P53-independent Functions Research Articles

A metabolic crosstalk between liposarcoma and muscle sustains tumor growth

Dedifferentiated and Well-differentiated liposarcoma are characterized by a systematic amplification of the Murine Double Minute 2 (MDM2) oncogene. We demonstrate that p53-independent metabolic functions of chromatin-bound MDM2 are exacerbated in liposarcoma and mediate an addiction to serine metabolism to sustain tumor growth. However, the origin of exogenous serine remains unclear. Here, we show that elevated serine levels in mice harboring liposarcoma-patient derived xenograft, released by distant muscle is essential for liposarcoma cell survival. Repressing interleukine-6 expression, or treating liposarcoma cells with Food and Drugs Administration (FDA) approved anti-interleukine-6 monoclonal antibody, decreases de novo serine synthesis in muscle, impairs proliferation, and increases cell death in vitro and in vivo. This work reveals a metabolic crosstalk between muscle and liposarcoma tumor and identifies anti-interleukine-6 as a plausible treatment for liposarcoma patients.

"The 10th International MDM2 Workshop": Opening up new avenues for MDM2 and p53 research, the First International MDM2 Workshop in Asia.

The 10th International MDM2 Workshop was held at the National Cancer Center Research Institute (NCCRI) in Tokyo, Japan, from October 15 to 18, 2023. It attracted 166 participants from 12 countries. The meeting featured 52 talks and 41 poster presentations. In the first special session, six invited speakers gave educational and outstanding talks on breakthroughs in MDM2 research. Three keynote speakers presented emerging p53-independent functions of MDM2/MDM4, functional association of MDM2/p53 with cancer immunity, and drug discovery targeting the MDM2/MDM4-p53 pathway. Additionally, 19 invited speakers introduced their new findings. Twenty-one presenters, many of whom were young investigators, postdocs, and students, were selected from submitted abstracts and reported their exciting and unpublished results. For poster presenters, outstanding poster awards were given to the best presenters. There were many inspiring questions and discussions throughout the meeting. Social events like a welcome party, a workshop dinner, and an optional tour enabled further scientific interactions among the participants. The meeting successfully provided an exciting platform for scientific exchange. The experience gained from organizing this meeting will be handed over to the next organizers of the 11th International MDM2 Workshop.

Abstract 2606: Requirement of MDM2 E3 ligase activity for regulating p53 during normal development, cell cycle regulation and genome integrity

Abstract p53 tumor suppressor, the most mutated gene in human cancer, is negatively regulated by MDM2 and MDM4 proteins. Two distinct mechanisms of MDM2/MDM4 regulation of p53 include, one, suppression of p53 transactivation activity by direct interaction of MDM2/MDM4 with p53 protein, thus blocking its transactivation interface and two, targeting p53 to proteasomal degradation by MDM2 E3 ligase activity. However, the importance of these mechanisms in normal p53 regulation in vivo has remained controversial. To genetically separate these two mechanisms of p53 regulation we generated a novel Mdm2 mutant mouse (Mdm2L466A) that lacks E3 ligase activity but retains the ability of Mdm2 to heterodimerize with Mdm4 and inhibit p53 transactivation. Homozygous Mdm2L466A mice are embryonic lethal due to dysregulated p53 activity, thus demonstrating Mdm2 E3 ligase mediated regulation of p53 is essential during embryonic development. Additionally, we uncovered novel p53-independent functions of MDM2 E3 ligase in cell cycle regulation and genome integrity in cells. Cells lacking MDM2 E3 ligase activity have defective G2-M transition during cell cycle and developed elevated levels of aneuploidy regardless of p53 status. This study unequivocally demonstrates the requirement of Mdm2 E3 ligase activity for normal regulation of p53 and uncovers previously unknown p53-independent role of Mdm2 in cell cycle regulation and maintaining genome integrity. A current therapeutic approach involves blocking MDM2-p53 interaction to promote p53 tumor suppression function in cancer cells. Our findings suggest that this approach alone is insufficient because MDM2 has other potential oncogenic mechanisms independent of its role as negative regulator of p53. Hence, targeting MDM2 E3 ligase activity is likely to be more effective cancer therapy as it addresses both p53-dependent and p53-independent oncogenic mechanisms of MDM2. Citation Format: Meenalakshmi Chinnam, Rati Lama, Chao Xu, Xiaojing Zhang, Carlos Cedeno, Yanqing Wang, Aimee B. Stablewski, David W. Goodrich, Xinjiang Wang. Requirement of MDM2 E3 ligase activity for regulating p53 during normal development, cell cycle regulation and genome integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2606.

