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Swietenia macrophylla King in Hook (SM) is known to have several medicinal properties. Chloroform extracts of SM seeds (SMCE) as well as two isolated limonoids swietenine (1) and swietenolide (2) showed significant in vitro anti-CRC activity in human colon carcinoma (HCT116) cell line. 2 (IC50 = 5.6 µM) was found to be two times more potent than 1 (IC50 = 10 µM). Both compounds showed anti-CRC activity through inhibition of the Mouse Double Minute 2 homolog (MDM2) of the MDM2-p53 pathway. The Selectivity Index (S.I.) of isolated compounds 1 and 2 for cancer cells were about 6.6 and 12.8 fold respectively which was significantly better than the S.I. of the extract (S.I. ∼1.5). [ABSTRACT FROM AUTHOR]

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors.

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors. [ABSTRACT FROM AUTHOR]

As the most common gene mutation found in cancers, p53 mutations are detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Meanwhile, mutant p53 overexpression is known to drive oncogenic phenotypes in cancer patients and to sustain the activation of EGFR signaling. Previously, we have demonstrated that the combined inhibition of EGFR and MDM2-p53 pathways, by gefitinib and JNJ-26854165, exerts a strong synergistic lethal effect on HGSOC cells. In this study, we investigated whether the gain-of-function p53 mutation (p53R248Q) overexpression could affect EGFR-related signaling and the corresponding drug inhibition outcome in HGSOC. The targeted inhibition responses of gefitinib and JNJ-26854165, in p53R248Q-overexpressing cells, were extensively evaluated. We found that the phosphorylation of AKT increased when p53R248Q was transiently overexpressed. Immunocytochemistry analysis further showed that upon p53R248Q overexpression, several AKT-related regulatory proteins translocated in unique intracellular patterns. Subsequent analysis revealed that, under the combined inhibition of gefitinib and JNJ-26854165, the cytonuclear trafficking of EGFR and MDM2 was disrupted. Next, we analyzed the gefitinib and JNJ-26854165 responses and found differential sensitivity to the single- or combined-drug inhibitions in p53R248Q-overexpressing cells. Our findings suggested that the R248Q mutation of p53 in HGSOC caused significant changes in signaling protein function and trafficking, under EGFR/MDM2-targeted inhibition. Such knowledge could help to advance our understanding of the role of mutant p53 in ovarian carcinoma and to improve the prognosis of patients receiving EGFR/MDM2-targeted therapies.

Alternative splicing (AS) enables a pre-mRNA to generate different functional protein variants. The change in AS has been reported as an emerging contributor to cellular senescence and aging. However, it remains to be elucidated which senescent AS variants are generated in and regulate senescence. Here, we observed commonly down-regulated SRSF7 in senescent cells, using publicly available RNA-seq datasets of several in vitro senescence models. We further confirmed SRSF7 deregulation from our previous microarray datasets of time-series replicative senescence (RS) and oxidative stress-induced senescence (OSIS) of human diploid fibroblast (HDF). We validated the time-course changes of SRSF mRNA and protein levels, developing both RS and OSIS. SRSF knockdown in HDF was enough to induce senescence, accompanied by p53 protein stabilization and MDM2 variants formation. Interestingly, expression of MDM2 variants showed similar patterns of p53 expression in both RS and OSIS. Next, we identified MDM2-C as a key functional AS variant generated specifically by SRSF7 depletion. Finally, we validated that MDM2-C overexpression induced senescence of HDF. These results indicate that SRSF7 down-regulation plays a key role in p53-mediated senescence by regulating AS of MDM2, a key negative regulator of p53, implying its critical involvement in the entry into cell senescence.

