Yanping Zhang - US grants

p53 and Rb mediate two major tumor suppression pathways that are believed to be functionally inactivated inmost, if not all, human cancers. Understanding how these two pathways are regulated has become a major goal of contemporary cancer cell biology. Along withp53, the ARF-INK4a locus is one of the two most frequently altered loci in human cancer. Functionally, p16INK4a inhibits the activity of cyclin D-dependent kinases (CDK4 and CDK6), thereby maintaining the retinoblastoma protein (Rb) in its growth suppressive state. ARF, on the other hand, mediates an oncogene- activated hyperproliferative checkpoint pathway through binding to and antagonizing the nuclear export of MDM2, thereby preventing cytoplasmic degradation of p53. With a focus on the connection between ARF and p53, I have tried to contribute to our understanding of these two major pathways and thereby cancer development. I had previously discovered that ARF stabilizes p53 through binding to and antagonizing the activity of MDM2-a negative regulator of p53, and thus revealed an ARF-MDM2-p53 tumor suppression pathway. Subsequently, I further elucidated the mechanism of ARFs p53 stabilization: ARF forms nuclear bodies in the nucleoplasm with MDM2 and p53, thereby blocking nuclear export of p53 and preventing its cytoplasmic degradation. I also demonstrated that frequently occurring tumor-derived mutations in the human ARF protein impair its function in blocking p53 nuclear export. More recently, I obtained new evidence showing that: (a) Association of MDM2 with ribosomal protein L5 is necessary for MDM2 nuclear export and this regulation is disrupted by frequent cancer-derived mutations in MDM2. (b) Ribosomal protein L5- binding deficient MDM2 failed to promote p53 degradation but retains its ability to suppress p53's transactivation activity. (c) ARF participates in a multipeptide complex, and (d) MDM2 is efficiently degraded by proteolysis. My current and future studies are aimed at several issues concerning the regulation of ARF-MDM2-p53 pathway: (a) Elucidate the mechanism of p53 and MDM2 nucleo-cytoplasmic shuttling controlled by ribosomal protein L5 and/or ARF. (b) Define the function of ARF-MDM2-p53 nuclear bodies by purifying the ARF complex, and (c) as a long-term goal to identify the mechanism and regulation of MDM2 ubiquitination and degradation. Together these experiments should advance understanding of the regulation of the ARF-MDM2-p53 pathway and the functional consequences of its alterations in human cancer.

DESCRIPTION (provided by applicant): The genomic loci for tumor suppressor p53 and ARF are the most frequently mutated in all types of human cancer. On the other hand, the proto-oncoprotein MDM2, a principle negative regulator of p53 in the cell, is frequently overexpressed in many types of human cancer, ARF, MDM2, and p53 constitute an important tumor suppression pathway (ARF-MDM2-p53 pathway) that safeguards cells from aberrant, uncontrolled growth. Recent studies have indicated that the ARF-MDM2-p53 pathway is not strictly linear but branches out to other targets and that the pathway crosstalks with other cellular pathways, presumably through the interaction with other cellular proteins. The targets of the branched-out interaction and the mechanisms of the crosstalk regulation, however, are largely elusive. We have recently found that MDM2 interacts with the ribosomal protein L11 through its zinc finger region. L11 forms in vivo complexes with ARF, MDM2, and p53. Enforced expression of L11 prevents MDM2-mediated p53 ubiquitination and degradation, restores MDM2-suppressed p53 transactivation activity and induces a p53-dependent G1 cell cycle arrest. More importantly, studies have shown that human cancer-associated mutations in the MDM2 gene preferentially target the zinc finger domain disrupting L11 binding. One of the cancer-derived MDM2 mutants we have examined, MDM2 (C305F),which has a Cys-to-Phe substitution in the central zinc finger motif, exhibited several distinct characteristics including the disruption of L11 binding. MDM2(C305F) retained the E3 ligase activity to ubiquitinate p53 but did not promote p53 degradation. It showed a stronger ability than the wild type MDM2 in suppressing p53's transactivation activity and cells expressing MDM2(C305F) escaped from L11 overexpression-induced growth inhibition. MDM2(C305F) represents a useful mutant to dissect the mechanism of L11-regulated MDM2 and p53 function. Based on our preliminary data, we hypothesize that the L11-MDM2-p53 connection constitutes a pathway that monitors the rate of protein synthesis and coordinates cell growth with cell cycle progression. To understand the function and regulation of the LI 1-MDM2-p53 pathway, we propose the following aims: Aim 1.To investigate the mechanism and regulation of the MDM2-L11 interaction. Aim 2. To investigate the in vivo function of the MDM2-L11 interaction using a MDM2 (C305F) knockin. Aim 3. To investigate the mechanism of ARF in the regulation of L11, MDM2, and p53.

