Mouse Double Minute 2 Expression Research Articles

KLF5/MDM2 Axis Modulates Oxidative Stress and Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells: The Role in Diabetic Cataract

Diabetic cataract (DC) is a common cause of visual loss in older diabetic subjects. Krüppel-like factor 5 (KLF5) plays an essential role in migration and the epithelial-mesenchymal transition (EMT) in diverse cells and is involved in oxidative stress. However, the effects of KLF5 on DC remain unknown. This study aimed to examine the biological function of KLF5 in DC and its underlying mechanism. The expression patterns of KLF5 were detected in vivo and in vitro. Then, KLF5 was knocked down in human lens epithelial cells (HLECs) to explore its functional roles and underlying mechanisms. Dual-luciferase reporter assay and chromatin immunoprecipitation analysis were used to detect whether KLF5 could bind the promoter of E3 ubiquitin ligase mouse double minute 2 (MDM2), a key regulator of EMT. Lastly, the regulation of KLF5 in the biological behaviors of HLECs via MDM2 was analyzed. We found a significant increase of KLF5 in the DC lens anterior capsular, diabetic rat lens, and high glucose (HG)-stimulated HLECs. Knockdown of KLF5 inhibited oxidative stress, inflammation, migration, and EMT of HG-stimulated HLECs. KLF5 silencing impeded MDM2 expression and restricted the activation of MARK1/FAK and NF-κB signaling pathways in HLECs under HG condition. Additionally, KLF5 was found to bind the MDM2 promoter and enhance the transcriptional activity of MDM2. The protective effects by silencing KLF5 on HG-cultured HLECs could be offset by MDM2 overexpression. We demonstrated that knockdown of KLF5 alleviated oxidative stress, migration, and EMT of HG-cultured HLECs by regulating MDM2, suggesting a potential therapeutic strategy for DC.

LncRNA PVT1 contributes to invasion and doxorubicin resistance of bladder cancer cells through promoting MDM2 expression and AURKB-mediated p53 ubiquitination.

In most bladder cancer (BC) patients, cancer cells will eventually develop chemical resistance causing increased mortality. This study aimed to explore the mechanism of lncRNA plasmacytoma variant translocation 1 (PVT1) in regulating doxorubicin (ADM) resistance of BC cells. We observed that PVT1 expression was upregulated in ADM-resistant BC cells compared with ADM-sensitive BC cells. Downregulation of PVT1 suppressed ADM-resistant BC cell proliferation and invasion, promoted apoptosis, and increased sensitivity to ADM, while PVT1 overexpression promoted ADM-sensitive BC cell growth and their resistance to ADM. Further study uncovered that PVT1 could interact with and promote mouse double minute 2 (MDM2) expression, and upregulated MDM2-mediated Aurora kinase B (AURKB). Furthermore, Nutlin-3, an inhibitor of MDM2, could counteract the promotive effects of PVT1 overexpression on ADM resistance of ADM-sensitive BC cell, the expression of multidrug-resistance-related proteins, and the inhibition of p53-mediated tumor suppressor genes. And, overexpression of MDM2 or AURKB reversed the promotive effects of PVT1 silence on the ADM sensitivity of ADM-resistant BC cell, and the inhibitory effect on expression multidrug resistance proteins. Mechanically, AURKB increased MDM2-mediated p53 ubiquitination. Taken together, PVT1 promoted BC cell proliferation and drug resistance via elevating MDM2 expression and AURKB-mediated p53 ubiquitination.

Expression profile of tumour suppressor protein p53 and its regulator MDM2 in a cohort of breast cancer patients in a Tertiary Hospital in Ghana.

