Turning P53 on in cancer cells

The P53 protein provides a first line of defense against cancers, causing cancer cells to commit apoptosis.  “p53 (also known as protein 53 or tumor protein 53), is a tumor suppressor protein that in humans is encoded by the TP53 gene.^[1][2][3] p53 is important in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer. As such, p53 has been described as “the guardian of the genome“, the “guardian angel gene”, and the “master watchman”, referring to its role in conserving stability by preventing genome mutation^[4](ref). ”  However, the guardian angel can’t do its job if is mutated in the cancer or if the cancer has evolved a method to turn it off – which is the case in about 50% of cancer types, those having “wild type” P-53.  Therefore, in recent years there has been considerable research on how to get the P53 going again in those cancers.  This blog entry reviews that and other research relevant to P53 and where it appears to be heading as a promising new anti-cancer approach.

The introduction to a 2010 review article Targeting p53 for Novel Anticancer Therapy sets the stage.  “Carcinogenesis is a multistage process, involving oncogene activation and tumor suppressor gene inactivation as well as complex interactions between tumor and host tissues, leading ultimately to an aggressive metastatic phenotype. Among many genetic lesions, mutational inactivation of p53 tumor suppressor, the “guardian of the genome,” is the most frequent event found in 50% of human cancers. p53 plays a critical role in tumor suppression mainly by inducing growth arrest, apoptosis, and senescence, as well as by blocking angiogenesis. In addition, p53 generally confers the cancer cell sensitivity to chemoradiation. Thus, p53 becomes the most appealing target for mechanism-driven anticancer drug discovery. This review will focus on the approaches currently undertaken to target p53 and its regulators with an overall goal either to activate p53 in cancer cells for killing or to inactivate p53 temporarily in normal cells for chemoradiation protection.”

The amazing P53 and cell metabolism

P53 plays other roles besides regulating cell cycle arrest and apoptosis in the presence of strong stress.  The 2009 publication Homeostatic functions of the p53 tumor suppressor: regulation of energy metabolism and antioxidant defense describes an additional role. “The p53 tumor suppressor plays pivotal role in the organism by supervising strict compliance of individual cells to needs of the whole organisms. It has been widely accepted that p53 acts in response to stresses and abnormalities in cell physiology by mobilizing the repair processes or by removing the diseased cells through initiating the cell death programs. Recent studies, however, indicate that even under normal physiological conditions certain activities of p53 participate in homeostatic regulation of metabolic processes and that these activities are important for prevention of cancer. These novel functions of p53 help to align metabolic processes with the proliferation and energy status, to maintain optimal mode of glucose metabolism and to boost the energy efficient mitochondrial respiration in response to ATP deficiency. Additional activities of p53 in non-stressed cells tune up the antioxidant defense mechanisms reducing the probability of mutations caused by DNA oxidation under conditions of daily stresses. The deficiency in the p53-mediated regulation of glycolysis and mitochondrial respiration greatly accounts for the deficient respiration of the predominance of aerobic glycolysis in cancer cells (the Warburg effect), while the deficiency in the p53-modulated antioxidant defense mechanisms contributes to mutagenesis and additionally boosts the carcinogenesis process.”  The suggestion is therefore that maintaining strong P53 activity is an important aspect of maintaining health. 

The role of P53 in cell respiration was described in the 2006 publication p53 aerobics: the major tumor suppressor fuels your workout.  “In addition to its role as the central regulator of the cellular stress response, p53 can regulate aerobic respiration via the novel transcriptional target SCO2, a critical regulator of the cytochrome c oxidase complex (Matoba et al., 2006). Loss of p53 results in decreased oxygen consumption and aerobic respiration and promotes a switch to glycolysis, thereby reducing endurance during physical exercise.” 