MDMX Regulates Transcriptional Activity of p53 and FOXO Proteins to Stimulate Proliferation of Melanoma Cells.

Simple SummaryWe have investigated the transcriptional changes occurring in uveal and cutaneous melanoma cell lines upon depletion of MDMX (aka:MDM4). Computational analyses of the mRNAs/genes affected upon MDMX depletion determined that many were containing a p53-bindingsite, but even more contained a FOX recognition site(s). Since connections between MDM2 and FOXO1 had already been published, we investigated whether indeed a subset of the MDMX-regulated genes are dependent on FOXO1/FOXO3 expression. Indeed, a number of such target genes, i.e., PIK3IP1, MXD4 and ZMAT3, were found to be FOXO target genes in our cell models. Some of these genes were recently identified as indirect p53-target genes, and their expression was found to be regulated by RFX7 transcription factor, which was found activated upon pharmacological activation of p53, e.g., by Nutlin-3. However, a clear involvement of RFX7 in our model could not be established, but an interplay between FOXO and RFX7 factors seems evident.The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity.

It's Getting Complicated-A Fresh Look at p53-MDM2-ARF Triangle in Tumorigenesis and Cancer Therapy.

Anti-tumorigenic mechanisms mediated by the tumor suppressor p53, upon oncogenic stresses, are our bodies’ greatest weapons to battle against cancer onset and development. Consequently, factors that possess significant p53-regulating activities have been subjects of serious interest from the cancer research community. Among them, MDM2 and ARF are considered the most influential p53 regulators due to their abilities to inhibit and activate p53 functions, respectively. MDM2 inhibits p53 by promoting ubiquitination and proteasome-mediated degradation of p53, while ARF activates p53 by physically interacting with MDM2 to block its access to p53. This conventional understanding of p53-MDM2-ARF functional triangle have guided the direction of p53 research, as well as the development of p53-based therapeutic strategies for the last 30 years. Our increasing knowledge of this triangle during this time, especially through identification of p53-independent functions of MDM2 and ARF, have uncovered many under-appreciated molecular mechanisms connecting these three proteins. Through recognizing both antagonizing and synergizing relationships among them, our consideration for harnessing these relationships to develop effective cancer therapies needs an update accordingly. In this review, we will re-visit the conventional wisdom regarding p53-MDM2-ARF tumor-regulating mechanisms, highlight impactful studies contributing to the modern look of their relationships, and summarize ongoing efforts to target this pathway for effective cancer treatments. A refreshed appreciation of p53-MDM2-ARF network can bring innovative approaches to develop new generations of genetically-informed and clinically-effective cancer therapies.

Differential p53-Mediated Cellular Responses to DNA-Damaging Therapeutic Agents.

The gene TP53, which encodes the tumor suppressor protein p53, is mutated in about 50% of cancers. In response to cell stressors like DNA damage and after treatment with DNA-damaging therapeutic agents, p53 acts as a transcription factor to activate subsets of target genes which carry out cell fates such as apoptosis, cell cycle arrest, and DNA repair. Target gene selection by p53 is controlled by a complex regulatory network whose response varies across contexts including treatment type, cell type, and tissue type. The molecular basis of target selection across these contexts is not well understood. Knowledge gained from examining p53 regulatory network profiles across different DNA-damaging agents in different cell types and tissue types may inform logical ways to optimally manipulate the network to encourage p53-mediated tumor suppression and anti-tumor immunity in cancer patients. This may be achieved with combination therapies or with p53-reactivating targeted therapies. Here, we review the basics of the p53 regulatory network in the context of differential responses to DNA-damaging agents; discuss recent efforts to characterize differential p53 responses across treatment types, cell types, and tissue types; and examine the relevance of evaluating these responses in the tumor microenvironment. Finally, we address open questions including the potential relevance of alternative p53 transcriptional functions, p53 transcription-independent functions, and p53-independent functions in the response to DNA-damaging therapeutics.