BackgroundInactivation or mutation of the tumour suppressor gene p53 or its regulator mouse double minute 2 (MDM2) is the commonest event in breast cancer. These altered genes usually express abnormally high levels of their proteins in many carcinomas. The phenotypic expression of p53 and MDM2 in breast cancer cases in our setting is not known. This study investigated the expression of the tumour suppressor protein p53 and its regulator MDM2, using immunohistochemistry in a Ghana breast cancer cohort.MethodA 9-year retrospective cross-sectional study on archived tissue blocks–formalin fixed paraffin embedded tissue (FFPE) was carried out. Demographic data were abstracted. Based on complete clinical data and availability of FFPE archived blocks 203 cases were selected for tissue micro array (TMA) construction. The TMA sections were subjected to immunohistochemistry (IHC) (ER, PR, HER2, p53, and MDM2). Expression of p53 and MDM2 were related to grade and molecular subtypes.ResultsThe age ranged from 17 to 92 years (mean = 49.34 ± 13.74). Most of the cases were high grade; grade II (34.9%) and grade III (55.7%). Fifty-four percent of the cases were triple negative. Invasive ductal carcinoma no special type was the commonest histotype (87.1%). Thirty-six percent (36%) of the cases expressed p53. Significant associations were found between p53 overexpression and histological grade (p = 0.034), triple negative (p = 0.0333) and luminal B (p<0.01) tumors. Most cases (93.1%) were negative for MDM2 expression. Significant association was found between MDM2 and HER2 over-expression as well as Ki-67. There was no significant positive correlation between MDM2 and p53 co-expression (p>0.05).ConclusionThe elevated level of p53 expression in the aggressive breast cancer phenotypes (high histological grade and triple negative) in our cohort suggest that P53 elevation may be a poor prognostic marker in our setting. High expression of MDM2 in our cohort with high Ki67; also in cases with Her2/neu overexpression known with predictable poor prognosis in the absence of target therapy suggest MDM2 may be associated with aggressive biological behaviour in our breast cancer cases. The non-significant association of p53 and MDM2 expression in the same cases as also documented by previous studies suggest independent genetic pathway in tumourigenesis.

VEGF-A/VEGF-B/VEGF-C expressions in non-hereditary, non-metastatic phaeochromocytoma.

Vascular endothelial growth factor (VEGF) is important in pathogenesis of different cancers. The aim of this study is to investigate the relationships between different VEGFs and clinicopathological factors in patients with phaeochromocytomas. Twenty patients (10 men; 10 women) with non-hereditary, non-metastatic phaeochromocytomas were examined for VEGF mRNA expressions by polymerase chain reaction. The expressions were correlated with the clinical and pathological factors of the patients. In addition, mouse double minute 2 (MDM2) expression in these tumours were studied by immunohistochemistry. High expressions of VEGF-A, VEGF-B, and VEGF-C mRNA were detected in 11 (55%), 9 (45%), and 9 (45%) of the tumours respectively. High expression of VEGF-A in phaeochromocytomas was significantly correlated with the tumour size (p=0.025) but did not correlate with patients' age, gender, and tumour laterality. Besides, there was a trend of VEGF-A expression correlated with MDM2 expression (p=0.064). On the other hand, expressions of VEGF-B and VEGF-C were not significantly correlated with tumour size, patients' age, gender, tumour laterality, and MDM2 expression. In addition, high expressions of VEGF-B and VEGF-A were associated with increase of tumour size (p=0.042). Co-expression of different VEGFs did not correlate with MDM2 expression. To conclude, there is a role for VEGF-A/VEGF-B/VEGF-C in the pathogenesis of non-hereditary, non-metastatic phaeochromocytomas.

RETRACTED: MicroRNA-498 disturbs the occurrence and aggression of colon cancer through targeting MDM2 to mediate PPARγ ubiquitination

MicroRNA (miR)-498 is indicative of diagnostic and prognostic significance in colon cancer (CC). On the basis of that, this study is initiated from miR-498, combined with mouse double minute 2 (MDM2)/peroxisome proliferator-activated receptor γ (PPARγ) ubiquitination axis to have an insight into CC progression. CC tissues and their adjacent tissues were harvested to determine miR-498, MDM2 and PPARγ expression. The interactions among these three factors were identified. The screened human CC cells were transfected with miR-498/MDM2-related sequences, followed by detection of the biological behaviors of CC cells. Xenografted tumors were taken to validate cell experimental outcomes. Bioinformatics and dual-luciferase report analysis verified the targeting relationship between miR-498 and MDM2. The relation between MDM2 and PPARγ was identified by immunoprecipitation and in vivo deubiquitination. Down-regulated miR-498 and PPARγ and up-regulated MDM2 were exhibited in CC. miR-498 targeted MDM2 while MDM2 mediated PPARγ ubiquitination. Elevated miR-498 or reduced MDM2 impaired cell viability, colony-forming, migratory and invasive activities and enhanced apoptosis in CC. Elevated MDM2 abolished the effects of up-regulated miR-498 on the biological behaviors of CC cells. Elevated miR-498 or reduced MDM2 depressed tumorigenic ability of CC cells in mice. It is conclusive that restoring miR-498 depresses MDM2 to modify PPARγ ubiquitination, thereby disturbing the tumorigenesis of CC. This work constructs the base for exploring novel agents in treating CC.

