MDM2 Research Articles

P53 Signaling Pathway Polymorphisms Associated With Emphysematous Changes in Patients With COPD

The p53 signaling pathway may be important for the pathogenesis of emphysematous changes in the lungs of smokers. Polymorphism of p53 at codon 72 is known to affect apoptotic effector proteins, and the polymorphism of mouse double minute 2 homolog (MDM2) single nucleotide polymorphism (SNP)309 is known to increase MDM2 expression. The aim of this study was to assess polymorphisms of the p53 and MDM2 genes in smokers and confirm the role of SNPs in these genes in the pathogenesis of pulmonary emphysema. This study included 365 patients with a smoking history, and the polymorphisms of p53 and MDM2 genes were identified. The degree of pulmonary emphysema was determined by means of CT scanning. SNPs, MDM2 mRNA, and p53 protein levels were assessed in human lung tissues from smokers. Plasmids encoding p53 and MDM2 SNPs were used to transfect human lung fibroblasts (HLFs) with or without cigarette smoke extract (CSE), and the effects on cell proliferation and MDM2 promoter activity were measured. The polymorphisms of the p53 and MDM2 genes were associated with emphysematous changes in the lung and were also associated with p53 protein and MDM2 mRNA expression in the lung tissue samples. Transfection with a p53 gene-coding plasmid regulated HLF proliferation, and the analysis of P2 promoter activity in MDM2 SNP309-coding HLFs showed the promoter activity was altered by CSE. Our data demonstrated that p53 and MDM2 gene polymorphisms are associated with apoptotic signaling and smoking-related emphysematous changes in lungs from smokers.

Specific amplifications and copy number decreases during human neural stem cells differentiation towards astrocytes, neurons and oligodendrocytes.

There is growing evidence that gene amplifications are an attribute of normal cells during development and differentiation. During neural progenitor cell differentiation half of the genome is involved in amplification process. To answer the question how specific amplifications occur at different stages and in different lineages of differentiation we analyzed the genes CDK4, MDM2, EGFR, GINS2, GFAP, TP53, DDB1 and MDM4 in human neural stem cells that were induced to differentiate towards astrocytes, neurons and oligodendrocytes. We found specific amplification pattern for each of the eight analyzed genes both in undifferentiated neural stem and progenitor cells and in cells that were induced for differentiation. Different amplification patterns were also found between adherently grown neural stem cells and cells that were grown as spheres. The most frequently amplified genes were MDM2 and CDK4 with the latter amplified in all three lineages at all analyzed stages. Amplification of the analyzed genes was also found in four glioma stem-like cells. The combined amplification data of stem cells and of tumor stem cells can help to define cell populations at the origin of the tumor. Furthermore, we detected a decrease of gene copies at specific differentiation stages most frequently for MDM4. This study shows specific amplification pattern in defined stem cell populations within specific time windows during differentiation processes indicating that amplifications occur in an orderly sequence during the differentiation of human neural stem and progenitor cells.

Assessment of changes in isoforms of MDM2 gene by 5-fluorouracil in esophageal cancer cell lines (KYSE-30)

Assessment of changes in isoforms of MDM2 gene by 5-fluorouracil in esophageal cancer cell lines (KYSE-30)

TargetingMDM4SplicinginCancers.

MDM4, an essential negative regulator of the P53 tumor suppressor, is frequently overexpressed in cancer cells that harbor a wild-type P53. By a mechanism based on alternative splicing, the MDM4 gene generates two mutually exclusive isoforms: MDM4-FL, which encodes the full-length MDM4 protein, and a shorter splice variant called MDM4-S. Previous results suggested that the MDM4-S isoform could be an important driver of tumor development. In this short review, we discuss a recent set of data indicating that MDM4-S is more likely a passenger isoform during tumorigenesis and that targeting MDM4 splicing to prevent MDM4-FL protein expression appears as a promising strategy to reactivate p53 in cancer cells. The benefits and risks associated with this strategy are also discussed.

Anatomy of Mdm2 and Mdm4 in evolution.

