Genes & Cancer

Since the discovery of Mdm2, the contribution of this RING E3 ubiquitin ligase to the pathobiology of cancer has focused almost exclusively on its role as a negative regulator of the p53 tumor suppressor. Under normal conditions, Mdm2 promotes the ubiquitin- and proteasome-dependent degradation of p53. Levels of p53 are thus kept sufficiently low to allow for cell survival and cell cycle progression. In the context of such insults as DNA damage or ribosomal stress, however, the Mdm2-p53 interaction is disrupted and p53 is stabilized. The myriad intracellular outcomes of p53 activation together comprise a robust program of tumor suppression that is short-circuited in cancer. Over half of all human malignancies are known to have lost p53 expression or sustained p53 mutation, whereas many of the remaining tumors harbor other alterations in key mediators of p53 activity that include overexpression of Mdm2. Therapies targeting the interaction between Mdm2 and p53 represent a possible means of pharmacologically reactivating the p53 pathway in this latter setting. The degree of overlap across the biological consequences of either p53 loss or Mdm2 overexpression, however, has not been thoroughly explored. Indeed, a body of evidence for several p53-independent functions of Mdm2 suggests that disrupting the Mdm2-p53 interaction may fail to address the full spectrum of oncogenic effects specific to tumors that overexpress Mdm2.

The MDM2 oncogene is a key negative regulator of the p53 tumor suppressor protein. MDM2 and p53 form an autoregulatory feedback loop to tightly control the proper cellular responses to various stress signals in order to prevent mutations and tumor formation. The levels and function of the MDM2 protein, an E3 ubiquitin ligase, are regulated by a wide variety of extracellular and intracellular stress signals through distinct signaling pathways and mechanisms. These signals regulate the E3 ubiquitin ligase activity of MDM2, the ability of MDM2 to interact with p53 and a number of other proteins, and the cellular localization of MDM2, which in turn impact significantly upon p53 function. This review provides an overview of the regulation of MDM2 activities by the signals and factors that regulate the MDM2 protein, including genotoxic stress signals, oncogenic activation, cell cycle transition, ribosomal stress, chronic stress, neurohormones, and microRNAs. Disruption of the proper regulation of the MDM2-p53 negative feedback loop impacts significantly upon the frequency of tumorigenesis in a host. A better understanding of the complex regulation of MDM2 and its impact upon p53 function in cells under different conditions will help to develop novel and more effective strategies for cancer therapy and prevention.

The p53 transcription factor regulates the expression of numerous genes whose products affect cell proliferation, senescence, cellular metabolism, apoptosis, and DNA repair. These p53-mediated effects can inhibit the growth of stressed or mutated cells and suppress tumorigenesis in the organism. However, the various growth-inhibitory properties of p53 must be kept in check in nondamaged cells in order to facilitate proper embryogenesis or the homeostatic maintenance of adult tissues. This requisite inhibition of p53 is performed primarily by the MDM oncoproteins, Mdm2 and MdmX. These p53-binding proteins limit p53 activity both in normal cells and in stressed cells seeking to promote resolution of their p53-stress response. Many mouse models bearing genetic alterations in Mdm2 or MdmX have been generated to explore the function and regulation of MDM-p53 signaling in development, in tissue homeostasis, in aging, and in cancer. These models not only have demonstrated a critical need for Mdm2 and MdmX in normal cell growth and in development but more recently have identified the MDM-p53 signaling axis as a key regulator of the cellular response to a wide variety of genetic or metabolic stresses. In this review, we discuss what has been learned from various studies of these Mdm2 and MdmX mouse models and highlight a few of the many important remaining questions.

Classically, p53 is considered to be an overarching tumor suppressor gene, important in its role as a transcription factor for a number of genes critical for cell cycle arrest, apoptosis, and senescence. More recently, the scope of p53 function has been further broadened, with evidence emerging that supports essential roles for p53 in reproduction and metabolism. The homologous proteins Mdm2 and MdmX function as the primary negative regulators of p53 stability and activity. Canonically, Mdm2 is thought to regulate p53 through 2 mechanisms: 1) through directly binding the p53 transactivation domain, suppressing p53 activity, and 2) through functioning as an E3 ubiquitin ligase capable of ubiquitinating p53, targeting it for nuclear export and degradation. MdmX similarly functions to bind the p53 transactivation domain; however, it is not characterized to harbor any intrinsic E3 ubiquitin ligase activity. Despite extensive study, the advent of a number of mouse models has brought to light the necessity of studying the p53 pathway at physiological levels and emphasized the major differences that can exist between in vitro and in vivo analysis. While many questions remain, a focus on the use of in vivo models in p53 study is providing a clearer view of how this pathway is regulated, with a newfound emphasis on the role of the Mdm2:MdmX heterodimer, and with that a better understanding of how this pathway could be better manipulated for therapeutic gains.

