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Xiong S, Zhang Y, Zhou X, Pant V, Mirani A, Gencel-Augusto J, Chau G, You MJ, Lozano G. Dependence on Mdm2 for Mdm4 inhibition of p53 activity. Cancer Lett 2025; 621:217622. [PMID: 40081463 DOI: 10.1016/j.canlet.2025.217622] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2025] [Revised: 03/06/2025] [Accepted: 03/09/2025] [Indexed: 03/16/2025]
Abstract
Both Mdm2 and Mdm4 inhibit p53 activity by masking of its transcriptional activation domain. In addition, Mdm2 functions as an E3 ubiquitin ligase, targeting p53 for degradation. The amino terminus of Mdm4 binds wild type and mutant p53 while its RING domain, which lacks E3 ligase activity, is required for heterodimerization with Mdm2. To determine how these domains of Mdm4 regulate p53, we generated mouse models with either a deletion of the Mdm4 RING domain (Mdm4ΔR) or all of Mdm4 (Mdm4─) on a hypomorphic (p53neo) background. Mdm4ΔR mice exhibited elevated p53 levels and activity, albeit to a lesser extent than mice with complete Mdm4 loss, indicating that the amino terminus of Mdm4 contributes to p53 inhibition. Moreover, in the absence of Mdm2, neither the deletion of the Mdm4 RING domain nor the complete loss of Mdm4 further increased p53 protein levels on a mutant p53 background, indicating that Mdm4 modulates Mdm2 in its regulation of p53 stability. Collectively, our findings suggest that Mdm4 contributes to p53 inhibition by modulating Mdm2 activity via both its amino terminus and RING domains.
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Affiliation(s)
- Shunbin Xiong
- Department of Genetics, UT M.D. Anderson Cancer Center, Houston, TX, USA
| | - Yun Zhang
- Department of Pharmaceutical Sciences, Joan M. Lafleur College of Pharmacy and Health Sciences, Texas Southern University, Houston, TX, 77004, USA
| | - Xin Zhou
- Department of Pediatrics, UT M.D. Anderson Cancer Center, Houston, TX, USA
| | - Vinod Pant
- Department of Genetics, UT M.D. Anderson Cancer Center, Houston, TX, USA
| | - Akshita Mirani
- Department of Genetics, UT M.D. Anderson Cancer Center, Houston, TX, USA
| | | | - Gilda Chau
- Department of Genetics, UT M.D. Anderson Cancer Center, Houston, TX, USA
| | - M James You
- Department of Hematopathology, UT M.D. Anderson Cancer Center, Houston, TX, USA
| | - Guillermina Lozano
- Department of Genetics, UT M.D. Anderson Cancer Center, Houston, TX, USA.
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Lama R, Fose JM, Martín D, Muñoz IG, Wang ES, Sung PJ, Chemler SR, Wang X. Novel Inhibitors for MDM2-MDM4 E3 Ligase Potently Induce p53-Indepedent Apoptosis in Drug-Resistant Leukemic Cells. Molecules 2025; 30:186. [PMID: 39795242 PMCID: PMC11722259 DOI: 10.3390/molecules30010186] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2024] [Revised: 12/31/2024] [Accepted: 01/04/2025] [Indexed: 01/13/2025] Open
Abstract
MDM2 and MDM4 are major negative regulators of tumor suppressor p53. Beyond regulating p53, MDM2 possesses p53-independent activity in promoting cell cycle progression and tumorigenesis via its RING domain ubiquitin E3 ligase activity. MDM2 and MDM4 form heterodimer polyubiquitin E3 ligases via their RING domain interaction. Inhibitors disrupting p53 interaction with MDM2/MDM4 are in clinical trials in patients bearing wild-type p53 cancers. However, these inhibitors are not designed to work for p53-null/mutant cancer cells. Owing to the importance of the E3 ligase of MDM2 in its p53-independent oncogenic activity, inhibitors targeting the E3 ligase activity of MDM2-MDM4 are desirable for p53-mutant cancer cells. Here, we report the development of such inhibitors with pro-apoptotic activity in p53-null leukemic cells. Among analogues of MDM2-MDM4 E3 ligase inhibitors, we initially identified MMRi36 as a potent pro-apoptotic compound in p53-null leukemic cells with acquired drug resistance. MMRi36 acts as an activator of MDM2-MDM4 E3 ligase by stabilizing MDM2-MDM4 heterodimers and promotes MDM2/MDM4 degradation in cells. Interestingly, replacement of the sulfur in 1,3,4-thiadiazole MMRi36 with a carbon led to identification of pyrazole MMRi36C that dissociates the MDM2-MDM4 RING heterodimers, inhibits the E3 ligase activity of the complex, and induces p53 protein accumulation, but retains the p53-independent pro-apoptotic activity. A brief SAR study identified a fluorine derivative of MMRi36C with improved pro-apoptotic activity. This study discovered a novel class of compound that targets MDM2-MDM4 ubiquitin E3 ligase activity for apoptosis induction in p53-mutant cancer cells.
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Affiliation(s)
- Rati Lama
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA; (R.L.); (P.J.S.)
| | - Joseph M. Fose
- Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260, USA; (J.M.F.); (S.R.C.)
| | - Diana Martín
- Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), 28009 Madrid, Spain; (D.M.); (I.G.M.)
| | - Inés G. Muñoz
- Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), 28009 Madrid, Spain; (D.M.); (I.G.M.)
| | - Eunice S. Wang
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA;
| | - Pamela J. Sung
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA; (R.L.); (P.J.S.)
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA;
| | - Sherry R. Chemler
- Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260, USA; (J.M.F.); (S.R.C.)
| | - Xinjiang Wang
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA; (R.L.); (P.J.S.)
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Lama R, Wu W, Mavis CK, Ruiz FM, Querol-García J, Martin D, Chemler SR, Chandra D, Goodrich DW, Hernandez-Ilizaliturri FJ, Muñoz IG, Wang X. Small-molecule MMRi36 induces apoptosis in p53-mutant lymphomas by targeting MDM2/MDM4/XIAP for degradation. Front Oncol 2024; 14:1462231. [PMID: 39763603 PMCID: PMC11700832 DOI: 10.3389/fonc.2024.1462231] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2024] [Accepted: 12/06/2024] [Indexed: 03/05/2025] Open
Abstract
Rituximab combined with systemic chemotherapy significantly improves the rate of complete response in B-cell lymphomas. However, acquired rituximab resistance develops in most patients leading to relapse. The mechanisms underlying rituximab resistance are not well-understood. MDM2 and MDM4 proteins are major negative regulators of p53, but they also have p53-independent activities in mouse models of lymphomagenesis. Whether MDM2 or MDM4 is involved in rituximab resistance has not been explored. Here we report that MDM2 and MDM4 are upregulated in p53-mutant rituximab-resistant cells by transcriptional and post-transcriptional mechanisms. Knockdown of MDM2 or MDM4 significantly hindered growth of rituximab-resistant cells. To explore whether targeting the RING-domain of MDM2-MDM4 heterodimers is a viable strategy for the treatment of rituximab-resistant lymphomas, we identified MMRi36 in a high throughput small-molecule screen. Here we show that MMRi36 binds and stabilizes MDM2-MDM4 RING heterodimers and acts as an activator of the MDM2-MDM4 E3 ligase complex in vitro and promotes proteasomal degradation of MDM2/MDM4 proteins in cells. MMRi36 potently induces p53-independent apoptosis in p53-mutant lymphoma cells and it exerts non-apoptotic anti-lymphoma effect in rituximab resistant cells. The pro-apoptotic mechanisms of MMRi36 involves activation of both caspase 3 and caspase 7 associated with increased polyubiquitination and degradation of XIAP. Therefore, MMRi36 is a novel prototype small-molecule for targeting MDM2/MDM4/XIAP for degradation and induction of apoptosis in p53-mutant lymphomas.
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Affiliation(s)
- Rati Lama
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States
| | - Wenjie Wu
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States
| | - Cory K. Mavis
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States
| | - Federico M. Ruiz
- Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Javier Querol-García
- Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Diana Martin
- Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Sherry R. Chemler
- Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY, United States
| | - Dhyan Chandra
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States
| | - David W. Goodrich
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States
| | | | - Inés G. Muñoz
- Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Xinjiang Wang
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States
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Merlino F, Pecoraro A, Longobardi G, Donati G, Di Leva FS, Brignola C, Piccarducci R, Daniele S, Martini C, Marinelli L, Russo G, Quaglia F, Conte C, Russo A, La Pietra V. Development and Nanoparticle-Mediated Delivery of Novel MDM2/MDM4 Heterodimer Peptide Inhibitors to Enhance 5-Fluorouracil Nucleolar Stress in Colorectal Cancer Cells. J Med Chem 2024; 67:1812-1824. [PMID: 38285632 DOI: 10.1021/acs.jmedchem.3c01312] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2024]
Abstract
Colorectal cancer (CRC) often involves wild-type p53 inactivation by MDM2 and MDM4 overexpression, promoting tumor progression and resistance to 5-fluoruracil (5-FU). Disrupting the MDM2/4 heterodimer can proficiently reactivate p53, sensitizing cancer cells to 5-FU. Herein, we developed 16 peptides based on Pep3 (1), the only known peptide acting through this mechanism. The new peptides, notably 3 and 9, showed lower IC50 values than 1. When incorporated into tumor-targeted biodegradable nanoparticles, these exhibited cytotoxicity against three different CRC cell lines. Notably, NPs/9 caused a significant increase in p53 levels associated with a strong increment of its main downstream target p21 inducing apoptosis. Also, the combined treatment of 9 with 5-FU caused the activation of nucleolar stress and a synergic apoptotic effect. Hence, the co-delivery of MDM2/4 heterodimer disruptors with 5-FU through nanoparticles might be a promising strategy to overcome drug resistance in CRC.
