MDM2 Research Articles

P53 promotes AKT and SP1-dependent metabolism through the pentose phosphate pathway that inhibits apoptosis in response to Nutlin-3a.

Nutlin-3a is a MDM2 antagonist and preclinical drug that activates p53. Cells with MDM2 gene amplification are especially prone to Nutlin-3a-induced apoptosis, though the basis for this is unclear. Glucose metabolism can inhibit apoptosis in response to Nutlin-3a through mechanisms that are incompletely understood. Glucose metabolism through the pentose phosphate pathway (PPP) produces NADPH that can protect cells from potentially lethal reactive oxygen species (ROS). We compared apoptosis and glucose metabolism in cancer cells with and without MDM2 gene amplification treated with Nutlin-3a. Apoptosis in MDM2-amplified cells was associated with a reduction in glycolysis and the PPP, reduced NADPH, increased ROS, and depletion of the transcription factor SP1, which normally promotes PPP gene expression. In contrast, glycolysis and the PPP were maintained or increased in MDM2 non-amplified cells treated with Nutlin-3a. This was dependent on p53-mediated AKT activation and was associated with maintenance of SP1 and continued expression of PPP genes. Knockdown or inhibition of AKT, SP1, or the PPP sensitized MDM2-non-amplified cells to apoptosis. The data indicate that p53 promotes AKT and SP1-dependent activation of the PPP that protects cells from Nutlin-3a-induced apoptosis. These findings provide insight into how glucose metabolism reduces Nutlin-3a-induced apoptosis, and also provide a mechanism for the heightened sensitivity of MDM2-amplified cells to apoptosis in response to Nutlin-3a.

Detection of a MicroRNA molecular signature of ultraviolet radiation in the superficial regions of melanocytic nevi on sun-exposed skin

How melanocytes transform into melanoma cells remains largely unknown. However, prolonged ultraviolet radiation exposure is linked with melanoma, and the DNA of melanomas arising in chronically sun-exposed skin is characterized by an elevated number of pyrimidine transitions, mainly C>T (predominantly caused by ultraviolet B), and transversions of GC>TA or AT>CG (caused by ultraviolet A over indirect mechanisms). Since ultraviolet penetrates mostly only the superficial dermis, we sought to determine the extent to which superficial and deep melanocytes of nevi in sun-exposed skin differ in their miRNA expression and consider the changes as likely secondary to ultraviolet radiation-induced damage. The differentially expressed miRNAs were analyzed for known potential oncomiRs or linked to potential oncogenes or tumor suppressors. Superficial and deep melanocytes were microdissected from the nevi of 14 patients. The suspensions were processed for hybridization to a ribonucleotide protection system with 2280 total probes, including 2256 miRNA probes targeting 2083 human miRNAs. A comprehensive analysis of all human miRNAs registered in miRBase 11.0 was performed using the HTG Molecular Diagnostic database. Statistical analysis of these data yielded for 14 samples a statistically relevant profile of 39 miRNA species at FDR<0.1 that were differentially expressed between superficial and deep melanocytes. Ingenuity Pathway Analysis based on the expression data of these 39 miRNAs suggested the gene transcripts AR, MDM2, SMAD2/3, and YBX1 as the most probable miRNA targets, which were validated at the protein level. Our findings suggest that superficial ultraviolet radiation-damaged melanocytes can be differentiated from deep melanocytes on the basis of the expression of 39 miRNAs, the most probable gene transcript and protein targets of which are AR, MDM2, SMAD2/3, and YBX1, with YBX1 expression validating the best the molecular signature in immunohistochemical analysis.

Analysis of 12 variants in the development of gastric and colorectal cancers.