Mdm2 is an essential regulator of the p53 tumor suppressor. Mdm2 is modified at transcriptional, post-transcriptional, and post-translational levels to control p53 activity in normal versus stressed cells. Importantly, errors in these regulatory mechanisms can result in aberrant Mdm2 expression and failure to initiate programmed cell death in response to DNA damage. Such errors can have severe consequences as evidenced by tumor phenotypes resulting from amplification at the Mdm2 locus and changes in post-transcriptional and post-translational regulation of Mdm2. Although Mdm2 mediated inhibition of p53 is well characterized, Mdm2 interacts with many additional proteins and also targets many of these for proteosomal degradation. Mdm2 also has E3-ligase independent functions and p53-independent functions that have important implications for genome stability and cancer.

MDM2 oncogenic protein is the principal cellular antagonist of the p53 tumor suppresser gene. p53 activity needs exquisite control to elicit appropriate responses to differential cellular stress conditions. p53 becomes stabilized and active upon various types of stresses. However, too much p53 is not beneficial to cells and causes lethality. At the steady state, p53 activity needs to be leashed for cell survival. Early studies suggested that the MDM2 oncoprotein negatively regulates p53 activity through the induction of p53 protein degradation. MDM2 serves as an E3 ubiquitin ligase of p53; it catalyzes polyubiquitination and subsequently induces proteasome degradation to downregulate p53 protein level. However, the mechanism by which MDM2 represses p53 is not a single mode. Emerging evidence reveals another cellular location of MDM2-p53 interaction. MDM2 is recruited to chromatin, specifically the p53 responsive promoter regions, in a p53 dependent manner. MDM2 is proposed to directly inhibit p53 transactivity at chromatin. This article provides an overview of the mechanism by which p53 is repressed by MDM2 in both ubiquitination dependent and ubiquitination independent pathways.

The transcription factor p53 regulates numerous cellular processes to guard against tumorigenesis. Cell-cycle inhibition, apoptosis, and autophagy are all regulated by p53 in a cell- and context-specific manner, underscoring the need for p53 activity to be kept low in most circumstances. p53 is kept in check primarily through its regulated ubiquitination and degradation by a number of different factors, whose contributions may reflect complex context-specific needs to restrain p53 activity. Chief among these E3 ubiquitin ligases in p53 homeostasis is the ubiquitously expressed proto-oncogene MDM2, whose loss renders vertebrates unable to limit p53 activity, resulting in early embryonic lethality. MDM2 has been validated as a critical, universal E3 ubiquitin ligase for p53 in numerous tissues and organisms to date, but additional E3 ligases have also been identified for p53 whose contribution to p53 activity is unclear. In this review, we summarize the recent advances in our knowledge regarding how p53 activity is apparently controlled by a multitude of ubiquitin ligases beyond MDM2.

p53 is an important tumor suppressor, functioning as a transcriptional activator and repressor. Upon receiving signals from multiple stress related pathways, p53 regulates numerous activities such as cell cycle arrest, senescence, and cell death. When p53 activities are not required, the protein is held in check by interacting with 2 key homologous regulators, Mdm2 and MdmX, and a search for inhibitors of these interactions is well underway. However, it is now recognized that Mdm2 and MdmX function beyond simple inhibition of p53, and a complete understanding of Mdm2 and MdmX functions is ever more important. Indeed, increasing evidence suggests that Mdm2 and MdmX affect p53 target gene specificity and influence the activity of other transcription factors, and Mdm2 itself may even function as a transcription co-factor through post-translational modification of chromatin. Additionally, Mdm2 affects post-transcriptional activities such as mRNA stability and translation of a variety of transcripts. Thus, Mdm2 and MdmX influence the expression of many genes through a wide variety of mechanisms, which are discussed in this review.

The p53 tumor suppressor is highly responsive to different physiological stresses such as abnormal cell proliferation, nutrient deprivation, and DNA damage. Distinct signaling mechanisms have evolved to activate p53, which in turn modulate numerous pathways to enhance fitness and survival of the organism. Elucidating the molecular mechanisms of these signaling events is critical for understanding tumor suppression by p53 and development of novel therapeutics. Studies in the past decade have established that MDM2 and MDMX are important targets of signaling input from different pathways. Here, we focus our discussion on MDM2 and MDMX phosphorylation, which is important for p53 activation by DNA damage. Investigations in this area have generated new insight into the inner workings of MDM2 and MDMX and underscore the importance of allosteric communication between different domains in achieving an efficient response to phosphorylation. It is likely that MDM2 and MDMX regulation by phosphorylation will share mechanistic similarities to other signaling hub molecules. Phosphorylation-independent p53 activators such as ARF and ribosomal proteins ultimately achieve the same outcome as phosphorylation, suggesting that they may induce similar changes in the structure and function of MDM2 and MDMX through protein-protein interactions.