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Affiliation(s)
- Francesco Merlino
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Annalisa Pecoraro
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Giuseppe Longobardi
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Greta Donati
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | | | - Chiara Brignola
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Rebecca Piccarducci
- Department of Pharmacy, University of Pisa, via Bonanno, 6, 56126 Pisa, PI, Italy
| | - Simona Daniele
- Department of Pharmacy, University of Pisa, via Bonanno, 6, 56126 Pisa, PI, Italy
| | - Claudia Martini
- Department of Pharmacy, University of Pisa, via Bonanno, 6, 56126 Pisa, PI, Italy
| | - Luciana Marinelli
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Giulia Russo
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Fabiana Quaglia
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Claudia Conte
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Annapina Russo
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
| | - Valeria La Pietra
- Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli, NA, Italy
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Byroju VV, Nadukkandy AS, Cordani M, Kumar LD. Retinoblastoma: present scenario and future challenges. Cell Commun Signal 2023; 21:226. [PMID: 37667345 PMCID: PMC10478474 DOI: 10.1186/s12964-023-01223-z] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Accepted: 07/12/2023] [Indexed: 09/06/2023] Open
Abstract
With an average incidence of 1 in every 18,000 live births, retinoblastoma is a rare type of intraocular tumour found to affect patients during their early childhood. It is curable if diagnosed at earlier stages but can become life-threateningly malignant if not treated timely. With no racial or gender predisposition, or even environmental factors known to have been involved in the incidence of the disease, retinoblastoma is often considered a clinical success story in pediatric oncology. The survival rate in highly developed countries is higher than 95% and they have achieved this because of the advancement in the development of diagnostics and treatment techniques. This includes developing the already existing techniques like chemotherapy and embarking on new strategies like enucleation, thermotherapy, cryotherapy, etc. Early diagnosis, studies on the etiopathogenesis and genetics of the disease are the need of the hour for improving the survival rates. According to the Knudson hypothesis, also known as the two hit hypothesis, two hits on the retinoblastoma susceptibility (RB) gene is often considered as the initiating event in the development of the disease. Studies on the molecular basis of the disease have also led to deciphering the downstream events and thus in the discovery of biomarkers and related targeted therapies. Furthermore, improvements in molecular biology techniques enhanced the development of efficient methods for early diagnosis, genetic counseling, and prevention of the disease. In this review, we discuss the genetic and molecular features of retinoblastoma with a special emphasis on the mutation leading to the dysregulation of key signaling pathways involved in cell proliferation, DNA repair, and cellular plasticity. Also, we describe the classification, clinical and epidemiological relevance of the disease, with an emphasis on both the traditional and innovative treatments to tackle retinoblastoma. Video Abstract.
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Affiliation(s)
- Vishnu Vardhan Byroju
- Department of Biochemistry, American International Medical University, Gros Islet, St. Lucia, USA
| | | | - Marco Cordani
- Department of Biochemistry and Molecular Biology, Complutense University of Madrid, and Instituto de Investigaciones Sanitarias San Carlos (IdISSC), Madrid, Spain.
| | - Lekha Dinesh Kumar
- CSIR-Centre for Cellular and Molecular Biology, Habsiguda, Uppal Road, Hyderabad, India.
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Wang P, Liu X, Chen Y, Jun-Hao ET, Yao Z, Min-Wen JC, Yan-Jiang BC, Ma S, Ma W, Luo L, Guo L, Song D, Shyh-Chang N. Adult progenitor rejuvenation with embryonic factors. Cell Prolif 2023; 56:e13459. [PMID: 37177849 DOI: 10.1111/cpr.13459] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2022] [Revised: 03/10/2023] [Accepted: 03/14/2023] [Indexed: 05/15/2023] Open
Abstract
During ageing, adult stem cells' regenerative properties decline, as they undergo replicative senescence and lose both their proliferative and differentiation capacities. In contrast, embryonic and foetal progenitors typically possess heightened proliferative capacities and manifest a more robust regenerative response upon injury and transplantation, despite undergoing many rounds of mitosis. How embryonic and foetal progenitors delay senescence and maintain their proliferative and differentiation capacities after numerous rounds of mitosis, remains unknown. It is also unclear if defined embryonic factors can rejuvenate adult progenitors to confer extended proliferative and differentiation capacities, without reprogramming their lineage-specific fates or inducing oncogenic transformation. Here, we report that a minimal combination of LIN28A, TERT, and sh-p53 (LTS), all of which are tightly regulated and play important roles during embryonic development, can delay senescence in adult muscle progenitors. LTS muscle progenitors showed an extended proliferative capacity, maintained a normal karyotype, underwent myogenesis normally, and did not manifest tumorigenesis nor aberrations in lineage differentiation, even in late passages. LTS treatment promoted self-renewal and rescued the pro-senescence phenotype of aged cachexia patients' muscle progenitors, and promoted their engraftment for skeletal muscle regeneration in vivo. When we examined the mechanistic basis for LIN28A's role in the LTS minimum combo, let-7 microRNA suppression could not fully explain how LIN28A promoted muscle progenitor self-renewal. Instead, LIN28A was promoting the translation of oxidative phosphorylation mRNAs in adult muscle progenitors to optimize mitochondrial reactive oxygen species (mtROS) and mitohormetic signalling. Optimized mtROS induced a variety of mitohormetic stress responses, including the hypoxic response for metabolic damage, the unfolded protein response for protein damage, and the p53 response for DNA damage. Perturbation of mtROS levels specifically abrogated the LIN28A-driven hypoxic response in Hypoxia Inducible Factor-1α (HIF1α) and glycolysis, and thus LTS progenitor self-renewal, without affecting normal or TS progenitors. Our findings connect embryonically regulated factors to mitohormesis and progenitor rejuvenation, with implications for ageing-related muscle degeneration.
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Affiliation(s)
- Peng Wang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Xupeng Liu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Yu Chen
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Elwin Tan Jun-Hao
- NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore
- Institute of Molecular and Cell Biology, Genome Institute of Singapore, Agency for Science Technology and Research, Singapore, Singapore
- Laboratory of Cancer Therapeutics, Program in Cancer and Stem Cell Biology, Duke-National University of Singapore Medical School, Singapore, Singapore
- Laboratory of Cancer Epigenome, Division of Medical Science, National Cancer Centre Singapore, Singapore, Singapore
| | - Ziyue Yao
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Jason Chua Min-Wen
- NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore
- Institute of Molecular and Cell Biology, Genome Institute of Singapore, Agency for Science Technology and Research, Singapore, Singapore
- Laboratory of Cancer Therapeutics, Program in Cancer and Stem Cell Biology, Duke-National University of Singapore Medical School, Singapore, Singapore
- Laboratory of Cancer Epigenome, Division of Medical Science, National Cancer Centre Singapore, Singapore, Singapore
| | - Benjamin Chua Yan-Jiang
- NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore
- Institute of Molecular and Cell Biology, Genome Institute of Singapore, Agency for Science Technology and Research, Singapore, Singapore
- Laboratory of Cancer Therapeutics, Program in Cancer and Stem Cell Biology, Duke-National University of Singapore Medical School, Singapore, Singapore
- Laboratory of Cancer Epigenome, Division of Medical Science, National Cancer Centre Singapore, Singapore, Singapore
| | - Shilin Ma
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Wenwu Ma
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Lanfang Luo
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Luyao Guo
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Dan Song
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Ng Shyh-Chang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
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7
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Pant V, Sun C, Lozano G. Tissue specificity and spatio-temporal dynamics of the p53 transcriptional program. Cell Death Differ 2023; 30:897-905. [PMID: 36755072 PMCID: PMC10070629 DOI: 10.1038/s41418-023-01123-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Revised: 10/30/2022] [Accepted: 11/15/2022] [Indexed: 02/10/2023] Open
Abstract
Transcription factors regulate hundreds of genes and p53 is no exception. As a stress responsive protein, p53 transactivates an array of downstream targets which define its role in maintaining physiological functions of cells/tissues. Despite decades of studies, our understanding of the p53 in vivo transcriptional program is still incomplete. Here we discuss some of the physiological stressors that activate p53, the pathological and physiological implications of p53 activation and the molecular profiling of the p53 transcriptional program in maintaining tissue homeostasis. We argue that the p53 transcriptional program is spatiotemporally regulated in a tissue-specific manner and define a p53 target signature that faithfully depicts p53 activity. We further emphasize that additional in vivo studies are needed to refine the p53 transactivation profile to harness it for therapeutic purposes.
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Affiliation(s)
- Vinod Pant
- Department of Genetics, 1515 Holcombe Blvd, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - Chang Sun
- Department of Genetics, 1515 Holcombe Blvd, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - Guillermina Lozano
- Department of Genetics, 1515 Holcombe Blvd, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.
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8
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Szwarc MM, Guarnieri AL, Joshi M, Duc HN, Laird MC, Pandey A, Khanal S, Dohm E, Bui AK, Sullivan KD, Galbraith MD, Andrysik Z, Espinosa JM. FAM193A is a positive regulator of p53 activity. Cell Rep 2023; 42:112230. [PMID: 36897777 PMCID: PMC10164416 DOI: 10.1016/j.celrep.2023.112230] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Revised: 12/21/2022] [Accepted: 02/21/2023] [Indexed: 03/11/2023] Open
Abstract
Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.
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Affiliation(s)
- Maria M Szwarc
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Anna L Guarnieri
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Molishree Joshi
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Functional Genomics Facility, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Huy N Duc
- Functional Genomics Facility, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Madison C Laird
- Functional Genomics Facility, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Ahwan Pandey
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia
| | - Santosh Khanal
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Emily Dohm
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Aimee K Bui
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Kelly D Sullivan
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Pediatrics, Section of Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Matthew D Galbraith
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Zdenek Andrysik
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
| | - Joaquin M Espinosa
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Functional Genomics Facility, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
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9
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Geiger-Schuller K, Eraslan B, Kuksenko O, Dey KK, Jagadeesh KA, Thakore PI, Karayel O, Yung AR, Rajagopalan A, Meireles AM, Yang KD, Amir-Zilberstein L, Delorey T, Phillips D, Raychowdhury R, Moussion C, Price AL, Hacohen N, Doench JG, Uhler C, Rozenblatt-Rosen O, Regev A. Systematically characterizing the roles of E3-ligase family members in inflammatory responses with massively parallel Perturb-seq. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.01.23.525198. [PMID: 36747789 PMCID: PMC9900845 DOI: 10.1101/2023.01.23.525198] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comβVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.
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10
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MDMX elevation by a novel Mdmx-p53 interaction inhibitor mitigates neuronal damage after ischemic stroke. Sci Rep 2022; 12:21110. [PMID: 36473920 PMCID: PMC9726886 DOI: 10.1038/s41598-022-25427-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Accepted: 11/29/2022] [Indexed: 12/12/2022] Open
Abstract
Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein-Protein Interaction (PPI) inhibitors, K-181, on Mdmx-p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.
