Developing methodological approaches for discovering novel pathways is a key challenge in the life science research. Biological pathways are regulated-in higher eukaryotes-by a vast diversity of linear peptide motifs that mediate combinatorial specificity in signal transduction pathways. The E3 ubiquitin ligase component (MDM2) is such a protein that interacts with target proteins containing linear motifs such as p53. Drug leads, such as Nutlin-3, that bind to the MDM2 hydrophobic pocket mimic p53 and can release p53 from MDM2 control and this can lead to cell death. However, these drug leads act allosterically, having agonist effects on MDM2's functions and there are other proteins whose steady state levels can be altered by Nutlin-3. As cell density can alter the proliferation state of cell populations, we examined the impact of Nutlin-3 on levels of newly synthesized proteins using pulse-SILAC mass spectrometry. The data demonstrate that at differing cell densities or population-wide proliferation rates, different newly synthesized proteins dominate the proteome landscape in a Nutlin-3 dependent manner. These data further confirm that the cell state in a population of cells can in turn impact on the MDM2 signalling landscape. This methodology forms a blueprint for biomarker discovery using clinical samples that can detect changes in the synthesis rate of proteins in cell populations treated with specific agents. Broader implications highlight tools that can be used to study allosteric regulation of protein-drug combinations.
- MeSH
- Imidazoles * pharmacology MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Piperazines * pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Proteome * metabolism MeSH
- Proteomics methods MeSH
- Proto-Oncogene Proteins c-mdm2 metabolism MeSH
- Signal Transduction drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The transcription factor p53 is the most frequently impaired tumor suppressor in human cancers. In response to various stress stimuli, p53 activates transcription of genes that mediate its tumor-suppressive functions. Distinctive characteristics of p53 outlined here enable a well-defined program of genes involved in cell cycle arrest, apoptosis, senescence, differentiation, metabolism, autophagy, DNA repair, anti-viral response, and anti-metastatic functions, as well as facilitating autoregulation within the p53 network. This versatile, anti-cancer network governed chiefly by a single protein represents an immense opportunity for targeted cancer treatment, since about half of human tumors retain unmutated p53. During the last two decades, numerous compounds have been developed to block the interaction of p53 with the main negative regulator MDM2. However, small molecule inhibitors of MDM2 only induce a therapeutically desirable apoptotic response in a limited number of cancer types. Moreover, clinical trials of the MDM2 inhibitors as monotherapies have not met expectations and have revealed hematological toxicity as a characteristic adverse effect across this drug class. Currently, combination treatments are the leading strategy for enhancing efficacy and reducing adverse effects of MDM2 inhibitors. This review summarizes efforts to identify and test therapeutics that work synergistically with MDM2 inhibitors. Two main types of drugs have emerged among compounds used in the following combination treatments: first, modulators of the p53-regulated transcriptome (including chromatin modifiers), translatome, and proteome, and second, drugs targeting the downstream pathways such as apoptosis, cell cycle arrest, DNA repair, metabolic stress response, immune response, ferroptosis, and growth factor signaling. Here, we review the current literature in this field, while also highlighting overarching principles that could guide target selection in future combination treatments.
- MeSH
- Molecular Targeted Therapy * MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 * metabolism genetics antagonists & inhibitors MeSH
- Neoplasms * drug therapy metabolism genetics MeSH
- Antineoplastic Agents * therapeutic use pharmacology MeSH
- Proto-Oncogene Proteins c-mdm2 antagonists & inhibitors metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
PURPOSE: Pediatric sarcomas are bone and soft tissue tumors that often exhibit high metastatic potential and refractory stem-like phenotypes, resulting in poor outcomes. Aggressive sarcomas frequently harbor a disrupted p53 pathway. However, whether pediatric sarcoma stemness is associated with abrogated p53 function and might be attenuated via p53 reactivation remains unclear. METHODS: We utilized a unique panel of pediatric sarcoma models and tumor tissue cohorts to investigate the correlation between the expression of stemness-related transcription factors, p53 pathway dysregulations, tumorigenicity in vivo, and clinicopathological features. TP53 mutation status was assessed by next-generation sequencing. Major findings were validated via shRNA-mediated silencing and functional assays. The p53 pathway-targeting drugs were used to explore the effects and selectivity of p53 reactivation against sarcoma cells with stem-like traits. RESULTS: We found that highly tumorigenic stem-like sarcoma cells exhibit dysregulated p53, making them vulnerable to drugs that restore wild-type p53 activity. Immunohistochemistry of mouse xenografts and human tumor tissues revealed that p53 dysregulations, together with enhanced expression of the stemness-related transcription factors SOX2 or KLF4, are crucial features in pediatric osteosarcoma, rhabdomyosarcoma, and Ewing's sarcoma development. p53 dysregulation appears to be an important step for sarcoma cells to acquire a fully stem-like phenotype, and p53-positive pediatric sarcomas exhibit a high frequency of early metastasis. Importantly, reactivating p53 signaling via MDM2/MDMX inhibition selectively induces apoptosis in aggressive, stem-like Ewing's sarcoma cells while sparing healthy fibroblasts. CONCLUSIONS: Our results indicate that restoring canonical p53 activity provides a promising strategy for developing improved therapies for pediatric sarcomas with unfavorable stem-like traits.
- MeSH
- Child MeSH
- Kruppel-Like Factor 4 * MeSH
- Humans MeSH
- Adolescent MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neoplastic Stem Cells * metabolism pathology MeSH
- Tumor Suppressor Protein p53 * metabolism genetics MeSH
- Child, Preschool MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Sarcoma * genetics pathology metabolism MeSH
- Signal Transduction MeSH
- Xenograft Model Antitumor Assays MeSH
- Animals MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Mice MeSH
- Child, Preschool MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.
The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.
- MeSH
- Leukemia, Myeloid, Acute * genetics MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Myelodysplastic Syndromes * genetics MeSH
- Mice MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Cell Cycle Proteins genetics metabolism MeSH
- Proto-Oncogene Proteins c-mdm2 genetics metabolism MeSH
- Proto-Oncogene Proteins genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Leiomyosarcoma with adipocytic differentiation or lipoleiomyosarcoma is an uncommon sarcoma of the female genital tract with only a few individual reports in the literature. We therefore performed a morphologic, immunohistochemical, MDM2 gene amplification and RNA and DNA sequencing analysis of a series of gynecologic lipoleiomyosarcoma to better define the clinicopathologic spectrum. Six tumors from 6 patients were identified and classified as spindled lipoleiomyosarcoma (n = 2), mixed spindled and myxoid lipoleiomyosarcoma (n = 1), epithelioid lipoleiomyosarcoma with focal myxoid features (n = 1) and mixed spindled and epithelioid lipoleiomyosarcoma (n = 2). Patient age ranged from 41 to 64 years (mean: 49; median: 50). Primary location included uterine corpus (3), uterine corpus/cervix (2) and broad ligament (1). Tumor size ranged from 4.5 to 22 cm (mean: 11.2; median: 9.8). Four patients had metastasis at presentation or subsequently developed recurrent or distant disease. Patient status was known for 5: 2 dead of disease, 2 alive with disease and 1 alive without evidence of disease. Immunohistochemical expression of smooth muscle markers, ER, PR and WT-1 showed patterns similar to non-adipocytic gynecologic leiomyosarcomas. MDM2 amplification fluorescence in situ hybridization performed on 2 tumors was negative in 1 and equivocal in 1. Sequencing studies performed on 3 tumors found TP53 mutations in 3, with 1 tumor also having an ATRX alteration. No gene fusions were identified. Although lipoleiomyosarcomas have a diverse morphologic spectrum, our findings suggest the smooth muscle component shares morphologic and immunohistochemical features with female genital tract non-adipocytic leiomyosarcomas. Lipoleiomyosarcomas also have genetic alterations associated with non-adipocytic gynecologic leiomyosarcomas.
- MeSH
- Adult MeSH
- In Situ Hybridization, Fluorescence MeSH
- Immunohistochemistry MeSH
- Leiomyosarcoma * pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Molecular Biology MeSH
- Smooth Muscle Tumor * pathology MeSH
- Biomarkers, Tumor genetics analysis MeSH
- Proto-Oncogene Proteins c-mdm2 genetics MeSH
- Genitalia, Female chemistry pathology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Leiomyomas with adipocytic differentiation typically occur in the uterus although they may arise at several sites in the female genital tract. While these are most commonly spindled leiomyomas with a component of adipocytic tissue ("conventional lipoleiomyomas"), there is a relatively ill-defined assortment of leiomyoma variants with adipocytic differentiation. We performed a morphologic, immunohistochemical and MDM2 gene amplification analysis of a large series of gynecologic leiomyomas with adipocytic differentiation to better define the clinicopathologic spectrum. Forty four tumors from 44 patients were identified and classified as conventional lipoleiomyoma (n = 21), adipocyte-rich lipoleiomyoma (defined as tumor volume >80 % adipocytes, n = 9); cellular lipoleiomyoma (n = 9); hydropic lipoleiomyoma (n = 3); and lipoleiomyoma with bizarre nuclei (n = 2). Patient age ranged from 32 to 83 years (mean 63; median 63). Primary location included uterine corpus (35), uterine cervix (3), uterine corpus/cervix (1), broad ligament (2), parametrium (2), and round ligament (1). Tumor size was 0.6-30 cm (mean 8; median 6). None of the 34 patients with follow up developed further disease (range 1-311 months; mean 65; median 41). Immunohistochemical expression of ER, PR, HMB45, Melan A, Cathepsin K and WT-1 in lipoleiomyomas and variants was similar to patterns in non-adipocytic gynecologic leiomyomas. MDM2 amplification fluorescence in situ hybridization performed on 14 tumors was negative in all. Our findings suggest female genital tract conventional lipoleiomyomas and lipoleiomyoma variants largely parallel their non-adipocytic counterparts in morphology and immunophenotype, and may be categorized using non-adipocytic leiomyoma histologic criteria.
