G. d’Annunzio” Chieti, Italy) further explored the effects of zinc on mutant p53 cancer cells [4]. In this study, a zinc-curcumin conjugate was utilized to achieve the same functional restoration of mutant p53 cancer cells as in the previous studies which used zinc chloride. While the previous studies showed the ability of zinc to restore normal functionality to mutant p53, this study focused on the ability of the zinc conjugate to decrease levels of mutant p53 in SKBR3 human breast cancer cells (TP53 R175H mutation). The degradation of mutant p53 following zinc exposure was found to be dependent on autophagy, and chemical autophagy inhibition with either 3-methyladenine or chloroquine prevented the zinc-induced mutant p53 degradation. Genetic knockdown of the essential autophagy gene ATG5 also completely prevented zinc-induced mutant p53 degradation, confirming the role of autophagy in this process. Furthermore it was found that after the addition of the zinc conjugate, the functional p53 induced various autophagy-related proteins.

Further experimentation showed that autophagy inhibition with chloroquine prevented the restoration of normal p53 function to the p53-mutant SKBR3 cells. The authors summarize these findings with the statements “Thus, our hypothesis is that zinc addition reactivates wt [wild-type] p53 function by acting on the folding of most (but likely not all) of the mutant p53 proteins within a cell, which then triggers the degradation of the remaining mutant p53 proteins by p53-induced autophagy… Viewed as a whole, these data indicate that autophagy inhibition represents an obstacle to ability of Zn(II)-curc [zinc-curcumin conjugate] to fully reactivate p53-R175H mutant…These data also suggest a greater caution in the use of autophagy inhibitors as antitumor drug, especially in the context of tumors carrying p53-R175H mutant.”

Zinc supplementation required for immunogenic effects of chemotherapy in p53-mutant cancer cells

Yet another study [5] by the same group in Italy (corresponding author Gabriella D’Orazi) focused on the requirement for zinc to induce immunogenic responses and maturation of dendritic cells following chemotherapy in p53-mutant cell lines. Mature dendritic cells are essential in the generation of effective anti-cancer immune responses. The researchers co-cultured immature dendritic cells with p53 deficient cell lines (ADF) or a p53-mutant glioblastoma cell line (U373) and exposed these cells to chemotherapy (cisplatin and Adriamycin/doxorubicin) with or without 100 micromolar concentration of zinc. They found that zinc supplementation was necessary for the maturation of dendritic cells in combination with chemotherapy.

Calreticulin is a protein that translocates to the cell surface following endoplasmic reticulum stress within the cell, and there functions as a danger signal leading to the cell’s destruction by the immune system, in a process known as immunogenic cell death. The addition of zinc to the p53-deficient or mutant cell cultures significantly increased the presence of calreticulin on the cell surface, with or without chemotherapy. This zinc-induced translocation of calreticulin to the cell surface was preceded by the activation of autophagy. When an inhibitor of calrecticulin translocation was added to the culture, the combination of chemotherapy plus zinc failed to activate dendritic cells that were co-cultured with the p53-deficient cancer cells, showing the importance of calreticulin exposure to dendritic cell activation.

In contrast, when p53-normal cancer cells were exposed to Adriamycin (doxorubicin), calreticulin was translocated to the cell surface and dendritic cells matured, without the requirement for zinc. This experiment showed that the requirement of zinc for the immunogenic effects of chemotherapy is particular to p53-deficient or mutant cells. Prior studies (6) had shown that autophagy is required for the release of ATP, another requirement of immunogenic cell death. One of the functions of p53 is to initiate transcription of autophagy-related genes, such as DRAM (DNA-damage regulated autophagy modulator). Based on this evidence, we may speculate that the addition of zinc, by restoring normal functions to mutant or defective p53, allowed the initiation of autophagy as a response to Adriamycin and cisplatin, and the subsequent release of ATP as an immunogenic signal, followed by maturation of dendritic cells. The mechanism behind the translocation of calreticulin to the cell surface following zinc supplementation to p53-deficient cells is unknown. Prior studies [6] have shown that autophagy does not affect this translocation of calreticulin.