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11
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Nagpal I, Yuan ZM. p53-mediated metabolic response to low doses of ionizing radiation. Int J Radiat Biol 2022; 99:934-940. [DOI: 10.1080/09553002.2022.2142983] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Isha Nagpal
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Zhi-Min Yuan
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA, USA
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12
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Lama R, Galster SL, Xu C, Davison LW, Chemler SR, Wang X. Dual Targeting of MDM4 and FTH1 by MMRi71 for Induced Protein Degradation and p53-Independent Apoptosis in Leukemia Cells. MOLECULES (BASEL, SWITZERLAND) 2022; 27:molecules27227665. [PMID: 36431769 PMCID: PMC9695299 DOI: 10.3390/molecules27227665] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/05/2022] [Revised: 10/31/2022] [Accepted: 11/03/2022] [Indexed: 11/09/2022]
Abstract
MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored. Here, we describe the synthesis and characterization of quinolinol derivatives for the identification of analogs that are capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, the targeted degradation of MDM4 and FTH1 in cells, and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity that conventional MDM2-p53 disrupting inhibitors lack. This study provides a prototype structure for developing MDM4/FTH1 dual-targeting inhibitors as potential cancer therapeutics.
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Affiliation(s)
- Rati Lama
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA
| | - Samuel L. Galster
- Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260, USA
| | - Chao Xu
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA
| | - Luke W. Davison
- Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260, USA
| | - Sherry R. Chemler
- Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260, USA
- Correspondence: (S.R.C.); (X.W.)
| | - Xinjiang Wang
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA
- Correspondence: (S.R.C.); (X.W.)
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13
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Chinnam M, Xu C, Lama R, Zhang X, Cedeno CD, Wang Y, Stablewski AB, Goodrich DW, Wang X. MDM2 E3 ligase activity is essential for p53 regulation and cell cycle integrity. PLoS Genet 2022; 18:e1010171. [PMID: 35588102 PMCID: PMC9119546 DOI: 10.1371/journal.pgen.1010171] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Accepted: 03/27/2022] [Indexed: 12/12/2022] Open
Abstract
MDM2 and MDM4 are key regulators of p53 and function as oncogenes when aberrantly expressed. MDM2 and MDM4 partner to suppress p53 transcriptional transactivation and polyubiquitinate p53 for degradation. The importance of MDM2 E3-ligase-mediated p53 regulation remains controversial. To resolve this, we generated mice with an Mdm2 L466A mutation that specifically compromises E2 interaction, abolishing MDM2 E3 ligase activity while preserving its ability to bind MDM4 and suppress p53 transactivation. Mdm2L466A/L466A mice exhibit p53-dependent embryonic lethality, demonstrating MDM2 E3 ligase activity is essential for p53 regulation in vivo. Unexpectedly, cells expressing Mdm2L466A manifest cell cycle G2-M transition defects and increased aneuploidy even in the absence of p53, suggesting MDM2 E3 ligase plays a p53-independent role in cell cycle regulation and genome integrity. Furthermore, cells bearing the E3-dead MDM2 mutant show aberrant cell cycle regulation in response to DNA damage. This study uncovers an uncharacterized role for MDM2’s E3 ligase activity in cell cycle beyond its essential role in regulating p53’s stability in vivo. The most frequently mutated protein in human cancer, the p53 tumor suppressor protein, is negatively regulated by the potentially oncogenic proteins MDM2 and MDM4. MDM2/MDM4 regulates p53 through two mechanisms, MDM2 E3 ubiquitin ligase activity marks p53 for degradation while MDM2/MDM4 can bind p53 to inhibit its ability to promote RNA transcription. Whether these mechanisms contribute to normal p53 regulation in vivo remains controversial. Using a newly developed mouse model that genetically separates these two mechanisms, we find that mice expressing MDM2 deficient specifically for E3 ubiquitin ligase activity do not survive embryonic development because unregulated p53 is lethal. In contrast to prior reports, MDM2 E3 ubiquitin ligase activity is thus required for p53 regulation during embryonic development. In addition, cells lacking MDM2 E3 ubiquitin ligase activity have cell cycle defects regardless of p53 status, uncovering a p53-independent function for MDM2 in regulating the cell cycle. Activating p53 by blocking physical interaction with MDM2/MDM4 is one currently pursued approach for cancer therapy, but this approach does not account for cancer-promoting activities of MDM2/MDM4 independent of p53. Findings reported here suggest targeting MDM2 E3 ligase activity directly may be advantageous as it would inhibit both p53-dependent and p53-independent oncogenic mechanisms.
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Affiliation(s)
- Meenalakshmi Chinnam
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
| | - Chao Xu
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
| | - Rati Lama
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
| | - Xiaojing Zhang
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
| | - Carlos D. Cedeno
- Flow and Image Cytometry Shared Resource, Buffalo, New York, United States of America
| | - Yanqing Wang
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
| | - Aimee B. Stablewski
- Department of Molecular & Cellular Biology, Buffalo, New York, United States of America
- Gene Targeting and Transgenic Shared Resource, Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States of America
| | - David W. Goodrich
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
- * E-mail: (XW); (DWG)
| | - Xinjiang Wang
- Department of Pharmacology and Therapeutics, Buffalo, New York, United States of America
- * E-mail: (XW); (DWG)
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14
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Pant V, Aryal NK, Xiong S, Chau GP, Fowlkes NW, Lozano G. Alterations of the MDM2 C-terminus differentially impact its function in vivo. Cancer Res 2022; 82:1313-1320. [PMID: 35078816 PMCID: PMC8983537 DOI: 10.1158/0008-5472.can-21-2381] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Revised: 12/07/2021] [Accepted: 01/20/2022] [Indexed: 01/10/2023]
Abstract
Murine double minute 2 (Mdm2) is the principal E3-ubiquitin ligase for p53 and contains a C2H2C4 type RING domain wherein the last cysteine residue is followed by an evolutionarily conserved 13 amino acid C-terminal tail. Previous studies have indicated that integrity of the C-terminal tail is critical for Mdm2 function. Recently, a mutation extending the MDM2 length by five amino acids was identified and associated with enhanced p53 response in fibroblasts and premature aging in a human patient. To investigate the importance of the conserved Mdm2 C-terminal length on p53 regulatory function in vivo, we engineered three novel mouse alleles using CRISPR-Cas9 technology. Genetic studies with these murine models showed that curtailing Mdm2 C-terminal length by even a single amino acid leads to p53-dependent embryonic lethality. Extension of the Mdm2 C-terminal length by five amino acids (QLTCL) yielded viable mice that are smaller in size, exhibit fertility problems, and have a shortened life span. Analysis of early passage mouse embryonic fibroblasts indicated impaired Mdm2 function correlates with enhanced p53 activity under stress conditions. Furthermore, analysis in mice showed tissue-specific alterations in p53 target gene expression and enhanced radiosensitivity. These results confirm the physiological importance of the evolutionarily conserved Mdm2 C-terminus in regulating p53 functions. SIGNIFICANCE This in vivo study highlights that alterations to the C-terminus of Mdm2 perturb its regulation of the tumor suppressor p53.
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Affiliation(s)
- Vinod Pant
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Neeraj K. Aryal
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
- Current address: Oncology R&D, AstraZeneca, 35 Gatehouse Park, Waltham, MA 02451
| | - Shunbin Xiong
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Gilda P Chau
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Natalie W. Fowlkes
- Department of Veterinary Medicine & Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Guillermina Lozano
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
- Corresponding author: Guillermina Lozano, PhD, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, Tel. 713 834 6386,
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15
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The roles of mouse double minute 2 (MDM2) oncoprotein in ocular diseases: A review. Exp Eye Res 2022; 217:108910. [PMID: 34998788 DOI: 10.1016/j.exer.2021.108910] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Revised: 12/03/2021] [Accepted: 12/21/2021] [Indexed: 12/19/2022]
Abstract
Mouse double minute 2 (MDM2), an E3 ubiquitin ligase and the primary negative regulator of the tumor suppressor p53, cooperates with its structural homolog MDM4/MDMX to control intracellular p53 level. In turn, overexpression of p53 upregulates and forms an autoregulatory feedback loop with MDM2. The MDM2-p53 axis plays a pivotal role in modulating cell cycle control and apoptosis. MDM2 itself is regulated by the PI3K-AKT and RB-E2F-ARF pathways. While amplification of the MDM2 gene or overexpression of MDM2 (due to MDM2 SNP T309G, for instance) is associated with various malignancies, numerous studies have shown that MDM2/p53 alterations may also play a part in the pathogenetic process of certain ocular disorders (Fig. 1). These include cancers (retinoblastoma, uveal melanoma), fibrocellular proliferative diseases (proliferative vitreoretinopathy, pterygium), neovascular diseases, degenerative diseases (cataract, primary open-angle glaucoma, age-related macular degeneration) and infectious/inflammatory diseases (trachoma, uveitis). In addition, MDM2 is implicated in retinogenesis and regeneration after optic nerve injury. Anti-MDM2 therapy has shown potential as a novel approach to treating these diseases. Despite major safety concerns, there are high expectations for the clinical value of reformative MDM2 inhibitors. This review summarizes important findings about the role of MDM2 in ocular pathologies and provides an overview of recent advances in treating these diseases with anti-MDM2 therapies.
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16
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Wu J, Lu G, Wang X. MDM4 alternative splicing and implication in MDM4 targeted cancer therapies. Am J Cancer Res 2021; 11:5864-5880. [PMID: 35018230 PMCID: PMC8727814] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Accepted: 10/31/2021] [Indexed: 06/14/2023] Open
Abstract
The oncogenic MDM4, initially named MDMX, has been identified as a p53-interacting protein and a key upstream negative regulator of the tumor suppressor p53. Accumulating evidence indicates that MDM4 plays critical roles in the initiation and progression of multiple human cancers. MDM4 is frequently amplified and upregulated in human cancers, contributing to overgrowth and apoptosis inhibition by blocking the expression of downstream target genes of p53 pathway. Disruptors for MDM4-p53 interaction have been shown to restore the anti-tumor activity of p53 in cancer cells. MDM4 possesses multiple splicing isoforms whose expressions are driven by the presence of oncogenes in cancer cells. Some of the MDM4 splicing isoforms lack p53 binding domain and may exhibit p53-independent oncogenic functions. These features render MDM4 to be an attractive therapeutic target for cancer therapy. In the present review, we primarily focus on the detailed molecular structure of MDM4 splicing isoforms, candidate regulators for initiating MDM4 splicing, deregulation of MDM4 isoforms in cancer and potential therapy strategies by targeting splicing isoforms of MDM4.