- MeSH
- Adult MeSH
- In Situ Hybridization, Fluorescence MeSH
- Leiomyoma * pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Lipoma * genetics pathology MeSH
- Smooth Muscle Tumor * MeSH
- Uterine Neoplasms * pathology MeSH
- Proto-Oncogene Proteins c-mdm2 genetics MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Uterus pathology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Specific C-terminal nucleophosmin (NPM) mutations are related to the acute myeloid leukaemia and cause mistargeting of mutated NPM (NPMmut) to the cytoplasm. Consequently, multiple NPM-interacting partners, e.g., the tumour suppressor p53, become also mislocalized. We found that ubiquitin ligase Mdm2 mislocalizes to the cytoplasm in the presence of NPMmut as well. Since p53 interacts with Mdm2, we searched for the NPMmut-p53-Mdm2 complex and interactions of its constituents in live cells and cell lysates using fluorescently tagged proteins, fluorescence lifetime imaging and immunoprecipitation. We proved existence of the ternary complex, which likely adopts a chain-like configuration. Interaction between Mdm2 and NPMmut was not detected, even under conditions of upregulated Mdm2 and p53 induced by Actinomycin D. We assume that p53 serves in the complex as a bridging link between Mdm2 and NPMmut. This conclusion was supported by disruption of the Mdm2-p53 interaction by Nutlin-3A, which resulted in relocalization of Mdm2 to the nucleus, while both NPMmut and p53 remained in the cytoplasm. Importantly, silencing of p53 also prevented mislocalization of Mdm2 in the presence of NPMmut.
- MeSH
- Cytoplasm genetics metabolism MeSH
- Nuclear Proteins * genetics metabolism MeSH
- Mutation MeSH
- Tumor Suppressor Protein p53 * genetics metabolism MeSH
- Nucleophosmin MeSH
- Proto-Oncogene Proteins c-mdm2 genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Activation of p53 by small molecule MDM2 inhibitors can induce cell cycle arrest or death in p53 wildtype cancer cells. However, cancer cells exposed to hypoxia can develop resistance to other small molecules, such as chemotherapies, that activate p53. Here, we evaluated whether hypoxia could render cancer cells insensitive to two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin. Inhibitor efficacy and potency were evaluated under short-term hypoxic conditions in human and mouse cancer cells expressing different p53 genotypes (wild-type, mutant, or null). Treatment of wild-type p53 cancer cells with MDM2 inhibitors reduced cell growth by > 75% in hypoxia through activation of the p53-p21 signaling pathway; no inhibitor-induced growth reduction was observed in hypoxic mutant or null p53 cells except at very high concentrations. The concentration of inhibitors needed to induce the maximal p53 response was not significantly different in hypoxia compared to normoxia. However, inhibitor efficacy varied by species and by cell line, with stronger effects at lower concentrations observed in human cell lines than in mouse cell lines grown as 2D and 3D cultures. Together, these results indicate that MDM2 inhibitors retain efficacy in hypoxia, suggesting they could be useful for targeting acutely hypoxic cancer cells.
- MeSH
- Apoptosis MeSH
- Hypoxia genetics MeSH
- Humans MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Neoplasms * drug therapy genetics MeSH
- Antineoplastic Agents * pharmacology MeSH
- Proto-Oncogene Proteins c-mdm2 metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
HDMX and its homologue HDM2 are two essential proteins for the cell; after genotoxic stress, both are phosphorylated near to their RING domain, specifically at serine 403 and 395, respectively. Once phosphorylated, both can bind the p53 mRNA and enhance its translation; however, both recognize p53 protein and provoke its degradation under normal conditions. HDM2 has been well-recognized as an E3 ubiquitin ligase, whereas it has been reported that even with the high similarity between the RING domains of the two homologs, HDMX does not have the E3 ligase activity. Despite this, HDMX is needed for the proper p53 poly-ubiquitination. Phosphorylation at serine 395 changes the conformation of HDM2, helping to explain the switch in its activity, but no information on HDMX has been published. Here, we study the conformation of HDMX and its phospho-mimetic mutant S403D, investigate its E3 ligase activity and dissect its binding with p53. We show that phospho-mutation does not change the conformation of the protein, but HDMX is indeed an E3 ubiquitin ligase in vitro; however, in vivo, no activity was found. We speculated that HDMX is regulated by induced fit, being able to switch activity accordingly to the specific partner as p53 protein, p53 mRNA or HDM2. Our results aim to contribute to the elucidation of the contribution of the HDMX to p53 regulation.
- MeSH
- Nuclear Proteins genetics MeSH
- RNA, Messenger metabolism MeSH
- Tumor Suppressor Protein p53 * genetics metabolism MeSH
- Cell Cycle Proteins metabolism MeSH
- Proto-Oncogene Proteins c-mdm2 * genetics metabolism MeSH
- Proto-Oncogene Proteins genetics MeSH
- Serine metabolism MeSH
- Ubiquitin genetics MeSH
- Ubiquitination MeSH
- Ubiquitin-Protein Ligases genetics metabolism MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