Targeting MDM2 for Neuroblastoma Therapy: In Vitro and In Vivo Anticancer Activity and Mechanism of Action.

Simple SummaryNeuroblastoma is a malignant tumor of the sympathetic nervous system that causes aggressive disease in children. The overall survival rate of high-risk patients is very low, therefore developing effective and safe therapies for neuroblastoma is an urgent unmet medical need. The mouse double minute 2 (MDM2) homolog gene is amplified and overexpressed in neuroblastoma and contributes to the poor response to treatment and poor prognosis in patients with high-risk neuroblastoma. Therefore, targeting MDM2 provides a promising approach to neuroblastoma therapy, especially for advanced disease. In the present study, we tested a unique MDM2 inhibitor, SP141, for its therapeutic efficacy and safety in neuroblastoma tumor models. We found that SP141 has significant anti- neuroblastoma activity in cell culture and inhibits tumor growth in animal models of human neuroblastoma, without any noticeable host toxicity. These results provide the basis for targeting MDM2 to treat high-risk neuroblastoma.Background: Neuroblastoma is an aggressive pediatric solid tumor with an overall survival rate of <50% for patients with high-risk disease. The majority (>98%) of pathologically-diagnosed neuroblastomas have wild-type p53 with intact functional activity. However, the mouse double minute 2 (MDM2) homolog, an E3 ubiquitin ligase, is overexpressed in neuroblastoma and leads to inhibition of p53. MDM2 also exerts p53-independent oncogenic functions. Thus, MDM2 seems to be an attractive target for the reactivation of p53 and attenuation of oncogenic activity in neuroblastoma. Methods: In this study, we evaluated the anticancer activities and underlying mechanisms of action of SP141, a first-in-class MDM2 inhibitor, in neuroblastoma cell lines with different p53 backgrounds. The findings were confirmed in mouse xenograft models of neuroblastoma. Results: We demonstrate that SP141 reduces neuroblastoma cell viability, induces apoptosis, arrests cells at the G2/M phase, and prevents cell migration, independent of p53. In addition, in neuroblastoma xenograft models, SP141 inhibited MDM2 expression and suppressed tumor growth without any host toxicity at the effective dose. Conclusions: MDM2 inhibition by SP141 results in the inhibition of neuroblastoma growth and metastasis, regardless of the p53 status of the cells and tumors. These findings provide proof-of-concept that SP141 represents a novel treatment option for both p53 wild-type and p53 null neuroblastoma.

Targeting the p53-MDM2 pathway for neuroblastoma therapy: Rays of hope.

Despite being the subject of extensive research and clinical trials, neuroblastoma remains a major therapeutic challenge in pediatric oncology. The p53 protein is a central safeguard that protects cells against genome instability and malignant transformation. Mutated TP53 (the gene encoding p53) is implicated in many human cancers, but the majority of neuroblastomas have wild type p53 with intact transcriptional function. In fact, the TP53 mutation rate does not exceed 1-2% in neuroblastomas. However, overexpression of the murine double minute 2 (MDM2) gene in neuroblastoma is relatively common, and leads to inhibition of p53. It is also associated with other non-canonical p53-independent functions, including drug resistance and increased translation of MYCN and VEGF mRNA. The p53-MDM2 pathway in neuroblastoma is also modulated at several different molecular levels, including via interactions with other proteins (MYCN, p14ARF). In addition, the overexpression of MDM2 in tumors is linked to a poorer prognosis for cancer patients. Thus, restoring p53 function by inhibiting its interaction with MDM2 is a potential therapeutic strategy for neuroblastoma. A number of p53-MDM2 antagonists have been designed and studied for this purpose. This review summarizes the current understanding of p53 biology and the p53-dependent and -independent oncogenic functions of MDM2 in neuroblastoma, and also the regulation of the p53-MDM2 axis in neuroblastoma. This review also highlights the use of MDM2 as a molecular target for the disease, and describes the MDM2 inhibitors currently being investigated in preclinical and clinical studies. We also briefly explain the various strategies that have been used and future directions to take in the development of effective MDM2 inhibitors for neuroblastoma.

Targeting Mouse Double Minute 2: Current Concepts in DNA Damage Repair and Therapeutic Approaches in Cancer.

Defects in DNA damage repair may cause genome instability and cancer development. The tumor suppressor gene p53 regulates cell cycle arrest to allow time for DNA repair. The oncoprotein mouse double minute 2 (MDM2) promotes cell survival, proliferation, invasion, and therapeutic resistance in many types of cancer. The major role of MDM2 is to inhibit p53 activity and promote its degradation. In this review, we describe the influence of MDM2 on genomic instability, the role of MDM2 on releasing p53 and binding DNA repair proteins to inhibit repair, and the regulation network of MDM2 including its transcriptional modifications, protein stability, and localization following DNA damage in genome integrity maintenance and in MDM2-p53 axis control. We also discuss p53-dependent and p53 independent oncogenic function of MDM2 and the outcomes of clinical trials that have been used with clinical inhibitors targeting p53-MDM2 to treat certain cancers.

Replication Stress Response and CDKN1A Engagement Constrain Fetal Hematopoietic Stem Cell Pool Size in Fanconi Anemia

Bone marrow (BM) failure is the principal source of morbidity and mortality in Fanconi Anemia (FA) patients. Recessively inherited germline mutations in one of 25 genes lead to deficits by in a pathway central to DNA crosslink repair. Functionally, FA proteins protect adult hematopoietic stem cells (HSC) from p53 mediated apoptosis elicited by alkylating agents, a range of experimental inflammatory cues or aldehyde exposure. However, these mechanisms do not seem to account for depleted hematopoietic stem and progenitor cell pools in very young FA patients, or the spontaneous, non-apoptotic and p53-independent fetal HSC deficits observed in murine models. Building on our previous observation of a quantitatively constrained fetal HSC pool in FA mice (Fancd2-/-), the current experiments reveal the specific developmental timeframe for the onset of stem cell deficits during HSC expansion in the fetal liver (FL). Cell cycle studies using an EdU/BrdU pulse chase protocol reveal delays in S-phase entry and progression at E13.5. Building on the role of FA proteins (FANCM, FANCI and FANCD2) in countering experimental replication stress (RS) in cell line models, we reasoned that rapid rates of proliferation required during expansion in the FL may similarly confer RS on the FA HSC pool. Experiments in E13.5 FL HSC confirmed the predicted increase in single stranded DNA and accumulation of nuclear replication associated protein (pRpa), along with activation of pChk1, a critical cell cycle checkpoint in cells under RS. For comparison, pChk1 in unperturbed adult cells was no different between Fancd2-/- and WT. The data are also consistent with gains in RAD51 and alkaline comet assays we previously published (Yoon et al., Stem Cell Reports 2016). The cell cycle regulator Cdkn1a (p21) is considered a canonical downstream component of the p53 response in adult FA HSC, but it also performs p53 independent functions in the RS response that coincide with its role in the nucleus. Here, we observed an increase in nuclear localization of p21 in Fancd2-/- FL HSC. TGF-β is a critical developmental morphogen that regulates p21 activity, and TGF-β inhibitors can partially reverse adult FA HSC function along with suppression of NHEJ mediated DNA repair. To test regulation of p21 in fetal HSC under RS, we first treated WT FL HSC with aphidicolin to experimentally simulate RS and found that SD208, a small molecule TGF-β-R1 inhibitor, completely rescued the p21 nuclear localization. We then went on to demonstrate that pharmacological inhibition of TGF-β signaling also reversed the nuclear p21 translocation in FA FL HSC (under physiological RS) and functionally rescued the primitive myeloid progenitor colony formation (CFU-GEMM) in vitro. Altogether, our data show that HSC deficits in FA first emerge in the fetal liver, where rapid fetal expansion causes RS that elicits pChk1 activation and nuclear p21 translocation, which restrain cell cycle progression and act as principal mechanisms limiting fetal HSC pool size in FA. Our experiments suggest a central and p53-independent role for p21 in fetal FA HSC regulation. Detailed knowledge of the physiological role of FA proteins in fetal phenotype HSC has the potential to lead to new therapies that delay or rescue hematopoietic failure in FA patients. Disclosures No relevant conflicts of interest to declare.