Mouse double minute (Mdm) genes span an evolutionary timeframe from the ancient eukaryotic placozoa Trichoplax adhaerens to Homo sapiens, implying a significant and possibly conserved cellular role throughout history. Maintenance of DNA integrity and response to DNA damage involve many key regulatory pathways, including precise control over the tumour suppressor protein p53. In most vertebrates, degradation of p53 through proteasomal targeting is primarily mediated by heterodimers of Mdm2 and the Mdm2-related protein Mdm4 (also known as MdmX). Both Mdm2 and Mdm4 have p53-binding regions, acidic domains, zinc fingers, and C-terminal RING domains that are conserved throughout evolution. Vertebrates typically have both Mdm2 and Mdm4 genes, while analyses of sequenced genomes of invertebrate species have identified single Mdm genes, suggesting that a duplication event occurred prior to emergence of jawless vertebrates about 550–440 million years ago. The functional relationship between Mdm and p53 in T. adhaerens, an organism that has existed for 1 billion years, implies that these two proteins have evolved together to maintain a conserved and regulated function.

Regulation of kidney development by the Mdm2/Mdm4-p53 axis.

While p53 activity is required for tumour suppression, unconstrained p53 activity on the other hand is detrimental to the organism, resulting in inappropriate cellular death or proliferation defects. Unimpeded p53 activity is lethal in the developing embryo, underlining the need for maintaining a tight control on p53 activity during this period. The critical role of the negative regulators of p53, Mdm2 and Mdm4, in vertebrate development came to light by fatal disruption of embryogenesis that was observed with Mdm2 and Mdm4 gene deletions in mice. Embryonic lethality was rescued only by superimposing p53 removal. Here we summarize the contribution of the Mdm2/Mdm4–p53 axis that occurs at multiple steps of kidney development. Conditional, cell type-specific deletions reveal distinct functions of these proteins in renal morphogenesis. The severe impact on the renal phenotype from targeted gene deletions underscores the critical role played by the Mdm2/Mdm4–p53 nexus on nephrogenesis, and emphasizes the need to monitor patients with aberrations in this pathway for kidney function defects and associated cardiovascular dysfunction.

Invasive Cardiac Lipoma: a case report and review of literature

BackgroundCardiac lipomas are rare benign tumors of the heart. They are usually asymptomatic and are thus most often diagnosed on autopsies. Symptoms, when present, depend upon the location within the heart. Typical locations are the endocardium of the right atrium and the left ventricle. Diagnostic modality of choice is cardiac MRI. Treatment guidelines have not yet been established due to the very low prevalence of these tumors and are thus guided by the patient’s symptomatology.Case presentationWe describe a case of an invasive cardiac lipoma, wherein the initial symptom of the patient was shortness of breath. Although the echocardiogram visualized the tumor in the right atrium, a cardiac MRI was performed for better tissue characterization. The MRI revealed a large, fat containing, septated mass in the right atrium with invasion into the inter-atrial septum and inferior cavoatrial junction. There was also invasion of the coronary sinus along the inferior and left lateral aspect of the posterior atrioventricular groove. Although the mass appeared to represent a lipoma by imaging characteristics, the unusual extension into the coronary sinus led to consideration of a low-grade liposarcoma in the differential. Thus a pre-operative biopsy was performed along with MDM2 gene amplification to rule out a liposarcoma preceding surgical excision.ConclusionCardiac lipomas are well-characterized on cardiac MRI, which is the diagnostic modality of choice. Typical locations are the right atrium and the left ventricle. However, in those with atypical features such as invasion of the coronary sinus, pre-operative biopsy for histopathologic confirmation is imperative to exclude well-differentiated liposarcoma. Our patient with a simple lipoma underwent partial resection to relieve symptoms. We discuss prognosis and treatment of symptomatic cardiac lipomas.

MDM2 gene polymorphisms and risk of classic Kaposi's sarcoma among Iranian patients.