AIMTo evaluate the relation between 12 polymorphisms and the development of gastric cancer (GC) and colorectal cancer (CRC).METHODSIn this study, we included 125 individuals with GC diagnosis, 66 individuals with CRC diagnosis and 475 cancer-free individuals. All participants resided in the North region of Brazil and authorized the use of their samples. The 12 polymorphisms (in CASP8, CYP2E1, CYP19A1, IL1A, IL4, MDM2, NFKB1, PAR1, TP53, TYMS, UGT1A1 and XRCC1 genes) were genotyped in a single PCR for each individual, followed by fragment analysis. To avoid misinterpretation due to population substructure, we applied a previously developed set of 61 ancestry-informative markers that can also be genotyped by multiplex PCR. The statistical analyses were performed in Structure v.2.3.4, R environment and SPSS v.20.RESULTSAfter statistical analyses with the control of confounding factors, such as genetic ancestry, three markers (rs79071878 in IL4, rs3730485 in MDM2 and rs28362491 in NFKB1) were positively associated with the development of GC. One of these markers (rs28362491) and the marker in the UGT1A1 gene (rs8175347) were positively associated with the development of CRC. Therefore, we investigated whether the joint presence of the deleterious alleles of each marker could affect the development of cancer and we obtained positive results in all analyses. Carriers of the combination of alleles RP1 + DEL (rs79071878 and rs28361491, respectively) are at 10-times greater risk of developing GC than carriers of other combinations. Similarly, carriers of the combination of DEL + RARE (rs283628 and rs8175347) are at about 12-times greater risk of developing CRC than carriers of other combinations.CONCLUSIONThese findings are important for the comprehension of gastric and CRC development, particularly in highly admixed populations, such as the Brazilian population.

Cardiac-specific ablation of the E3 ubiquitin ligase Mdm2 leads to oxidative stress, broad mitochondrial deficiency and early death.

The maintenance of normal heart function requires proper control of protein turnover. The ubiquitin-proteasome system is a principal regulator of protein degradation. Mdm2 is the main E3 ubiquitin ligase for p53 in mitotic cells thereby regulating cellular growth, DNA repair, oxidative stress and apoptosis. However, which of these Mdm2-related activities are preserved in differentiated cardiomyocytes has yet to be determined. We sought to elucidate the role of Mdm2 in the control of normal heart function. We observed markedly reduced Mdm2 mRNA levels accompanied by highly elevated p53 protein expression in the hearts of wild type mice subjected to myocardial infarction or trans-aortic banding. Accordingly, we generated conditional cardiac-specific Mdm2 gene knockout (Mdm2f/f;mcm) mice. In adulthood, Mdm2f/f;mcm mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction with early mortality post-tamoxifen. A decreased polyubiquitination of myocardial p53 was observed, leading to its stabilization and activation, in the absence of acute stress. In addition, transcriptomic analysis of Mdm2-deficient hearts revealed that there is an induction of E2f1 and c-Myc mRNA levels with reduced expression of the Pgc-1a/Ppara/Esrrb/g axis and Pink1. This was associated with a significant degree of cardiomyocyte apoptosis, and an inhibition of redox homeostasis and mitochondrial bioenergetics. All these processes are early, Mdm2-associated events and contribute to the development of pathological hypertrophy. Our genetic and biochemical data support a role for Mdm2 in cardiac growth control through the regulation of p53, the Pgc-1 family of transcriptional coactivators and the pivotal antioxidant Pink1.

MDM2 SNP309 polymorphism in Turkish population

Amac: Murine double minute 2 ( MDM2 ) geni p53 tumor supressor yolaginin duzenlenmesinde kritik bir role sahiptir. MDM2, p53 fonksiyonunda buyuk bir negatif regulatordur. MDM2 proteini birbirinden bagimsiz bircok yolla p53 fonksiyonunu duzenler. Bu calismada bir Turk Populasyonunda MDM2 geni SNP309 (rs2279744) polimorfizm sikliginin belirlenmesi amaclanmistir. Yontem: Bu calismada, MDM2 rs2279744 polimorfizm sikligi toplam 301 (kadin: 159, % 53,0 ve erkek: 141, % 47,0) Turk saglikli bireyde incelendi. T309G MDM2 rs2279744 polimorfizim sikligi 301 saglikli kontrolde RFLP-PCR metoduyla belirlendi. Bulgular: Calisma grubunun yas ortalamasi 49,80±16,4 yil (erkek, 51,97±15,98; kadin, 47,58±16,82) idi. MDM2 rs2279744 polimorfizmine ait gozlenen genotip frekanslari % 17.9 TT, % 55.1 TG ve %26.9 GG olarak tespit edildi. Sonuc: Bu calisma, Turk toplumunda MDM2 geni rs2279744 polimorfizminin allel ve genotip frekans dagilimlari hakkinda bilgi vermektedir. Bu allel ve genotip dagilimlari kanser basta olmak uzere bircok hastaliga yatkinligin arastirilmasinda referans parametre olacaktir.

