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Protein oligomerization plays a very important role in many physiological processes. p53 acts as a key tumor suppressor by regulating cell cycle arrest, DNA repair, and apoptosis, and its antitumor activity is regulated by the hetero- and homo-oligomerization of MDMX and MDM2 proteins. So far, some traditional methods have been utilized to study the oligomerization of MDMX and MDM2 in vitro, but they have not clarified some controversial issues or whether the extracellular results can represent the intracellular results. Here, we put forward an in situ method for studying protein homo- and hetero-oligomerization in single living cells by using fluorescence correlation spectroscopy. In this study, MDMX and MDM2 were labeled with fluorescent proteins using lentiviral transfection. Autocorrelation spectroscopy and cross-correlation spectroscopy methods were used to study the oligomerization of MDMX and MDM2 in situ and the effect of regulation of MDMX oligomerization on p53-MDMX interactions in single living cells. We observed the homo- and hetero-oligomerization of MDMX and MDM2 in living cells. Meanwhile, the levels of the homo-oligomers of MDMX and MDM2 were increased due to the lack of hetero-oligomerization. Finally, the binding affinity of MDMX for p53 was improved with an increase in the level of MDMX hetero-oligomerization.

Pigs have been increasingly recognized as a relevant model for studying many human diseases. However, functions and regulations of numerous critical molecules involved in human diseases are not well characterized in pigs, including the prominent tumor suppressor p53, a transcription factor involved in various anti-proliferative processes. In this study, we systematically characterized porcine p53 (p-p53) in its transcriptional activity and regulation by the E3 ligase Mdm2, in comparison with that of human p53 (h-p53). p-p53 is highly homologous to h-p53 with the N-terminal region showing relative divergence. p-p53 exhibits a comparable transcriptional activity to that of h-p53 towards a diverse range of known target genes, and is subject to ubiquitination and degradation by both human and porcine Mdm2 (h-/p-Mdm2). Utilization of the h-Mdm2 targeting compound Nutlin-3 and protein RPL11 inhibits the negative effect of p-Mdm2 on p-p53. These results suggest that the transcription activity and regulation of p-p53 is very similar to that of h-p53, and that the developed agents targeting the h-p53 pathway could be used in the study of p53 related processes and diseases in pigs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Aims: Murine double minute-2 (MDM2) has been poorly studied in cardiovascular diseases. The aim of the present study was to determine the biological role of MDM2 in inflammation activation and mitochondrial damage in human aortic endothelial cells (HAECs) stimulated with oxidized low-density lipoprotein (ox-LDL)., Methods: The expression of MDM2 in the aortas of atherosclerotic mice was determined. An adenoviral vector for MDM2 overexpression and siRNA for MDM2 downregulation were constructed and used to transfect HAECs. The functional changes in HAECs stimulated by ox-LDL were observed., Results: The protein expression of MDM2 was increased in atherosclerotic mice and ox-LDL-treated HAECs. In addition, ox-LDL-induced mRNA expression and secretion of TNF-α, IL-6 and IL-1β were significantly decreased by MDM2 downregulation and increased by MDM2 overexpression, and activation of NF-κB and caspase-1 was involved in the activity of MDM2. The ox-LDL-induced mitochondrial damage, indicated as increase in mitochondrial ROS production, decrease in mitochondrial membrane potential and elevation of mitochondrial DNA release, was significantly reversed by MDM2 downregulation and worsened by MDM2 overexpression. The ox-LDL-induced activation of TLR9/NF-κB and NLRP3/caspase-1 pathway was inhibited by MDM2 downregulation and worsened by MDM2 overexpression. The aggravation caused by MDM2 overexpression was abolished by mito-TEMPO. Treatment with mito-TEMPO significantly reduced the increase in mRNA expression and secretion of TNF-α, IL-6 and IL-1β induced by MDM2 overexpression in ox-LDL treated HAECs., Conclusions: These findings suggest that MDM2 contributes to ox-LDL-induced inflammation via regulating mitochondrial damage., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Loss-of-function mutations in the E3 ubiquitin ligase parkin have been implicated in the death of dopaminergic neurons in the substantia nigra, which is the root cause of dopamine deficit in the striatum in Parkinson's disease. Parkin ubiquitinates proteins on mitochondria that lost membrane potential, promoting the elimination of damaged mitochondria. Neuroprotective activity of parkin has been linked to its critical role in the mitochondria maintenance. Here we report a novel regulatory mechanism: another E3 ubiquitin ligase Mdm2 directly binds parkin and enhances its enzymatic activity in vitro and in intact cells. Mdm2 translocates to damaged mitochondria independently of parkin, enhances parkin-dependent ubiquitination of the outer mitochondria membrane protein mitofusin1. Mdm2 facilitates and its knockdown reduces parkin-dependent mitophagy. Thus, ubiquitously expressed Mdm2 might enhance cytoprotective parkin activity. The data suggest that parkin activation by Mdm2 could be targeted to increase its neuroprotective functions, which has implications for anti-parkinsonian therapy.