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Affiliation(s)
- Jin Wu
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer CenterBuffalo, NY, USA
| | - Guanting Lu
- Department of Pathology, Key Laboratory of Tumor Molecular Research, People’s Hospital of Deyang City173 Tai Shan North Road, Deyang 618000, Sichuan, P. R. China
| | - Xinjiang Wang
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer CenterBuffalo, NY, USA
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17
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Sanford JD, Yang J, Han J, Tollini LA, Jin A, Zhang Y. MDMX is essential for the regulation of p53 protein levels in the absence of a functional MDM2 C-terminal tail. BMC Mol Cell Biol 2021; 22:46. [PMID: 34551723 PMCID: PMC8459461 DOI: 10.1186/s12860-021-00385-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Accepted: 08/20/2021] [Indexed: 01/21/2023] Open
Abstract
BACKGROUND MDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation. RESULTS Here, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression. CONCLUSIONS These results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.
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Affiliation(s)
- Jack D Sanford
- Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
| | - Jing Yang
- Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Jiangsu Province Key Laboratory of Immunity and Metabolism and Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221002, Jiangsu, China
| | - Jing Han
- Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Jiangsu Province Key Laboratory of Immunity and Metabolism and Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221002, Jiangsu, China
| | - Laura A Tollini
- Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
| | - Aiwen Jin
- Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA
| | - Yanping Zhang
- Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA.
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA.
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA.
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA.
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18
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Márquez-Cantudo L, Ramos A, Coderch C, de Pascual-Teresa B. Proteasomal Degradation of Zn-Dependent Hdacs: The E3-Ligases Implicated and the Designed Protacs That Enable Degradation. Molecules 2021; 26:molecules26185606. [PMID: 34577077 PMCID: PMC8467390 DOI: 10.3390/molecules26185606] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 09/10/2021] [Accepted: 09/13/2021] [Indexed: 12/21/2022] Open
Abstract
Protein degradation by the Ubiquitin-Proteasome System is one of the main mechanisms of the regulation of cellular proteostasis, and the E3 ligases are the key effectors for the protein recognition and degradation. Many E3 ligases have key roles in cell cycle regulation, acting as checkpoints and checkpoint regulators. One of the many important proteins involved in the regulation of the cell cycle are the members of the Histone Deacetylase (HDAC) family. The importance of zinc dependent HDACs in the regulation of chromatin packing and, therefore, gene expression, has made them targets for the design and synthesis of HDAC inhibitors. However, achieving potency and selectivity has proven to be a challenge due to the homology between the zinc dependent HDACs. PROteolysis TArgeting Chimaera (PROTAC) design has been demonstrated to be a useful strategy to inhibit and selectively degrade protein targets. In this review, we attempt to summarize the E3 ligases that naturally ubiquitinate HDACs, analyze their structure, and list the known ligands that can bind to these E3 ligases and be used for PROTAC design, as well as the already described HDAC-targeted PROTACs.
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19
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Manfredi JJ. Mdm2 and MdmX: Partners in p53 Destruction. Cancer Res 2021; 81:1633-1634. [PMID: 34003788 DOI: 10.1158/0008-5472.can-21-0145] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Accepted: 01/14/2021] [Indexed: 11/16/2022]
Abstract
Mdm2 and MdmX are two closely related proteins that have been well-characterized as negative regulators of the tumor suppressor p53. Their interplay and especially respective roles in ubiquitination and subsequent degradation of p53 have lacked clarity. Yang and colleagues now demonstrate an obligate role for MdmX in recruitment of the E2 ubiquitin ligase UbcH5c to the Mdm2-MdmX hetero-oligomer. The use of elegant genetically engineered mouse models ensures the biological relevance of their findings that have important implications for targeted therapies involving these key players in the p53 pathway.See related article by Yang et al., Cancer Res 2021;81:898-909.
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Affiliation(s)
- James J Manfredi
- Department of Oncological Sciences and Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.
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20
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Zhang S, Lou J, Li Y, Zhou F, Yan Z, Lyu X, Zhao Y. Recent Progress and Clinical Development of Inhibitors that Block MDM4/p53 Protein-Protein Interactions. J Med Chem 2021; 64:10621-10640. [PMID: 34286973 DOI: 10.1021/acs.jmedchem.1c00940] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
MDM4 is a homologue of MDM2, serving cooperatively as the negative regulator of tumor suppressor p53. Under the shadow of MDM2 inhibitors, limited efforts had been put into the discovery of MDM4 modulators. Recent studies of the experimental drug ALRN-6924, a dual MDM4 and MDM2 inhibitor, suggest that concurrent inhibition of MDM4 and MDM2 might be beneficial over only MDM2 inhibition. In view of the present research progress, we summarized published inhibitors of MDM4/p53 interactions including both peptide-based compounds and small molecules. Cocrystal structures of ligand/MDM4 complexes have been examined, and their structural features were compiled and compared in order to show the molecular basis required for high MDM4 binding affinities. Representative examples of small-molecule MDM4 inhibitors were discussed, followed by clinical results of ALRN-6924, together, providing a consolidated reference for further development of MDM4 inhibitors, either dual or selective.
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Affiliation(s)
- Shiyan Zhang
- University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Jianfeng Lou
- University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Yafang Li
- Nano Science and Technology Institute, University of Science and Technology of China, Suzhou, Jiangsu 215123, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Feilong Zhou
- State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Ziqin Yan
- State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Xilin Lyu
- State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Yujun Zhao
- University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China.,School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
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21
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Luwang JW, Nair AR, Natesh R. Stability of p53 oligomers: Tetramerization of p53 impinges on its stability. Biochimie 2021; 189:99-107. [PMID: 34197865 DOI: 10.1016/j.biochi.2021.06.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 06/21/2021] [Accepted: 06/24/2021] [Indexed: 10/21/2022]
Abstract
The p53 protein has been known to exist structurally in three different forms inside the cells. Earlier studies have reported the predominance of the lower oligomeric forms of p53 over its tetrameric form inside the cells, although only the tetrameric p53 contributes to its transcriptional activity. However, it remains unclear the functional relevance of the existence of other p53 oligomers inside the cells. In this study, we characterize the stability and conformational state of tetrameric, dimeric and monomeric p53 that spans both DNA Binding Domain (DBD) and Tetramerization Domain (TD) of human p53 (94-360 amino acid residues). Intriguingly, our studies reveal an unexpected drastic reduction in tetrameric p53 thermal stability in comparison to its dimeric and monomeric form with a higher propensity to aggregate at physiological temperature. Our EMSA study suggests that tetrameric p53, not their lower oligomeric counterpart, exhibit rapid loss of binding to their consensus DNA elements at the physiological temperature. This detrimental effect of destabilization is imparted due to the tetramerization of p53 that drives the DBDs to misfold at a faster pace when compared to its lower oligomeric form. This crosstalk between DBDs is achieved when it exists as a tetramer but not as dimer or monomer. Our findings throw light on the plausible reason for the predominant existence of p53 in dimer and monomer forms inside the cells with a lesser population of tetramer form. Therefore, the transient disruption of tetramerization between TDs could be a potential cue for the stabilization of p53 inside the cells.
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Affiliation(s)
- Johnson Wahengbam Luwang
- School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, Thiruvananthapuram, 695551, Kerala, India
| | - Aadithye R Nair
- School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, Thiruvananthapuram, 695551, Kerala, India
| | - Ramanathan Natesh
- School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, Thiruvananthapuram, 695551, Kerala, India.
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22
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Timmerman DM, Remmers TL, Hillenius S, Looijenga LHJ. Mechanisms of TP53 Pathway Inactivation in Embryonic and Somatic Cells-Relevance for Understanding (Germ Cell) Tumorigenesis. Int J Mol Sci 2021; 22:ijms22105377. [PMID: 34065345 PMCID: PMC8161298 DOI: 10.3390/ijms22105377] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 05/14/2021] [Accepted: 05/15/2021] [Indexed: 01/10/2023] Open
Abstract
The P53 pathway is the most important cellular pathway to maintain genomic and cellular integrity, both in embryonic and non-embryonic cells. Stress signals induce its activation, initiating autophagy or cell cycle arrest to enable DNA repair. The persistence of these signals causes either senescence or apoptosis. Over 50% of all solid tumors harbor mutations in TP53 that inactivate the pathway. The remaining cancers are suggested to harbor mutations in genes that regulate the P53 pathway such as its inhibitors Mouse Double Minute 2 and 4 (MDM2 and MDM4, respectively). Many reviews have already been dedicated to P53, MDM2, and MDM4, while this review additionally focuses on the other factors that can deregulate P53 signaling. We discuss that P14ARF (ARF) functions as a negative regulator of MDM2, explaining the frequent loss of ARF detected in cancers. The long non-coding RNA Antisense Non-coding RNA in the INK4 Locus (ANRIL) is encoded on the same locus as ARF, inhibiting ARF expression, thus contributing to the process of tumorigenesis. Mutations in tripartite motif (TRIM) proteins deregulate P53 signaling through their ubiquitin ligase activity. Several microRNAs (miRNAs) inactivate the P53 pathway through inhibition of translation. CCCTC-binding factor (CTCF) maintains an open chromatin structure at the TP53 locus, explaining its inactivation of CTCF during tumorigenesis. P21, a downstream effector of P53, has been found to be deregulated in different tumor types. This review provides a comprehensive overview of these factors that are known to deregulate the P53 pathway in both somatic and embryonic cells, as well as their malignant counterparts (i.e., somatic and germ cell tumors). It provides insights into which aspects still need to be unraveled to grasp their contribution to tumorigenesis, putatively leading to novel targets for effective cancer therapies.
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23
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Yang J, Jin A, Han J, Chen X, Zheng J, Zhang Y. MDMX Recruits UbcH5c to Facilitate MDM2 E3 Ligase Activity and Subsequent p53 Degradation In Vivo. Cancer Res 2021; 81:898-909. [PMID: 33277368 PMCID: PMC8026549 DOI: 10.1158/0008-5472.can-20-0790] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Revised: 10/21/2020] [Accepted: 12/01/2020] [Indexed: 11/16/2022]
Abstract
MDM2 regulates p53 degradation by functioning as an E3 ubiquitin ligase. The role of MDMX, an MDM2 homolog that lacks E3 ligase activity, in the regulation of p53 degradation remains incompletely understood and sometime controversial. This confusion is due at least in part to studies of p53 degradation mainly carried out in in vitro settings, as elimination of either MDM2 or MDMX from mice results in p53-dependent embryonic lethality, thus obfuscating in vivo studies of the individual roles of MDM2 and MDMX in p53 degradation. To overcome this problem, we generated mice expressing an inducible p53 allele under various MDM2 and MDMX deletion and mutation statuses and studied in vivo p53 degradation. Degradation of p53 in vivo was largely prevented in mice and mouse embryonic fibroblast retaining MDM2 but lacking MDMX. Although MDM2 and MDMX interacted with p53 in the absence of each other, they bound p53 more efficiently as a heterodimer. MDMX, but not MDM2, interacted with ubiquitin-conjugating enzyme UbcH5c, an interaction that was essential for MDMX to enable MDM2 E3 ligase activity for p53 degradation. Grafting the C-terminal residues of MDMX to the C-terminus of MDM2 allowed MDM2 to interact with UbcH5c and enhanced MDM2-mediated p53 degradation in the absence of MDMX. Together, these data indicate that MDMX plays an essential role for p53 degradation in vivo by recruiting UbcH5c to facilitate MDM2 E3 ligase function. SIGNIFICANCE: This study provides the first in vivo evidence of MDMX facilitating MDM2-mediated p53 degradation, clarifying its role in the regulation of this critical tumor suppressor.