 The glycolysis provides an ideal environment for carcinogenesis.  As stated in the 2006 paper p53 regulates mitochondrial respiration, “The energy that sustains cancer cells is derived preferentially from glycolysis. This metabolic change, the Warburg effect, was one of the first alterations in cancer cells recognized as conferring a survival advantage. Here, we show that p53, one of the most frequently mutated genes in cancers, modulates the balance between the utilization of respiratory and glycolytic pathways. We identify Synthesis of Cytochrome c Oxidase 2 (SCO2) as the downstream mediator of this effect in mice and human cancer cell lines.  SCO2 is critical for regulating the cytochrome c oxidase (COX) complex, the major site of oxygen utilization in the eukaryotic cell. Disruption of the SCO2 gene in human cancer cells with wild-type p53 recapitulated the metabolic switch toward glycolysis that is exhibited by p53-deficient cells. That SCO2 couples p53 to mitochondrial respiration provides a possible explanation for the Warburg effect and offers new clues as to how p53 might affect aging and metabolism.”

Recapitulating in simple terms, deficiency or mutation of P53 switches the respiratory environment in cells to glycolysis favoring cancer development.  This is in addition to inactivated or mutated P53 being unable to kill off cancer cells by apoptosis.  The research literature of cancer metabolism and its relationship to mitochondrial signaling is very rich and interesting and I was tempted to cite more publications in that area.  However, I choose to focus on P53 here.

Mutations of P53 in cancers

The 2007 paper Restoration of wild-type p53 function in human tumors: strategies for efficient cancer therapy points out “The p53 tumor suppressor gene is mutated in around 50% of all human tumors. Most mutations inactivate p53’s specific DNA binding, resulting in failure to activate transcription of p53 target genes. As a consequence, mutant p53 is unable to trigger a p53-dependent biological response, that is cell cycle arrest and apoptosis. Many tumors express high levels of nonfunctional mutant p53. Several strategies for restoration of wild-type p53 function in tumors have been designed. Wild-type p53 reconstitution by adenovirus-mediated gene transfer has shown antitumor efficacy in clinical trials. Screening of chemical libraries has allowed identification of small molecules that reactivate mutant p53 and trigger mutant p53-dependent apoptosis. These novel strategies raise hopes for more efficient cancer therapy.”  As will be explained, not only is there the issue of mutant P53 in some cancers, but there is also an issue of wild-type (normal) P53 in other cancers being inactivated by the cancer.

MDM2 and MDMX

Two key proteins are known to play roles in both normal and cancer P53 homeostasis MDM2 and MDMX.  Regulation of these proteins may offer an important cancer therapy approach, not only in cells with mutated P53 but also in cancer cells with wild-type P53.  The 2010 review paper The regulation of MDM2 by multisite phosphorylation–opportunities for molecular-based intervention to target tumours? explains: “The p53 tumour suppressor is a tightly controlled transcription factor that coordinates a broad programme of gene expression in response to various cellular stresses leading to the outcomes of growth arrest, senescence, or apoptosis. MDM2 is an E3 ubiquitin ligase that plays a key role in maintaining p53 at critical physiological levels by targeting it for proteasome-mediated degradation. Expression of the MDM2 gene is p53-dependent and thus p53 and MDM2 operate within a negative feedback loop in which p53 controls the levels of its own regulator. Induction and activation of p53 involves mainly the uncoupling of p53 from its negative regulators, principally MDM2 and MDMX, an MDM2-related and -interacting protein that inhibits p53 transactivation function. MDM2 is tightly regulated through various mechanisms including gene expression, protein turnover (mediated by auto-ubiquitylation), protein-protein interaction with key regulators, and post-translational modification, mainly, but not exclusively, by multisite phosphorylation.–. This analysis also provides an opportunity to consider the signalling pathways regulating MDM2 as potential targets for non-genotoxic therapies aimed at restoring p53 function in tumour cells.”

Many cancers have in the course of evolution developed a strategy for inactivating P53 using MDM2.  Reactivating MDM2 has therefore been considered as an anti-cancer strategy.  The 2008 publication Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer states “MDM2 oncoprotein binds directly to the p53 tumor suppressor and inhibits its function in cancers retaining wild-type p53. Blocking this interaction using small molecules is a promising approach to reactivate p53 function and is being pursued as a new anticancer strategy.– This study suggests that p53 activation by a potent and specific spiro-oxindole MDM2 antagonist may represent a promising therapeutic strategy for the treatment of colon cancer and should be further evaluated in vivo and in the clinic.”