DESCRIPTION (provided by applicant): The transcription factor p53 responds to variety of cellular stressors by inducing cell cycle arrest or apoptosis, playing a critical role in tumor suppression. Mutations in the p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of two p53 inhibitors Mdm2 and Mdm4 occur in most of the rest. Current dogma holds that Mdm2 mainly regulates p53 stability via its RING finger E3 ubiquitin ligase and Mdm4 mainly controls p53 transcriptional activity through concealing the p53 transcriptional activation domain. In vitro data have shown that Mdm2's RING E3 is also responsible for degradation of Mdm4 and itself, and a model is proposed that switch from Mdm2 degradation of p53 to self-degradation is responsible for p53 accumulation and activation after stress. Many p53 inducers including tumor suppressor p14ARF and ribosomal protein L11 stabilize and activate p53 through inhibition of Mdm2's E3 function. Thus, theoretically anticancer strategies targeting Mdm2 E3 function could cooperate with strategies targeting the Mdm2-p53 interaction to activate p53 in the millions of patients diagnosed with p53-positive cancers each year. Importantly however, detailed knowledge of the molecular mechanisms of Mdm2 E3 regulation will be required to achieve this goal. We have recently generated mice bearing a single-residue substitution in the Mdm2 RING finger domain abolishing its E3 function without affecting p53 binding. Unexpectedly however, in contrast to current notion our data have shown that 1) the Mdm2-p53 interaction, in the absence of Mdm2-mediated p53 ubiquitination, cannot control p53 activity, and 2) Mdm2 auto-ubiqutination is not the principle mechanism for Mdm2 degradation in vivo. Our analysis reveals yet another disconnect between hypotheses generated by in vitro transfection studies and mouse models. Based on this mouse model we will test three hypotheses: 1) Mdm2-Mdm4 interaction augments or necessitates Mdm2's E3 ligase function, 2) the binding of Mdm2 suppresses p53's apoptotic but not cell cycle arrest function, and 3) there is an unknown novel E3 ubiquitin ligase for Mdm2 degradation in vivo. Our specific aims are: Aim 1. To investigate the role of Mdm2 RING E3 in regulation of Mdm4 Aim 2. To investigate the non-redundant roles of Mdm2 and Mdm4 in regulation of p53. Aim 3. To investigate the role of Mdm2 in regulation of p53-induced cell cycle arrest and apoptosis. PUBLIC HEALTH RELEVANCE: Mutations in the tumor suppressor p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of two p53 inhibitors Mdm2 and Mdm4 occur in most of the rest. This project proposes to investigate the function and mechanism of the Mdm2- Mdm4-p53 regulatory loop using novel mouse models. Detailed knowledge of the molecular mechanisms and regulation of the Mdm2 E3 ubiqutin ligase is critically important for the design of future p53-based anticancer strategies.