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Affiliation(s)
- Jing Yang
- Department of Radiation Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
- Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Jiangsu Province Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China
| | - Aiwen Jin
- Department of Radiation Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Jing Han
- Department of Radiation Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Jiangsu Province Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China
| | - Xin Chen
- Department of Radiation Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Jiangsu Province Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China
| | - Junnian Zheng
- Center of Clinical Oncology, Affiliated Hospital of Xuzhou Medical University, Xuzhou Medical University, Xuzhou, Jiangsu, China.
| | - Yanping Zhang
- Department of Radiation Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
- Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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24
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Humpton TJ, Nomura K, Weber J, Magnussen HM, Hock AK, Nixon C, Dhayade S, Stevenson D, Huang DT, Strathdee D, Blyth K, Vousden KH. Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice. Genes Dev 2021; 35:117-132. [PMID: 33334825 PMCID: PMC7778261 DOI: 10.1101/gad.341875.120] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Accepted: 11/06/2020] [Indexed: 12/17/2022]
Abstract
The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.
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Affiliation(s)
- Timothy J Humpton
- The Francis Crick Institute, London NW1 1AT, United Kingdom
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Koji Nomura
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Julia Weber
- The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Helge M Magnussen
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Andreas K Hock
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Colin Nixon
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Sandeep Dhayade
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - David Stevenson
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Danny T Huang
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
- Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, United Kingdom
| | - Douglas Strathdee
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
| | - Karen Blyth
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, United Kingdom
- Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, United Kingdom
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25
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Gnanasundram SV, Malbert-Colas L, Chen S, Fusée L, Daskalogianni C, Muller P, Salomao N, Fåhraeus R. MDM2's dual mRNA binding domains co-ordinate its oncogenic and tumour suppressor activities. Nucleic Acids Res 2020; 48:6775-6787. [PMID: 32453417 PMCID: PMC7337897 DOI: 10.1093/nar/gkaa431] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 05/05/2020] [Accepted: 05/11/2020] [Indexed: 12/16/2022] Open
Abstract
Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.
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Affiliation(s)
| | - Laurence Malbert-Colas
- Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St. Louis, F-75010 Paris, France
| | - Sa Chen
- Department of Medical Biosciences, Building 6M, Umeå University, 901 85 Umeå, Sweden
| | - Leila Fusée
- Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St. Louis, F-75010 Paris, France
| | - Chrysoula Daskalogianni
- Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St. Louis, F-75010 Paris, France
| | - Petr Muller
- RECAMO, Masaryk Memorial Cancer Institute, Zlutykopec 7, 65653 Brno, Czech Republic
| | - Norman Salomao
- Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St. Louis, F-75010 Paris, France
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26
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Wang B, Rasmussen-Ivey C, Little JB, Yuan ZM. The MDM2/MDMX/p53 axis in the adaptive stress response. Transl Cancer Res 2020; 9:1993-1997. [PMID: 35117546 PMCID: PMC8797865 DOI: 10.21037/tcr.2019.12.89] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2019] [Accepted: 12/20/2019] [Indexed: 11/06/2022]
Abstract
Via regulation of cellular stress responses, p53 contributes to the maintenance of homeostasis. Contrary to its well-established pro-death function, p53 is also implicated in promoting cell survival by mediating the adaptive stress response. Emerging data reveal that the adaptive stress response is coupled with p53 decline that is a prerequisite for the induction of pro-survival pathways augmenting cell fitness. However, if the adaptive stress responses persist or become chronic, the sustained p53 downregulation would result in a permanent loss of p53 function and p53-dependent homeostasis. The available information suggests a model in which cells respond to different levels of stress by governing the activity and abundance of p53 that, in turn, determines the cell fate dependent on not only the intensity but also the duration of stress.
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Affiliation(s)
- Bing Wang
- Key Laboratory of Space Radiobiology of Gansu Province & Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- John B. Little Center for Radiation Sciences, Harvard T. H. Chan School of Public Health, Boston, MA, USA
| | - Cody Rasmussen-Ivey
- John B. Little Center for Radiation Sciences, Harvard T. H. Chan School of Public Health, Boston, MA, USA
| | - John B. Little
- John B. Little Center for Radiation Sciences, Harvard T. H. Chan School of Public Health, Boston, MA, USA
| | - Zhi-Min Yuan
- John B. Little Center for Radiation Sciences, Harvard T. H. Chan School of Public Health, Boston, MA, USA
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27
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Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells. Int J Mol Sci 2020; 21:ijms21041309. [PMID: 32075226 PMCID: PMC7072982 DOI: 10.3390/ijms21041309] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Revised: 02/07/2020] [Accepted: 02/09/2020] [Indexed: 12/22/2022] Open
Abstract
Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway.
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28
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Bang S, Kaur S, Kurokawa M. Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway. Int J Mol Sci 2019; 21:E261. [PMID: 31905981 PMCID: PMC6981958 DOI: 10.3390/ijms21010261] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Accepted: 12/24/2019] [Indexed: 12/25/2022] Open
Abstract
The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.
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Affiliation(s)
| | | | - Manabu Kurokawa
- Department of Biological Sciences, Kent State University, Kent, OH 44242, USA; (S.B.); (S.K.)
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29
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Guven-Maiorov E, Tsai CJ, Nussinov R. Oncoviruses Can Drive Cancer by Rewiring Signaling Pathways Through Interface Mimicry. Front Oncol 2019; 9:1236. [PMID: 31803618 PMCID: PMC6872517 DOI: 10.3389/fonc.2019.01236] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Accepted: 10/28/2019] [Indexed: 01/17/2023] Open
Abstract
Oncoviruses rewire host pathways to subvert host immunity and promote their survival and proliferation. However, exactly how is challenging to understand. Here, by employing the first and to date only interface-based host-microbe interaction (HMI) prediction method, we explore a pivotal strategy oncoviruses use to drive cancer: mimicking binding surfaces-interfaces-of human proteins. We show that oncoviruses can target key human network proteins and transform cells by acquisition of cancer hallmarks. Experimental large-scale mapping of HMIs is difficult and individual HMIs do not permit in-depth grasp of tumorigenic virulence mechanisms. Our computational approach is tractable and 3D structural HMI models can help elucidate pathogenesis mechanisms and facilitate drug design. We observe that many host proteins are unique targets for certain oncoviruses, whereas others are common to several, suggesting similar infectious strategies. A rough estimation of our false discovery rate based on the tissue expression of oncovirus-targeted human proteins is 25%.
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Affiliation(s)
- Emine Guven-Maiorov
- Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, United States
| | - Chung-Jung Tsai
- Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, United States
| | - Ruth Nussinov
- Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, United States
- Department of Human Genetics and Molecular Medicine, Sackler Institute of Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
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30
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Polireddy K, Singh K, Pruski M, Jones NC, Manisundaram NV, Ponnela P, Ouellette M, Van Buren G, Younes M, Bynon JS, Dar WA, Bailey JM. Mutant p53R175H promotes cancer initiation in the pancreas by stabilizing HSP70. Cancer Lett 2019; 453:122-130. [DOI: 10.1016/j.canlet.2019.03.047] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Revised: 03/25/2019] [Accepted: 03/25/2019] [Indexed: 12/25/2022]
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31
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Haupt S, Mejía-Hernández JO, Vijayakumaran R, Keam SP, Haupt Y. The long and the short of it: the MDM4 tail so far. J Mol Cell Biol 2019; 11:231-244. [PMID: 30689920 PMCID: PMC6478121 DOI: 10.1093/jmcb/mjz007] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2018] [Revised: 12/16/2018] [Accepted: 01/21/2019] [Indexed: 12/27/2022] Open
Abstract
The mouse double minute 4 (MDM4) is emerging from the shadow of its more famous relative MDM2 and is starting to steal the limelight, largely due to its therapeutic possibilities. MDM4 is a vital regulator of the tumor suppressor p53. It restricts p53 transcriptional activity and also, at least in development, facilitates MDM2's E3 ligase activity toward p53. These functions of MDM4 are critical for normal cell function and a proper response to stress. Their importance for proper cell maintenance and proliferation identifies them as a risk for deregulation associated with the uncontrolled growth of cancer. MDM4 tails are vital for its function, where its N-terminus transactivation domain engages p53 and its C-terminus RING domain binds to MDM2. In this review, we highlight recently identified cellular functions of MDM4 and survey emerging therapies directed to correcting its dysregulation in disease.
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Affiliation(s)
- Sue Haupt
- Tumor Suppression Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria, Australia
- Department of Clinical Pathology, The University of Melbourne, Parkville, Victoria, Australia
| | | | - Reshma Vijayakumaran
- Tumor Suppression Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria, Australia
| | - Simon P Keam
- Tumor Suppression Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria, Australia
| | - Ygal Haupt
- Tumor Suppression Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria, Australia
- Department of Clinical Pathology, The University of Melbourne, Parkville, Victoria, Australia
- Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Victoria, Australia
- Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia
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32
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Howard TP, Arnoff TE, Song MR, Giacomelli AO, Wang X, Hong AL, Dharia NV, Wang S, Vazquez F, Pham MT, Morgan AM, Wachter F, Bird GH, Kugener G, Oberlick EM, Rees MG, Tiv HL, Hwang JH, Walsh KH, Cook A, Krill-Burger JM, Tsherniak A, Gokhale PC, Park PJ, Stegmaier K, Walensky LD, Hahn WC, Roberts CWM. MDM2 and MDM4 Are Therapeutic Vulnerabilities in Malignant Rhabdoid Tumors. Cancer Res 2019; 79:2404-2414. [PMID: 30755442 DOI: 10.1158/0008-5472.can-18-3066] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2018] [Revised: 12/28/2018] [Accepted: 02/07/2019] [Indexed: 12/21/2022]
Abstract
Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.