A single-nucleotide polymorphism (SNP) in the promoter region of MDM2 (SNP309T>G, rs2279744) has been shown to increase the expression of the MDM2 protein in various cancer types. However, only one study has analyzed the role of the MDM2 polymorphism in the development of Kaposi's sarcoma (KS). The association of MDM2 SNP309 with classic KS risk was evaluated in 79 Iranian patients with classic KS and 123 healthy controls. The MDM2 SNP309 was genotyped using PCR and restriction fragment length polymorphism methods. No significant correlation was found between the SNP309 polymorphism in MDM2 promoter and classic KS risk. There was no significant correlation between gender and disease stage. However, a significant association was found between SNP309 GG genotype and younger age (≤50years) (odds ratio 9.5, 95% confidence intervals 1.5-60, p = 0.03). Our findings support no major role for the MDM2 SNP309 in KS development although it might influence the clinical outcome of KS in younger patients.

Epoxy clerodane diterpene inhibits MCF-7 human breast cancer cell growth by regulating the expression of the functional apoptotic genes Cdkn2A, Rb1, mdm2 and p53.

Systematic analyses of plants that are used in traditional medicine may lead to the discovery of novel cytotoxic secondary metabolites. Diterpene possesses multiple bioactivities; here, epoxy clerodane diterpene (ECD) was isolated from Tinospora cordifolia (Willd.) stem and shown potential antiproliferative effect in MCF-7 human breast cancer cells. The antiproliferative effect of ECD on MCF-7 cells was systematically analyzed by cell and nuclear morphology, alterations in oxidative stress, and the expression of tumor suppressor and mitochondria-mediated apoptosis-related genes. We found that the IC50 value of ECD was 3.2μM at 24h and 2.4μM at 48h. We observed that the cytotoxicity of ECD was specific to MCF-7 cells, whereas ECD was nontoxic to normal Vero and V79 cells. ECD significantly triggered intracellular ROS generation even from the lower doses of 0.6 and 1.2μM; and it is relative to higher dose of 2.4μM. Further, we used 0.6μM, 1.2μM and 2.4μM as experimental doses to analyze the relative dose-dependent effects. Nuclear staining revealed that cells treated with the 2.4μM dose exhibited characteristic apoptotic morphological changes and that 46% of the cells were apoptotic and 4% were necrotic after 48h. ECD significantly increased the expression of mitochondria-dependent apoptotic pathway-related genes after 48h; we observed significantly (p≤0.05) increased expression of CYP1A, GPX, GSK3β and TNF-α and downregulated expression of NF-κB. ECD also increased the expression of tumor suppressor genes such as Cdkn2A, Rb1 and p53. In addition, we observed that ECD treatment significantly (p≤0.001) upregulated the expression of apoptotic genes such as Bax, cas-3, cas-8, cas-9 and p21 and downregulated the expression of BCL-2, mdm2 and PCNA. In conclusion, ECD regulates the expression of Cdkn2A, p53 and mdm2 and induces apoptosis via the mitochondrial pathway in MCF-7 human breast cancer cells.

Polymorphisms in apoptosis-related genes in cutaneous melanoma prognosis: sex disparity.

Cutaneous melanoma (CM) cells are resistant to apoptosis, and steroid hormones are involved in this process through regulation of TP53, MDM2, BAX, and BCL2 expression. We analyzed herein sex differences in outcomes of CM patients associated with TP53 c.215G>C, MDM2 c.309T>G, BAX c.-248G>A, and BCL2 c.-717C>A polymorphisms. DNA from 121 men and 116 women patients was analyzed by polymerase chain reaction and enzymatic digestion assays. At 60months of follow-up, shorter progression-free survival (PFS) was seen in males with MDM2 GG+BCL2 AA (20.0 vs. 62.6%, P=0.0008) genotype. Men carriers of the genotype had poor PFS (HR 3.78, 95% CI 1.30-11.0) than others. For women, shorter PFS was associated with TP53 GC or CC (61.4 vs. 80.8%, P=0.01) and TP53 GC or CC+MDM2 TG or GG (59.1 vs. 85.4%, P=0.01) genotypes at the same time. Women carriers of the genotypes had poor PFS (HR 2.46, 95% CI 1.19-5.09; HR 9.49, 95% CI 1.14-78.50) than others, respectively. Our data present, for the first time, preliminary evidence that inherited abnormalities on TP53, MDM2 and BCL2 genes, enrolled in apoptosis pathways, have a pivotal role in differences of outcomes in women and men with CM.