Thyroid hormone upregulates MDM2 in rat type I fibre: Implications for skeletal muscle mass regulation.

Based upon a microarray assay, we have identified that triiodothyronine (T3) upregulates MDM2 gene expression in the rat skeletal muscle. As MDM2 protein is an E3 ligase, we hypothesized that this enzyme could play a role in T3 effects on skeletal muscle mass control. To test our hypothesis, male rats (2 months old) were randomly assigned into the following groups: intact controls, treated with 20 physiological doses of T3 for 0.5, 1 and 7 days, or with 5, 20 and 50 physiological doses of T3 for 7 days. For in vitro experiments, myotubes and C2C12 cells were treated with T3 for 3 days. After validation of the microarray finding throughout RT-PCR and confirmation that T3 induces increases in MDM2 protein expression in a dose-dependent manner, we observed that MDM2 was upregulated by T3 exclusively in fibre type I. Moreover, detailed histological evaluation showed that MDM2 overexpression distributes punctiformily along the cross section of the fibre and also inside nuclei. MDM2 colocalizes with PAX7 in control muscle and T3 downregulates this myogenic factor. Pharmacological inhibition of MDM2 in cultured myotubes caused a severe decrease in their diameter (~35%, P < .001 vs Control), enhancing the effect of T3 (from ~12% to ~35%, P < .001) alone upon myotube diameter and mRNA levels of atrogenes. Finally, we observed that FOXO3 (MDM2 target) is kept outside the nucleus under T3 stimulation. Our results indicate that MDM2 might be involved in the pro-trophic effects of T3 in skeletal muscle.

Amplification of 1q32.1 Refines the Molecular Classification of Endometrial Carcinoma.

Purpose: Molecular classification of endometrial cancer identified distinct molecular subgroups. However, the largest subset of endometrial cancers remains poorly characterized and is referred to as the "nonspecific molecular profile" (NSMP) subgroup. Here, we aimed at refining the classification of this subgroup by profiling somatic copy-number aberrations (SCNAs).Experimental Design: SCNAs were analyzed in 141 endometrial cancers using whole-genome SNP arrays and pooled with 361 endometrial cancers from The Cancer Genome Atlas. Genomic Identification of Significant Targets in Cancer (GISTIC) identified statistically enriched SCNAs and penalized Cox regression assessed survival effects. The prognostic significance of relevant SCNAs was validated using multiplex ligation-dependent probe amplification in 840 endometrial cancers from the PORTEC-1/2 trials. Copy-number status of genes was correlated with gene expression to identify potential cancer drivers. One plausible oncogene was validated in vitro using antisense oligonucleotide-based strategy.Results: SCNAs affecting chromosome 1q32.1 significantly correlated with worse relapse-free survival (RFS) in the NSMP subgroup (HR, 2.12; 95% CI, 1.26-3.59; P = 0.005). This effect was replicated in NSMP endometrial cancers from PORTEC-1/2 (HR, 2.34; 95% CI, 1.17-4.70; P = 0.017). A new molecular classification including the 1q32.1 amplification improved risk prediction of recurrence. MDM4 gene expression strongly correlated with 1q32.1 amplification. Silencing MDM4 inhibited cell growth in cell lines carrying 1q32.1 amplification, but not in those without MDM4 amplification. Vice versa, increasing MDM4 expression in nonamplified cell lines stimulated cell proliferation.Conclusions: 1q32.1 amplification was identified as a prognostic marker for poorly characterized NSMP endometrial cancers, refining the molecular classification of this subgroup. We functionally validated MDM4 as a potential oncogenic driver in the 1q32.1 region. Clin Cancer Res; 23(23); 7232-41. ©2017 AACR.

Individual and combined effect of TP53, MDM2, MDM4, MTHFR, CCR5, and CASP8 gene polymorphisms in lung cancer.