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Affiliation(s)
- Thomas P Howard
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts.,Harvard Medical School, Boston, Massachusetts
| | - Taylor E Arnoff
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Melinda R Song
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Andrew O Giacomelli
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Xiaofeng Wang
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts
| | - Andrew L Hong
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Neekesh V Dharia
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Su Wang
- Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts
| | | | - Minh-Tam Pham
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Ann M Morgan
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Harvard Medical School, Boston, Massachusetts.,Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Franziska Wachter
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Gregory H Bird
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | | | - Elaine M Oberlick
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Matthew G Rees
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Hong L Tiv
- Experimental Therapeutics Core and Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Justin H Hwang
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Katherine H Walsh
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - April Cook
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | | | - Aviad Tsherniak
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Prafulla C Gokhale
- Experimental Therapeutics Core and Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Peter J Park
- Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts
| | - Kimberly Stegmaier
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts.,Harvard Medical School, Boston, Massachusetts
| | - Loren D Walensky
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.,Harvard Medical School, Boston, Massachusetts.,Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - William C Hahn
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. .,Broad Institute of Harvard and MIT, Cambridge, Massachusetts.,Harvard Medical School, Boston, Massachusetts
| | - Charles W M Roberts
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts. .,Broad Institute of Harvard and MIT, Cambridge, Massachusetts.,Harvard Medical School, Boston, Massachusetts.,Department of Oncology, Comprehensive Cancer Center, St. Jude Children's Research Hospital, Memphis, Tennessee
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33
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He P, Sun X, Cheng HJ, Zou YB, Wang Q, Zhou CL, Liu WQ, Hao YM, Meng XW. UBA2 promotes proliferation of colorectal cancer. Mol Med Rep 2018; 18:5552-5562. [PMID: 30387828 PMCID: PMC6236309 DOI: 10.3892/mmr.2018.9613] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Accepted: 08/13/2018] [Indexed: 12/02/2022] Open
Abstract
Small ubiquitin-like modifier proteins are involved in tumorigenesis; however, the potential effects and functions of the family member ubiquitin-like modifier-activating enzyme 2 (UBA2) on colorectal cancer are not clear. The present study aimed to examine the effects of UBA2 on the proliferation of colorectal cancer cells in vitro and in vivo. The mRNA and protein expression levels of UBA2 in patients with colorectal cancer were measured by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. UBA2 expression levels in colorectal cancer tissues were significantly increased compared with the paracancerous normal tissues. The expression of UBA2 was also associated with higher stage colorectal cancer and poor prognosis. MTT and colony formation assays were used to examine proliferation in colorectal cancer cell lines. Flow cytometry was performed to examine the effects of UBA2 on the cell cycle and apoptosis of colorectal cancer cell lines and protein expression levels were examined by western blotting. Athymic nude mice were used to examine the ability of transfected colorectal cancer cells to form tumors in vivo. Downregulation of UBA2 inhibited the proliferation of colorectal cancer cell lines in vitro and in vivo through the regulation of cell cycle associated protein expression and apoptosis. Furthermore, downregulation of UBA2 decreased the expression levels of cyclin B1, B-cell lymphoma-2, phosphorylated protein kinase B and E3 ubiquitin-protein ligase MDM2 in colorectal cancer cells, whereas the expression levels of p21 and p27 were increased. UBA2 was demonstrated to serve an essential role in the proliferation of colorectal cancer and may be used as a potential biomarker to predict prognosis and as a therapeutic target in colorectal cancer.
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Affiliation(s)
- Ping He
- Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Xun Sun
- Department of Pathology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Hong-Jing Cheng
- Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Ya-Bin Zou
- Department of Pathology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Quan Wang
- Department of Gastrointestinal Surgery, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Chang-Li Zhou
- Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Wan-Qi Liu
- Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Yue-Ming Hao
- Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
| | - Xiang-Wei Meng
- Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
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34
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Li D, Tavana O, Sun SC, Gu W. Peli1 Modulates the Subcellular Localization and Activity of Mdmx. Cancer Res 2018; 78:2897-2910. [PMID: 29523541 PMCID: PMC5984691 DOI: 10.1158/0008-5472.can-17-3531] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Revised: 01/12/2018] [Accepted: 03/05/2018] [Indexed: 12/21/2022]
Abstract
Mdm2 and Mdmx, both major repressors of p53 in human cancers, are predominantly localized to the nucleus and cytoplasm, respectively. The mechanism by which subcellular localization of Mdmx is regulated remains unclear. In this study, we identify the E3 ligase Peli1 as a major binding partner and regulator of Mdmx in human cells. Peli1 bound Mdmx in vitro and in vivo and promoted high levels of ubiquitination of Mdmx. Peli1-mediated ubiquitination was degradation-independent, promoting cytoplasmic localization of Mdmx, which in turn resulted in p53 activation. Consistent with this, knockdown or knockout Peli1 in human cancer cells induced nuclear localization of Mdmx and suppressed p53 activity. Myc-induced tumorigenesis was accelerated in Peli1-null mice and associated with downregulation of p53 function. Clinical samples of human cutaneous melanoma had decreased Peli1 expression, which was associated with poor overall survival. Together, these results demonstrate that Peli1 acts as a critical factor for the Mdmx-p53 axis by modulating the subcellular localization and activity of Mdmx, thus revealing a novel mechanism of Mdmx deregulation in human cancers.Significance: Peli1-mediated regulation of Mdmx, a major inhibitor of p53, provides critical insight into activation of p53 function in human cancers. Cancer Res; 78(11); 2897-910. ©2018 AACR.
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Affiliation(s)
- Dawei Li
- Institute for Cancer Genetics, Department of Pathology and Cell Biology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, New York
| | - Omid Tavana
- Institute for Cancer Genetics, Department of Pathology and Cell Biology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, New York
| | - Shao-Cong Sun
- Department of Immunology, the University of Texas MD Anderson Cancer Center, MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, Texas
| | - Wei Gu
- Institute for Cancer Genetics, Department of Pathology and Cell Biology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, New York.
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35
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Bulatov E, Zagidullin A, Valiullina A, Sayarova R, Rizvanov A. Small Molecule Modulators of RING-Type E3 Ligases: MDM and Cullin Families as Targets. Front Pharmacol 2018; 9:450. [PMID: 29867461 PMCID: PMC5951978 DOI: 10.3389/fphar.2018.00450] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2017] [Accepted: 04/18/2018] [Indexed: 12/14/2022] Open
Abstract
Ubiquitin-proteasome system (UPS) is a primary signaling pathway for regulation of intracellular protein levels. E3 ubiquitin ligases, substrate-specific members of the UPS, represent highly attractive protein targets for drug discovery. The importance of E3 ligases as prospective targets for small molecule modulation is reinforced by ever growing evidence of their role in cancer and other diseases. To date the number of potent compounds targeting E3 ligases remains rather low and their rational design constitutes a challenging task. To successfully address this problem one must take into consideration the multi-subunit nature of many E3 ligases that implies multiple druggable pockets and protein-protein interfaces. In this review, we briefly cover the current state of drug discovery in the field of RING-type E3 ligases with focus on MDM and Cullin families as targets. We also provide an overview of small molecule chimeras that induce RING-type E3-mediated proteasomal degradation of substrate proteins of interest.
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Affiliation(s)
- Emil Bulatov
- Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
| | - Almaz Zagidullin
- A.E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, Kazan, Russia
| | - Aygul Valiullina
- Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
| | - Regina Sayarova
- Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
| | - Albert Rizvanov
- Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
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36
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MDMX acidic domain inhibits p53 DNA binding in vivo and regulates tumorigenesis. Proc Natl Acad Sci U S A 2018; 115:E3368-E3377. [PMID: 29581299 DOI: 10.1073/pnas.1719090115] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
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.
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37
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Medina-Medina I, Martínez-Sánchez M, Hernández-Monge J, Fahraeus R, Muller P, Olivares-Illana V. p53 promotes its own polyubiquitination by enhancing the HDM2 and HDMX interaction. Protein Sci 2018. [PMID: 29524278 DOI: 10.1002/pro.3405] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N-terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C-terminal RING domains but here we show that the presence of p53 induces an N-terminal interface under normal cellular conditions. This results in an increase in HDM2-mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin-3 binds the N-terminal p53 binding pocket and is sufficient to induce the HDM2-HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX-HDM2-p53) and help to explain the molecular mechanisms of HDM2-inhibitory drugs.
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Affiliation(s)
- Ixaura Medina-Medina
- Laboratorio de Interacciones Biomoleculares y Cáncer, Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, 78290, SLP, México
| | - Mayra Martínez-Sánchez
- Laboratorio de Interacciones Biomoleculares y Cáncer, Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, 78290, SLP, México
| | - Jesús Hernández-Monge
- Laboratorio de Interacciones Biomoleculares y Cáncer, Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, 78290, SLP, México
| | - Robin Fahraeus
- Équipe Labellisée Ligue Contre le Cancer, INSERM UMRS1162, Institut de Génétique Moléculaire, Université Paris 7, IUH Hôpital St. Louis, Paris, 75010, France
| | - Petr Muller
- RECAMO, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53, Brno, Czech Republic
| | - Vanesa Olivares-Illana
- Laboratorio de Interacciones Biomoleculares y Cáncer, Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, 78290, SLP, México
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38
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Pant V, Larsson CA, Aryal N, Xiong S, You MJ, Quintas-Cardama A, Lozano G. Tumorigenesis promotes Mdm4-S overexpression. Oncotarget 2018; 8:25837-25847. [PMID: 28460439 PMCID: PMC5432220 DOI: 10.18632/oncotarget.15552] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2016] [Accepted: 02/06/2017] [Indexed: 11/29/2022] Open
Abstract
Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.