The tumor suppressor protein p53 is inactivated in a large variety of cancer cells. Cellular p53 inhibitors like the mouse double minute 2 homolog (MDM2) commonly suppress the p53 function in acute myeloid leukemia (AML). Moreover, fms like tyrosine kinase 3 (FLT3) growth factor signaling pathways including the mitogen-activated kinase (MAPK) cascade (RAS-RAF-MEK-ERK) are highly active in AML cells. Consequently, the combined administration of MDM2 and MEK inhibitors may present a promising anti-leukemic treatment strategy. Here we assessed the MDM2 antagonist idasanutlin and the MEK1 inhibitor cobimetinib as single agents and in combination in a variety of AML cell lines and primary AML blast cells for their ability to induce apoptosis and cell death. AML cell lines and blast cells comprised all major AML subtypes based on the mutational status of TP53, FLT3 and NPM1 genes. We observed a considerably varying anti-leukemic efficacy of idasanutlin and cobimetinib. AML cells with high sensitivity to the single compounds as well as to the combined treatment emerged with normal karyotype, wild-type TP53 and elevated FLT3 and MDM2 protein levels. Our data indicate that AML cells with normal karyotype (NK) and wild-type status of TP53 with elevated FLT3 and MDM2 expression emerge to be most sensitive to the combined treatment with cobimetinib and idasanutlin. FLT3 and MDM2 are biomarkers for treatment response to idasanutlin and cobimetinib in AML.

Background: Although the incidence of glioma is relatively low, it is the most malignant tumor of the central nervous system. The prognosis of high-grade glioma patient is very poor due to the difficulties in complete resection and resistance to radio-/chemotherapy. Therefore, it is worth investigating the molecular mechanisms involved in glioma drug resistance. MicroRNAs have been found to play important roles in tumor progression and drug resistance. Our previous work showed that miR-181b is involved in the regulation of temozolomide resistance. In the current study, we investigated whether miR-181b also plays a role in antagonizing the effect of teniposide. Methods: MiR-181b expression was measured in 90 glioma patient tissues and its relationship to prognosis of these patients was analyzed. Cell sensitivity to teniposide was tested in 48 primary cultured glioma samples. Then miR-181b stably overexpressed U87 cells were generated. The candidate genes of miR-181b from our previous study were reanalyzed, and the interaction between miR-181b and target gene MDM2 was confirmed by dual luciferase assay. Cell sensitivity to teniposide was detected on miR-181b over expressed and MDM2 down regulated cells. Results: Our data confirmed the low expression levels of miR-181b in high-grade glioma tissues, which is related to teniposide resistance in primary cultured glioma cells. Overexpression of miR-181b increased glioma cell sensitivity to teniposide. Through target gene prediction, we found that MDM2 is a candidate target of miR-181b. MDM2 knockdown mimicked the sensitization effect of miR-181b. Further study revealed that miR-181b binds to the 3'-UTR region of MDM2 leading to the decrease in MDM2 levels and subsequent increase in teniposide sensitivity. Partial restoration of MDM2 attenuated the sensitivity enhancement by miR-181b. Conclusions: MiR-181b is an important positive regulator on glioma cell sensitivity to teniposide. It confers glioma cell sensitivity to teniposide through binding to the 3'-UTR region of MDM2 leading to its reduced expression. Our findings not only reveal the novel mechanism involved in teniposide resistance, but also shed light on the optimization of glioma treatment in the future. [ABSTRACT FROM AUTHOR]

Abstract: Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK–VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK–VEGFR3 protein–protein interaction inhibitors. [Copyright &y& Elsevier]

Abstract: Preclinical animal models are useful tools to better understand tumor initiation and progression and to predict the activity of an anticancer agent in the clinic. Ideally, these models should recapitulate the biological characteristics of the tumor and of the related tumor microenvironment (e.g. vasculature, immune cells) in patients. Even if several examples of translational success have been reported it is a matter of fact that clinical trials in oncology often fail to meet their primary endpoints despite encouraging preclinical data. For this reason, there is an increasing need of improved and more predictive models. This review aims to give an overview on existing mouse models for preclinical evaluation of cancer therapeutics and their applicability. Different types of mouse models commonly used for the evaluation of cancer therapeutics are described and considerations for a “fit-for purpose” application of these models for the evaluation of different cancer therapeutics dependent on their mode of action are outlined. Furthermore, considerations for study design and data interpretation to translatability of findings into the clinics are given. Conclusion: Detailed knowledge of the molecular/biological properties of the respective model, diligent experimental setup, and awareness of its limitations are indispensable prerequisites for the successful translational use of animal models. [Copyright &y& Elsevier]

Abstract: p53 is a transcriptional regulator in the nucleus that functions as a tumor suppressor and its mutations are frequently found in human tumors. It has been reported that p53 with R213Q mutation is exist in certain tumor cell lines and its methylation on R213 as well. However, the mechanisms and consequences of these modifications on p53 function are not fully understood. Mutations of p53 at R213Q (R/Q) and R213K (R/K) were respectively constructed and transfected into the p53 null H1299 cells. As shown in luciferase reporter assays, either R/Q or R/K disrupted the efficiency of p53 transactivation. EMSA and ChIP assays revealed that these mutants were less efficient in targeting the consensus binding sequences of p53 in the regulatory region of p21 gene. In addition, R/Q and R/K mutants attenuated the expression of p21 gene and counteracted the p53 mediated G1/S arrest to deliver a normal cell cycle progression as in the mock H1299 cells. Through this study, we have provided the first evidence on the pivotal role of arginine 213 that determines the p53 mediated functions of p21 in human cancer cells. [Copyright &y& Elsevier]

Abstract: T-cell acute lymphoblastic leukemia (T-ALL) is characterized as a high-risk stratified disease associated with frequent relapse, chemotherapy resistance, and a poorer prognostic outlook than B-precursor ALL. Many of the challenges in treating T-ALL reflect the lack of prognostic cytogenetic or molecular abnormalities on which to base therapy, including targeted therapy. Notch1 activating mutations were identified in more than 50% of T-ALL cases and can be therapeutically targeted with γ-secretase inhibitors (GSIs). Mutant Notch1 can activate cMyc and PI3K–AKT–mTOR1 signaling in T-ALL. In T-ALLs with wild-type phosphatase and tensin homolog deleted on chromosome ten (PTEN), Notch1 transcriptionally represses PTEN, an effect reversible by GSIs. Notch1 also promotes growth factor receptor (IGF1R and IL7Rα) signaling to PI3K–AKT. Loss of PTEN is common in primary T-ALLs due to mutation or posttranslational inactivation and results in chronic activation of PI3K–AKT–mTOR1 signaling, GSI-resistance, and repression of p53-mediated apoptosis. Notch1 itself might regulate posttranslational inactivation of PTEN. PP2A is activated by Notch1 in PTEN-null T-ALL cells, and GSIs reduce PP2A activity and increase phosphorylation of AKT, AMPK, and p70S6K. This review focuses on the central role of the PI3K–AKT–mTOR1 signaling in T-ALL, including its regulation by Notch1 and potential therapeutic interventions, with emphasis on GSI-resistant T-ALL. [Copyright &y& Elsevier]

Summary: Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients’ survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC. [ABSTRACT FROM AUTHOR]

Abstract: 3β-Hydroxysterol Δ24-reductase (DHCR24) catalyzes the conversion of desmosterol to cholesterol. This ultimate step of cholesterol biosynthesis appears to be remarkable in its diverse functions and the number of diseases it is implicated in from vascular disease to Hepatitis C virus (HCV) infection to cancer to Alzheimer’s disease. This review summarizes the present knowledge on the DHCR24 gene, sterol Δ24-reductase protein and the regulation of both. In addition, the functions of desmosterol, DHCR24 and their roles in human diseases are discussed. It is apparent that DHCR24 exerts more complex effects than what would be expected based on the enzymatic activity of sterol Δ24-reduction alone, such as its influence in modulating oxidative stress. Increasing information about DHCR24 membrane association, processing, enzymatic regulation and interaction partners will provide further fundamental insights into DHCR24 and its many and varied biological roles. [Copyright &y& Elsevier]