Lung cancer (LC) is the second common and with the highest mortality oncological disease. Specific biomarkers for its diagnostics, treatment, and prognosis are still under the investigations. Aim of our study was to evaluate the relationship between the polymorphisms of TP53 pathway genes TP53, MDM2, MDM4, the polymorphisms of HPV-associated genes MTHFR, CASP8, CCR5, and HPV infection with survival of LC patients. SNPs were genotyped using PCR-RFLP. qRT-PCR was used to detect, identify, and quantify HPV. No statistically significant differences were detected between individual SNPs and patient survival with stage I-IV LC. Cluster analysis of SNPs in genes MDM4 A/A, CCR5 wt/Δ32, MTHFR C/T, MDM2 T/T showed possible association with the worse survival. Patients who were diagnosed with C/T polymorphic variant of gene MTHFR tend not to survive stage III-IV LC (P = .12). There is a tendency between MDM2 gene T/T variant and worse survival of patients diagnosed with late stage LC (P = .11). HPV infection is very rear among LC patients (3 of 92). Overall, there is a link, although statistically insignificant, between specific SNPs and LC patient survival frequency and time, meanwhile the combination of specific SNPs showed a statistically significant measure. In conclusion, we determined statistically significant (P = .04) link between the poor survival of LC patients after surgery and the combination of polymorphic variants C/T of the MTHFR and T/T of the MDM2 genes, whereas individually these SNPs do not show significant relationship with the survival of patients after surgery.

Micro RNA-518 inhibits gastric cancer cell growth by inducing apoptosis via targeting MDM2

Gastric cancer is a most common malignancy and the third leading cause of cancer mortality worldwide. So it is important to identify the prognostic markers and effective therapeutic targets against gastric cancer. miRNA plays an important role in tumor cell cycle, differentiation, apoptosis, invasion and metastasis. Many studies devote to the mechanism of miRNA regulates gastric cancer carcinoma and progression. In the present study, we found that the level of miR-518 in gastric cancer and cell lines were lower than the control or the adjacent tissues by qRT-PCR. Transfected with miR-518 mimic trigger apoptosis in MKN45 and HGC27 gastric cancer cell in vitro and in vivo. Moreover, we found that MDM2 was negatively regulated by miR-518 via targeting 26–32 site of 3’UTR using luciferase reporter assay. The western blot assay showed that miR-518 up-regulated the expression of p53, pro-apoptotic protein Bax and active the activity of cleaved caspase-3, down-regulated expression of anti-apoptotic protein Bcl-2 via targeting MDM2. Thus, our study suggested that miR-518 acted as a new tumor suppressor by targeting MDM2 gene and trigger apoptosis in vivo and in vitro. The findings of the study first established the role of miR-518 in gastric cancer and may be a potential therapeutic target against gastric cancer in the further.

The association of polymorphic markers Arg399Gln of XRCC1 gene, Arg72Pro of TP53 gene and T309G of MDM2 gene with breast cancer in Kyrgyz females

BackgroundThe association of genes XRCC1, TP53 and MDM2 with breast cancer (BC) has never been tested in Kyrgyz population. We, therefore, aimed to identify an association of alleles and genotypes of polymorphic markers Arg399Gln of gene XRCC1, Arg72Pro of gene TP53, and T309G of gene MDM2 with the risk of BC in Kyrgyz women.MethodsThis was a case-control study of 219 women of Kyrgyz origin with morphologically verified BC (N = 117) and 102 controls, age-matched with BC cases. The mean age of subjects in this study was 52.2 ± 10.8 years. We extracted DNA from the venous blood and genotyped polymorphic markers Arg399Gln of gene XRCC1, Arg72Pro of gene TP53 and T309G of gene MDM2 using polymerase chain reaction and the method of restriction fragment polymorphism.ResultsAllele 399Gln (OR 1.57; 95% CI 1.05–2.35), Arg399Gln of gene XRCC1 heterozygous genotype (OR 2.77; 95% CI 1.60–4.80), the combination of Arg399Gln/Arg72Pro of genes XRCC1/TP53 heterozygous genotype (OR 3.98; 95% CI 1.57–10.09), Arg399Gln/T309G of genes XRCC1/MDM2 (OR 3.0; 95% CI 1.18–7.56), as well as Arg399Gln/Arg72Pro/T309G of genes XRCC1/TP53/MDM2 (OR 6.40; 95% CI 1.18–34.63) were associated with BC in Kyrgyz women.ConclusionsThis is the first study to identify the inter-loci interaction and to find molecular markers of individual risk of BC in Kyrgyz women.