DESCRIPTION (provided by applicant): In vivo function and mechanism of the r-protein-Mdm2-p53 pathway Mdm2 interacts with a large number of proteins and plays a critical role in regulating cell growth, proliferation and apoptosis. One of the most significant breakthroughs in the study of Mdm2 in the past several years is the demonstration of Mdm2's interaction with ribosomal proteins (r-proteins). We discovered that three r-proteins, L5, L11 and L23, bind Mdm2 and block Mdm2-mediated p53 degradation, leading to p53-dependent growth inhibition. We demonstrated that events inhibiting ribosomal biogenesis cause nucleolar stress and release nucleolar r-proteins, which interact with Mdm2 in the nucleus and trigger activation of p53. However, the physiological function of the r-protein- Mdm2 interaction has not been addressed in vivo, and the biological role of the interaction has not been established in a mouse model. A number of Mdm2 inhibiting molecules, including the three r-proteins and the tumor suppressor ARF, are known to bind Mdm2 in the central acidic domain. Mutations targeting Mdm2 the central acidic domain have been reported in human cancers. We recently found that mutations in the Mdm2' central acidic domain disrupt r-protein interaction and attenuate p53's response to nucleolar stress. However, despite implications in human cancer the role of the r-protein-Mdm2-p53 pathway in tumor suppression has not been proven in vivo, and mouse models are essential for the further study of these proteins. We have recently generated a knockin mouse model expressing a mutant Mdm2 deficient in r-protein binding and begun to dissect the biological functions of the mutant mice. During a discovery screen for novel Mdm2 binding partners we identified the mitochondrial Hep27 as a potential Mdm2 binding protein. Hep27 binds to Mdm2's central acidic region-in concert with the other Mdm2 inhibitors, and mechanistically is an inhibitor of Mdm2 function. Building upon a large amount of biochemical, biological and genetic information, we have formulated two hypotheses: 1) the r-protein-Mdm2 interaction constitutes surveillance for ribosomal biogenesis and is critical for the activation of p53 and suppression of tumorigenesis. 2) The Hep27- Mdm2 interaction represents a novel mitochondrial retrograde signaling pathway that transduces mitochondrial stress through Mdm2 to amplify p53 function. Our Specific Aims are: 1. To determine the biological function and biochemical mechanism of Mdm2 C4 zinc finger. 2. To investigate how the r-protein-Mdm2-p53 pathway interacts with other p53 signaling pathways. 3. To define a mitochondrial stress-activated, Hep27-mediated p53 pathway. PUBLIC HEALTH RELEVANCE: Mutations in the tumor suppressor p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of a p53 inhibitor Mdm2 occur in most of the rest. This project proposes to investigate the function and mechanism of the Mdm2-p53 regulatory loop using novel mouse models. Detailed knowledge of the molecular mechanisms and regulation of the Mdm2 and p53 is critically important for the design of future p53-based anticancer strategies.

DESCRIPTION (provided by applicant): A mitochondria-localized protein, p32, was recently identified by our lab as a binding partner for the tumor suppressor protein p14ARF. Preliminary data indicated that p32 is essential for p14ARF, a critical mediator for transducing hyperproliferative, oncogenic stress signals to the Mdm2-p53 tumor suppression pathway, to localize to mitochondria and to induce p53-dependent apoptosis. Importantly, human cancer-derived p14ARF mutations that disrupt p32 binding can impair both of these functions. Recently, our preliminary studies have shown that p32 is in fact essential for apoptosis induced by a broad range of apoptotic stimuli. The overall hypothesis behind the proposed research is that p32 specifies an essential factor for a surveillance system that monitors the integrity of mitochondrial function and promotes apoptosis in response to irreparable mitochondrial damage. The rationale for the hypothesis is based on the following observations. (1) p32 is a mitochondrial protein. (2) Knockdown p32 desensitizes cells to apoptosis induced by a broad range of apoptotic stimuli. (3) Like cytochrome c, p32 accumulates in the cytoplasm during apoptosis. (4) Ectopically expressed, cytoplasm- localized p32 induces apoptosis. (5) p32 is reported to have a role in oxidative phosphorylation. The experimental focus of the proposal is on dynamics and outcome of p32 localization, function and mechanism of p32 in regulating apoptosis, and genetics and biology of p32 in metabolic regulation and tumorigenesis. Based on these observations, the proposed research will focus on characterizing the function and mechanism of p32 in regulating apoptosis by a combination of biochemical, cellular, and genetic approaches. The specific aims are designed to assess p32's role in regulating apoptosis under a broad range of apoptotic conditions and define the dynamics of p32 subcellular localization, to Investigate mechanisms by which p32 promotes apoptotic cell death, and to investigate p32's physiological function using p32 conditional knockout mice. If successful, the proposed study will ascribe new functions to p32. It will also aid in our understanding of apoptotic cell death - a process that is critical during development and in the pathogenesis of diseases such as cancer, rheumatoid arthritis, and neurodegenerative diseases - and may eventually lead to additional drug targets for controlling apoptosis in treatment of these diseases.