Overexpression of MDM2 oncoprotein has been detected in a large number of diverse human malignancies and has been shown to play both p53-dependent and p53-independent roles in oncogenesis. Our study was designed to explore the impact of MDM2 overexpression on the levels of various cell cycle regulatory proteins including Aurora kinase-B (AURK-B), CDC25C and CDK1, which are known to promote tumor progression and increase metastatic potential. Our data from human cell cycle RT 2 profiler PCR array experiments revealed significant changes in the expression profile of genes that are involved in different phases of cell cycle regulation in LNCaP-MST (MDM2 transfected) prostate cancer cells. Our current study has demonstrated a significant increase in the expression level of AURK-B, CDC25C, Cyclin A2, Cyclin B and CDK1 in LNCaP-MST cells as compared with wild type LNCaP cells that were modulated by MDM2 specific inhibitor Nutlin-3. In fact, the expression levels of the above- mentioned proteins were significantly altered at both mRNA and protein levels after treating the cells with 20 μM Nutlin-3 for 24h. Additionally, the pro-apoptotic proteins including p53, p21, and Bax were elevated with the concomitant decrease in the key anti-apoptotic proteins following MDM2 inhibitor treatment. Also, Nutlin-3 treated cells demonstrated caspase-3 activation was observed with an in-vitro caspase-3 fluorescent assay performed with caspase 3/7 specific DEVD-amc substrate. Our results offer significant evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest via blocking the transmission of signals through AURKB-CDK1 axis and inducing apoptosis in LNCaP-MST cancer cells. It is evident from our data that MDM2 overexpression probably is the primary cause for CDK1 up-regulation in the LNCaP-MST cells, which might have occurred possibly through activation of AURK-B. However, further studies in this direction should shed more light on the intracellular mechanisms involved in the regulation of Aurora kinase-B and CDK1 axis in MDM2 positive cancers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The respiratory lineage initiates from the specification of NKX2-1 + progenitor cells that ultimately give rise to a vast gas-exchange surface area. How the size of the progenitor pool is determined and whether this directly impacts final lung size remains poorly understood. Here, we show that epithelium-specific inactivation of Mdm2 , which encodes an E3 ubiquitin ligase, led to lethality at birth with a striking reduction of lung size to a single vestigial lobe. Intriguingly, this lobe was patterned and contained all the appropriate epithelial cell types. The reduction of size can be traced to the progenitor stage, when p53, a principal MDM2 protein degradation target, was transiently upregulated. This was followed by a brief increase of apoptosis. Inactivation of the p53 gene in the Mdm2 mutant background effectively reversed the lung size phenotype, allowing survival at birth. Together, these findings demonstrate that p53 protein turnover by MDM2 is essential for the survival of respiratory progenitors. Unlike in the liver, in which genetic reduction of progenitors triggered compensation, in the lung, respiratory progenitor number is a key determinant factor for final lung size., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53 R178E mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53 -/- mice. Surprisingly, stabilization of p53 R178E in Mdm2 -/- mice nevertheless triggers extensive apoptosis, indicative of residual wild-type activities. Although this apoptotic activity suffices to trigger lethality of Trp53 R178E ;Mdm2 -/- embryos, it proves insufficient for suppression of spontaneous and oncogene-driven tumorigenesis. Trp53 R178E mice develop tumors indistinguishably from Trp53 -/- mice and tumors retain and even stabilize the p53 R178E protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53 R178E tumors exhibit remarkably better chemotherapy responses than Trp53 -/- ones, resulting in enhanced eradication of p53-mutated tumor cells. Together, this provides genetic proof-of-principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Seizures can induce endoplasmic reticulum (ER) stress, and sustained ER stress contributes to neuronal death after epileptic seizures. Despite the recent debate on whether inhibiting ER stress can reduce neuronal death after seizures, whether and how ER stress impacts neural activity and seizures remain unclear. In this study, we discovered that the acute ER stress response functions to repress neural activity through a protein translation-dependent mechanism. We found that inducing ER stress promotes the expression and distribution of murine double minute-2 (Mdm2) in the nucleus, leading to ubiquitination and down-regulation of the tumor suppressor p53. Reduction of p53 subsequently maintains protein translation, before the onset of translational repression seen during the latter phase of the ER stress response. Disruption of Mdm2 in an Mdm2 conditional knockdown (cKD) mouse model impairs ER stress-induced p53 down-regulation, protein translation, and reduction of neural activity and seizure severity. Importantly, these defects in Mdm2 cKD mice were restored by both pharmacological and genetic inhibition of p53 to mimic the inactivation of p53 seen during ER stress. Altogether, our study uncovered a novel mechanism by which neurons respond to acute ER stress. Further, this mechanism plays a beneficial role in reducing neural activity and seizure severity. These findings caution against inhibition of ER stress as a neuroprotective strategy for seizures, epilepsies, and other pathological conditions associated with excessive neural activity., Competing Interests: The authors have declared that no competing interests exist.

Genome instability is a common feature of tumor cells, and the persistent presence of genome instability is a potential mechanism of tumorigenesis. The E3 ubiquitin ligase MDM2 is intimately involved in genome instability, but its mechanisms are unclear. Our data demonstrated that the transcription factor HBP1 is a target of MDM2. MDM2 facilitates HBP1 proteasomal degradation by ubiquitinating HBP1, regardless of p53 status, thus attenuating the transcriptional inhibition of HBP1 in the expression of its target genes, such as the DNA methyltransferase DNMT1 and histone methyltransferase EZH2, which results in global DNA hypermethylation and histone hypermethylation and ultimately genome instability. The repression of HBP1 by MDM2 finally promotes cell growth and tumorigenesis. Next, we thoroughly explored the regulatory mechanism of the MDM2/HBP1 axis in DNA damage repair following ionizing radiation. Our data indicated that MDM2 overexpression-mediated repression of HBP1 delays DNA damage repair and causes cell death in a p53-independent manner. This investigation elucidated the mechanism of how MDM2 promotes genome instability and enhances tumorigenesis in the absence of p53, thus providing a theoretical and experimental basis for targeting MDM2 as a cancer therapy.

Functions of tumor suppressor p53 and its negative regulator mouse double minute 2 homolog (Mdm2) in ovarian granulosa cells remain to be elucidated, and the current study aims at clarifying this issue. Mice with Mdm2 deficiency in ovarian granulosa cells [ Mdm2-loxP/ progesterone receptor ( Pgr)-Cre mice] were infertile as a result of impairment of oocyte maturation, ovulation, and fertilization, and those with Mdm2/p53 double deletion in granulosa cells ( Mdm2-loxP/ p53-loxP/ Pgr-Cre mice) showed normal fertility, suggesting that p53 induction in the ovarian granulosa cells is detrimental to ovarian function by disturbing oocyte quality. Another model of Mdm2 deletion in ovarian granulosa cells ( Mdm2-loxP/ anti-Mullerian hormone type 2 receptor-Cre mice) also showed subfertility as a result of the failure of ovulation and fertilization, indicating critical roles of ovarian Mdm2 in ovulation and fertilization. Mdm2-p53 pathway in cumulus granulosa cells transcriptionally controlled an orphan nuclear receptor steroidogenic factor 1 (SF1), a key regulator of ovarian function. Importantly, MDM2 and SF1 levels in human cumulus granulosa cells were positively associated with the outcome of oocyte maturation and fertilization in patients undergoing infertility treatment. These findings suggest that the Mdm2-p53-SF1 axis in ovarian cumulus granulosa cells directs ovarian function by affecting their neighboring oocyte quality.-Haraguchi, H., Hirota, Y., Saito-Fujita, T., Tanaka, T., Shimizu-Hirota, R., Harada, M., Akaeda, S., Hiraoka, T., Matsuo, M., Matsumoto, L., Hirata, T., Koga, K., Wada-Hiraike, O., Fujii, T., Osuga, Y. Mdm2-p53-SF1 pathway in ovarian granulosa cells directs ovulation and fertilization by conditioning oocyte quality.