The mouse double minute 2 polymorphism is associated with both decreased p53 expression and poor clinicopathological outcomes of gastric cancer.

This study aimed to determine the mouse double minute 2 (MDM2) SNP309 polymorphism and to evaluate MDM2 and p53 expression and the association of MDM2 positivity in gastric cancer and clinicopathological outcomes. A total of 400 patients with chronic gastritis, precancerous lesions, and gastric cancer were used to identify the MDM2 SNP309 polymorphism by using the Taq Man SNP Genotyping assay. Immunohistochemistry was performed to evaluate MDM2 and p53 expression. The associations of polymorphisms, protein expression, clinicopathological outcomes, and gastric cancer risk were calculated by multivariate Cox proportional hazards regression model analysis and expressed by odds ratios (ORs) and 95% confidence intervals (CIs). The MDM2 SNP309 G/G homozygous polymorphism was significantly associated with expressed MDM2 in gastric cancer (OR = 1.57, 95% CI = 1.39-2.03, P = 0.039). Moreover, in gastric cancer, p53 was significantly decreased compared to MDM2 (P = 0.007). However, MDM2 and p53 expression were not significantly different among genotypes, and the G/G genotype can result in the altered protein expression of p53 in gastric cancer. Clinicopathological outcome was significantly associated with MDM2 expression, including tumor location in the upper gastric region (OR = 1.48, 95% CI = 1.25-3.54, P = 0.037), undifferentiated type (OR = 2.47, 95% CI = 1.38-4.14, P = 0.016), presence of lymphatic invasion (OR = 1.96, 95% CI = 1.22-3.19, P = 0.014), and unresectable tumor (OR = 3.39, 95% CI = 1.61-4.94, P = 0.017). Our study indicated associations of the MDM2 SNP309 G/G homozygous polymorphism, MDM2 and p53 expression. Therefore, G/G-associated MDM2 revealed that P53 expression was decreased in gastric cancer and poor clinicopathological outcomes. Understanding the genetic polymorphisms and expression of MDM2 may help explain gastric cancer risk.

Targeting MDM2 for Neuroblastoma Therapy: In Vitro and In Vivo Anticancer Activity and Mechanism of Action.

Simple SummaryNeuroblastoma is a malignant tumor of the sympathetic nervous system that causes aggressive disease in children. The overall survival rate of high-risk patients is very low, therefore developing effective and safe therapies for neuroblastoma is an urgent unmet medical need. The mouse double minute 2 (MDM2) homolog gene is amplified and overexpressed in neuroblastoma and contributes to the poor response to treatment and poor prognosis in patients with high-risk neuroblastoma. Therefore, targeting MDM2 provides a promising approach to neuroblastoma therapy, especially for advanced disease. In the present study, we tested a unique MDM2 inhibitor, SP141, for its therapeutic efficacy and safety in neuroblastoma tumor models. We found that SP141 has significant anti- neuroblastoma activity in cell culture and inhibits tumor growth in animal models of human neuroblastoma, without any noticeable host toxicity. These results provide the basis for targeting MDM2 to treat high-risk neuroblastoma.Background: Neuroblastoma is an aggressive pediatric solid tumor with an overall survival rate of <50% for patients with high-risk disease. The majority (>98%) of pathologically-diagnosed neuroblastomas have wild-type p53 with intact functional activity. However, the mouse double minute 2 (MDM2) homolog, an E3 ubiquitin ligase, is overexpressed in neuroblastoma and leads to inhibition of p53. MDM2 also exerts p53-independent oncogenic functions. Thus, MDM2 seems to be an attractive target for the reactivation of p53 and attenuation of oncogenic activity in neuroblastoma. Methods: In this study, we evaluated the anticancer activities and underlying mechanisms of action of SP141, a first-in-class MDM2 inhibitor, in neuroblastoma cell lines with different p53 backgrounds. The findings were confirmed in mouse xenograft models of neuroblastoma. Results: We demonstrate that SP141 reduces neuroblastoma cell viability, induces apoptosis, arrests cells at the G2/M phase, and prevents cell migration, independent of p53. In addition, in neuroblastoma xenograft models, SP141 inhibited MDM2 expression and suppressed tumor growth without any host toxicity at the effective dose. Conclusions: MDM2 inhibition by SP141 results in the inhibition of neuroblastoma growth and metastasis, regardless of the p53 status of the cells and tumors. These findings provide proof-of-concept that SP141 represents a novel treatment option for both p53 wild-type and p53 null neuroblastoma.