Assessment of the expression of the BRCA1, BRCA2, TP53, MDM2, BAX and CASP-3 genes in normal and tumor tissues for patients with breast cancer in Morocco

Nowadays, many genes are associated with Breast Cancer. In order to understand the cause behind this particular type of cancer, we chose to study crucial genes previously reported to be involved in its development and mechanisms. Therefore, we worked on BRCA1 and BRCA2 as the main predisposition genes related to 5 to 10% of familial cases and assessed TP53 since it's associated to >50% types of cancer. Additionally, we measured MDM2, BAX and CASP3 giving their important role in the internal pathway of apoptosis. For a molecular characterization, we quantified BRCA1, BRCA2, TP53, MDM2, BAX and CASP-3 mRNA using the qRT-PCR approach in large series of tumor and normal breast tissues of Moroccan patients. We found a significant correlation between the expression of BRCA1 and BRCA2 in normal and tumor tissue. The results showed a positive correlation in the expression of MDM2 between normal and tumor tissue. Furthermore, we detected an elevation of expression of CASP-3 and BAX in tumor and normal in breast tissue. These data can be critical in understanding Breast Cancer and possibly leading to a therapeutic pathway for patients worldwide in general and Moroccans in particular.

487O - Tumour histology and primary site but not MDM2 amplification levels are prognostic for clinical outcomes in well differentiated and dedifferentiated liposarcoma

Background:MDM2, an oncogene and p53 inhibitor is consistently amplified in well differentiated (WD) and dedifferentiated (DD) liposarcomas (LPS). Although previous reports have deemed fluorescent in situ hybridization (FISH) of MDM2 as the diagnostic gold standard for WD/DDLPS, the prognostic utility of this biomarker is less well established. This study aims to examine MDM2 amplification as a prognostic factor in LPS. Methods: A retrospective review of clinical records was done to identify patients (pts) with primary LPS that were curatively resected and assessed for MDM2 via FISH. Dual color MDM2/CEP 12 probes were applied to FFPE tissue sections. Signals from 60 non–overlapping nuclei were counted for copy numbers of MDM2 and CEP 12. MDM2 gene amplification was defined as the ratio of MDM2 signals to CEP 12 signals. Receiver operating curve analysis was used to determine a cut-off for high MDM2 ratio (>9.1). Progression free survival (PFS) was assessed from time of resection to relapse or death. Survival analysis was done using Kaplan-Meier and multivariable cox models. Results: 55 pts consisting of 20 DDLPS and 35 WDLPS were identified. Higher levels of MDM2 ratio was associated with retroperitoneal tumour site (8.1 vs 5.4, Mann-Whitney; p=0.023). No statistical difference in MDM2 ratio was seen in other clinical features; including age of diagnosis, performance status, resection margins, tumour histology and size. On univariate survival analysis, DDLPS (HR 11.163; 95%CI 3.935-31.663; p<0.001), positive resection margins (HR 3.138; 95%CI 1.21-8.110; p=0.034) and retroperitoneal primary (HR 6.884; 95%CI 2.324-20.387; p<0.001) were predictive of poorer PFS. However, MDM2 amplification level was not prognostic for PFS (HR 2.191; 95%CI 0.599-8.016; p=0.130). On multivariable analysis, only retroperitoneal primary (HR 3.400; 95%CI 1.015-11.388; p=0.047) and DDLPS (HR 6.993; 95%CI 1.762-27.757, p=0.006) remain prognostic for worse outcomes. Conclusions: In this cohort of WD and DD LPS pts, tumour histology and primary site but not MDM2 amplification level, were prognostic for clinical outcomes. Legal entity responsible for the study: SingHealth Centralised Institutional Review Board Funding: None Disclosure: All authors have declared no conflicts of interest.

A Case of Upper Gastrointestinal Bleeding Due to a Large Gastric Lipoma Within a Hiatal Hernia