Pterygium is a fibrovascular subepithelial growth of degenerative tissue over the limbus. It is a common condition worldwide that is especially prevalent in tropical countries within the "pterygium belt." Its exact etiology remains to be elucidated; however, it is strongly associated with exposure to ultraviolet light. The high expression levels of tumor protein p53 (TP53) observed in laboratory studies of pterygium seem to contradict the fast-growing nature of its clinical behavior, and TP53 mutations have been suggested. We demonstrated that mouse double minute 2 (MDM2), a TP53-binding protein, contributes to the inhibition of TP53 activity in human pterygium. Thus, disruption of the MDM2-TP53 interaction should attenuate human pterygium cell growth. For primary pterygium, treatment is relatively straightforward and involves surgical excision. To minimize the risk of recurrence, many adjunctive therapies are adopted, including antimetabolites, such as mitomycin C and 5-fluorouracil, amniotic membrane, different variations on conjunctival and/or limbal conjunctival grafts, and other medications such as anti-vascular endothelial growth factor. In the future, MDM2 antagonists may help further lower the recurrence rates after the treatment of pterygium.

Aims: To confirm that mouse double minute 2 (MDM2) could inhibit p53 activity in human pterygium. And to show the disruption of MDM2-p53 interaction could reactive the functions of p53 in pterygium., Method: Pterygium and corresponding conjunctiva tissues were collected for establishment of primary cell lines. Expression patterns of MDM2 and p53 were detected by immunofluorescence. Protein localization of p53 and MDM2, and transcriptional activity of p53 in both untreated and MDM2 antagonist (Nutlin) treated pterygium cells were quantified., Results: In pterygium, p53 was highly expressed in cytoplasm and slightly expressed in the nuclei. MDM2 was localized in the nuclei. A p53 transcriptional regulated target gene, p21, was not expressed in pterygium tissues, suggesting the p53 transcriptional activity was not active in pterygium. After treatment with Nutlin, increased nuclear localization of p53 (4.05%-80.56%) was observed in pterygium cells along with increasing Nutlin dosages (from 0 to 50 μM, p < 0.001). The expression of p21 was increased after Nutlin treatments in pterygium cells (2.49 folds in 20 μM Nutlin treated cells compared to control treated cells, p = 0.012)., Conclusion: We discovered a novel mechanism in pterygium whereby MDM2 suppresses p53 transcriptional activity despite abundant p53 in pterygium. Disruption of MDM2-p53 interaction by Nutlin could be a potential treatment for pterygium., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

ARF is a tumor suppressor protein that has a pivotal role in the prevention of cancer development through regulating cell proliferation, senescence, and apoptosis. As a factor that induces senescence, the role of ARF as a tumor suppressor is closely linked to the p53-MDM2 axis, which is a key process that restrains tumor formation. Thus, many cancer cells either lack a functional ARF or p53, which enables them to evade cell oncogenic stress-mediated cycle arrest, senescence, or apoptosis. In particular, the ARF gene is a frequent target of genetic and epigenetic alterations including promoter hyper-methylation or gene deletion. However, as many cancer cells still express ARF, pathways that negatively modulate transcriptional or post-translational regulation of ARF could be potentially important means for cancer cells to induce cellular proliferation. These recent findings of regulators affecting ARF protein stability along with its low levels in numerous human cancers indicate the significance of an ARF post-translational mechanism in cancers. Novel findings of regulators stimulating or suppressing ARF function would provide new therapeutic targets to manage cancer- and senescence-related diseases. In this review, we present the current knowledge on the regulation and alterations of ARF expression in human cancers, and indicate the importance of regulators of ARF as a prognostic marker and in potential therapeutic strategies.

The MDM2 RING domain harbors E3 ubiquitin ligase activity critical for regulating the degradation of tumor suppressor p53, which controls many cellular pathways. The MDM2 RING domain also is required for an interaction with MDMX. Mice containing a substitution in the MDM2 RING domain, MDM2 C462A , disrupting MDM2 E3 function and the MDMX interaction, die during early embryogenesis that can be rescued by p53 deletion. To investigate whether MDM2 C462A , which retains p53 binding, has p53-suppressing activity, we generated Mdm2 C462A/C462A ; p53 ER /- mice, in which we replaced the endogenous p53 alleles with an inducible p53 ER /- allele, and compared survival with that of similarly generated Mdm2 -/- ; p53 ER /- mice. Adult Mdm2 -null mice died ∼7 days after tamoxifen-induced p53 activation, indicating that in the absence of MDM2, MDMX cannot suppress p53. Surprisingly, Mdm2 C462A/C462A ; p53 ER /- mice died ∼5 days after tamoxifen injection, suggesting that p53 activity is higher in the presence of MDM2 C462A than in the absence of MDM2. Indeed, in MDM2 C462A -expressing mouse tissues and embryonic fibroblasts, p53 exhibited higher transcriptional activity than in those expressing no MDM2 or no MDM2 and MDMX. This observation indicated that MDM2 C462A not only is unable to suppress p53 but may have gained the ability to enhance p53 activity. We also found that p53 acetylation, a measure of p53 transcriptional activity, was higher in the presence of MDM2 C462A than in the absence of MDM2. These results reveal an unexpected role of MDM2 C462A in enhancing p53 activity and suggest the possibility that compounds targeting MDM2 RING domain function could produce even more robust p53 activation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Caveolin-1 (Cav-1), as an important structural protein of caveolae, has been proven to be correlated with several signal transduction pathways. Recent studies have shown that Cav-1 may play a critical role in response to DNA damage in irradiated pancreatic cancer cells. However, it is not known whether down-regulation of Cav-1 is required to enhance the damage of other kinds of human cells exposed to X-radiation. In this study, the role of Cav-1 in Chang liver cell line (CHL) exposed to X-radiation was investigated. Cav-1 knockdown cell line (CHL-CAV7) was stably established by the siRNA plasmids transfection, and Cav-1 expression was suppressed by 60%, compared with that of control group (CHL-C) which was transfected with non-targeting plasmids. Cellular survival ability and the expressions of proteins related to DNA damage and repair were examined by colony formation assay and Western blot, respectively. Down-regulation of Cav-1 expression induced a significant decrease of the survival rate in CHL-CAV7 cells exposed to 8 and 10 Gy X-radiation. Compared with CHL-C cells, CHL-CAV7 cells showed increased γH2AX expression, as well as decreased p-ATM, DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) and p53 protein expressions when treated with X-radiation. Meanwhile, the colocalization of Mdm2 and Cav-1 was decreased in CHL-CAV7 cells compared with that in CHL-C cells. These results suggest that the down-regulation of Cav-1 may aggravate DNA damage of CHL cells through reducing the interaction of Cav-1 and Mdm2, which results in the promotion of p53 degradation.