Classification of Bladder Cancer Patients via Penalized Linear Discriminant Analysis

Objectives:In order to identify genes with the greatest contribution to bladder cancer, we proposed a sparse model making the best discrimination from other patients.Methods:In a cross-sectional study, 22 genes with a key role in most cancers were considered in 21 bladder cancer patients and 14 participants of the same age (± 3 years) without bladder cancer in Shiraz city, Southern Iran. Real time-PCR was carried out using SYBR Green and for each of the 22 target genes 2-Δct as a quantitative index of gene expression was reported. We determined the most affective genes for the discriminant vector by applying penalized linear discriminant analysis using LASSO penalties. All the analyses were performed using SPSS version 18 and the penalized LDA package in R.3.1.3 software.Results:Using penalized linear discriminant analysis led to elimination of 13 less important genes. Considering the simultaneous effects of 22 genes with important influence on many cancers, it was found that TGFβ, IL12A, Her2, MDM2, CTLA-4 and IL-23 genes had the greatest contribution in classifying bladder cancer patients with the penalized linear discriminant vector. The receiver operating characteristic (ROC) curve revealed that the proposed vector had good performance with minimal (only 3) mis-classification. The area under the curve (AUC) of our proposed test was 96% (95%CI:83%-100%) and sensitivity, specificity, positive and negative predictive values were 90.5%, 85.7%, 90.5% and 85.7%, respectively.Conclusions:The penalized discriminant method can be considered as appropriate for classifying bladder cancer cases and searching for important biomarkers.

P16 Immunohistochemistry is Less Useful Than MDM2 and CDK4 to Distinguish Dedifferentiated Liposarcomas From Other Retroperitoneal Mimics.

Dedifferentiated liposarcoma (DDL) frequently involves the retroperitoneum. In the absence of a lipogenic component histologically, the differential diagnosis of a retroperitoneal DDL includes other sarcomas and, if the tumor has visceral involvement, sarcomatoid carcinoma. DDL demonstrates amplification of chromosome subregion 12q13-q15. Detection of the amplification itself, or the resulting overexpression of the MDM2 and CDK4 genes by genetic and immunohistochemical methods, is a useful ancillary test in the diagnosis of DDL. More recently, immunohistochemistry for p16, the product of the CDKN2A gene, was shown to be a useful adjunct in differentiating well-differentiated liposarcoma from benign adipocytic tumors. In the present study, we examined the utility of p16 immunohistochemistry to distinguish DDL (n=44) from other high-grade and low-grade retroperitoneal mimics (n=73). p16 positivity was observed in 43/44 (98%) DDLs, with the majority of these showing strong, diffuse, staining. The rate of p16 positivity in other retroperitoneal tumors was lower (37/73, 51%) and staining was not as consistently diffuse or intense. Furthermore, p16 positivity varied between the control sarcomas based on tumor type as follows: 11/11 leiomyosarcomas, 8/11 pleomorphic undifferentiated sarcomas, 9/39 sarcomatoid carcinomas, 7/7 desmoid tumors, 1/3 endometrial stromal sarcomas, and 1/2 malignant gastrointestinal stromal tumors. On the basis of these findings, we conclude that p16 is highly sensitive for retroperitoneal DDL. However, the lack of specificity limits the diagnostic utility compared with the more established markers MDM2 and CDK4.

Mutant p53 talks to proteasomes—is there a feedback loop between Nrf2 and mutant p53?