Gastric lipomas are rare mesenchymal tumors with a prevalence of 0.03-0.18% in the general population. They account for 5% of gastrointestinal tract lipomas and represent 3% of benign gastric tumors. They are usually asymptomatic, but can cause symptoms of abdominal discomfort, obstruction, intussusception, or bleeding. Most gastric lipomas (75%) are located in the antrum. We present a case of a large, proximal lesser curvature gastric lipoma contained within a hiatal hernia that presented as an upper gastrointestinal hemorrhage. A 78 female with a remote history of stroke on clopidogrel was admitted after two days of melena. She was hemodynamically stable with mild tachycardia. Melena was preceded by two days of mid-epigastric discomfort. She endorsed rare use of aspirin for headaches. Hemoglobin dropped from baseline of 14 gm/dL to 6.8 gm/dL requiring transfusion. Upper endoscopy revealed a 4-5 cm, smooth, soft, submucosal mass on the proximal lesser curvature of the stomach with a 1 cm clean based ulcer overlying the mass. The mass was located within a large hiatal hernia. Mucosal biopsies obtained during the procedure showed fundic type mucosa with active gastritis, necroinflammatory changes, and were negative for Helicobacter pylori. A CT scan of the chest, abdomen, and pelvis showed a lipomatous, lower mediastinal mass within a hiatal hernia measuring 11.5 x 9.4 x 8.2 cm concerning for gastric lipoma versus low grade liposarcoma. The patient underwent laparoscopic mobilization of a large Type III hiatal hernia with conversion to open for subsequent wedge resection of the gastric mass and hiatal hernia repair. Pathologic examination revealed a well-differentiated, mature, encapsulated, fatty neoplasm consistent with gastric lipoma with necrosis and ulceration of the overlying gastric mucosa. MDM2 gene amplification was not detected, confirming the diagnosis of benign lipoma. The patient had an uneventful recovery and is doing well post-operatively. While gastric lipomas harbor no malignant potential, they should be treated when they present with complications. Traditionally, symptomatic gastric lipomas are managed with surgical resection or enucleation. Endoscopic resection and submucosal dissection have been used to manage these tumors, but these techniques are technically challenging and carry an increased risk of perforation with larger masses.Figure: Axial CT scan image showing the large lipomatous mass in the lower mediastinum within a hiatal hernia.Figure: Large submucosal mass with overlying clean based ulcer as seen on retroflexion during endoscopy.Figure: Endoscopic image of submucosal mass and ulcer appreciated along the proximal lesser curvature with the diaphragmatic hiatus seen in the background.

TruSeq-Based Gene Expression Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Cutaneous T-Cell Lymphoma Samples: Subgroup Analysis Results and Elucidation of Biases from FFPE Sample Processing on the TruSeq Platform.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of malignancies with courses ranging from indolent to potentially lethal. We recently studied in a 157 patient cohort gene expression profiles generated by the TruSeq targeted RNA gene expression sequencing. We observed that the sequencing library quality and depth from formalin-fixed paraffin-embedded (FFPE) skin samples were significantly lower when biopsies were obtained prior to 2009. We also observed that the fresh CTCL samples clustered together, even though they included stage I–IV disease. In this study, we compared TruSeq gene expression patterns in older (≤2008) vs. more recent (≥2009) FFPE samples to determine whether these clustering analyses and earlier described differentially expressed gene findings are robust when analyzed based on the year of biopsy. We also explored biases found in FFPE samples when subjected to the TruSeq analysis of gene expression. Our results showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, importantly, both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1) stage I–IV and (2) stage IV (alone) CTCL samples. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Furthermore, by using subgroup analyses, we were able to identify additional novel differentially expressed genes (DEGs), which did not reach statistical significance in the prior full data set analysis. Those included CTCL-upregulated BCL11A, SELL, IRF1, SMAD1, CASP1, BIRC5, and MAX and CTCL-downregulated MDM4, SERPINB3, and THBS4 genes. With respect to sample biases, no matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While principal component analysis revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained in the proximity to other normal/benign and FFPE CTCL samples and were not clustering as outliers by themselves. Notably, this did not affect the determination of DEGs when analyzing ≥2009 samples (fresh and FFPE biopsies) vs. ≥2009 FFPE samples alone.

High amplification levels of MDM2 and CDK4 correlate with poor outcome in patients with dedifferentiated liposarcoma: A cytogenomic microarray analysis of 47 cases

Dedifferentiated liposarcoma (DDLS) is characterized at the molecular level by amplification of genes within 12q13-15 including MDM2 and CDK4. However, other than FNCLCC grade, prognostic markers are limited. We aim to identify molecular prognostic markers for DDLS to help risk stratify patients. To this end, we studied 49 cases of DDLS in our institutional archives and performed cytogenomic microarray analysis on 47 cases. Gene copy numbers for 12 loci were evaluated and correlated with outcome data retrieved from our institutional electronic medical records. Using cut point analysis and comparison of Kaplan-Meier survival curves by log rank tests, high amplification levels of MDM2 (>38 copies) and CDK4 (>30 copies) correlated with decreased disease free survival (DFS) (P = .0168 and 0.0169 respectively) and disease specific survival (DSS) (P = .0082 and 0.0140 respectively). Additionally, MDM2 and CDK4 showed evidence of a synergistic effect so that each additional copy of one enhances the effect on prognosis of each additional copy of the other for decreased DFS (P = .0227, 0.1% hazard). High amplification of JUN (>16 copies) also correlated with decreased DFS (P = .0217), but not DSS. The presence of copy number alteration at 3q29 correlated with decreased DSS (P = .0192). The presence of >10 mitoses per 10 high power fields and FNCLCC grade 3 also correlated with decreased DFS (P = .0310 and 0.0254 respectively). MDM2 and CDK4 gene amplification levels, along with JUN amplification and copy alterations at 3q29, can be utilized for predicting outcome in patients with DDLS.