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.

Inflammation is recently recognized as one of the hallmarks of human cancer. Chronic inflammatory response plays a critical role in cancer development, progression, metastasis, and resistance to chemotherapy. Conversely, the oncogenic aberrations also generate an inflammatory microenvironment, enabling the development and progression of cancer. The molecular mechanisms of action that are responsible for inflammatory cancer and cancer-associated inflammation are not fully understood due to the complex crosstalk between oncogenic and pro-inflammatory genes. However, molecular mediators that regulate both inflammation and cancer, such as NF-κB and STAT have been considered as promising targets for preventing and treating these diseases. Recent works have further demonstrated an important role of oncogenes (e.g., NFAT1, MDM2) and tumor suppressor genes (e.g., p53) in cancer-related inflammation. Natural products that target these molecular mediators have shown anticancer and anti-inflammatory activities in preclinical and clinical studies. Sesquiterpenoids (STs), a class of novel plant-derived secondary metabolites have attracted great interest in recent years because of their diversity in chemical structures and pharmacological activities. At present, we and other investigators have found that dimeric sesquiterpenoids (DSTs) may exert enhanced activity and binding affinity to molecular targets due to the increased number of alkylating centers and improved conformational flexibility and lipophilicity. Here, we focus our discussion on the activities and mechanisms of action of STs and DSTs in treating inflammation and cancer as well as their structure-activity relationships., (Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution., (Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.)

Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

The tumor suppressor p53 is a central regulator of signaling pathways that controls the cell cycle and maintains the integrity of the human genome. p53 level is regulated by mouse double minute 2 homolog (Mdm2), which marks p53 for proteasomal degradation. The p53-Mdm2 circuitry is subjected to complex regulation by a variety of mechanisms, including microRNAs (miRNAs). We found a novel effector of this regulatory circuit, namely, miR-122*, the passenger strand of the abundantly expressed liver-specific miR-122. Here, we demonstrate that miR-122* levels are reduced in human hepatocellular carcinoma (HCC). We found that miR-122* targets Mdm2, thus participating as an important player in the p53-Mdm2 circuitry. Moreover, we observed significant negative correlation between levels of miR-122* and Mdm2 in a large set of human HCC samples. In vivo tumorigenicity assays demonstrate that miR-122* is capable of inhibiting tumor growth, emphasizing the tumor-suppressor characteristics of this miRNA. Furthermore, we show that blocking miR-122 in murine livers with an antagomiR-122 (miRNA inhibitor) results in miR-122* accumulation, leading to Mdm2 repression followed by elevated p53 protein levels., Conclusion: miR-122*, the passenger strand of miR-122, regulates the activity of p53 by targeting Mdm2. Importantly, similarly to miR-122, miR-122* is significantly down-regulated in human HCC. We therefore propose that miR-122* is an important contributor to the tumor suppression activity previously attributed solely to miR-122. (Hepatology 2016;64:1623-1636)., (© 2016 by the American Association for the Study of Liver Diseases.)

Retinoic acid receptor alpha (RARα) has a critical role in the differentiation process of osteosarcoma cells induced by all-trans retinoic acid (ATRA). However, degradation of RARα through ubiquitin proteasome pathway weakens the differentiation efficiency of osteosarcoma cells. In this study, we discover that murine double minute-2 (MDM2) acts as an E3 ubiquitin ligase to target RARα for degradation. We observe that MDM2 is required for RARα polyubiquitination and proteasomal degradation because downregulation of MDM2 by short hairpin RNA results in the accumulation of RARα, and MDM2 overexpression promotes the degradation of RARα. We also demonstrate that the N-terminal domain of MDM2 (amino acids 1-109) is the major RARα-binding site. Importantly, endogenous MDM2 levels are not only upregulated in human primary osteosarcoma blasts but are also inversely correlated with the level of osteopontin, which is a marker of bone differentiation. Moreover, MDM2 impairs the ATRA-induced osteoblastic differentiation of osteosarcoma cells, whereas an inhibitor of the MDM2 ubiquitin ligase synergizes with ATRA to enhance the differentiation of osteosarcoma cells and primary osteosarcoma blasts. Therefore, our study indicates that MDM2 serves as an E3 ubiquitin ligase to regulate the degradation of RARα and suggests that MDM2 is a novel therapeutic target for ATRA-based differentiation therapeutic approaches in osteosarcoma.

The atypical E3 ubiquitin ligase RNF31 is highly expressed in human breast cancer, the most frequent neoplastic lethality among women. Here, RNF31 depletion in breast cancer cells in combination with global gene expression profiling revealed p53 (TP53) signaling as a potential RNF31 target. Interestingly, RNF31 decreased p53 stability, whereas depletion of RNF31 in breast cancer cells caused cell cycle arrest and cisplatin-induced apoptosis in a p53-dependent manner. Furthermore, RNF31 associated with the p53/MDM2 complex and facilitated p53 polyubiquitination and degradation by stabilizing MDM2, suggesting a molecular mechanism by which RNF31 regulates cell death. Analysis of publically available clinical data sets displayed a negative correlation between RNF31 and p53 target genes, including IGFBP3 and BTG1, consistent with RNF31 regulating p53 function in vivo as well. Together, our findings suggest RNF31 as a potential therapeutic target to restore p53 function in breast cancer.