DESCRIPTION (provided by applicant): TP53 is a critical tumor suppressor gene capable of inducing cell cycle arrest, senescence, and apoptosis. Canonically, the primary negative regulator of the TP53 protein product p53, Mdm2, is considered to regulate p53 through two mechanisms; 1) through direct binding to 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. In addition to Mdm2, a homologous protein, MdmX also functions in p53 regulation, primarily through binding and blocking the p53 transactivation domain in a similar mechanism to Mdm2. Both Mdm2 and MdmX knockout mice are embryonically lethal, and rescued completely with concomitant deletion of p53, indicative of their critical role in p53 regulation. The development of an Mdm2C462A knock-in mouse model that maintains Mdm2-p53 binding, but disrupts Mdm2 E3 ligase activity, was found to result in embryonic lethality, rescued with simultaneous deletion of p53. Surprisingly, this study suggests that Mdm2-p53 binding alone is not sufficient for p53 regulation, and implicates the Mdm2 RING finger domain as critical in p53 regulation. Along with disrupting Mdm2 E3 ubiquitin activity, the mutation also disrupts Mdm2-MdmX heterodimerization. Because the Mdm2C462A mutation disrupts both functions of the RING finger domain, the E3 ubiquitin ligase activity and the MdmX binding, it cannot be deduced which of these changes is causing the observed misregulation of p53. Despite intensive study, much remains unknown about how Mdm2 and MdmX function in vivo to regulate p53. In vitro this binding has been demonstrated to amplify or rescue Mdm2 E3 ligase activity towards p53, but its role in vivo is not yet clear. Recent development of an Mdm2Y487A knock-in mouse, which maintains the ability to bind to MdmX and p53, but has disrupted E3 ubiquitin ligase activity has allowed for the separation of these two Mdm2 RING finger domain functions. Through utilizing this model, we hope to further elucidate the function of the Mdm2 RING finger domain in p53 regulation, as further understanding p53 regulation is critical in the development of effective therapeutics.

ABSTRACT The tumor suppressor gene TP53 is the most commonly mutated gene in human cancers. Understanding the full breadth of p53 function and regulation is crucial to our understanding of tumor initiation and development. The MDM2 proto-oncoprotein is a primary negative regulator for p53 by promoting p53 polyubiquitination and proteosomal degradation. The MDM2 homologous protein MDMX also functions as a negative regulator for p53. While many questions remain, a focus on the use of in vivo models is allowing for a closer view of how this MDM2/MDMX-p53 pathway is regulated, with a newfound emphasis on how this pathway can be better manipulated for therapeutic gains. During the last few years, our experiments have been focused on generating and examining MDM2 mutation knock-in mice that are deficient in MDM2 E3 ligase function or MDM2-MDMX binding. We have generated MDM2C462A and MDM2Y487A mutant mice, both lacking MDM2 E3 ligase function. Studies with the MDM2C462A mice, which also lack MDM2-MDMX interaction, and the MDM2Y487A mice, which retain MDM2- MDMX interaction, have generated new insight into the in vivo regulation of p53 by MDM2 E3 ligase and the MDM2-MDMX heterooligomer. Recently, we have generated a number of new mouse models expressing an inducible p53 under various MDM2 and MDMX backgrounds. Our preliminary data generated with these inducible p53 mice have revealed several surprising findings. We found that MDMX can suppress p53 expression independent of MDM2 E3 ligase function; we found that in vivo MDMX is required for MDM2 mediated p53 degradation; and we found that the MDM2C462A mutant has gained a neomorphic function for stimulating p53 transcriptional activity. Experiments proposed in this application are designed to study (1) whether MDMX regulates p53 expression through modulating its translation; (2) what is the mechanism by which MDMX impacts on MDM2 mediated p53 degradation; (3) how the RING finger mutant MDM2 gained a function to activate p53. These experiments provide proof-of-principle evidence for exploring inhibition of MDM2 RING E3 ligase function and disruption of MDM2-MDMX interaction as potential drug development strategies targeting cancers expressing high levels of MDM2 and MDMX and WT p53.