Death-associated protein kinase 1 phosphorylates MDM2 and inhibits its protein stability and function.

Breast cancer is one of the major malignancies in women, and most related deaths are due to recurrence, drug resistance, and metastasis. The expression of the mouse double minute 2 (MDM2) oncogene is upregulated in breast cancer; however, its regulatory mechanism has yet to be fully elucidated. Herein, we identified the tumor suppressor death-associated protein kinase 1 (DAPK1) as a novel MDM2 regulator by unbiased peptide library screening. DAPK1 is directly bound to MDM2 and phosphorylates it at Thr419. DAPK1-mediated MDM2 phosphorylation promoted its protein degradation via the ubiquitin-proteasome pathway, resulting in upregulated p53 expression. DAPK1 overexpression, but not its kinase activity-deficient form, decreased colony formation and increased doxorubicin-induced cell death; however, DAPK1 knockdown produced the opposite effects in human breast cancer cells. In a xenograft tumorigenesis assay, DAPK1 overexpression significantly reduced tumor formation, whereas inhibition of DAPK1 kinase activity reduced its antitumorigenic effect. Finally, DAPK1 expression was negatively correlated with MDM2 levels in human breast cancer tissues. Thus, these results suggest that DAPK1-mediated MDM2 phosphorylation and its protein degradation may contribute to its antitumorigenic function in breast cancer.

MDM2-Based Proteolysis-Targeting Chimeras (PROTACs): An Innovative Drug Strategy for Cancer Treatment.

Proteolysis-targeting chimeras (PROTACs) are molecules that selectively degrade a protein of interest (POI). The incorporation of ligands that recruit mouse double minute 2 (MDM2) into PROTACs, forming the so-called MDM2-based PROTACs, has shown promise in cancer treatment due to its dual mechanism of action: a PROTAC that recruits MDM2 prevents its binding to p53, resulting not only in the degradation of POI but also in the increase of intracellular levels of the p53 suppressor, with the activation of a whole set of biological processes, such as cell cycle arrest or apoptosis. In addition, these PROTACs, in certain cases, allow for the degradation of the target, with nanomolar potency, in a rapid and sustained manner over time, with less susceptibility to the development of resistance and tolerance, without causing changes in protein expression, and with selectivity to the target, including the respective isoforms or mutations, and to the cell type, overcoming some limitations associated with the use of inhibitors for the same therapeutic target. Therefore, the aim of this review is to analyze and discuss the characteristics of MDM2-based PROTACs developed for the degradation of oncogenic proteins and to understand what potential they have as future anticancer drugs.

Design, Synthesis, Chemical and Biochemical Insights Into Novel Hybrid Spirooxindole-Based p53-MDM2 Inhibitors With Potential Bcl2 Signaling Attenuation.

The tumor resistance to p53 activators posed a clinical challenge. Combination studies disclosed that concomitant administration of Bcl2 inhibitors can sensitize the tumor cells and induce apoptosis. In this study, we utilized a rapid synthetic route to synthesize two novel hybrid spirooxindole-based p53-MDM2 inhibitors endowed with Bcl2 signaling attenuation. The adducts mimic the thematic features of the chemically stable potent spiro [3H-indole-3,2'-pyrrolidin]-2(1H)-ones p53-MDM2 inhibitors, while installing a pyrrole ring via a carbonyl spacer inspired by the natural marine or synthetic products that efficiently inhibit Bcl2 family functions. A chemical insight into the two synthesized spirooxindoles including single crystal x-ray diffraction analysis unambiguously confirmed their structures. The synthesized spirooxindoles 2a and 2b were preliminarily tested for cytotoxic activities against normal cells, MDA-MB 231, HepG-2, and Caco-2 via MTT assay. 2b was superior to 5-fluorouracil. Mechanistically, 2b induced apoptosis-dependent anticancer effect (43%) higher than that of 5-fluorouracil (34.95%) in three studied cancer cell lines, activated p53 (47%), downregulated the Bcl2 gene (1.25-fold), and upregulated p21 (2-fold) in the treated cancer cells. Docking simulations declared the possible binding modes of the synthesized compounds within MDM2.

Ubiquitin-specific protease 7 mediates platelet-derived growth factor-induced pulmonary arterial smooth muscle cells proliferation.

Pulmonary arterial hypertension is a devastating pulmonary vascular disease, in which the pathogenesis is complicated and unclear. Pulmonary arterial smooth muscle cells (PASMCs) proliferation is a key pathological feature of pulmonary arterial hypertension. It has been shown that ubiquitin-specific protease 7 (USP7) is involved in cancer cell proliferation via deubiquitinating and stabilizing E3 ubiquitin ligase mouse double minute 2 (MDM2). However, the effect of USP7 and MDM2 on platelet-derived growth factor (PDGF)-induced PASMCs proliferation is uncertain. This study aims to explore this issue. Our results indicated that PDGF up-regulated USP7 protein expression and stimulated PASMCs proliferation; this was accompanied with the increase of MDM2, forkhead box O4 (FoxO4) reduction and elevation of CyclinD1. While prior transfection of USP7 siRNA blocked PDGF-induced MDM2 up-regulation, FoxO4 down-regulation, increase of CyclinD1 and cell proliferation. Pre-depletion of MDM2 by siRNA transfection reversed PDGF-induced reduction of FoxO4, up-regulation of CyclinD1 and PASMCs proliferation. Furthermore, pre-treatment of cells with proteasome inhibitor MG-132 also abolished PDGF-induced FoxO4 reduction, CyclinD1 elevation and cell proliferation. Our study suggests that USP7 up-regulates MDM2, which facilitates FoxO4 ubiquitinated degradation, and subsequently increases the expression of CyclinD1 to mediate PDGF-induced PASMCs proliferation.

p53 p.Pro72Arg (rs1042522) and Mouse Double Minute 2 (MDM2) Single-Nucleotide Polymorphism (SNP) 309 Variants and Their Interaction in Chronic Lymphocytic Leukemia(CLL): A Survey in CLL Patients from Western Iran.

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The MDM2 and p53 are interacting proteins that play crucial roles in cell biology. Genetic variations of p53 and MDM2 have been identified in many cancers including CLL; among which are SNP309 in the promoter of MDM2 and SNP codon72 in p53. Materials and Methods: In this study, we sought to find the impact of two SNPs of p53 and MDM2 in the pathogenesis of CLL. A total of 100 CLL patients and 102 healthy controls were recruited. Genomic DNA was extracted, and genotyping was performed using the PCR-RFLP method. The allele and genotype associations were analyzed using the χ2 test. The gene-gene interaction analysis was studied using GMDR v0.9. Results: Our study found the absence of a significant difference between CLL patients and controls related to the allelic frequencies or genotypic distributions for both MDM2 SNP309 and p53 codon72. A significantly higher frequency of p53 C allele was found in patients with disease duration of more than 36 compared to those less than 36 months. However, GMDR analysis suggests genetic interaction between the genes under study. Conclusion: Our findings indicated each polymorphism of p53 codon72 and MDM2 (SNP309) was not a risk factor for CLL but the p53 C allele could be associated with the disease duration. Besides, the interaction between p53/MDM2 genotypes may confer susceptibility to CLL. Our study could be useful in genetic association studies of CLL and the role of gene-gene interactions in the susceptibility to the disease.

The proto-oncogene function of Mdm2 in bone.

Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.

Exposure to High-Dose Diesel Exhaust Particles Induces Intracellular Oxidative Stress and Causes Endothelial Apoptosis in Cultured In Vitro Capillary Tube Cells.

Previous studies suggest a direct correlation between exposure to diesel exhaust particles (DEP) and the onset of vascular permeability, presumably through the disruption of the adherens junctions. This would lead to deleterious effects on vasculature, such as acute myocardial infarction and atherosclerosis. Although the mechanism remains unclear, we demonstrate DEP-induced mitochondrial reactive oxygen species generation, which may be a central cause of the above vascular disorders. In vitro capillary-like HUVEC tube cells are used in this study and show that acute DEP exposure stimulates ATP depletion, followed by depolarization of their actin cytoskeleton, which sequentially inhibits PI3K/Akt activity and induces endothelial apoptosis. These events are accompanied by induction of p53/Mdm2 feedback regulation at 10 µg/mL DEP and produce 20 % cell apoptosis. Nevertheless, 100 µg/mL DEP augments tube cell apoptosis up to 70 % but disrupts the p53 negative regulator Mdm2. Addition of N-acetylcysteine provides substantial protection against the cytotoxic effects of DEP. In summary, exposure to a low dose of DEP actin triggers cytoskeleton depolarization, reduces PI3K/Akt activity, and induces a p53/Mdm2 feedback loop, and a high dose causes apoptosis by depleting Mdm2.