LINCRNA-P21 IS REGULATED BY CYCLIC MECHANICAL STRETCH AND UPREGULATED IN AORTIC ANEURYSMS

Emerging evidence suggests that long noncoding RNAs (lncRNAs) may represent a cellular hub for coordination of cellular processes involved in cardiovascular health and disease. Alterations in the characteristics of vascular smooth muscle cells, triggered largely by mechanical stress, play a critical role in the pathophysiology of vascular diseases. We hypothesized that lncRNAs are involved in the pathogenesis of mechanical stretch-induced changes in human aortic smooth muscle cells (HASMCs). HASMCs were subjected to 20% elongation (1 Hz) for 24 h (Flexcell® FX4000™). Total RNA was extracted with TRIzol. LncRNAs were profiled using the Arraystar Human LncRNA Microarray V3.0. Human aneurysmal and non-aneurysmal aortic samples were collected from patients undergoing aortic arch repair and coronary artery bypass grafting, respectively. Aneurysmal and non-aneurysmal samples were also collected from the aorta of ApoE-/- mice after 4-week infusion of angiotensin II and saline, respectively. Gene expression was quantified via qRT-PCR. For knockdown studies, siLincRNA-p21 and scrambled control were transfected into HASMCs at 10 nM and 5nM final concentration, respectively, using Dharmafect 2 (Dharmacon). Apoptosis was assessed using Annexin V/PI double staining. Of the 30,586 human lncRNAs screened, 580 were differentially expressed (P < 0.05) in the stretched vs. static group. Human long intergenic non-coding RNA-p21 (lincRNA-p21) was significantly upregulated in stretched HASMCs (N = 3, P < 0.05) and human aneurysmal samples (N = 10, P < 0.05). The murine ortholog of lincRNA-p21 was upregulated in aortic tissues from angiotensin-II-infused ApoE-/- mice (N = 3, P < 0.05). Upon silencing of lincRNA-p21 in stretched HASMCs, there was a decrease in total apoptosis (N = 3, P < 0.05) and expression of the p53-downstream genes Bax, Puma, Noxa, and Mdm2 (N = 3, P < 0.05). Our data provides the first transcriptome profile of stretched HASMCs and implicates lincRNA-p21 as a mechanoresponsive regulator of aneurysm formation in mice and humans. This data provides novel insights into the regulatory switches governing aberrant VSMC remodeling, which may have importance in the pathogenesis of aneurysms and hypertension.

Investigation of the association between the MDM2 T309G polymorphism and gastric cancer.

Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. The association between the MDM2 polymorphism and gastric cancer (GC) has been investigated in Turkish population. In the present case-control study, the aim was to investigate the association between genetic polymorphisms of the MDM2 gene (a major regulator of p53 function) and primary GC risk in a Turkish population. The polymorphism, T309G (rs2279744) in the MDM2 gene was determined in patients with GC (n=65) and in healthy control subjects (n=67) using the polymerase chain reaction-restriction fragment length polymorphism method. The findings were evaluated using logistic regression and χ2 tests. No statistically significant differences were observed between the control subjects and patients with GC regarding smoking status. A comparison between GC cases and control subjects indicated a statistically significant difference for family history of cancer [odds ratio (OR)=0.17; 95% confidence interval (CI), 0.05-0.56; χ2=0.19; P=0.01]. A significant difference was identified in the GG genotype distribution between GC patients and control subjects (OR=4.58; 95% CI, 1.18-17.79; P=0.022). Thus, the results of the present study indicate that the MDM2 gene T309G intron (GG) genotype may be an important risk factor for GC development in the Turkish population.