The p53 protein is a powerful tumor suppressor, often termed as ‘guardian of the genome’, whose function is lost in many types of human cancers (1). The loss of function is impelled either by de-regulation of the E3 ubiquitin ligase, MDM2 protein (2) or by the TP53 gene mutations of missense type (3). Mutations in TP53 gene occur at very high frequency and range from 10% to 97% depending on the cancer type (4). Single amino acids substitutions result in the loss of tumor suppression transcription function, promote the change of the conformation and loss of sequence-specific DNA binding function of mutant p53 (mtp53) (5).

AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX-MDM2 complex.

Deregulation of the tyrosine kinase signalling is often associated with tumour progression and drug resistance, but its underlying mechanisms are only partly understood. In this study, we investigated the effects of the receptor tyrosine kinase AXL on the stability of the MDMX-MDM2 heterocomplex and the activity of p53 in melanoma cells. Our data demonstrated that AXL overexpression or activation through growth arrest-specific 6 (Gas6) ligand stimulation increases MDMX and MDM2 protein levels and decreases p53 activity. Upon activation, AXL stabilizes MDMX through a post-translational modification that involves phosphorylation of MDMX on the phosphosite Ser314, leading to increased affinity between MDMX and MDM2 and favouring MDMX nuclear translocation. Ser314 phosphorylation can also protect MDMX from MDM2-mediated degradation, leading to stabilization of the MDMX-MDM2 complex. We identified CDK4/6 and p38 MAPK as the two kinases mediating AXL-induced modulation of the MDMX-MDM2 complex, and demonstrated that suppression of AXL, either through siRNA silencing or pharmacological inhibition, increases expression levels of p53 target genes P21, MDM2, and PUMA, improves p53 pathway response to chemotherapy, and sensitizes cells to both Cisplatin and Vemurafenib. Our findings offer an insight into a novel signalling axis linking AXL to p53 and provide a potentially druggable pathway to restore p53 function in melanoma.

Clinical and Biological Role of Accumulation of the NPM-1 Splice Variant R2 in AML, MDS and sAML