A somewhat broader view of the same situation is offered in the previously-mentioned 2007 paper Restoration of wild-type p53 function in human cancer: relevance for tumor therapy.  “BACKGROUND: In the majority of human cancers, the tumor suppressor activity of p53 is impaired because of mutational events or interactions with other proteins (i.e., MDM2). The loss of p53 function is responsible for increased aggressiveness of cancers, while tumor chemoresistance and radioresistance are dependent upon the expression of mutant p53 proteins. METHODS: Review of the literature indicates that p53 acts primarily as a transcription factor whose function is subject to a complex and diverse array of covalent post-translational modifications that markedly influence the expression of p53 target genes responsible for cellular responses such as growth arrest, senescence, or apoptosis. The ability of p53 to induce apoptosis in cancer cells is believed essential for cancer therapy. RESULTS: Numerous data indicate that p53 dependent apoptosis is a relevant factor in determining the efficacy of anticancer treatments. Thus, the development of new strategies for restoration of p53 function in human tumors is considered an important issue. Two main approaches for restoration of p53 function have been pursued that impact anticancer treatments: (a) de novo expression of wild-type p53 (wt-p53) through gene therapy and (b) identification of small molecules reactivating wt-p53 function. CONCLUSIONS: The extensive body of knowledge acquired has identified manipulations of p53 signaling as a relevant issue for successful therapies. In this context, the recognition of p53 status in cancer cells is significant and would help considerably in the selection of an appropriate therapeutic approach. p53 manipulations for cancer therapy have revealed the need for specificity of p53 activation and ability to spare body tissues. Furthermore, the promising results obtained by using molecules competent to reactivate wt-p53 functions in cancer cells provide the basis for the design of new molecules with lower side effects and higher anti-tumor efficiency. The reexpression and reactivation of p53 protein in human cancer cells would increase tumor susceptibility to radiation or chemotherapy enhancing the efficacy of standard therapeutic protocols.”

Numerous other publications have been concerned with reactivation of the P53 pathway in cancers including the 2005 publication Nongenotoxic activation of the p53 pathway as a therapeutic strategy for multiple myeloma.  “Mutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53-murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death.”    

The 2010 publication Controlling the Mdm2-Mdmx-p53 Circuit offers a note of caution “Two human family members, Mdm2 and Mdmx, are primarily responsible for inactivating p53 transcription and targeting p53 protein for ubiquitin-mediated degradation. — In tumors that harbor wild-type p53, reactivation of p53 by modulating both Mdm2 and Mdmx signaling is well suited as a therapeutic strategy. However, the rationale for development of kinase inhibitors that target the Mdm2-Mdmx-p53 axis must be carefully considered since modulation of certain kinase signaling pathways has the potential to destabilize and inactivate p53.”  The interactions are quite complex.

Enter Nutlins 

There is great interest in a new class of MDM2 inhibitors called Nutlins.  “Nutlins are cis–imidazoline analogs which inhibit the interaction between MDM2 and p53, and were discovered by screening a chemical library by Vassiliev et al. Nutlin-1, Nutlin-2 and Nutlin-3 were all identified in the same screen,^[1] however Nutlin-3 is the compound most commonly used in anti-cancer studies.^[2]  Inhibiting the interaction between MDM2 and p53 stabilizes p53 and is thought to selectively kill cancer cells. These compounds are therefore thought to work best on tumors that contain normal or wild type p53(ref).”