MiR-509-5p inhibits cellular proliferation and migration via targeting MDM2 in pancreatic cancer cells

ObjectiveThis study aimed to explore the effect of miR-509-5p on pancreatic cancer progression and clarify the underlying mechanisms.MethodsReal-time quantitative reverse transcription polymerase chain reaction was employed to determine miR-509-5p expression in pancreatic cancer tissues and noncancerous adjacent tissues. CCK-8 and Transwell experiments were employed to examine cellular proliferation, migration, and invasion after miR-509-5p mimic or inhibitor transfection. Bioinformatics tools were used to identify the target gene of miR-509-5p, and cotransfection of the target gene and miR-509-5p mimic was performed to determine the effect on the proliferation and migration of pancreatic cancer cells. A xenograft mouse model and histological analysis were also used to test the effect of miR-509-5p on tumor growth in vivo.ResultsmiR-509-5p expression was dramatically downregulated in pancreatic cancer tissues and in pancreatic cancer cell lines. miR-509-5p mimic markedly inhibited PANC-1 cell proliferation, migration, and invasion. Conversely, miR-509-5p inhibitor promoted PANC-1 cell proliferation, migration, and invasion. Furthermore, the 3′UTR–specific target site luciferase reporter assay also showed that miR-509-5p negatively regulated MDM2 at the post-transcriptional level. miR-509-5p effectively reversed the MDM2 overexpression-induced increase in PANC-1 cell proliferation and invasion. Moreover, miR-509-5p inhibited tumor growth and accelerated cell death in the tumor samples.ConclusionsOur results suggested that miR-509-5p served as a new tumor suppressor via targeting the MDM2 gene, inhibiting pancreatic cancer progression.

The p53–Mdm2 regulation relationship under different radiation doses based on the continuous–discrete extended Kalman filter algorithm

The tumour suppressor gene p53 plays a key role in cell response to DNA damage, and the p53–Mdm2 regulation relationship is crucial for the expression of p53. In this paper, based on gene expression time-series data of human leukaemia cells after exposure to ionising radiation, with ionising radiation as input, a nonlinear continuous time-delay dynamic stochastic mathematical model of a p53–Mdm2 network is established using a continuous–discrete extended Kalman filter algorithm. The accuracy of the established model is then validated. Numerical simulation is used to simulate the dynamic regulation in p53–Mdm2 networks for low, medium, and high ionising radiation doses. The results show that the proposed algorithm is convergent and that the error rate of the model is only 1.19%. In addition, the model can simulate accurately the response of the p53–Mdm2 network to different doses of ionising radiation. The methods proposed in this paper can supply the foundation for research on the dynamics of the p53–Mdm2 gene regulation relationship and play a guiding role in research on the p53–Mdm2 response process after DNA damage under different doses of ionising radiation.

Mouse double minute homologue 2 (MDM2) downregulation by miR-661 impairs human endometrial epithelial cell adhesive capacity.

Human blastocysts that fail to implant following IVF secrete elevated levels of miR-661, which is taken up by primary human endometrial epithelial cells (HEECs) and impairs their adhesive capability. MicroRNA miR-661 downregulates mouse double minute homologue 2 (MDM2) and MDM4 in other epithelial cell types to activate p53; however, this has not been examined in the endometrium. In this study MDM2 protein was detected in the luminal epithelium of the endometrium, the site of blastocyst attachment, during the mid secretory receptive phase of the menstrual cycle. The effects of miR-661 on gene expression in and adhesion of endometrial cells was also examined. MiR-661 overexpression consistently downregulated MDM2 but not MDM4 or p53 gene expression in the Ishikawa endometrial epithelial cell line and primary HEEC. Adhesion assays were performed on the real-time monitoring xCELLigence system and by co-culture using Ishikawa cells and HEECs with HTR8/SVneo trophoblast spheroids. Targeted siRNA-mediated knockdown of MDM2 in endometrial epithelial cells reduced Ishikawa cell adhesion (P<0.001) and also reduced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.05) and HEECs (P<0.05). MDM2 overexpression using recombinant protein treatment resulted in enhanced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.01) and HEECs (P<0.05). This study highlights a potential new mechanism by which human blastocyst-secreted miR-661 reduces endometrial epithelial cell adhesion; via downregulation of MDM2. These findings suggest that MDM2 contributes to endometrial-blastocyst adhesion, implantation and infertility in women.