Abstract: Members of the DEAD box family of RNA helicases, which are characterised by the presence of twelve conserved motifs (including the signature D-E-A-D motif) within a structurally conserved ‘helicase’ core, are involved in all aspects of RNA metabolism. Apart from unwinding RNA duplexes, which established these proteins as RNA helicases, DEAD box proteins have been shown to also catalyse RNA annealing and to displace proteins from RNA. DEAD box proteins generally act as components of large multi-protein complexes and it is thought that interactions, via their divergent N- and C-terminal extensions, with other factors in the complexes may be responsible for the many different functions attributed to these proteins. In addition to their established crucial roles in the manipulation of RNA structure, it is becoming increasingly clear that several members of the DEAD box family act as regulators of transcription. In this review I shall focus on DDX5 (p68) and the highly related DDX17 (p72), two proteins for which there is a large body of evidence demonstrating that they function in transcriptional regulation. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life. [Copyright &y& Elsevier]

Abstract: MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions. [Copyright &y& Elsevier]

As the most common gene mutation found in cancers, p53 mutations are detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Meanwhile, mutant p53 overexpression is known to drive oncogenic phenotypes in cancer patients and to sustain the activation of EGFR signaling. Previously, we have demonstrated that the combined inhibition of EGFR and MDM2-p53 pathways, by gefitinib and JNJ-26854165, exerts a strong synergistic lethal effect on HGSOC cells. In this study, we investigated whether the gain-of-function p53 mutation (p53R248Q) overexpression could affect EGFR-related signaling and the corresponding drug inhibition outcome in HGSOC. The targeted inhibition responses of gefitinib and JNJ-26854165, in p53R248Q-overexpressing cells, were extensively evaluated. We found that the phosphorylation of AKT increased when p53R248Q was transiently overexpressed. Immunocytochemistry analysis further showed that upon p53R248Q overexpression, several AKT-related regulatory proteins translocated in unique intracellular patterns. Subsequent analysis revealed that, under the combined inhibition of gefitinib and JNJ-26854165, the cytonuclear trafficking of EGFR and MDM2 was disrupted. Next, we analyzed the gefitinib and JNJ-26854165 responses and found differential sensitivity to the single- or combined-drug inhibitions in p53R248Q-overexpressing cells. Our findings suggested that the R248Q mutation of p53 in HGSOC caused significant changes in signaling protein function and trafficking, under EGFR/MDM2-targeted inhibition. Such knowledge could help to advance our understanding of the role of mutant p53 in ovarian carcinoma and to improve the prognosis of patients receiving EGFR/MDM2-targeted therapies. [ABSTRACT FROM AUTHOR]

Background: Metallothionein 1M (MT1M) functions to regulate cell proliferation and cancer metastasis. This study assessed the effects of MT1M overexpression and mouse double minute 2 homolog (MDM2) knockdown on the regulation of non-small cell lung cancer A549 cell viability, migration, and protein expression in vitro and explored the underlying molecular events., Methods: A549 cells were stably infected with lentivirus carrying MT1M cDNA or transiently transfected MDM2 siRNA and/or treated with the p53 inhibitor for the assessment of changes in cell viability, wound healing, Transwell migration, and qRT-PCR and Western blot assays. Luciferase reporter assay was performed to investigate p53 binding to the MT1M promoter., Results: The data showed that MT1M overexpression inhibited A549 cell viability and migration capacity in vitro , whereas the p53 inhibitor reversed the inhibition of A549 cell viability and migration caused by MT1M overexpression as well as the expression of MMP2, MMP9, and MMP14. Furthermore, knockdown of MDM2, an upstream inhibitor of p53 activity, was able to reduce A549 cell viability, migration, and protein expression. Thus, MDM2 knockdown had synergistic effects with MT1M overexpression on the suppression of A549 cell viability, migration, and protein expression., Conclusions: In conclusion, MDM2 can bind to and phosphorylate p53 protein to inactivate the protein, thereby reducing MT1M expression and leading to tumor cell proliferation and migration., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2020.02.61). The authors have no conflicts of interest to declare., (2020 Translational Cancer Research. All rights reserved.)

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kβ, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR., (© 2019 Han et al.)

Prolyl hydroxylase 3 (PHD3) has initially been reported to hydroxylase hypoxia-inducible factor α (HIFα) and mediate HIFα degradation. More recent studies have shown that, in addition to HIFα, PHD3 has also other substrates. Moreover, pHD3 is believed to act as a tumor suppressor, but the underlying mechanism remains to be elucidated. Here, we demonstrate that PHD3 stabilizes p53 in a hydroxylase-independent manner. We found that PHD3 overexpression increases and PHD3 knockdown decreases p53 levels. Mechanistically, PHD3 bound MDM2 proto-oncogene (MDM2) and prevented MDM2 from interacting with p53, thereby inhibiting MDM2-mediated p53 degradation. Interestingly, we found that PHD3 overexpression could enhance p53 in the presence of the prolyl hydroxylase inhibitor dimethyloxalylglycine, and the prolyl hydroxylase activity-deficient variant PHD3-H196A also inhibited the p53-MDM2 interaction and stabilized p53. Genetic ablation of PHD3 decreased p53 protein levels in mice intestinal epithelial cells, but a genetic knockin of PHD3-H196A did not affect p53 protein levels in vivo These results suggest that the prolyl hydroxylase activity of PHD3 is dispensable for its ability to stabilize p53. We found that both PHD3 and PHD3-H196A suppress the expression of the stem cell-associated gene NANOG and inhibited the properties of colon cancer stem cells through p53. Our results reveal an additional critical mechanism underlying the regulation of p53 expression and highlight that PHD3 plays a role in the suppression of colon cancer cell stemness in a hydroxylase-independent manner., (© 2019 Xu et al.)

Autophagy promotes cancer cell survival in response to p53 activation by the anticancer agent Nutlin-3a (Nutlin). We reported previously that Nutlin kills MDM2-amplified cancer cells and that this killing is associated with an inhibition of glucose metabolism, reduced α-ketoglutarate (α-KG) levels, and reduced autophagy. In the current report, using SJSA1, U2OS, A549, and MHM cells, we found that Nutlin alters histone methylation in an MDM2 proto-oncogene-dependent manner and that this, in turn, regulates autophagy-related gene (ATG) expression and cell death. In MDM2-amplified cells, Nutlin increased histone (H) 3 lysine (K) 9 and K36 trimethylation (me3) coincident with reduced autophagy and increased apoptosis. Blocking histone methylation restored autophagy and rescued these cells from Nutlin-induced killing. In MDM2-nonamplified cells, H3K9me3 and H3K36me3 levels were either reduced or not changed by the Nutlin treatment, and this coincided with increased autophagy and cell survival. Blocking histone demethylation reduced autophagy and sensitized these cells to Nutlin-induced killing. Further experiments suggested that MDM2 amplification increases histone methylation in Nutlin-treated cells by causing depletion of the histone demethylase Jumonji domain-containing protein 2B (JMJD2B). Finally, JMJD2B knockdown or inhibition increased H3K9/K36me3 levels, decreased ATG gene expression and autophagy, and sensitized MDM2-nonamplified cells to apoptosis. Together, these results support a model in which MDM2- and JMJD2B-regulated histone methylation levels modulate ATG gene expression, autophagy, and cell fate in response to the MDM2 antagonist Nutlin-3a., (© 2019 Duan et al.)

The tumor suppressor protein p53 is inactivated in a large variety of cancer cells. Cellular p53 inhibitors like the mouse double minute 2 homolog (MDM2) commonly suppress the p53 function in acute myeloid leukemia (AML). Moreover, fms like tyrosine kinase 3 (FLT3) growth factor signaling pathways including the mitogen-activated kinase (MAPK) cascade (RAS-RAF-MEK-ERK) are highly active in AML cells. Consequently, the combined administration of MDM2 and MEK inhibitors may present a promising anti-leukemic treatment strategy. Here we assessed the MDM2 antagonist idasanutlin and the MEK1 inhibitor cobimetinib as single agents and in combination in a variety of AML cell lines and primary AML blast cells for their ability to induce apoptosis and cell death. AML cell lines and blast cells comprised all major AML subtypes based on the mutational status of TP53, FLT3 and NPM1 genes. We observed a considerably varying anti-leukemic efficacy of idasanutlin and cobimetinib. AML cells with high sensitivity to the single compounds as well as to the combined treatment emerged with normal karyotype, wild-type TP53 and elevated FLT3 and MDM2 protein levels. Our data indicate that AML cells with normal karyotype (NK) and wild-type status of TP53 with elevated FLT3 and MDM2 expression emerge to be most sensitive to the combined treatment with cobimetinib and idasanutlin. FLT3 and MDM2 are biomarkers for treatment response to idasanutlin and cobimetinib in AML. [ABSTRACT FROM AUTHOR]

Hepatocelluar carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide and among the leading causes of cancer-related death. Although deregulation of microRNAs has been frequently described in HCC, imperfection is known about the precise molecular mechanisms by which microRNAs modulate the process of tumorogenesis and behavior of cancer cells. In this study, we demonstrated that miR-758-3p could suppress cell proliferation, migration and invasion in hepatocellular carcinoma cells. We screened and identified two novel miR-758-3p targets, MDM2 and mTOR. Up-regulation of miR-758-3p could specifically and markedly down-regulate the expression of MDM2 and mTOR. Additionally, miR-758-3p over-expression displayed significant suppression in HCC development. To identify the mechanisms, we further investigated the P53 and mTOR pathway and found that p-p70S6 kinase(Ser371), p-p70 S6 kinase(Thr389) and p-4E-BP1were dramatically down-regulated after miR-758-3p transfection, while an enhanced expression of P53, AKT and PRAS40 were visualized, thus suggesting that the role of miR-758-3p in HCC progression may be associated with MDM2-p53 and mTOR signaling pathways. Collectively, our results indicate that miR-758-3pserves as a tumor suppressor and plays a crucial role in inhibiting the proliferation, migration and invasion of HCC via targeting MDM2 and mTOR and implicate its potential application in cancer therapy., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Utilizing the TCGA PANCAN12 dataset we discovered that cancer patients with mutations in TP53 tumor suppressor and overexpression of MDM2 oncogene exhibited decreased survival post treatment. Interestingly, in the case of breast cancer patients, this phenomenon correlated with high expression level of several molecular chaperones belonging to the HSPA, DNAJB and HSPC families. To verify the hypothesis that such a genetic background may promote chaperone-mediated chemoresistance, we employed breast and lung cancer cell lines that constitutively overexpressed heat shock proteins and have shown that HSPA1A/HSP70 and DNAJB1/HSP40 facilitated the binding of mutated p53 to the TAp73α protein. This chaperone-mediated mutated p53-TAp73α complex induced chemoresistance to DNA damaging reagents, like Cisplatin, Doxorubicin, Etoposide or Camptothecin. Importantly, when the MDM2 oncogene was overexpressed, heat shock proteins were displaced and a stable multiprotein complex comprising of mutated p53-TAp73α-MDM2 was formed, additionally amplifying cancer cells chemoresistance. Our findings demonstrate that molecular chaperones aid cancer cells in surviving the cytotoxic effect of chemotherapeutics and may have therapeutic implications., Competing Interests: CONFLICTS OF INTEREST The authors claim no conflicts of interest.

Deregulated receptor tyrosine kinase (RTK) signaling is frequently associated with tumorigenesis and therapy resistance, but its underlying mechanisms still need to be elucidated. In this study, we have shown that the RTK human epidermal growth factor receptor 4 (Her4, also known as Erbb4) can inhibit the tumor suppressor p53 by regulating MDMX-mouse double minute 2 homolog (MDM2) complex stability. Upon activation by either overexpression of a constitutively active vector or ligand binding (Neuregulin-1), Her4 was able to stabilize the MDMX-MDM2 complex, resulting in suppression of p53 transcriptional activity, as shown by p53-responsive element-driven luciferase assay and mRNA levels of p53 target genes. Using a phospho-proteomics approach, we functionally identified a novel Her4-induced posttranslational modification on MDMX at Ser-314, a putative phosphorylation site for the CDK4/6 kinase. Remarkably, inhibition of Ser-314 phosphorylation either with Ser-to-Ala substitution or with a specific inhibitor of CDK4/6 kinase blocked Her4-induced stabilization of MDMX-MDM2 and rescued p53 activity. Our study offers insights into the mechanisms of deregulated RTK-induced carcinogenesis and provides the basis for the use of inhibitors targeting RTK-mediated signals for p53 restoration., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

MDM2 and p53 form a negative feedback loop, in which p53 as a transcription factor positively regulates MDM2 and MDM2 negatively regulates tumor suppressor p53 through promoting its degradation. However, the mechanism of the feedback loop is poorly understood in cancers. We had reported previously that the oncoprotein hepatitis B X-interacting protein (HBXIP) is a key oncoprotein in the development of cancer. Thus, we supposed that HBXIP might be involved in the event. Here, we observed that the expression levels of HBXIP were positively correlated to those of MDM2 in clinical breast cancer tissues. Interestingly, HBXIP was able to up-regulate MDM2 at the levels of mRNA and protein in MCF-7 breast cancer cells. Mechanically, HBXIP increased the promoter activities of MDM2 through directly binding to p53 in the P2 promoter of MDM2. Strikingly, we identified that the acetyltransferase p300 was recruited by HBXIP to p53 in the promoter of MDM2. Moreover, we validated that HBXIP enhanced the p53 degradation mediated by MDM2. Functionally, the knockdown of HBXIP or/and p300 inhibited the proliferation of breast cancer cells in vitro, and the depletion of MDM2 or overexpression of p53 significantly blocked the HBXIP-promoted growth of breast cancer in vitro and in vivo. Thus, we concluded that highly expressed HBXIP accelerates the MDM2-mediated degradation of p53 in breast cancer through modulating the feedback loop of MDM2/p53, resulting in the fast growth of breast cancer cells. Our findings provide new insights into the mechanism of the acceleration of the MDM2/p53 feedback loop in the development of cancer., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G1 to S phase transition of cells by enhancing CDK inhibitor p21(Cip-1/Waf1) expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB(Ser-780)) followed by the up-regulation of E2F1 target genes required to promote G1 to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB(Ser-780) levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G1 phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21(Cip-1/Waf1) pathway., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while down-regulating the satellite cell marker Pax7. CCAAT/Enhancer-binding protein β (C/EBPβ) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPβ protein is rapidly down-regulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPβ protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPβ for degradation by the 26 S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPβ levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin-injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPβ, suggesting that the regulation of C/EBPβ turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPβ for degradation by the 26 S proteasome., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Myeloid Elf-1-like factor (MEF) or Elf4 is an ETS transcription factor that activates innate immunity-associated genes such as lysozyme (LYZ), human β-defensin 2 (HβD2), and interleukin-8 (IL-8) in epithelial cells and is also known to influence cell cycle progression. MEF is transcriptionally activated by E2F1, but the E2F1-mediated transcriptional activation is inhibited by p53 through E2F1-p53 protein interaction. Although the transcriptional activation of MEF has been investigated in depth, its post-translational regulation is not well explored. By overexpressing MEF cDNA in human cell lines, here we show that MEF protein expression is suppressed by p53. By screening a number of E3 ligases regulated by p53, we found that MDM2 is involved in the effect of p53 on MEF. MDM2 is transcriptionally activated by p53 and interacts with MEF protein to enhance MEF degradation. MDM2 reduces MEF protein expression, as well as stability and function of MEF as transcriptional activator. Furthermore, MDM2 was able to down-regulate MEF in the absence of p53, indicating a p53-independent effect on MEF. Notably, MEF transcriptionally activates MDM2, which was previously demonstrated to be the mechanism by which MEF suppresses the p53 protein. These results reveal that in addition to the potential of MEF to down-regulate p53 by transcriptionally activating E3 ligase MDM2, MEF participates with MDM2 in a novel autoregulatory feedback loop to regulate itself. Taken together with the findings on the effect of p53 on MEF, these data provide evidence that the p53-MDM2-MEF axis is a feedback mechanism that exquisitely controls the balance of these transcriptional regulators., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)