The 2008 publication The MDM2 inhibitor Nutlins as an innovative therapeutic tool for the treatment of haematological malignancies tells the story. “At variance to solid tumors, which show percentage of p53 deletions and/or mutations close to 50%, more than 80% of haematological malignancies express wild-type p53 at diagnosis. Therefore, activation of the p53 pathway by antagonizing its negative regulator murine double minute 2 (MDM2) might offer a new therapeutic strategy for the great majority of haematological malignancies. Recently, potent and selective small-molecule MDM2 inhibitors, the Nutlins, have been identified. Studies with these compounds have strengthened the concept that selective, non-genotoxic p53 activation might represent an alternative to the current cytotoxic chemotherapy. Interestingly, Nutlins not only are able to induce apoptotic cell death when added to primary leukemic cell cultures, but also show a synergistic effect when used in combination with the chemotherapeutic drugs commonly used for the treatment of haematological malignancies. Of interest, Nutlins also display non-cell autonomous biological activities, such as inhibition of vascular endothelial growth factor, stromal derived factor-1/CXCL12 and osteprotegerin expression and/or release by primary fibroblasts and endothelial cells. Moreover, Nutlins have a direct anti-angiogenic and anti-osteoclastic activity. Thus, Nutlins might have therapeutic effects by two distinct mechanisms: a direct cytotoxic effect on leukemic cells and an indirect non-cell autonomous effect on tumor stromal and vascular cells, and this latter effect might be therapeutically relevant also for treatment of haematological malignancies carrying p53 mutations.”

A number of other 2010 papers are also concerned with the use of Nutlins as P-53 activating cancer therapies, including Nutlin-3 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through up-regulation of death receptor 5 (DR5) in human sarcoma HOS cells and human colon cancer HCT116 cells and Pharmacological activation of the p53 pathway in haematological malignancies.  “p53 gene mutations are rarely detected at diagnosis in common haematological cancers such as multiple myeloma (MM), acute myeloid leukaemia (AML), chronic lymphocytic leukaemia (CLL) and Hodgkin’s disease (HD), although their prevalence may increase with progression to more aggressive or advanced stages. Therapeutic induction of p53 might therefore be particularly suitable for the treatment of haematological malignancies. Some of the anti-tumour activity of current chemotherapeutics has been derived from activation of p53. However, until recently it was unknown whether p53 signalling is functional in certain haematological cancers including MM and if p53 activity is sufficient to trigger an apoptotic response. With the recent discovery of nutlins, which represent the first highly selective small molecule inhibitors of the p53-MDM2 interaction, pharmacological tools are now available to induce p53 irrespective of upstream signalling defects, and to functionally analyse the downstream p53 pathway in primary leukaemia and lymphoma cells. Combination therapy is emerging as a key factor, and development of non-genotoxic combinations seems very promising for tackling the problems of toxicity and resistance. This review will highlight recent findings in the research into molecules capable of modulating p53 protein activities and mechanisms that activate the p53 pathway, restoring response to therapy in haematological malignancies.” 

Nutlins and Vitamin D. 

What about supplements in the anti-aging firewalls regimen and activation of P-53 and the other pro-apoptotic channels in cancers?  There is much to say about that subject and it has to be the focus of a separate blog entry.  However, I stumbled across one paper relevant to the present discussion 1,25-dihydroxyvitamin D3 enhances the apoptotic activity of MDM2 antagonist nutlin-3a in acute myeloid leukemia cells expressing wild-type p53.  in leukemia cells expressing wild-type P-53 “Combination of nutlin-3a with 1,25D accelerated programmed cell death, likely because of enhanced nutlin-induced upregulation of the proapoptotic PIG-6 protein and downregulation of antiapoptotic BCL-2, MDMX, human kinase suppressor of Ras 2, and phosphorylated extracellular signal-regulated kinase 2.” 

The scope of the relevant research literature is overwhelming.  A search in Pubmed on Nutlin produces 220 references!  What I have included here should be enough to convey the general picture, however.  A search in clinicaltrials.gov on “nutlin and cancer” failed to reveal any trials, suggesting that nutlin-based cancer therapies are not yet in the clinical trials phase.  I expect such clinical trials will be launched soon. 

The bottom line               

Turning on a strong P53 defense is emerging as an important anticancer strategy in the advanced research stage.  A central approach for cancers with wild-type P53 is to inhibit the P53-controlling proteins MDM2 and MDMX using a new class of substances called Nutlins.  This approach is not yet in clinical trials but probably soon will be.  A separate blog entry will deal with the anti-cancer capabilities of supplements in the anti-aging firewalls regimen.