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Affiliation(s)
- Vinod Pant
- Department of Genetics, M.D. Anderson Cancer Center, Houston, Texas, 77030, USA
| | - Connie A Larsson
- Department of Genetics, M.D. Anderson Cancer Center, Houston, Texas, 77030, USA
| | - Neeraj Aryal
- Department of Genetics, M.D. Anderson Cancer Center, Houston, Texas, 77030, USA
| | - Shunbin Xiong
- Department of Genetics, M.D. Anderson Cancer Center, Houston, Texas, 77030, USA
| | - M James You
- Department of Hematopathology, M.D. Anderson Cancer Center, Houston, Texas, 77030, USA
| | | | - Guillermina Lozano
- Department of Genetics, M.D. Anderson Cancer Center, Houston, Texas, 77030, USA
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39
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Zhao Y, Tan M, Liu X, Xiong X, Sun Y. Inactivation of ribosomal protein S27-like confers radiosensitivity via the Mdm2-p53 and Mdm2-MRN-ATM axes. Cell Death Dis 2018; 9:145. [PMID: 29396424 PMCID: PMC5833388 DOI: 10.1038/s41419-017-0192-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2017] [Revised: 11/27/2017] [Accepted: 11/28/2017] [Indexed: 01/21/2023]
Abstract
RPS27L (ribosomal protein S27-like) is an evolutionarily conserved ribosomal protein and a direct p53 target. We recently reported that Rps27l disruption triggers ribosomal stress to induce p53, causing postnatal death, which can be rescued by Trp53+/−. Whether and how Rps27l modulates radiosensitivity is unknown. Here we report that Rps27l−/−; Trp53+/− mice are extremely sensitive to radiation due to reduced proliferation and massive induction of apoptosis in radiation-sensitive organs. Mechanistically, the radiation sensitivity is mediated by two signaling pathways: (1) activated p53 pathway due to imbalanced Mdm2/Mdm4 levels and reduced E3 ligase activity; and (2) reduced DNA damage response due to reduced MRN/Atm signal as a result of elevated Mdm2 binding of Nbs1 to inhibit Nbs1–Atm binding and subsequent Atm activation. Indeed, heterozygous deletion of Mdm2 restores the MRN/Atm signal. Collectively, our study revealed a physiological condition under which Rps27l regulates the Mdm2/p53 and MRN/Atm axes to maintain DNA damage response and to confer radioprotection in vivo.
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Affiliation(s)
- Yongchao Zhao
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China. .,Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA. .,Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
| | - Mingjia Tan
- Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA
| | - Xia Liu
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China
| | - Xiufang Xiong
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China. .,Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA.
| | - Yi Sun
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China. .,Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA. .,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China.
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40
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Kon N, Wang D, Li T, Jiang L, Qiang L, Gu W. Inhibition of Mdmx (Mdm4) in vivo induces anti-obesity effects. Oncotarget 2018; 9:7282-7297. [PMID: 29484110 PMCID: PMC5800902 DOI: 10.18632/oncotarget.23837] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2017] [Accepted: 12/08/2017] [Indexed: 01/22/2023] Open
Abstract
Although cell-cycle arrest, senescence and apoptosis remain as major canonical activities of p53 in tumor suppression, the emerging role of p53 in metabolism has been a topic of great interest. Nevertheless, it is not completely understood how p53-mediated metabolic activities are regulated in vivo and whether this part of the activities has an independent role beyond tumor suppression. Mdmx (also called Mdm4), like Mdm2, acts as a major suppressor of p53 but the embryonic lethality of mdmx-null mice creates difficulties to evaluate its physiological significance in metabolism. Here, we report that the embryonic lethality caused by the deficiency of mdmx, in contrast to the case for mdm2, is fully rescued in the background of p533KR/3KR, an acetylation-defective mutant unable to induce cell-cycle arrest, senescence and apoptosis. p533KR/3KR/mdmx-/- mice are healthy but skinny without obvious developmental defects. p533KR/3KR/mdmx-/- mice are resistant to fat accumulation in adipose tissues upon high fat diet. Notably, the levels of p53 protein are only slightly increased and can be further induced upon DNA damage in p533KR/3KR/mdmx-/- mice, suggesting that Mdmx is only partially required for p53 degradation in vivo. Further analyses indicate that the anti-obesity phenotypes in p533KR/3KR/mdmx-/- mice are caused by activation of lipid oxidation and thermogenic programs in adipose tissues. These results demonstrate the specific effects of the p53/Mdmx axis in lipid metabolism and adipose tissue remodeling and reveal a surprising role of Mdmx inhibition in anti-obesity effects beyond, commonly expected, tumor suppression. Thus, our study has significant implications regarding Mdmx inhibitors in the treatment of obesity related diseases.
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Affiliation(s)
- Ning Kon
- Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA
| | - Donglai Wang
- Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA
| | - Tongyuan Li
- Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA
| | - Le Jiang
- Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA
| | - Li Qiang
- Naomi Berrie Diabetes Center, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York, USA.,Department of Pathology and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York, USA
| | - Wei Gu
- Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA.,Department of Pathology and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York, USA
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41
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Qin JJ, Li X, Wang W, Zi X, Zhang R. Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen. Front Pharmacol 2017; 8:917. [PMID: 29311926 PMCID: PMC5735069 DOI: 10.3389/fphar.2017.00917] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2017] [Accepted: 11/30/2017] [Indexed: 12/31/2022] Open
Abstract
The MDM2 and MDMX oncogenes are overexpressed in various types of human cancer and are highly associated with the initiation, progression, metastasis and chemotherapeutic resistance of these diseases, including prostate cancer. The present study was designed to test a natural MDM2 inhibitor, Inulanolide A (InuA), for anti-prostate cancer activity and to determine the underlying mechanism(s) of action. InuA directly bound to the RING domains of both MDM2 and MDMX with high affinity and specificity and disrupted MDM2-MDMX binding, markedly enhancing MDM2 protein degradation. We further discovered that InuA bound to the DNA binding domain of NFAT1, resulting in marked inhibition of MDM2 transcription. InuA inhibited the proliferation, migration, and invasion of prostate cancer cells, regardless of their p53 status and AR responsiveness. Double knockdown of MDM2 and NFAT1 also revealed that the expression of both of these molecules is important for InuA’s inhibitory effects on the proliferation and invasion of prostate cancer cells. In summary, InuA represents a novel class of bifunctional MDM2 inhibitors, and should be further investigated as a candidate lead compound for prostate cancer prevention and therapy.
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Affiliation(s)
- Jiang-Jiang Qin
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, United States.,Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, United States
| | - Xin Li
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, United States.,Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, United States
| | - Wei Wang
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, United States.,Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, United States.,Center for Drug Discovery, University of Houston, Houston, TX, United States
| | - Xiaolin Zi
- Department of Urology, University of California, Irvine, Irvine, CA, United States.,Department of Pharmacology, University of California, Irvine, Irvine, CA, United States
| | - Ruiwen Zhang
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, United States.,Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, United States.,Center for Drug Discovery, University of Houston, Houston, TX, United States
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42
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Moscetti I, Cannistraro S, Bizzarri AR. Surface Plasmon Resonance Sensing of Biorecognition Interactions within the Tumor Suppressor p53 Network. SENSORS 2017; 17:s17112680. [PMID: 29156626 PMCID: PMC5713020 DOI: 10.3390/s17112680] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/05/2017] [Accepted: 11/16/2017] [Indexed: 12/15/2022]
Abstract
Surface Plasmon Resonance (SPR) is a powerful technique to study the kinetics of biomolecules undergoing biorecognition processes, particularly suited for protein-protein interactions of biomedical interest. The potentiality of SPR was exploited to sense the interactions occurring within the network of the tumor suppressor p53, which is crucial for maintaining genome integrity and whose function is inactivated, mainly by down regulation or by mutation, in the majority of human tumors. This study includes p53 down-regulators, p53 mutants and also the p53 family members, p63 and p73, which could vicariate p53 protective function. Furthermore, the application of SPR was extended to sense the interaction of p53 with anti-cancer drugs, which might restore p53 function. An extended review of previous published work and unpublished kinetic data is provided, dealing with the interaction between the p53 family members, or their mutants and two anticancer molecules, Azurin and its cell-penetrating peptide, p28. All the kinetic results are discussed in connection with those obtained by a complementary approach operating at the single molecule level, namely Atomic Force Spectroscopy and the related literature data. The overview of the SPR kinetic results may significantly contribute to a deeper understanding of the interactions within p53 network, also in the perspective of designing suitable anticancer drugs.
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Affiliation(s)
- Ilaria Moscetti
- Biophysics & Nanoscience Centre, DEB, Università della Tuscia, Largo dell'Università, 01100 Viterbo, Italy.
| | - Salvatore Cannistraro
- Biophysics & Nanoscience Centre, DEB, Università della Tuscia, Largo dell'Università, 01100 Viterbo, Italy.
| | - Anna Rita Bizzarri
- Biophysics & Nanoscience Centre, DEB, Università della Tuscia, Largo dell'Università, 01100 Viterbo, Italy.
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43
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Tian H, Tackmann NR, Jin A, Zheng J, Zhang Y. Inactivation of the MDM2 RING domain enhances p53 transcriptional activity in mice. J Biol Chem 2017; 292:21614-21622. [PMID: 29123033 DOI: 10.1074/jbc.ra117.000122] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Revised: 11/02/2017] [Indexed: 01/08/2023] Open
Abstract
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.
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Affiliation(s)
- Hui Tian
- From the Department of Radiation Oncology, Lineberger Comprehensive Cancer Center and.,the Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu 221002, China
| | - Nicole R Tackmann
- From the Department of Radiation Oncology, Lineberger Comprehensive Cancer Center and.,the Curriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 and
| | - Aiwen Jin
- From the Department of Radiation Oncology, Lineberger Comprehensive Cancer Center and
| | - Junnian Zheng
- the Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu 221002, China
| | - Yanping Zhang
- From the Department of Radiation Oncology, Lineberger Comprehensive Cancer Center and .,the Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu 221002, China
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Wang D, Ma L, Wang B, Liu J, Wei W. E3 ubiquitin ligases in cancer and implications for therapies. Cancer Metastasis Rev 2017; 36:683-702. [DOI: 10.1007/s10555-017-9703-z] [Citation(s) in RCA: 68] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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45
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Tackmann NR, Zhang Y. Mouse modelling of the MDM2/MDMX-p53 signalling axis. J Mol Cell Biol 2017; 9:34-44. [PMID: 28096294 PMCID: PMC5907827 DOI: 10.1093/jmcb/mjx006] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2016] [Accepted: 01/12/2017] [Indexed: 01/10/2023] Open
Abstract
It is evident that p53 activity is critical for tumour prevention and stress response through its transcriptional activation of genes affecting cellular senescence, apoptosis, cellular metabolism, and DNA repair. The regulation of p53 is highly complex, and MDM2 and MDMX are thought to be critical for deciding the fate of p53, both through inhibitory binding and post-translational modification. Many mouse models have been generated to study the regulation of p53 in vivo, and they have altered our interpretations of how p53 is regulated by MDM2 and MDMX. Although MDM2 is absolutely required for p53 regulation, certain functions are dispensable under unstressed conditions, including the ability of MDM2 to degrade p53. MDMX, on the other hand, may only be required in select situations, like embryogenesis. These models have also clarified how cellular stress signals modify the p53-inhibiting activities of MDM2 and MDMX in vivo. It is clear that more work will need to be performed to further understand the contexts for each of these signals and the requirements of various MDM2 and MDMX functions. Here, we will discuss what we have learned from mouse modelling of MDM2 and MDMX and underscore the ways in which these models could inform future therapies.