October 24, 2015
An important new study shows that metallothioneins are increased in shorter surviving GBM patients. Metallothioneins are a class of intracellular heavy metal binding proteins, which can bind 7 atoms of zinc per metallothionein molecule. Amazingly, both U251 (p53 mutant) and U87 (p53 wild-type) GBM cell lines were found to be mostly positive for unfolded, mutant-type p53 proteins, and the U87 cell line had a four-fold increased level of metallothionein 3 (MT3). This suggests that the zinc-binding activity of metallothionein is leading to unfolded mutant-type p53 conformation in the U87 cells, even in the absence of an actual TP53 mutation. Moreover, the addition of supplemental zinc reversed this pattern in vitro, increasing the level of normal conformation p53, and decreasing the level of mutant-type, unfolded, inactive p53 proteins. This evidence points towards a potential benefit of zinc supplementation in both p53-mutant and p53 non-mutant tumors.
High metallothionein predicts poor survival in glioblastoma multiforme

Zinc dosage

According to examine.com, “Superloading zinc by taking up to 100 mg zinc a day is confirmed to be safe in the short term (2-4 months), but because this dose is higher than the 40 mg Tolerable Upper Limit (TUL) of zinc, prolonged superloading is not advised. Zinc’s intestinal uptake is hindered by other minerals, including Calcium-D-Glucarate, Magnesium, and Iron Absorption, since they all use the same transporter. If the transporter’s uptake limit (800mg) is exceeded between these four minerals, absorption rates will fall. Taking less than 800 mg of these four minerals at the same time is fine.”

Mutant p53 and glucose deprivation

The following paragraphs were taken from the Diet page.

A study [7] published in 2012 in the journal Cell Cycle by a group from Georgetown University in Washington DC provides evidence that mutant p53 protein accumulation can be inhibited by a severely carbohydrate restricted diet, leading to significantly inhibited tumour growth in mice with mutant-p53 tumours.

The researchers first showed that the expression of mutant p53 proteins can be inhibited in vitro by depleting the glucose concentration in the cell medium. Conversely, mutant p53 expression was not affected by serum or amino acid depletion. This inhibition of mutant p53 accumulation following glucose depletion was found to be due to protein degradation (breakdown) via an autophagic process rather than by proteasome activity.

This breakdown of mutant p53 by glucose restriction was then tested in mice. p53-mutant (p53 A135V) transgenic mice and wild-type (non-mutant) mice were randomized to one of three diets: a normal mouse chow, a low-carbohydrate diet, and a high-carbohydrate diet. The low carbohydrate diet was 74% protein, 24% fat and 2% carbs by caloric content, or 71.7% protein, 10% fat, and 1.9% carbohydrates by weight (the remainder being fiber, vitamins and minerals). By caloric content, the standard diet and high-carb diets were 53% and 69% carbohydrate, 20% and 17% protein.

Blood tests showed that mice on the low-carb diet had significantly reduced fasting blood glucose levels: around 100 or <100 mg/dl versus about 130 mg/dl in the standard chow group. After four months on the various diets, the mice were sacrificed. Remarkably, mutant p53 levels were reduced in the mammary glands, ovaries and fat of the p53-mutant, low-carb diet group. Conversely, wild-type p53 was stabilized in the wild-type p53, low-carb diet group. Further testing was done to determine the effects of carbohydrate restriction on tumour growth. Mice were fed either the standard diet or the low-carb diet for two weeks, then implanted with mouse mammary cancer cells which contained either wild-type p53, mutant p53 (G242A), or lacking p53. Three to four weeks later, tumour volumes were assessed. On the standard diet, tumours were largest in the p53-mutant group, followed by p53 null. Tumours were smallest in the p53 wild-type group. The low-carb diet effectively inhibited tumours in all three groups, but most significantly in the p53-mutant and p53-null groups. The extracted tumours were analyzed for p53 levels. In agreement with the in vitro studies, the low-carb diet stabilized p53 in the wild-type group, but inhibited mutant p53 in the mutant group. This study is in agreement with a previous study by a different group which showed that colon cancer xenografts in which p53 was knocked out were inhibited by metformin treatment, while the same xenografts with normal p53 expression were completely resistant to metformin treatment [8]. Metformin is a diabetic drug which lowers and stabilizes blood glucose levels.

  1. Mutational landscape and clonal architecture in grade II and III gliomas. Suzuki et al. 2015.
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  2. The Somatic Genomic Landscape of Glioblastoma. Brennan et al. 2013.
  3. Restoring p53 active conformation by zinc increases the response of mutant p53 tumor cells to anticancer drugs. Puca et al. 2011.
  4. Degradation of mutant p53H175 protein by Zn(II) through autophagy. Garufi et al. 2014.
  5. Zinc supplementation is required for the cytotoxic and immunogenic effects of chemotherapy in chemoresistant p53-functionally deficient cells. Cirone et al. 2013.
  6. Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death. Martins et al. 2012.
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  7. Dietary downregulation of mutant p53 levels via glucose restriction: mechanisms and implications for tumor therapy. Rodriguez et al. 2012.
  8. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Buzzai et al. 2007.