Sex-determining region Y box 6 (SOX6) has been described as a tumor-suppressor gene in several cancers. Our previous work has suggested that SOX6 upregulated p21(Waf1/Cip1)(p21) expression in a p53-dependent manner; however, the underlying mechanism has remained elusive. In this study, we confirmed that SOX6 can suppress cell proliferation in vitro and in vivo by stabilizing p53 protein and subsequently upregulating p21. Co-immunoprecipitation and immunocytofluorescence assays demonstrated that SOX6 can promote formation of the p14ARF-HDM2-p53 ternary complex by promoting translocation of p14ARF (p14 alternate reading frame tumor suppressor) to the nucleoplasm, thereby inhibiting HDM2-mediated p53 nuclear export and degradation. Chromatin immunoprecipitation combined with PCR assay proved that SOX6 can bind to a potential binding site in the regulatory region of the c-Myc gene. Furthermore, we confirmed that SOX6 can downregulate the expression of c-Myc, as well as its direct target gene nucleophosmin 1 (NPM1), and that the SOX6-induced downregulation of NPM1 is linked to translocation of p14ARF to the nucleoplasm. Finally, we showed that the highly conserved high-mobility group (HMG) domain of SOX6 is required for SOX6-mediated p53 stabilization and tumor inhibitory activity. Collectively, these results reveal a new mechanism of SOX6-mediated tumor suppression involving p21 upregulation via the p14ARF-HDM2-p53 axis in an HMG domain-dependent manner.

Introduction: Radiation therapy, alone or in combination with chemotherapy, is effective for patients with locally advanced and recurrent pancreatic cancer. Ionizing radiation induces cell cycle arrest and cell apoptosis through enhancement several signals such as p53, p21(Waf1/Cip1), and caspase. However, the therapeutic efficacy is attenuated by radiation-induced activation of NF-κB. Nafamostat mesilate, a synthetic serine protease inhibitor, inhibits NF-κB activation in pancreatic cancer. Therefore, we hypothesized that nafamostat mesilate inhibited radiation-induced activation of NF-κB and improves therapeutic outcome., Results: In combination group, NF-κB activation was significantly inhibited in comparison with that of radiation group. Nafamostat mesilate obviously down-regulated the expression levels of Mdm2 compared with control cells or irradiated cells. Consequently, p53 expression was stabilized inversely in correlation with Mdm2 protein expression level. The expression levels of p53, p21(Waf1/Cip1), cleaved caspase-3 and -8 were the highest in the combination group. Nafamostat mesilate enhanced ionizing radiation-induced cell apoptosis and G2/M cell cycle arrest. In combination group, cell proliferation and tumor growth were significantly slower than those in other groups., Conclusion: Combination therapy of radiation with nafamostat mesilate exerts enhanced anti-tumor effect against human pancreatic cancer., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The field of toxicity testing for non-pharmaceutical chemicals is in flux with multiple initiatives in North America and the EU to move away from animal testing to mode-of-action based in vitro assays. In this arena, there are still obstacles to overcome, such as developing appropriate cellular assays, creating pathway-based dose-response models and refining in vitro-in vivo extrapolation (IVIVE) tools. Overall, it is necessary to provide assurances that these new approaches are adequately protective of human and ecological health. Another major challenge for individual scientists and regulatory agencies is developing a cultural willingness to shed old biases developed around animal tests and become more comfortable with mode-of-action based assays in human cells. At present, most initiatives focus on developing in vitro alternatives and assessing how well these alternative methods reproduce past results related to predicting organism level toxicity in intact animals. The path forward requires looking beyond benchmarking against high dose animal studies. We need to develop targeted cellular assays, new cell biology-based extrapolation models for assessing regions of safety for chemical exposures in human populations, and mode-of-action-based approaches which are constructed on an understanding of human biology. Furthermore, it is essential that assay developers have the flexibility to 'validate' against the most appropriate mode-of-action data rather than against apical endpoints in high dose animal studies. This chapter demonstrates the principles of fit-for-purpose assay development using pathway-targeted case studies. The projects include p53-mdm2-mediated DNA-repair, estrogen receptor-mediated cell proliferation and PPARα receptor-mediated liver responses.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

p53 plays a pivotal role in maintaining the genomic integrity of the cell and has an important role in cellular transformation. We isolated and cloned a full length p53 cDNA (Bp53) from water buffalo in expression vectors designed to generate tagged proteins with FLAG or GFP. Bp53 was found to be 1161 nucleotide long and codes for 386 amino acid residues with 79% homology with human p53 containing 393 amino acids. Although Bp53 has some inherent differences in amino acid composition in different functional domains as compared to human p53 but the total electrostatic charge of amino acids has been maintained. Bp53 cDNA was transiently transfected in a p53 null human NSCLC cell line and as expected, it was predominantly localized in the nucleus. Besides, Bp53 effectively transactivates a number of target genes similar to human p53 and exerts most of its anti-tumorigenic potential in culture as observed in clonogenic and cell viability assays. Like human p53 mutants, core domain mutant version of Bp53 was found to be mis-localized to cytoplasm with diminished tumor suppressor activity. However, Bp53 appeared to be more sensitive to mdm2 mediated degradation and as a result, this protein was less stable as compared to human p53. For the first time we have characterized a functionally efficient wild-type p53 from buffalo having lower stability than human p53 and thus, buffalo p53 could be used as a model system for further insight to the molecular basis of wild-type p53 instability., (Copyright © 2015. Published by Elsevier B.V.)

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.

Upregulated expression of nucleolar GTPase nucleostemin (NS) has been associated with increased cellular proliferation potential and tumor malignancy during cancer development. Recent reports attribute the growth regulatory effects of NS protein to its role in facilitating ribosome production. However, the oncogenic potential of NS remains unclear, as imbalanced levels of NS have been reported to exert growth inhibitory effect by modulating p53 tumor-suppressor activity. It also remains in questions if aberrant NS levels might have a p53-independent role in regulation of cell proliferation and growth. In this study, we performed affinity purification and mass spectrometry analysis to explore protein-protein interactions influencing NS growth regulatory properties independently of p53 tumor suppressor. We identified the alternative reading frame (ARF) protein as a key protein associating with NS and further verified the interaction through in vitro and in vivo assays. We demonstrated that NS is able to regulate cell cycle progression by regulating the stability of the ARF tumor suppressor. Furthermore, overexpression of NS suppressed ARF polyubiquitination by its E3 ligase Ubiquitin Ligase for ARF and elongated its half-life, whereas knockdown of NS led to the decrease of ARF levels. Also, we found that NS can enhance NPM stabilization of ARF. Thus, we propose that in the absence of p53, ARF can be stabilized by NS and nucleophosmin to serve as an alternative tumor-suppressor surveillance, preventing potential cellular transformation resulting from the growth-inducing effects of NS overexpression.

