Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.

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. Trp53R178E mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53-/- mice. Surprisingly, stabilization of p53R178E in Mdm2-/- mice nevertheless triggers extensive apoptosis, indicative of residual wild-type activities. Although this apoptotic activity suffices to trigger lethality of Trp53R178E ;Mdm2-/- embryos, it proves insufficient for suppression of spontaneous and oncogene-driven tumorigenesis. Trp53R178E mice develop tumors indistinguishably from Trp53-/- mice and tumors retain and even stabilize the p53R178E protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53R178E 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.

Micro(RNA) managing by mTORC1.

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.

An mTORC1-Mdm2-Drosha axis for miRNA biogenesis in response to glucose- and amino acid-deprivation.

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.

E3 ubiquitin ligase MDM2 acts through p53 to control respiratory progenitor cell number and lung size.

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.

Novel roles of ER stress in repressing neural activity and seizures through Mdm2- and p53-dependent protein translation.

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.

Studying Homo-oligomerization and Hetero-oligomerization of MDMX and MDM2 Proteins in Single Living Cells by Using In Situ Fluorescence Correlation Spectroscopy.

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.

Mdm2-p53-SF1 pathway in ovarian granulosa cells directs ovulation and fertilization by conditioning oocyte quality.

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.

Inactivation of the MDM2 RING domain enhances p53 transcriptional activity in mice.

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, MDM2C462A, disrupting MDM2 E3 function and the MDMX interaction, die during early embryogenesis that can be rescued by p53 deletion. To investigate whether MDM2C462A, which retains p53 binding, has p53-suppressing activity, we generated Mdm2C462A/C462A ;p53ER/- mice, in which we replaced the endogenous p53 alleles with an inducible p53ER/- allele, and compared survival with that of similarly generated Mdm2-/-;p53ER/- 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, Mdm2C462A/C462A ;p53ER/- mice died ∼5 days after tamoxifen injection, suggesting that p53 activity is higher in the presence of MDM2C462A than in the absence of MDM2. Indeed, in MDM2C462A-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 MDM2C462A 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 MDM2C462A than in the absence of MDM2. These results reveal an unexpected role of MDM2C462A in enhancing p53 activity and suggest the possibility that compounds targeting MDM2 RING domain function could produce even more robust p53 activation.

p53 inhibition by MDM2 in human 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.

The liver-specific microRNA-122*, the complementary strand of microRNA-122, acts as a tumor suppressor by modulating the p53/mouse double minute 2 homolog circuitry.

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).

Caveolin-1 is involved in DNA damage and repair signaling in X-irradiated Chang liver cells.

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.

Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1.

The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehyde-binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1(D88A)) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1(C91S)), but not Otub1(D88A), bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1(D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.

Mdm2 regulates p53 mRNA translation through inhibitory interactions with ribosomal protein L26.

Mdm2 regulates the p53 tumor suppressor by promoting its proteasome-mediated degradation. Mdm2 and p53 engage in an autoregulatory feedback loop that maintains low p53 activity in nonstressed cells. We now report that Mdm2 regulates p53 levels also by targeting ribosomal protein L26. L26 binds p53 mRNA and augments its translation. Mdm2 binds L26 and drives its polyubiquitylation and proteasomal degradation. In addition, the binding of Mdm2 to L26 attenuates the association of L26 with p53 mRNA and represses L26-mediated augmentation of p53 protein synthesis. Under nonstressed conditions, both mechanisms help maintain low cellular p53 levels by constitutively tuning down p53 translation. In response to genotoxic stress, the inhibitory effect of Mdm2 on L26 is attenuated, enabling a rapid increase in p53 synthesis. The Mdm2-L26 interaction thus represents an additional important component of the autoregulatory feedback loop that dictates cellular p53 levels and activity.

Pathway Based Toxicology and Fit-for-Purpose Assays.

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.

The role of ubiquitin modification in the regulation of p53.

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.

Tissue-specific and age-dependent effects of global Mdm2 loss.

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.

p53-independent effects of Mdm2.

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.

Mdm2 and MdmX involvement in human cancer.

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.

Targeting p53-MDM2-MDMX loop for cancer therapy.

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.

Reactivation of p53 as therapeutic intervention for malignant melanoma.

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.