The contribution of Mdm2, and its recently identified family member Mdmx (Mdm4), to tumorigenesis has primarily focused on their negative regulation of the p53 tumor suppressor. Although Mdm2 and Mdmx clearly inhibit p53, which can lead to tumor development, both have also been shown to affect tumorigenesis independent of p53. Given that Mdm2 and/or Mdmx overexpression is common and likely underestimated in human cancers, understanding the functions of these proteins beyond p53 control is critical. In recent years, new functions of Mdm2 and Mdmx that lead to genome instability, a hallmark of malignancy, have emerged. Specifically, roles in the DNA damage response that are distinct from their regulation of p53 have been identified. Inhibition of p53 as well as other components of the DNA damage response by Mdm2 and Mdmx can result in delayed DNA repair and increased genome instability, making Mdm2 and Mdmx a danger to the genome when aberrantly expressed. However, the genome instability caused by altered levels of Mdm2 and Mdmx could be used therapeutically for the treatment of cancer. Specifically, drugs/small molecules that target the interaction between Mdm2 and p53 can stabilize Mdm2, resulting in negative consequences on the genome that could be exploited for cancer treatment, particularly malignancies lacking functional p53.

The function and regulation of MDM2 as a component of a p53-dependent negative feedback loop has formed a core paradigm in the p53 field. This concept, now 20 years old, has been solidified by fields of protein science, transgenic technology, and drug discovery in human cancer. However, it has been noted that a simple negative feedback loop between p53 and MDM2 lacks an intrinsic “activating” step that counteracts this inhibition and permits oscillation of the feedback to occur as p53 is switched on and off. More recent work has identified a solution to the missing piece of the picture that counters the negative feedback loop, which is MDM2 itself. Under conditions of genotoxic stress, MDM2 helps to activate p53 by increasing its rate of protein synthesis. This simple observation makes certain aspects of the p53 response more comprehensible such as why MDM2 is upregulated by p53 early on following DNA damage and how phosphorylation of MDM2 at the C-terminal Ser395 by ATM translates into p53 activation. The latter acts by inducing allosteric changes in the RING domain of MDM2 that expose its RNA binding pocket, support p53 synthesis, and suppress its degradation. This allosteric nature of MDM2 in the C-terminus mirrors the allosteric effects of the binding of small molecules to the p53 interacting pocket at the N-terminus of MDM2, which opens the core domain of MDM2 to central domains of p53, which controls p53 ubiquitination. Thus, the highly allosteric nature of MDM2 provides the basis for dynamic protein-protein interactions and protein-RNA interactions through which MDM2’s activity is regulated in p53 protein destruction or in p53 protein synthesis. We discuss these mechanisms and how this information can be exploited for drug development programs aimed at activating p53 via targeting MDM2.

The oncoprotein MDM2 is both the transcriptional target and the predominant antagonist of the tumor suppressor p53. MDM2 inhibits the functions of p53 via a negative feedback loop that can be circumvented by several ribosomal proteins in response to nucleolar or ribosomal stress. Stress conditions in the nucleolus can be triggered by a variety of extracellular and intracellular insults that impair ribosomal biogenesis and function, such as chemicals, nutrient deprivation, DNA damaging agents, or genetic alterations. The past decade has witnessed a tremendous progress in understanding this previously underinvestigated ribosomal stress-MDM2-p53 pathway. Here, we review the recent progress in understanding this unique signaling pathway, discuss its biological and pathological significance, and share with readers our insight into the research in this field.

Cancer cells often have high expression of Mdm2. However, in many cancers mdm2 is alternatively spliced, with more than 40 mRNA variants identified. Many of the alternative spliced mdm2 mRNAs have the potential to encode truncated Mdm2 isoforms. These putative Mdm2 isoforms can theoretically increase the diversity of the cancer proteome. The 3 best characterized are Mdm2-A, Mdm2-B, and Mdm2-C. As described in this review, the exogenous expression of these isoforms results in paradoxical phenotypes of transformation-associated growth as well as the inhibition of growth. Interestingly, these Mdm2 isoforms contribute tumor-promoting capacity in p53-null backgrounds. Herein we describe how alternative splicing of mdm2 may result in Mdm2 protein products that alter signal transduction to promote tumorigenesis. The tumor promoting capacity of Mdm2 isoforms is discussed in the context of functions that do not require the inhibition of p53. When N-terminal portions of Mdm2 are missing, the biochemical functions encoded by exon 12 are proposed to become more important. This may result in growth promoting functions when wild-type p53 is absent or compromised. The p53-independent tumor promoting activity of Mdm2 is proposed to result from C-terminal biochemical contributions of DNA binding, RNA binding, nucleolar localization, and nucleotide binding.

While the presence, in the invertebrates, of genes related in sequence and function to the vertebrate p53 family has been known since the discovery of the fly Drosophila melanogaster Dmp53 and the worm Caenorhabditis elegans cep-1 gene, the failure to discover homologs of the essential vertebrate negative regulator of p53 Mdm2 in these species led to the false assumption that Mdm2 was only present in vertebrates. Very recently, clear homologs of Mdm2 have been discovered in a wide range of invertebrate species, raising a series of interesting questions about the evolution of the p53 pathway. Here, a personal account of the discovery of Mdm2-like genes in the Placozoa and Arthropoda is used to speculate on aspects of the evolution, structure, and function of the p53 pathway.