BackgroundAcute myelogenous leukemia (AML) represents a heterogeneous group of myeloid malignancies harboring different chromosomal abnormalities, gene mutations, and epigenetic modifications. Recent clinical and biological studies indicate that myelodysplastic syndromes (MDS) and AML could be considered as part of the same continuous disease spectrum rather than as distinct disorders. NPM1 is a multifunctional protein involved in both biological and pathological processes controlling development, cell proliferation, ribosome biogenesis, transformation and genomic stability. It interacts with many cellular proteins, including ARF and the tumor suppressor p53. Recently, we found that high expression of the NPM1 splice variant R2, which encodes a truncated form of NPM1, may provide prognostic value for CN-AML patients.AimsTherefore, our aim was evaluation of NPM1 R2 splice variant significance for MDS and sAML cases, as well as assignment if different expression levels of R2 might have influence on the expression pattern of each of the components of the ARF-MDM2-p53-p21 signaling pathway and additional downstream molecules (miR-34a, miR-34b and miR-34c). In order to determine the impact of NPM1 R2 on NPM1 localization and to compare it with the NPM1mut effect, transfection analyses and IHC stainings were performed.MethodsNPM1 R2, CDKN2A (encoding ARF), MDM2, TP53 and CDKN1A(encoding p21) genes expression levels were assessed for 128 samples (58 AML, 62 MDS and 8 sAML) using qRT-PCR. Additionally, expression level of miR-34a (n=29), miR-34b (n=20) and miR-34c (n=20) was measured in CD33+ cells derived from AML patient samples. WI-38 fibroblasts and HEK-293 cells were transfected with constructs containing eGFP-tagged NPM1-R2, NPM1-mut and NPM1-wt under a cytomegalovirus promoter, stained and visualized with confocal microscope. Immunochemistry analysis was performed for NPM1 in 23 AML bone marrow smears.ResultsNPM1 R2 expression levels differed between AML, sAML, MDS and healthy volunteers (HV) groups and were significantly higher in AML, sAML and MDS groups compared to HVs (median 0.023 vs 0.005, p<0.001, 0.025 vs 0.005, p<0.001 and 0.017 vs 0.005, p<0.001, respectively). CDKN2A, MDM2, TP53 and CDKN1A expression analysis in these sample groups showed also significant differences. Expression of TP53 was elevated in groups with high R2 expression in comparison to groups with low R2 expression in AML and MDS patients (median 0.01 vs 0.005, p<0.001 and 0.007 vs 0.004, p<0.001, respectively). Moreover, we found strong positive correlation of R2 expression with TP53 expression in AML (r=0.77, p<0.001) and MDS (r=0.68, p<0.001). We observed elevated expression of miR-34c in HVs group compared to AML (0.11 vs 0.07, p<0,001) and trend to decreased expression of miR-34a in AML in comparison with HVs. No differences were found in miR-34a, miR-34b and miR-34c expression between groups with high or low R2 expression.Transfection analyses showed various localization of each eGFP-tagged NPM1 forms. NPM1-wt localized mainly in nucleoli, NPM1-R2 was detected in the nucleoplasm and nucleoli, whereas eGFP-NPM1-mut displayed cytoplasmic localization. However, the IHC stainings for AML samples revealed that in cases with high R2 expression we were able to determine a cytoplasmic localization of NPM1 even in the absence of its concomitant mutation.ConclusionsThe elevated level of NPM1 R2 splice variant in AML, sAML and MDS groups versus HVs suggests that R2 might play some role in neoplasia process also in early stages of this hematological malignancy. Transfection analyses established that NPM1 R2 mostly localizes in the nucleoplasm, where it might interact with other proteins e.g. ARF and p53. Nucleolar localization of this NPM1 form might be determined both by lack of nucleolar localization signal present in the wt form of NPM1 and nuclear export signal occurring in mutated NPM1. Moreover, strong positive correlation between R2 and TP53 expression was found in AML and MDS groups suggesting biological link between these transcripts. In summary, the expression of NPM1 R2 might be of biological importance for AML as well as for transformation of MDS into sAML.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313). DisclosuresGrzasko:Celgene: Honoraria; Munipharma: Honoraria; Janssen: Honoraria.

Variation of the breast cancer susceptibility marker, rs4245739, is associated with differential miRNA binding and MDM4 expression

A polymorphism, rs4245739, has been associated with susceptibility of several cancers including ER-negative breast cancer. Rs4245739 is located at the 3’UTR of MDM4 gene, an oncogene that negatively regulates p53. The polymorphism has been associated with binding changes of miR-191. We studied, the influence of SNP rs4245739 to the binding of microRNAs, expression of microRNAs and MDM4 . Using FindTar software, we detected potential microRNAs affected by the SNP-flanking sequence. We then used RNA sequencing data from ER-negative breast cancer to compare expression of miR-184, miR-191, miR-193a, miR-378, and MDM4 in different genotypes. Comparison of ER-negative patients with and without expression of miR-191 as well as profile microRNAs (miR-184, miR-191, miR-193a and miR-378 altogether) can differentiate expression of MDM4 between different alleles. In addition, the number of lymphatic nodes affected in the individuals was also found to be significantly reduced in the risk group obtained by the miRNA profile method. We show our methods especially miRNA profile approach, are able to obtain new molecular and clinical features related to the rs4245739 SNP, a variant located in the 3’UTR of MDM4 gene and known to appear in different types of cancer. Keywords : ER negative breast cancer, rs4245739, microRNA, MDM4, p53

Next-generation repeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improving patient selection to MDM2-targeted inhibitors