EGFR Controls Hair Shaft Differentiation in a p53-Independent Manner

SummaryEpidermal growth factor receptor (EGFR) signaling controls skin development and homeostasis in mice and humans, and its deficiency causes severe skin inflammation, which might affect epidermal stem cell behavior. Here, we describe the inflammation-independent effects of EGFR deficiency during skin morphogenesis and in adult hair follicle stem cells. Expression and alternative splicing analysis of RNA sequencing data from interfollicular epidermis and outer root sheath indicate that EGFR controls genes involved in epidermal differentiation and also in centrosome function, DNA damage, cell cycle, and apoptosis. Genetic experiments employing p53 deletion in EGFR-deficient epidermis reveal that EGFR signaling exhibits p53-dependent functions in proliferative epidermal compartments, as well as p53-independent functions in differentiated hair shaft keratinocytes. Loss of EGFR leads to absence of LEF1 protein specifically in the innermost epithelial hair layers, resulting in disorganization of medulla cells. Thus, our results uncover important spatial and temporal features of cell-autonomous EGFR functions in the epidermis.

Loss of SET reveals both the p53-dependent and the p53-independent functions in vivo

Our previous study showed that the oncoprotein SET acts as a new reader of unacetylated p53 for transcriptional repression. To further elucidate the physiological significance of SET in vivo, we generated set knockout mice. Set knockout mice died during embryonic development between day 11.5 and day 12.5 post coitum, exhibiting cardiac edema and open neural tube, among other developmental defects. Further analyses revealed that loss of SET leads to upregulation of p53 target genes including p21 and puma without any obvious effect on p53 stability in set knockout embryos. Notably, the developmental defects of set knockout mice were significantly, but nonetheless partially, rescued by concomitant deletion of p53. The failure to obtain fully live set/p53 double knockout mice suggested that p53-independent targets of SET also contribute to the embryonic lethality of set knockout mice. Indeed, we found that FOXO1 acts as an important target of SET and that SET-mediated regulation of FOXO1 is also acetylation-dependent. Taken together, these data underscore the importance of SET oncoprotein during embryonic development and reveal both of the p53-dependent and the p53-independent functions of SET in vivo.

Abstract P3-06-03: Copy number analysis identifies ESR1 and MDM4 as drivers of progression in invasive lobular breast carcinoma

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer after invasive ductal carcinoma (IDC). While specific clinical and pathological features differ between ILC and IDC, both histologies are treated the same, due to a lack of knowledge of targetable pathways underlying the observed differences. To identify potential genetic drivers of ILC progression, we set out to identify genes with copy number (CN) alterations, comparing tumors with good outcome to those with poor outcome. Method: We designed probes for a total of 67 genes known to be frequently altered in breast cancer and used sensitive nanoString technology to comprehensively investigate CN alterations of these genes in 70 well-curated primary ILCs. ILC cell lines MDA-MB-134-VI, SUM44PE, and BCK4 were used for functional studies including proliferation, apoptosis, colony formation, and analysis of gene expression. Results: Our studies reveal that ESR1 is frequently amplified in primary ILC (14% gains and 10% amplification), and that tumors with amplified ESR1 are more likely to recur compared to those with normal CN. Our analysis also identified a subset of ILCs with HER2 amplification (19%) despite a negative clinical IHC score, and these tumors expressed high HER2 mRNA, protein, and demonstrated enrichment of a molecular HER2 signature. The other most frequently amplified genes included CCND1 (33%), MDM4 (17%), and MYC (17%), and most frequently lost genes were NCOR2 (7%), FGFR4 (6%) and TP53 (6%). MDM4, a negative regulator of p53, has previously been reported to play a role in breast cancer, though little is known about its role in ILC. We demonstrate that decreasing MDM4 levels in p53 wild type ILC cell lines results in increased apoptosis, decreased proliferation associated with cell cycle arrest, and activation of p53 target genes. Intriguingly, a similar induction of G0/G1 cell cycle arrest and increase in apoptosis was observed in p53 mutant ILC cells after MDM4 downregulation, suggesting a p53-independent function of MDM4. Conclusion: Sensitive detection of CN changes identified amplifications of ESR1 and MDM4 as potential drivers of ILC. Functional studies demonstrate that MDM4 has both p53 dependent and independent functions that warrant further study. Citation Format: Atkinson JM, Cao L, Basudan A, Sikora MJ, Bahreini A, Tasdemir N, Jankowitz RC, McAuliffe PF, Dabbs D, Haupt S, Haupt Y, Peter Lucas PC, Lee AV, Oesterreich S. Copy number analysis identifies ESR1 and MDM4 as drivers of progression in invasive lobular breast carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-03.

Inhibition of Noncanonical Murine Double Minute 2 Homolog Abrogates Ocular Inflammation through NF-κB Suppression

Uveitis is estimated to account for 10% of all cases of blindness in the United States, including 30,000 new cases of legal blindness each year. Intraocular and oral corticosteroids are the effective mainstay treatment, but they carry the risk of serious long-term ocular and systemic morbidity. New noncorticosteroid therapies with a favorable side effect profile are necessary for the treatment of chronic uveitis, given the paucity of existing treatment choices. We have previously demonstrated thatNutlin-3, a small-molecule inhibitor of murine double minute 2 (MDM2) homolog, suppresses pathologic retinal angiogenesis through a p53-dependent mechanism, but the noncanonical p53-independent functions have not been adequately elucidated. Herein, we demonstrate an unanticipated function of MDM2 inhibition, where Nutlin-3 potently abrogates lipopolysaccharide-induced ocular inflammation. Furthermore, we identified a mechanism by which transcription and translation of NF-κB is mediated byMDM2, independent of p53, in ocular inflammation. Small-molecule MDM2 inhibition is a novel noncorticosteroid strategy for inhibiting ocular inflammation, which may potentially benefit patients with chronic uveitis.

MDMX acidic domain inhibits p53 DNA binding in vivo and regulates tumorigenesis

The MDM2 homolog MDMX oncoprotein is indispensable for inhibition of p53 during normal embryonic development and malignant transformation, yet how MDMX harnesses p53 functions is unclear. In addition to a canonical N-terminal p53-binding domain, recent work suggests the central acidic domain of MDMX regulates p53 interaction through intramolecular mimicry and engages in second-site interaction with the p53 core domain in vitro. To test the physiological relevance of these interactions, we generated an MDMX knockin mouse having substitutions in a conserved WW motif necessary for these functions (W201S/W202G). Notably, MDMXSG cells have normal p53 level but increased p53 DNA binding and target gene expression, and rapidly senesce. In vivo, MDMXSG inhibits early-phase disease in Eµ-Myc transgenic mice but accelerates the onset of lethal lymphoma and shortens overall survival. Therefore, MDMX is an important regulator of p53 DNA binding, which complements the role of MDM2 in regulating p53 level. Furthermore, the results suggest that the WW motif has dual functions that regulate p53 and inhibit Myc-driven lymphomas independent of p53.

Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

The interplay between influenza A viruses (IAV) and the p53 pathway has been reported in several studies, highlighting the antiviral contribution of p53. Here, we investigated the impact of IAV on the E3-ubiquitin ligase Mdm2, a major regulator of p53, and observed that IAV targets Mdm2, notably via its non-structural protein (NS1), therefore altering Mdm2 stability, p53/Mdm2 interaction and regulatory loop during the time-course of infection. This study also highlights a new antiviral facet of Mdm2 possibly increasing the list of its many p53-independent functions. Altogether, our work contributes to better understand the mechanisms underlining the complex interactions between IAV and the p53 pathway, for which both NS1 and Mdm2 arise as key players.

Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53

Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) invitro and invivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

The p53 inhibitor Mdm4 cooperates with multiple genetic lesions in tumourigenesis.

The p53 inhibitor Mdm4 is present at high levels in multiple human cancers. Overexpression of Mdm4 in mice drives the spontaneous development of mostly lymphomas and sarcomas. In this study, we explored the ability of Mdm4 to cooperate with lesions in tumour development. The Mdm4 transgene contributed to mammary tumour development in a BALB/cJ background. High levels of Mdm4 enhanced tumour development in a mutant p53R172H heterozygous background, and reduced the need to lose the wild-type p53 allele, as compared with mice heterozygous only for the p53R172H mutation. Additionally, high levels of Mdm4 cooperated with an oncogenic K-ras mutation to drive lung tumourigenesis in vivo. Finally, we examined p53-independent functions of Mdm4 by studying the contribution of Mdm4 to tumour development in the absence of p53. Whereas the overall survival times of p53-null mice with and without the Mdm4 transgene were similar, male mice with both alterations showed significantly shorter survival than p53-null male mice, and showed differences in tumour spectrum, demonstrating a p53-independent function of Mdm4 in tumourigenesis. Furthermore, p53-null mice with the highest level of Mdm4 tended to have multiple tumours. Thus, a detailed analysis of Mdm4 transgenic mice in various genetic backgrounds shows synergy in tumour development in vivo. Mdm4 may thus serve as a therapeutic target in cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mdm2 as a chromatin modifier.

Mdm2 is the key negative regulator of the tumour suppressor p53, making it an attractive target for anti-cancer drug design. We recently identified a new role of Mdm2 in gene repression through its direct interaction with several proteins of the polycomb group (PcG) family. PcG proteins form polycomb repressive complexes PRC1 and PRC2. PRC2 (via EZH2) mediates histone 3 lysine 27 (H3K27) trimethylation, and PRC1 (via RING1B) mediates histone 2A lysine 119 (H2AK119) monoubiquitination. Both PRCs mostly support a compact and transcriptionally silent chromatin structure. We found that Mdm2 regulates a gene expression profile similar to that of PRC2 independent of p53. Moreover, Mdm2 promotes the stemness of murine induced pluripotent stem cells and human mesenchymal stem cells, and supports the survival of tumour cells. Mdm2 is recruited to target gene promoters by the PRC2 member and histone methyltransferase EZH2, and enhances PRC-dependent repressive chromatin modifications, specifically H3K27me3 and H2AK119ub1. Mdm2 also cooperates in gene repression with the PRC1 protein RING1B, a H2AK119 ubiquitin ligase. Here we discuss the possible implications of these p53-independent functions of Mdm2 in chromatin dynamics and in the stem cell phenotype. We propose that the p53-independent functions of Mdm2 should be taken into account for cancer drug design. So far, the majority of clinically tested Mdm2 inhibitors target its binding to p53 but do not affect the new functions of Mdm2 described here. However, when targeting the E3 ligase activity of Mdm2, a broader spectrum of its oncogenic activities might become druggable.

MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation.

Retinoblastomas can arise from cone photoreceptor precursors in response to the loss of pRB function. Cone precursor-specific circuitry cooperates with pRB loss to initiate this process and subsequently contributes to the malignancy. Intrinsic high level MDM2 expression is a key component of the cone precursor circuitry and is thought to inactivate p53-mediated tumor surveillance that could otherwise be induced in response to pRB loss. However, the MDM2-related MDM4 has also been proposed to abrogate p53-mediated tumor surveillance in the absence of detectable MDM2 in retinoblastoma cells, bringing into question the importance of high-level MDM2 versus MDM4 expression. Here we report that high-level MDM2 but not MDM4 has a consistent critical role in retinoblastoma cell proliferation in vitro as well as in orthotopic xenografts. Reduction of either MDM2 or MDM4 weakly induced p53, yet reduction of MDM2 but not MDM4 severely impaired proliferation and survival through a p53-independent mechanism. Specifically, MDM2 up-regulated the mRNA expression and translation of another component of the cone circuitry, MYCN, in retinoblastoma cells. Moreover, MYCN was essential to retinoblastoma cell growth and tumor formation, and ectopic MYCN partially reversed the effects of MDM2 depletion, indicating that MYCN is an important MDM2 target. These findings indicate that high-level MDM2 expression is needed in order to perform a critical p53-independent function and may obviate the need for genomic alterations to the p53 pathway during retinoblastoma tumorigenesis.