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Affiliation(s)
- Nicole R Tackmann
- Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.,Curriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA
| | - Yanping Zhang
- Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.,Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical College, Xuzhou 221002, China
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46
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Nomura K, Klejnot M, Kowalczyk D, Hock AK, Sibbet GJ, Vousden KH, Huang DT. Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity. Nat Struct Mol Biol 2017; 24:578-587. [PMID: 28553961 PMCID: PMC6205632 DOI: 10.1038/nsmb.3414] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2017] [Accepted: 05/01/2017] [Indexed: 02/08/2023]
Abstract
MDM2-MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2-MDMX-E2(UbcH5B)-ubiquitin complex, we designed MDM2 mutants that prevent E2-ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53's transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors.
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Affiliation(s)
- Koji Nomura
- Cancer Research-UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
| | - Marta Klejnot
- Cancer Research-UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
| | - Dominika Kowalczyk
- Cancer Research-UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
| | - Andreas K. Hock
- Cancer Research-UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
| | - Gary J. Sibbet
- Cancer Research-UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
| | | | - Danny T. Huang
- Cancer Research-UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
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47
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de Polo A, Luo Z, Gerarduzzi C, Chen X, Little JB, Yuan ZM. AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX-MDM2 complex. J Mol Cell Biol 2017; 9:154-165. [PMID: 27927748 PMCID: PMC5907837 DOI: 10.1093/jmcb/mjw045] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 10/31/2016] [Indexed: 12/14/2022] Open
Abstract
Deregulation of the tyrosine kinase signalling is often associated with tumour progression and drug resistance, but its underlying mechanisms are only partly understood. In this study, we investigated the effects of the receptor tyrosine kinase AXL on the stability of the MDMX-MDM2 heterocomplex and the activity of p53 in melanoma cells. Our data demonstrated that AXL overexpression or activation through growth arrest-specific 6 (Gas6) ligand stimulation increases MDMX and MDM2 protein levels and decreases p53 activity. Upon activation, AXL stabilizes MDMX through a post-translational modification that involves phosphorylation of MDMX on the phosphosite Ser314, leading to increased affinity between MDMX and MDM2 and favouring MDMX nuclear translocation. Ser314 phosphorylation can also protect MDMX from MDM2-mediated degradation, leading to stabilization of the MDMX-MDM2 complex. We identified CDK4/6 and p38 MAPK as the two kinases mediating AXL-induced modulation of the MDMX-MDM2 complex, and demonstrated that suppression of AXL, either through siRNA silencing or pharmacological inhibition, increases expression levels of p53 target genes P21, MDM2, and PUMA, improves p53 pathway response to chemotherapy, and sensitizes cells to both Cisplatin and Vemurafenib. Our findings offer an insight into a novel signalling axis linking AXL to p53 and provide a potentially druggable pathway to restore p53 function in melanoma.
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Affiliation(s)
- Anna de Polo
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA
| | - Zhongling Luo
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA,Department of Dermatology, Xiangya Hospital, Central South University, Changsha 410008, China
| | - Casimiro Gerarduzzi
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA
| | - Xiang Chen
- Department of Dermatology, Xiangya Hospital, Central South University, Changsha 410008, China
| | - John B. Little
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA
| | - Zhi-Min Yuan
- John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA,Correspondence to: Zhi-Min Yuan, E-mail:
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Mancini F, Teveroni E, Di Conza G, Monteleone V, Arisi I, Pellegrino M, Buttarelli M, Pieroni L, D'Onofrio M, Urbani A, Pontecorvi A, Mazzone M, Moretti F. MDM4 actively restrains cytoplasmic mTORC1 by sensing nutrient availability. Mol Cancer 2017; 16:55. [PMID: 28270148 PMCID: PMC5341177 DOI: 10.1186/s12943-017-0626-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Accepted: 02/27/2017] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions. METHOD MDM4-mTOR relationship was analysed through experiments of overexpression or silencing of endogenous proteins in cell culture and using purified proteins in vitro. Data were further confirmed in vivo using a transgenic mouse model overexpressing MDM4. Additionally, the Cancer Genome Atlas (TCGA) database (N = 356) was adopted to analyze the correlation between MDM4 and mTOR levels and 3D cultures were used to analyse the p53-independent activity of MDM4. RESULTS Following nutrient deprivation, MDM4 impairs mTORC1 activity by binding and inhibiting the kinase mTOR, and contributing to maintain the cytosolic inactive pool of mTORC1. This function is independent of p53. Inhibition of mTORC1 by MDM4 results in reduced phosphorylation of the mTOR downstream target p70S6K1 both in vitro and in vivo in a MDM4-transgenic mouse. Consistently, MDM4 reduces cell size and proliferation, two features controlled by p70S6K1, and, importantly, inhibits mTORC1-mediated mammosphere formation. Noteworthy, MDM4 transcript levels are significantly reduced in breast tumors characterized by high mTOR levels. CONCLUSION Overall, these data identify MDM4 as a nutrient-sensor able to inhibit mTORC1 and highlight its metabolism-related tumor-suppressing function.
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Affiliation(s)
- Francesca Mancini
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), 00143, Rome, Italy. .,PostGraduate School of Endocrinology and Metabolic Diseases, Institute of Pathology, Catholic University of Rome, 00168, Rome, Italy.
| | - Emanuela Teveroni
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), 00143, Rome, Italy
| | - Giusy Di Conza
- Laboratory of Molecular Oncology and Angiogenesis, Department of Oncology, KU Leuven, 3000, Leuven, Belgium.,Laboratory of Molecular Oncology and Angiogenesis, Vesalius Research Center, VIB, 3000, Leuven, Belgium
| | - Valentina Monteleone
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), 00143, Rome, Italy
| | - Ivan Arisi
- European Brain Research Institute (EBRI) Rita Levi-Montalcini, 00143, Rome, Italy
| | - Marsha Pellegrino
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), 00143, Rome, Italy
| | - Marianna Buttarelli
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), 00143, Rome, Italy
| | - Luisa Pieroni
- Proteomic and Metabonomic Laboratory, Fondazione Santa Lucia, 00143, Rome, Italy
| | - Mara D'Onofrio
- European Brain Research Institute (EBRI) Rita Levi-Montalcini, 00143, Rome, Italy
| | - Andrea Urbani
- Proteomic and Metabonomic Laboratory, Fondazione Santa Lucia, 00143, Rome, Italy.,Institute of Biochemistry and Biochemical Clinic, Catholic University of Rome, 00168, Rome, Italy
| | - Alfredo Pontecorvi
- PostGraduate School of Endocrinology and Metabolic Diseases, Institute of Pathology, Catholic University of Rome, 00168, Rome, Italy
| | - Massimiliano Mazzone
- Laboratory of Molecular Oncology and Angiogenesis, Department of Oncology, KU Leuven, 3000, Leuven, Belgium.,Laboratory of Molecular Oncology and Angiogenesis, Vesalius Research Center, VIB, 3000, Leuven, Belgium
| | - Fabiola Moretti
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), 00143, Rome, Italy.
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Abstract
The p53 tumor suppressor has been studied for decades, and still there are many questions left unanswered. In this review, we first describe the current understanding of the wild-type p53 functions that determine cell survival or death, and regulation of the protein, with a particular focus on the negative regulators, the murine double minute family of proteins. We also summarize tissue-, stress-, and age-specific p53 activities and the potential underlying mechanisms. Among all p53 gene alterations identified in human cancers, p53 missense mutations predominate, suggesting an inherent biological advantage. Numerous gain-of-function activities of mutant p53 in different model systems and contexts have been identified. The emerging theme is that mutant p53, which retains a potent transcriptional activation domain, also retains the ability to modify gene transcription, albeit indirectly. Lastly, because mutant p53 stability is necessary for its gain of function, we summarize the mechanisms through which mutant p53 is specifically stabilized. A deeper understanding of the multiple pathways that impinge upon wild-type and mutant p53 activities and how these, in turn, regulate cell behavior will help identify vulnerabilities and therapeutic opportunities.
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Affiliation(s)
- Yun Zhang
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
| | - Guillermina Lozano
- Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
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50
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Fan C, Wang X. Mdm2 Splice isoforms regulate the p53/Mdm2/Mdm4 regulatory circuit via RING domain-mediated ubiquitination of p53 and Mdm4. Cell Cycle 2017; 16:660-664. [PMID: 28166445 DOI: 10.1080/15384101.2017.1288327] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
p53 is regulated by heterodimer E3 ligase Mdm2-Mdm4 via RING domain interaction. Mdm2 transcripts undergo alternative splicing, and Mdm2 splice isoforms are increased in cancer and induced by DNA damage. Although 2 major Mdm2 splice isoforms that do not bind to p53 were reported to impact the p53 pathway, the underlying biochemical mechanisms were not understood. Here, we show that these Mdm2 splice isoforms ubiquitinate Mdm2 and Mdm4 in vivo and regulate the activity of Mdm2-Mdm4 E3 complex in cells. The Mdm2 isoforms are capable of promoting p53 ubiquitination in the absence of Mdm2 or Mdm4. The 2 isoforms stimulate Mdm2 or Mdm4 activity for p53 ubiquitination in vivo and promote degradation of p53 and Mdm4 in cells. However, the Mdm2 isoforms have opposing effects on the steady-state p53 levels depending on the stoichiometric ratios of Mdm2, Mdm4 and the isoforms, causing either decreased or increased p53 levels in cells. Our data indicate that the Mdm2 splice isoforms can act as independent E3 ligases for p53 when Mdm2 and Mdm4 are absent, form potent heterodimer E3 ligases with either Mdm2 or Mdm4 for targeting p53 degradation, or act as inhibitory regulators of Mdm2-Mdm4 E3 ligase activity by downregulating Mdm4. These findings suggest that Mdm2 splice isoforms may play critical roles in the regulatory loop of p53/Mdm2-Mdm4 via a RING domain-mediated biochemical mechanism.
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Affiliation(s)
- Chuandong Fan
- a Department of Pharmacology and Therapeutics , Roswell Park Cancer Institute , Buffalo , NY , USA
| | - Xinjiang Wang
- a Department of Pharmacology and Therapeutics , Roswell Park Cancer Institute , Buffalo , NY , USA
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