In this issue of Molecular Cell, Ye et al. (2015) demonstrate that mTORC1 globally regulates miRNA biogenesis under nutrient-rich conditions via the E3 ubiquitin ligase Mdm2, which promotes Drosha degradation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

mTOR senses nutrient and energy status to regulate cell survival and metabolism in response to environmental changes. Surprisingly, targeted mutation of Tsc1, a negative regulator of mTORC1, caused a broad reduction in miRNAs due to Drosha degradation. Conversely, targeted mutation of Raptor, an essential component of mTORC1, increased miRNA biogenesis. mTOR activation increased expression of Mdm2, which is hereby identified as the necessary and sufficient ubiquitin E3 ligase for Drosha. Drosha was induced by nutrient and energy deprivation and conferred resistance to glucose deprivation. Using a high-throughput screen of a miRNA library, we identified four miRNAs that were necessary and sufficient to protect cells against glucose-deprivation-induced apoptosis. These miRNA was regulated by glucose through the mTORC1-MDM2-DROSHA axis. Taken together, our data reveal an mTOR-Mdm2-Drosha pathway in mammalian cells that broadly regulates miRNA biogenesis as a response to alteration in cellular environment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The MYC oncoprotein and transcription factor is dysregulated in a majority of human cancers and is considered a major driver of the malignant phenotype. As such, developing drugs for effective inhibition of MYC in a manner selective to malignancies is a 'holy grail' of transcription factor-based cancer therapy. Recent advances in elucidating MYC biology in both normal cells and pathological settings were anticipated to bring inhibition of tumorigenic MYC function closer to the clinic. However, while the extensive array of cellular pathways that MYC impacts present numerous fulcrum points on which to leverage MYC's therapeutic potential, identifying the critical target(s) for MYC-specific cancer therapy has been difficult to achieve. Somewhat unexpectedly, MYC's fundamental role in regulating the 'housekeeping' process of ribosome biogenesis, one of the most ubiquitously required and conserved cell functions, may provide the Achilles' heel for therapeutically targeting MYC-driven tumors.

Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that Rad54B--a SNF2 helicase-like DNA-repair protein--limits the strength of both the G1/S and G2/M checkpoints. We find that Rad54B functions as a scaffold for p53 degradation via its direct interaction with the MDM2-MDMX ubiquitin-ligase complex. During the early phases of the DDR, Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the p53-mediated checkpoint is established, Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.

Thymic stromal lymphopoietin (TSLP) is known to promote T helper type 2 cell-associated inflammation. Mast cells are major effector cells in allergic inflammatory responses. We noted that the population and maturation of mast cells were reduced in TSLP-deficient mice (TSLP-/-). Thus, we hypothesized that TSLP might affect mast cell development. We found that TSLP induced the proliferation and differentiation of mast cells from bone marrow progenitors. TSLP-induced mast cell proliferation was abolished by depletion of mouse double minute 2 (MDM2) and signal transducers and activators of transcription 6 (STAT6), as an upstream activator of MDM2. TSLP-/-, in particular, had a considerable deficit in the expression of MDM2 and STAT6. Also, the TSLP deficiency attenuated mast cell-mediated allergic reactions through the downregulation of STAT6 and MDM2. In an antibody microarray chip analysis, MDM2 expression was increased in atopic dermatitis patients. These observations indicate that TSLP is a factor for mast cell development, and that it aggravates mast cell-mediated immune responses.

Mdm2, an E3 ubiquitin ligase, negatively regulates the tumour suppressor p53. In this study we utilized a conditional Mdm2 allele, Mdm2(FM) , and a CAG-CreER tamoxifen-inducible recombination system to examine the effects of global Mdm2 loss in adult mice. Two different tamoxifen injection regimens caused 100% lethality of Mdm2(FM) (/-) ;CAG-CreER mice; both radio-sensitive and radio-insensitive tissues were impaired. Strikingly, a large number of radio-insensitive tissues, including the kidney, liver, heart, retina and hippocampus, exhibited various pathological defects. Similar tamoxifen injections in older (16-18 month-old) Mdm2(FM) (/-) ;CAG-CreER mice yielded abnormalities only in the kidney. In addition, transcriptional activation of Cdkn1a (p21), Bbc3 (Puma) and multiple senescence markers in young (2-4 month-old) mice following loss of Mdm2 was dampened in older mice. All phenotypes were p53-dependent, as Mdm2(FM) (/-) ;Trp53(-/-) ;CAG-CreER mice subjected to the same tamoxifen regimens were normal. Our findings implicate numerous possible toxicities in many normal tissues upon use of cancer therapies that aim to inhibit Mdm2 in tumours with wild-type p53., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The tumour suppressor p53 is activated to induce cell-cycle arrest or apoptosis in the DNA damage response (DDR). p53 phosphorylation at Ser46 by HIPK2 (homeodomain-interacting protein kinase 2) is a critical event in apoptosis induction. Interestingly, HIPK2 is degraded by Mdm2 (a negative regulator of p53), whereas Mdm2 is downregulated by HIPK2 through several mechanisms. Here, we develop a four-module network model for the p53 pathway to clarify the role of interplay between Mdm2 and HIPK2 in the DDR evoked by ultraviolet radiation. By numerical simulations, we reveal that Mdm2-dependent HIPK2 degradation promotes cell survival after mild DNA damage and that inhibition of HIPK2 degradation is sufficient to trigger apoptosis. In response to severe damage, p53 phosphorylation at Ser46 is promoted by the accumulation of HIPK2 due to downregulation of nuclear Mdm2 in the later phase of the response. Meanwhile, the concentration of p53 switches from moderate to high levels, contributing to apoptosis induction. We show that the presence of three mechanisms for Mdm2 downregulation, i.e. repression of mdm2 expression, inhibition of its nuclear entry and HIPK2-induced degradation, guarantees the apoptosis of irreparably damaged cells. Our results agree well with multiple experimental observations, and testable predictions are also made. This work advances our understanding of the regulation of p53 activity in the DDR and suggests that HIPK2 should be a significant target for cancer therapy., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

The tumor suppressor protein p53 is a multifunctional transcription factor involved in the control of cell survival and death. p53 is inactivated by mutation of the p53 gene in approximately 50% of human cancers. While the rest (including hematological malignancies) encode wild-type p53, p53 is frequently inhibited by its negative regulator human double minute 2 (HDM2). HDM2 is a p53-specific E3 ubiquitin ligase. Therefore, there has been considerable interest in identifying compounds for disrupting the p53-HDM2 interaction. Small-molecule antagonist of HDM2, which binds HDM2 in the p53-binding pocket, negatively controls the activity of HDM2 and prevents p53 degradation. This stabilization of p53 results in its activation, leading to cell cycle arrest, growth inhibition, and apoptosis in wild type p53-haboring hematological malignant cells. Biology of p53-HDM2 interaction and anti-tumor effects of the HDM2 inhibitor in hematological malignant cells are described in this review.

The molecular basis of nephron progenitor cell renewal and differentiation into nascent epithelial nephrons is an area of intense investigation. Defects in these early stages of nephrogenesis lead to renal hypoplasia, and eventually hypertension and chronic kidney disease. Terminal nephron differentiation, the process by which renal epithelial precursor cells exit the cell cycle and acquire physiological functions is equally important. Failure of terminal epithelial cell differentiation results in renal dysplasia and cystogenesis. Thus, a better understanding of the transcriptional frameworks that regulate early and late renal cell differentiation is of great clinical significance. In this review, we will discuss evidence implicating the MDM2-p53 pathway in cell fate determination during development. The emerging central theme from loss- and gain-of-function studies is that tight regulation of p53 levels and transcriptional activity is absolutely required for nephrogenesis. We will also discuss how post-translational modifications of p53 (e.g., acetylation and phosphorylation) alter the spatiotemporal and functional properties of p53 and thus cell fate during kidney development. Mutations and polymorphisms in the MDM2-p53 pathway are present in more than 50 % of cancers in humans. This raises the question of whether sequence variants in the MDM2-p53 pathway increase the susceptibility to renal dysgenesis, hypertension or chronic kidney disease. With the advent of whole exome sequencing and other high throughput technologies, this hypothesis is testable in cohorts of children with renal dysgenesis.

p53 is a tumor suppressor and the p53 dynamics displays stimulus dependent patterns. Recent evidence suggests a bimodal p53 switch in cell fate decision. However, no theoretical studies have been proposed to investigate bimodal p53 induction. Here we constructed a model and showed that MDM2-p53 mRNA binding might contribute to bimodal p53 switch through an intrinsic positive feedback loop. Lower damage favored pulsing while monotonic increasing was generated with higher damage. Bimodal p53 dynamics was largely influenced by cellular MDM2 and elevated p53/MDM2 ratios with increasing etoposide favor mono-ubiquitination. Our model replicated recent experiments and provided potential insights into dynamic mechanisms of bimodal switch., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage-induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM.

The tumor-suppressor p53 provides a critical brake on tumor development. HDM2 (human double-minute 2), a p53 E3 ubiquitin ligase, is the principal cellular antagonist of p53. Mounting evidence has suggested that ribosomal proteins (RPs) modulate HDM2-p53 as a novel pathway for regulating p53 signaling. However, the upstream regulators that mediate RP-HDM2-p53 circuits remain poorly understood. Here we identify human coilin-interacting nuclear ATPase protein (hCINAP) as an interacting partner of ribosomal protein S14 (RPS14). RPS14 stabilized and activated p53 by inhibiting HDM2-mediated p53 polyubiquitination and degradation. More importantly, RPS14 was specifically modified with NEDD8 and hCINAP inhibited RPS14 NEDDylation by recruiting NEDD8-specific protease 1. The decrease in RPS14 NEDDylation led to reduced stability and incorrect localization of RPS14, thereby attenuating the interaction between RPS14 and HDM2. Free HDM2 stimulated p53 polyubiquitination and degradation. In conclusion, we demonstrate that hCINAP acts as a novel regulator of RPS14-HDM2-p53 by regulating the interaction between RPS14 and HDM2 through the control of RPS14 NEDDylation. These findings suggest that hCINAP is an important regulator of RP-HDM2-p53 pathway and a potential anticancer drug target.

The p53 tumor suppressor protein is involved in regulating a wide variety of stress responses, from senescence and apoptosis to more recently discovered roles in allowing adaptation to metabolic and oxidative stress. After 34years of research, significant progress has been made in unraveling the complexity of the p53 network, and it is clear that the regulation of p53 protein stability is critical in the control of p53 activity. This article focuses on our current understanding of how the level and activity of p53 is controlled by this seemingly simple mechanism. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose of Review: Targeted therapy of malignant melanoma recently experienced remarkable advances with gene mutation-based therapies with signaling pathway inhibitors (kinase inhibitors). The treatments prolong patients' survival, but in general resistance is acquired and progression of disease occurs. Therefore, additional therapeutic targets are desperately needed., Recent Findings: The p53 tumor suppressor gene is rarely mutated in melanoma, but its functional attenuation is needed for tumor development. Recently, it was found that the essential p53 inhibitor Mdmx is very frequently overexpressed in melanoma. Mdmx displays both p53-dependent and p53-independent oncogenic effects needed for melanoma growth, Summary: Current melanoma therapy based upon kinase inhibitors shows robust initial clinical effect, but the duration of effect is limited. Inactivation of Mdmx in melanoma inhibits tumor growth also of kinase-inhibitor-resistant tumors. An observed synergistic effect of kinase-inhibition and Mdmx targeting can lead to better and more durable treatment of melanoma patients.

Discovered in 1987 and 1997 respectively, Mdm2 and MdmX represent two critical cellular regulators of the p53 tumor suppressor. This chapter reviews each from initial discovery to our current understanding of their deregulation in human cancer with a focus on how each regulator impacts p53 function. While p53 independent activities of Mdm2 and MdmX are noted the reader is directed to other reviews on this topic. The chapter concludes with an examination of the various mechanisms of Mdm-deregulation and an assessment of the current therapeutic approaches to target Mdm2 and MdmX overexpression.

Mdm2 is best known as the primary negative regulator of p53, but a growing body of evidence suggests that Mdm2 also has a number of functions independent of its role in regulating p53. Although these functions are not yet well-characterized, they have been implicated in regulating of a number of cellular processes, including cell-cycle control, apoptosis, differentiation, genome stability, and transcription, among others. It appears that Mdm2 exerts these functions through a surprisingly wide variety of mechanisms. For example, it has been shown that Mdm2 can ubiquitinate alternative targets, can stimulate the activity of transcription factors, and can directly bind to mRNA to regulate its stability. Dysregulation of p53-independent functions could be responsible for the oncogenic properties of Mdm2 seen even in the absence of p53, and may explain why approximately 10 % of human tumors overexpress Mdm2 instead of inactivating p53 through other mechanisms. As the p53-independent functions of Mdm2 present novel targets for potential therapeutic interventions, fully characterizing these cellular and pathogenic roles of Mdm2 will be important in the study of tumor biology and the treatment of cancer.

The tumor suppressor p53 plays a central role in anti-tumorigenesis and cancer therapy. It has been described as "the guardian of the genome", because it is essential for conserving genomic stability by preventing mutation, and its mutation and inactivation are highly related to all human cancers. Two important p53 regulators, MDM2 and MDMX, inactivate p53 by directly inhibiting its transcriptional activity and mediating its ubiquitination in a feedback fashion, as their genes are also the transcriptional targets of p53. On account of the importance of the p53-MDM2-MDMX loop in the initiation and development of wild type p53-containing tumors, intensive studies over the past decade have been aiming to identify small molecules or peptides that could specifically target individual protein molecules of this pathway for developing better anti-cancer therapeutics. In this chapter, we review the approaches for screening and discovering efficient and selective MDM2 inhibitors with emphasis on the most advanced synthetic small molecules that interfere with the p53-MDM2 interaction and are currently on Phase I clinical trials. Other therapeutically useful strategies targeting this loop, which potentially improve the prospects of cancer therapy and prevention, will also be discussed briefly.

E3 ubiquitin ligases and deubiquitylating enzymes (DUBs) are the key components of ubiquitin proteasome system which plays a critical role in cellular protein homeostasis. Any shortcoming in their biological roles can lead to various diseases including cancer. The dynamic interplay between ubiquitylation and deubiquitylation determines the level and activity of several proteins including p53, which is crucial for cellular stress response and tumor suppression pathways. In this review, we describe the different types of E3 ubiquitin ligases including those targeting tumor suppressor p53, SCF ligases and RING type ligases and accentuate on biological functions of few important E3 ligases in the cellular regulatory networks. Tumor suppressor p53 level is tightly regulated by multiple E3 ligases including Mdm2, COP1, Pirh2, etc. SCF ubiquitin ligase complexes are key regulators of cell cycle and signal transduction. BRCA1 and VHL RING type ligases function as tumor suppressors and play an important role in DNA repair and hypoxia response respectively. Further, we discuss the biological consequences of deregulation of the E3 ligases and the implications for cancer development. We also describe deubiquitylases which reverse the process of ubiquitylation and regulate diverse cellular pathways including metabolism, cell cycle control and chromatin remodelling. As the E3 ubiquitin ligases and DUBs work in a substrate specific manner, an improved understanding of them can lead to better therapeutics for cancer., (Copyright © 2013 UICC.)

The molecular mechanisms that underpin invasive ductal breast cancer (IDC) invasion and metastasis are incompletely understood. The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers, where it stimulates the expression of matrix metalloproteinase 9 (MMP9), an important enzyme in the breakdown of the extracellular matrix. However, its role in breast cancer remains poorly understood. This study assessed the clinical significance of MDM2 expression in IDC and used in vitro expression assays to determine the molecular roles of MDM2. Immunohistochemical staining for MMP9 and MDM2 was performed using archived tumor blocks from 321 women who underwent surgical resection for IDC at the First Affiliated Hospital of Nanjing Medical University, China between January 2002 and December 2003. MCF-7 and MDA-MD-231 cell lines were transfected with siRNA targeted against MDM2, or MDM2 was overexpressed using transiently expressed vectors. The invasion, cell migration and proteolytic capabilities of cells that over- or underexpressed MDM2 was then assessed and compared against control cells, in addition to the consequent effects on MMP9 expression using RT-PCR. In vivo, 54.9% and 49.6% of samples were positive for MMP9 and MDM2 expression, respectively, and their expression was significantly correlated (r² = 0.171, P = 0.012). Moreover, MDM2 expression was markedly correlated with disease-free survival (HR 2.56, 95% CI 1.02-6.40, P = 0.038). In vitro, MDM2 overexpression significantly enhanced cell invasion, migration and proteolysis compared with control cells, and the converse effects were observed after MDM2-siRNA treatment. MDM2 overexpression induced MMP9 expression in a dose-dependent manner. Taken together, these results suggest that high levels of MDM2 are associated with a poorer prognosis in IDC. This might result from increased tumor invasiveness due to enhanced MMP9 expression causing increased extracellular matrix breakdown.

Our recent study showed critical roles of Dmp1 as a sensor of oncogenic Ras, HER2/neu signaling and activation of the Arf-p53 pathway. To elucidate the role of human DMP1 (hDMP1) in breast cancer, one hundred and ten pairs of human breast cancer specimen were studied for the alterations of the hDMP1-ARF-Hdm2-p53 pathway with follow up of clinical outcomes. Loss of heterozygosity (LOH) of the hDMP1 locus was found in 42% of human breast carcinomas, while that of INK4a/ARF and p53 were found in 20 and 34%, respectively. Hdm2 amplification was found in 13% of the same sample, which was found independently of LOH for hDMP1. Conversely, LOH for hDMP1 was found in mutually exclusive fashion with that of INK4a/ARF and p53, and was associated with low Ki67 index and diploid karyotype. Consistently, LOH for hDMP1 was associated with luminal A category and longer relapse-free survival, while that of p53 was associated with non-luminal A and shorter survival. Thus, loss of hDMP1 could define a new disease category associated with prognosis of breast cancer patients. Human breast epithelial cells/cancer cells with wild-type p53 were sensitive to growth inhibition by activated Dmp1:ER while those that delete p14(ARF) or p53, and/or Hdm2 amplification showed partial or nearly complete resistance, indicating that p53 is a critical target for hDMP1 to exhibit its biological activity.