A next-generation FISH probe mapping to the MDM2 locus-specific region has recently been designed. The level of MDM2 gene amplification (high versus low) may allow selection of patients for cancer treatment with MDM2 inhibitors and may predict their responsiveness. We investigated the spectrum of MDM2 gene alterations using the new probes in vivo after visualizing single neoplastic cells in situ from a series of glioblastomas. Signals from next-generation repeat-free FISH interphase probes were identified in tissue microarrays that included 3 spots for each of the 48 cases. The murine double minutes (MDM2)-specific DNA probe and the satellite enumeration probe for chromosome 12 were used. Three cases (6%) showed more than 25 signals (high gene amplification), and 7 (15%) showed 3-10 signals (gains); among these, 4 cases (8%) had an equal number of MDM2 and centromeric signals on chromosome 12 (polyploidy). Genomic heterogeneity was observed only in 3 cases with low gene amplification. In our series, 6% of glioblastomas exhibited high MDM2 amplification (in vivo) with a pattern related to the known double minutes/chromothripsis phenomenon (in situ), and only cases with low amplification showed genomic heterogeneity. We concluded that the rate of MDM2 gene amplification can be a useful predictive biomarker to improve patient selection.

MET-inhibitors meet MET mutations in lung cancer

Mesenchymal-to-epithelial transition ( MET ) exon 14 mutation in non-small cell lung cancer (NSCLC) has been recognized. However, clinical, molecular, and pathologic features have not been well understood. Awad et al . described NSCLC patients with MET exon 14 mutations precisely in the ‘ Journal of Clinical Oncology 2016;34:721-30’. Among 933 non-squamous NSCLC patients, they found 28 (3.0%) patients who represented a unique clinical and molecular subtype of NSCLC. Median age of the 28 patients was 72.5 years, 68% were women, 36% were never-smokers, 64% had stage IV, 100% were white, non-Hispanic, and 64% had adenocarcinoma and 14% had pleomorphic carcinoma. Genomic deletions and point mutations occurred in 17 and 11, respectively, occurred of the 28 patients. Although none of the 28 patients with MET exon 14 mutations had KRAS , epidermal growth factor receptor ( EGFR ), ERBB2 , anaplastic lymphoma kinase ( ALK ), ROS1 , or RET alterations, mutations of TP53 , CDKN2A/B , BRAF600E , PIK3CA , PTEN , RB1 , ATM , BRCA2 , NF1 , or ARID2 were co-existed. Amplification of MDM2 was observed in 13 (46%), and 6 (21%) and 8 (29%) had high- and low-level MET copy gain, respectively. To date, two MET inhibitors, onartuzumab or tivantinib, combination with erlotinib in previously treated NSCLC were investigated in phase III trials. However, neither showed prolonged overall survival (OS) compared with erlotinib alone in molecularly unselected patients. Several publications including the report of Awad et al . revealed that patients with MET exon 14 mutation were successfully treated with MET-tyrosine kinase inhibitors (TKIs) such as crizotinib. Prospective trials using MET-TKIs in MET exon 14 mutated NSCLC are ongoing. Concerning translational research, significance of co-existed other mutations or amplifications and mechanism of acquired resistance to MET-TKIs remain to be clarified. Finally, the therapeutic strategies against the MET-TKI resistance and intracranial metastasis in NSCLC with MET exon 14 mutation should be elucidated.

Molecular effects of 1-naphthyl-methylcarbamate and solar radiation exposures on human melanocytes

Carbaryl (1-naphthyl-methylcarbamate), a broad-spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, the main etiologic factor for skin carcinogenesis. We hypothesized that carbaryl exposure may increase deleterious effects of UV solar radiation on skin melanocytes. This study aimed to characterize human melanocytes after individual or combined exposure to carbaryl (100μM) and solar radiation (375mJ/cm2). In a microarray analysis, carbaryl, but not solar radiation, induced an oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of Microphtalmia-associated Transcription Factor (MITF), the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Carbaryl and solar radiation induced a gene response suggestive of DNA damage and cell cycle alteration. The expression of CDKN1A, BRCA1/2 and MDM2 genes was notably more intense in the combined treatment group, in a synergistic manner. Flow cytometry assays demonstrated S-phase cell cycle arrest, reduced apoptosis levels and faster induction of cyclobutane pyrimidine dimers (CPD) lesions in carbaryl treated groups. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation.

Systems biology analysis reveals role of MDM2 in diabetic nephropathy.

To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers.