Transfected SARS-CoV-2 spike DNA for mammalian cell expression inhibits p53 activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2 proteins in cancer cells and increases cancer cell viability after chemotherapy exposure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike were found to have increased cell viability as compared to control cells. Further observations on γ-H2AX expression in spike-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway. The preliminary observations reported here warrant further studies to unravel the impact of SARS-CoV-2 and its various encoded proteins including spike on pathways of tumorigenesis and response to cancer therapeutics. More efforts should be directed at studying the effects of the SARS-CoV-2 spike and other viral proteins on host DNA damage sensing, response and repair mechanisms. A goal would be to understand the structural basis for maximal anti-viral immunity while minimizing suppression of host defenses including the p53 DNA damage response and tumor suppression pathway. Such directions are relevant and important including not only in the context of viral infection and mRNA vaccines in general but also for patients with cancer who may be receiving cytotoxic or other cancer treatments.

MDM2 Inhibitors for Cancer Therapy: The Past, Present, and Future.

Since its discovery over 35 years ago, MDM2 has emerged as an attractive target for the development of cancer therapy. MDM2's activities extend from carcinogenesis to immunity to the response to various cancer therapies. Since the report of the first MDM2 inhibitor more than 30 years ago, various approaches to inhibit MDM2 have been attempted, with hundreds of small-molecule inhibitors evaluated in preclinical studies and numerous molecules tested in clinical trials. Although many MDM2 inhibitors and degraders have been evaluated in clinical trials, there is currently no Food and Drug Administration (FDA)-approved MDM2 inhibitor on the market. Nevertheless, there are several current clinical trials of promising agents that may overcome the past failures, including agents granted FDA orphan drug or fast-track status. We herein summarize the research efforts to discover and develop MDM2 inhibitors, focusing on those that induce MDM2 degradation and exert anticancer activity, regardless of the p53 status of the cancer. We also describe how preclinical and clinical investigations have moved toward combining MDM2 inhibitors with other agents, including immune checkpoint inhibitors. Finally, we discuss the current challenges and future directions to accelerate the clinical application of MDM2 inhibitors. In conclusion, targeting MDM2 remains a promising treatment approach, and targeting MDM2 for protein degradation represents a novel strategy to downregulate MDM2 without the side effects of the existing agents blocking p53-MDM2 binding. Additional preclinical and clinical investigations are needed to finally realize the full potential of MDM2 inhibition in treating cancer and other chronic diseases where MDM2 has been implicated. SIGNIFICANCE STATEMENT: Overexpression/amplification of the MDM2 oncogene has been detected in various human cancers and is associated with disease progression, treatment resistance, and poor patient outcomes. This article reviews the previous, current, and emerging MDM2-targeted therapies and summarizes the preclinical and clinical studies combining MDM2 inhibitors with chemotherapy and immunotherapy regimens. The findings of these contemporary studies may lead to safer and more effective treatments for patients with cancers overexpressing MDM2.

The MDM2-p53 Axis Represents a Therapeutic Vulnerability Unique to Glioma Stem Cells.

The prevention of tumor recurrence by the successful targeting of glioma stem cells endowed with a tumor-initiating capacity is deemed the key to the long-term survival of glioblastoma patients. Glioma stem cells are characterized by their marked therapeutic resistance; however, recent evidence suggests that they have unique vulnerabilities that may be therapeutically targeted. We investigated MDM2 expression levels in glioma stem cells and their non-stem cell counterparts and the effects of the genetic and pharmacological inhibition of MDM2 on the viability of these cells as well as downstream molecular pathways. The results obtained showed that MDM2 expression was substantially higher in glioma stem cells than in their non-stem cell counterparts and also that the inhibition of MDM2, either genetically or pharmacologically, induced a more pronounced activation of the p53 pathway and apoptotic cell death in the former than in the latter. Specifically, the inhibition of MDM2 caused a p53-dependent increase in the expression of BAX and PUMA and a decrease in the expression of survivin, both of which significantly contributed to the apoptotic death of glioma stem cells. The present study identified the MDM2-p53 axis as a novel therapeutic vulnerability, or an Achilles' heel, which is unique to glioma stem cells. Our results, which suggest that non-stem, bulk tumor cells are less sensitive to MDM2 inhibitors, may help guide the selection of glioblastoma patients suitable for MDM2 inhibitor therapy.

Microfluidic Fabricated Liposomes for Nutlin-3a Ocular Delivery as Potential Candidate for Proliferative Vitreoretinal Diseases Treatment.

Purpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.

Synthesis and biological evaluation of 4-imidazolidinone-containing compounds as potent inhibitors of the MDM2/p53 interaction.

Inhibition of MDM2/p53 interaction with small-molecule inhibitors stabilizes p53 from MDM2 mediated degradation, which is a promising strategy for the treatment of cancer. In this report, a novel series of 4-imidazolidinone-containing compounds have been synthesized and tested in MDM2/p53 and MDM4/p53 FP binding assays. Upon SAR studies, compounds 2 (TB114) and 22 were identified as the most potent inhibitors of MDM2/p53 but not MDM4/p53 interactions. Both 2 and 22 exhibited strong antiproliferative activities in HCT-116 and MOLM-13 cell lines harboring wild type p53. Mechanistic studies show that 2 and 22 dose-dependently activated p53 and its target genes and induced apoptosis in cells based on the Western blot, qPCR, and flow cytometry assays. In addition, the antiproliferative activities of 2 and 22 were dependent on wild type p53, while they were not toxic to HEK-293 kidney cells. Furthermore, the on-target activities of 2 were general and applicable to other cancer cell lines with wild type p53. These attributes make 2 a good candidate for future optimization to discover a potential treatment of wild-type p53 cancer.

Discovery of ganoderic acid A (GAA) PROTACs as MDM2 protein degraders for the treatment of breast cancer.

Breast cancer is one of the most common female malignant tumors, with triple-negative breast cancer (TNBC) being the most specific, highly invasive, metastatic and associated with a poor prognosis. Our previous study showed that the natural product ganoderic acid A (GAA) has a certain affinity for MDM2. In this study, two series of novel GAA PROTACs C1-C10 and V1-V10 were designed and synthesized for the treatment of breast cancer. The antitumor activity of these compounds was evaluated against four human tumor cell lines (MCF-7, MDA-MB-231, SJSA-1, and HepG2). Among them, V9 and V10 showed stronger anti-proliferative effects against breast cancer cells, and V10 showed the best selectivity in MDA-MB-231 cells (TNBC), which was 5-fold higher than that of the lead compound GAA. Preliminary structure-activity analysis revealed that V-series GAA PROTACs had better effects than C-series, and the introduction of 2O-4O PEG linkers could significantly improve the antitumor activity. Molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and Western blot researches showed that both V9 and V10 could bind with MDM2, and degrade the protein through the ubiquitin-proteasome system. Molecular dynamics simulation (MD) revealed that V10 is a bifunctional molecule that can bind to von Hippel-Lindau (VHL) at one end and target MDM2 at the other. In addition, V10 promoted the upregulation of p21 in p53-mutant MDA-MB-231 cells, and induced apoptosis via down-regulation of the bcl-2/bax ratio and the expression of cyclin B1. Finally, in vivo experiments showed that, V10 also exhibited good tumor inhibitory activity in xenografted TNBC zebrafish models, with an inhibition rate of 27.2% at 50 µg/mL. In conclusion, our results suggested that V10 has anti-tumor effects on p53-mutant breast cancer in vitro and in vivo, and may be used as a novel lead compound for the future development of TNBC.

Primary hepatic pleomorphic liposarcoma: Case report and literature review.

Primary hepatic liposarcoma is an extremely rare malignant tumour derived from adipocytes and is part of the group of mesenchymal tumours. We present the case of a 43-year-old Hispanic male patient with a pleomorphic hepatic liposarcoma and absence of MDM2 gene amplification. Two years and six months after surgery, the patient is asymptomatic. The present case is the first report of this entity with positive immunohistochemical testing for p16, p53, S100, vimentin and absence of MDM2 gene amplification.

Xinnaotongluo liquid protects H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3.

Xinnaotongluo liquid has been used to improve the clinical symptoms of patients with myocardial infarction. However, the molecular mechanism of Xinnaotongluo liquid is not completely understood. H9c2 cells exposed to hypoxia/reoxygenation (H/R) was used to simulate damage to cardiomyocytes in myocardial infarction in vitro. The biological indicators of H9c2 cells were measured by cell counting kit-8, enzyme linked immunoabsorbent assay, and western blot assay. In H/R-induced H9c2 cells, a markedly reduced murine double minute 2 (MDM2) was observed. However, the addition of Xinnaotongluo liquid increased MDM2 expression in H/R-induced H9c2 cells. And MDM2 overexpression strengthened the beneficial effects of Xinnaotongluo liquid on H9c2 cells from the perspective of alleviating oxidative damage, cellular inflammation, apoptosis and ferroptosis of H/R-induced H9c2 cells. Moreover, MDM2 overexpression reduced the protein expression of p53 and Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3). Whereas, STEAP3 overexpression hindered the function of MDM2-overexpression in H/R-induced H9c2 cells. Our results insinuated that Xinnaotongluo liquid could protect H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3, which provide a potential theoretical basis for further explaining the working mechanism of Xinnaotongluo liquid.

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation.

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity; however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with the inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.

Well-differentiated liposarcomas and dedifferentiated liposarcomas: Systemic treatment options for two sibling neoplasms.

Well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPS) account for 60 % of all liposarcomas, reflecting the heterogeneity of this type of sarcoma. Genetically, both types of liposarcomas are characterized by the amplification of MDM2 and CDK4 genes, which indicates an important molecular event with diagnostic and therapeutic relevance. In both localized WDLPS and DDLPS of the retroperitoneum and the extremities, between 25 % and 30 % of patients have local or distant recurrence, even when perioperatively treated, with clear margins present. The systemic treatment of WDLPS and DDLPS remains a challenge, with anthracyclines as the gold standard for first-line treatment. Several regimens have been tested with modest results regarding their efficacy. Herein we discuss the systemic treatment options for WDLPS and DDLPS and review their reported clinical efficacy results.

[Detection of MDM2 gene amplification by fluorescence in situ hybridization and its diagnostic value in low-grade osteosarcoma].

Objective: To investigate the diagnostic value of detecting MDM2 gene amplification by fluorescence in situ hybridization (FISH) in low-grade osteosarcoma (LGOS). Methods: Thirty cases of parosteal osteosarcoma (POS) and 14 cases of low-grade central osteosarcoma (LGCOS) from April 2009 to August 2022 at Beijing Jishuitan Hospital, Capital Medical University were analyzed for the presence of MDM2 gene amplification by FISH. Fifty-eight additional cases were used as negative controls (including 28 cases of fibrous dysplasia, 5 cases of giant cell tumor, 4 cases of conventional osteosarcoma, 2 cases each of periosteal osteosarcoma, reparative changes after fracture, pleomorphic undifferentiated sarcoma, low grade myofibroblastic sarcoma, fibrous dysplasia with malignant transformation, one case each of leiomyosarcoma, sclerosing epithelioid fibrosarcoma, malignant peripheral nerve sheath tumor, desmoplastic fibroma of bone, solitary fibrous tumor, aneurysmal bone cyst, clear cell chondrosarcoma, osteofibrous dysplasia, and 3 cases of unclassified spindle cell tumor). Results: Among the 30 patients with POS, 15 were male and 15 were female, ranging in age from 10 to 59 years (mean 35 years, median 30.5 years). Among the 14 patients with LGCOS, four were male and 10 were female, ranging in age from 15 to 56 years (mean 37 years, median 36 years). All except one case were successfully detected by FISH. MDM2 gene amplification was detected in 27 cases of POS (27/29,91.3%) and 8 cases of LGCOS (8/14). All the negative controls were negative for MDM2 gene amplification. The positive rate of MDM2 gene amplification was significantly different between the case group and the control group (P<0.05). The sensitivity and specificity of MDM2 gene amplification in diagnosing POS and LGCOS were 91.3% and 100.0%; and 57.1% and 100.0%, respectively. The sensitivity and specificity of MDM2 gene amplification in diagnosing LGOS (including POS and LGCOS) were 81.3% and 100.0%, respectively. In cases where MDM2 gene was amplified, the MDM2 amplified signal was clustered. Nine cases showed increased CEP12 signal different from polyploidy which was displayed as small and weak signal points or cloud flocculent and cluster signals. Conclusions: Detection of MDM2 gene amplification by FISH is a highly sensitive and specific marker for LGOS. The interpretation criteria for FISH detection of MDM2 amplification are currently not unified. The signal characteristics need more attention when interpreting.

Deciphering the Multi-state Conformational Equilibrium of HDM2 in the Regulation of p53 Binding: Perspectives from Molecular Dynamics Simulation and NMR Analysis.

HDM2 negatively regulates the activity of the tumor suppressor p53. Previous NMR studies have shown that apo-HDM2 interconverts between an "open" state in which the N-terminal "lid" is disordered and a "closed" state in which the lid covers the p53-binding site in the core region. Molecular dynamics (MD) simulation studies have been performed to elucidate the conformational dynamics of HDM2, but the direct relevance of the experimental and computational analyses is unclear. In addition, how the phosphorylation of S17 in the lid contributes to the inhibition of p53 binding remains controversial. Here, we used both NMR and MD simulations to investigate the conformational dynamics of apo-HDM2. The NMR analysis revealed that apo-HDM2 exists in a fast-exchanging equilibrium within two closed states, closed 1 and closed 2, in addition to a previously demonstrated slow-exchanging "open-closed" equilibrium. MD simulations visualized two characteristic closed states, where the spatial orientation of the key residues corresponds well to the chemical shift changes of the NMR spectra. Furthermore, the phosphorylation of S17 induced an equilibrium shift toward closed 1, thereby suppressing the binding of p53 to HDM2. This study reveals a multi-state equilibrium of apo-HDM2 and provides new insights into the regulation mechanism of HDM2-p53 interactions.

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma.

Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.

DDIT3-amplified or low-polysomic pleomorphic sarcomas without MDM2 amplification: Clinicopathological review and immunohistochemical profile of nine cases.

Several high-grade pleomorphic sarcoma cases that cannot be classified into any existing established categories have been reported. These cases were provisionally classified into undifferentiated pleomorphic sarcoma (UPS). Some dedifferentiated liposarcoma (DDLS) cases may also have been classified into the UPS category due to the absence of MDM2 amplification or an atypical lipomatous tumor/well-differentiated liposarcoma component. We retrieved and reviewed 77 high-grade pleomorphic sarcoma cases, initially diagnosed as UPS in 66 cases and DDLS in 11 cases. Fluorescence in situ hybridization (FISH) analyses of DDIT3 and MDM2 were performed for available cases. Of the cases successfully subjected to DDIT3 FISH (n = 56), nine (7 UPS and 2 DDLS) showed DDIT3 amplification but no MDM2 amplification. Two UPS cases showed both telomeric (5') and centromeric (3') amplification of DDIT3 or low polysomy of chromosome 12, whereas 5 UPS and 2 DDLS cases showed 5'-predominant DDIT3 amplification. Histopathologically, all cases showed UPS-like proliferation of atypical pleomorphic tumor cells. Immunohistochemically, only one case showed focal nuclear positivity for DDIT3, supporting the previous finding that DDIT3 expression was not correlated with DDIT3 amplification. All three cases with focal MDM2 expression involved 5'-predominant amplification, two of which showed DDLS-like histological features. The majority of cases (7/9) showed decreased expression in p53 staining, suggesting that DDIT3 amplification regulates the expression of TP53 like MDM2. From a clinicopathological perspective, we hypothesize that DDIT3-amplified sarcoma, especially with 5'-predominant amplification, can be reclassified out of the UPS category.

Superficial dedifferentiated liposarcoma: A clinicopathologic study.

INTRODUCTION: Dedifferentiation occurs in approximately 10% of atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDLPS), primarily in retroperitoneal or deep-seated tumors, conferring metastatic potential. Superficial dedifferentiated liposarcoma (sDDLPS) is rare, and its progression and natural history are poorly documented. METHODS: We performed a 15-year retrospective review of our pathology database to identify cases of DDLPS in the skin or subcutaneous tissue. Diagnosis of primary sDDLPS required evidence of non-lipogenic sarcoma in the skin or subcutis, with concurrent ALT/WDLPS and/or MDM2 amplification. RESULTS: We identified 14 cases of DDLPS involving skin or subcutis: 7 primary sDDLPS and 7 secondary lesions (3 from recurrent deep DDLPS and 4 from metastasis). Primary sDDLPS cases (4 females, 3 males; median age: 74) mainly presented as undifferentiated spindle cell or pleomorphic sarcoma. Tumor grades were grade 2 (5 cases) and grade 3 (2 cases), with three cases also showing grade 1 areas. MDM2 amplification was confirmed in 6 sDDLPSs for which FISH was successfully performed. Follow-up available for 6 sDDLPS patients showed 2 local recurrences, treated with re-excision and radiation therapy, with all disease-free at last follow-up (5-126 months). Of the 7 secondary cases, 2 had ongoing disease after multiple recurrences, 1 was disease-free, and all 4 with cutaneous metastasis died of disease (follow-up range: 24-263 months). CONCLUSION: These findings emphasize the importance of distinguishing between primary sDDLPS and secondary lesions due to their distinct prognoses. Metastasis or superficial extensions from deep DDLP correlate with a considerably worse prognosis than those originating in superficial tissues.

Nanoencapsulation of MDM2 Inhibitor RG7388 and Class-I HDAC Inhibitor Entinostat Enhances their Therapeutic Potential Through Synergistic Antitumor Effects and Reduction of Systemic Toxicity.

Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells. We investigated whether coencapsulation of RG7388 and Entinostat within polymeric nanoparticles (NPs) could overcome efficacy and toxicity limitations of this drug combination. Combinations of RG7388 and Entinostat across a range of different molar ratios resulted in synergistic increases in cell death when delivered in both free drug and nanoencapsulated formats in all colorectal cell lines tested. Importantly, we also explored the in vivo impact of the drug combination on murine blood leukocytes, showing that the leukopenia induced by the free drugs could be significantly mitigated by nanoencapsulation. Taken together, this study demonstrates that formulating these agents within a single nanoparticle delivery platform may provide clinical utility beyond use as nonencapsulated agents.

Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 µM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Association of MDM2 Overexpression in Ameloblastomas with MDM2 Amplification and BRAFV600E Expression.

Ameloblastoma is a rare tumor but represents the most common odontogenic neoplasm. It is localized in the jaws and, although it is a benign, slow-growing tumor, it has an aggressive local behavior and high recurrence rate. Therefore, alternative treatment options or complementary to surgery have been evaluated, with the most promising one among them being a targeted therapy with the v-Raf murine sarcoma viral oncogene homologue B (BRAF), as in ameloblastoma the activating mutation V600E in BRAF is common. Studies in other tumors have shown that the synchronous inhibition of BRAF and human murine double minute 2 homologue (MDM2 or HDM2) protein is more effective than BRAF monotherapy, particularly in the presence of wild type p53 (WTp53). To investigate the MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAFV600E and p53 expression. Forty-four cases of ameloblastoma fixed in 10% buffered formalin and embedded in paraffin were examined for MDM2 overexpression and BRAFV600E and p53 expression by immunohistochemistry, and for MDM2 ploidy with fluorescence in situ hybridization. Sixteen of forty-four (36.36%) cases of ameloblastoma showed MDM2 overexpression. Seven of sixteen MDM2-positive ameloblastomas (43.75%) were BRAFV600E positive and fifteen of sixteen MDM2-positive ameloblastomas (93.75%) were p53 negative. All MDM2 overexpressing tumors did not show copy number alterations for MDM2. Overexpression of MDM2 in ameloblastomas is not associated with MDM2 amplification, but most probably with MAPK activation and WTp53 expression. Further verification of those findings could form the basis for the use of MDM2 expression as a marker of MAPK activation in ameloblastomas and the trial of dual BRAF/MDM2 inhibition in the management of MDM2-overexpressing/BRAFV600E-positive/WTp53 ameloblastomas.

miR-34c-5p inhibited fibroblast proliferation, differentiation and epithelial-mesenchymal transition in benign airway stenosis via MDMX/p53 pathway.

Benign airway stenosis (BAS) means airway stenosis or obstruction that results from a variety of non-malignant factors, including tuberculosis, trauma, benign tumors, etc. In consideration of the currently limited research on microRNAs in BAS, this study aimed to explore the role and mechanism of miR-34c-5p in BAS. The expression of miR-34c-5p in BAS granulation tissues showed a significant down-regulation compared with the normal control group. Moreover, miR-34c-5p mimics suppressed the proliferation and differentiation of human bronchial fibroblasts (HBFs) and the epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells (HBE). Conversely, miR-34c-5p inhibitors aggravated those effects. A dual-luciferase reporter assay confirmed that miR-34c-5p can target MDMX rather than Notch1. The over-expression of MDMX can reverse the inhibiting effect of miR-34c-5p on HBFs proliferation, differentiation and EMT. Furthermore, the expressions of tumor protein (p53) and PTEN were down-regulated following the over-expression of MDMX. In addition, the expressions of PI3K and AKT showed an up-regulation. In conclusion, miR-34c-5p was down-regulated in BAS and may inhibit fibroblast proliferation differentiation and EMT in BAS via the MDMX/p53 signaling axis. These findings expand the understanding of the role of miR-34c-5p and will help develop new treatment strategies for BAS.