CircTP53 promotes the proliferation of thyroid cancer via targeting miR-1233-3p/MDM2 axis.

Follicular cells give rise to thyroid cancer. Worldwide thyroid cancer incidence continues to rise in recent decades but the mortality rate remains at a stable level. The discovery of novel molecular mechanisms in the pathogenesis of thyroid cancer will promote new diagnostic or therapeutic strategies. Circular RNA (circRNA) is a type of noncoding RNA which is characterized by the covalently closed loop and non-protein coding capacity. The abnormal expression of circRNAs is an important part during the pathogenesis and development of thyroid cancer. CircTP53 is a novel circRNA, and we aimed to investigate its function in the pathogenesis of thyroid cancer and to further demonstrate the underlying molecular mechanism. The levels of circTP53, miR-1233-3p, and other relative mRNA were analyzed by qRT-PCR. Protein levels were shown by Western blot. RNA-pulldown assay and luciferase assay were employed to examine the interaction between circTP53 and miR-1233-3p. Cell proliferation was analyzed by the MTT assay. CircTP53 was a circRNA highly expressed in thyroid cancer tissues. CircTP53 promoted cell proliferation and cell viability of TPC-1 cells. Knockdown of circTP53 inhibited the expression of Mouse double minute 2 (MDM2) and increased the protein level of p53. CircTP53 acted as a target of miR-1233-3p to increase MDM2 expression. p53 expression in thyroid cancer tissue exhibited a negative correlation with circTP53 expression. In thyroid cancer, overexpressed circTP53 decreased the protein level of p53 via targeting miR-1233-3p/MDM2 axis and promoted cancer cell proliferation.

Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

Upregulation of circular RNA circ‑FAM53B predicts adverse prognosis and accelerates the progression of ovarian cancer via the miR‑646/VAMP2 and miR‑647/MDM2 signaling pathways.

Ovarian cancer (OC) is a common cancer of the human genital system. Circular RNAs (circRNAs) play an important role in carcinogenesis and progression of various cancers. The present study aimed to clarify the expression profile and functions of circ‑FAM53B in the progression of OC and reveal its underlying mechanisms. Relative levels of circ‑FAM53B in OC specimens and cell lines were determined by quantitative real‑time polymerase chain reaction (qRT‑PCR). The clinical significance of circ‑FAM53B in OC patients was analyzed through Fisher's exact test, Kaplan‑Meier curves, and Cox regression analysis. Subsequently, the regulatory effects of circ‑FAM53B on the proliferative, apoptotic, migratory, and invasive potential of OC cells were determined by loss/gain‑of‑function experiments. Mechanistically, bioinformatics analysis and luciferase reporter gene assay were used to reveal the potential molecular mechanisms of circ‑FAM53B in OC. circ‑FAM53B was overexpressed in OC specimens and cells and correlated with clinical severity and poor prognosis of OC patients. The overexpression of circ‑FAM53B accelerated the proliferation, migration, and invasion of HO8910 cells; however, it decreased the number of apoptotic cells. Silencing of circ‑FAM53B induced the opposite effect. Through dual‑luciferase reporter gene assay and functional experiments, the potential functions of circ‑FAM53B/miRNA‑646/vesicle‑associated membrane protein 2 (VAMP2) and circ‑FAM53B/miRNA‑647/mouse double minute 2 (MDM2) in mediating the progression of OC were identified. Collectively, the present results indicated that circ‑FAM53B could be a competing endogenous RNA (ceRNA) to competitively sponge miR‑646 and miR‑647 to upregulate VAMP2 and MDM2 expression at the post‑transcriptional level, thus mediating the cellular behaviors of OC cells.

Differential expressions of MDM2 and TAP73 in cancer and cancer-adjacent tissues in patients with non-small-cell lung carcinoma

AimTo investigate the differences in mRNA and protein expressions of MDM2 (mouse double minute 2 homolog) and P73 in cancer and cancer-adjacent tissues in patients with non-small-cell lung carcinoma (NSCLC). Materials and methodsWe compared the protein expressions of MDM2 and P73 in lung cancer and cancer-adjacent tissues in NSCLC patients by IHC (immunohistochemistry) and WB (Western blot). We divided the NSCLC patients into two subgroups, adenocarcinoma and squamous carcinoma. The mRNA expressions of two main isoforms of P73, TAP73 and DNP73, as well as the ratio of DNP73/TAP73 were analyzed by qPCR (quantitative real-time PCR) in the two tissues in all NSCLC patients and in patients with adenocarcinoma or squamous carcinoma, respectively. ResultsWB results did not show significant differences in MDM2 and P73 protein expressions in lung cancer and cancer-adjacent tissues. However, IHC results indicated that MDM2 expression significantly increased in cancer tissues in female patients, but not male patients. In addition, TAP73 mRNA expression significantly increased in cancer tissues in all NSCLC patients (p=0.002) and in patients with adenocarcinoma (p=0.01); while there was no significant difference in DNP73 mRNA expression. Hence the fold-change of DNP73/TAP73 ratio significantly decreased (p=0.0003) in cancer tissues in all NSCLC patients and in patients with adenocarcinoma. ConclusionsTAP73 mRNA expression significantly increased in cancer tissues than cancer-adjacent tissues in all NSCLC patients and in patients with adenocarcinoma. Meanwhile, the fold-change of DNP73/TAP73 ratio was similar to TAP73. MDM2 protein expression significantly increased in cancer tissues in female NSCLC patients.

Modifying effect of mouse double minute-2 promoter variants on risk of recurrence for patients with squamous cell carcinoma of oropharynx

Functional mouse double minute-2 (MDM2) promoter variants may alter MDM2 expression and thus affect radiotherapy response and prognosis of squamous cell carcinoma of oropharynx (SCCOP). Thus we assessed association of 2 functional MDM2 promoter variants with recurrence risk of SCCOP. The disease-free survival (DFS) of patients with MDM2rs2279744 TT or MDM2rs937283 AA genotypes was significantly reduced compared with that of patients with corresponding GT/GG or AG/GG genotypes. Multivariable analysis showed patients with TT or AA genotypes had a significantly higher risk of SCCOP recurrence than those with corresponding GT/GG or AG/GG genotypes did. Furthermore, patients with combined risk genotypes of the 2 polymorphisms had significantly worse DFS and a higher recurrence risk than patients with fewer combined risk genotypes did (Ptrend < 0.001). Compared with patients with 0 risk genotypes, patients with 1 or 2 risk genotypes had an approximately 3- or 11-fold increased risk of SCCOP recurrence, respectively. Notably, for both individual and combined polymorphisms, the above similar recurrence risks were particularly higher among patients with human papilloma virus (HPV)-positive tumors. Taken together, our findings suggest that MDM2 promoter variants individually, or more likely jointly, play a role in determining the risk of recurrence of SCCOP, particularly HPV-positive SCCOP.

Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer.

Mouse double minute 2 (MDM2) protein functionally inactivates the tumor suppressor p53 in human cancer. Conventional MDM2 inhibitors provide limited clinical application as they interfere only with the MDM2-p53 interaction to release p53 from MDM2 sequestration but do not prevent activated p53 from transcriptionally inducing MDM2 expression. Here, we report a rationally synthesized chalcone-based pyrido[ b ]indole, CPI-7c, as a unique small-molecule inhibitor of MDM2, which not only inhibited MDM2-p53 interaction but also promoted MDM2 degradation. CPI-7c bound to both RING and N-terminal domains of MDM2 to promote its ubiquitin-mediated degradation and p53 stabilization. CPI-7c-induced p53 directly recruited to the promoters of DR4 and DR5 genes and enhanced their expression, resulting in sensitization of TNF-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells toward TRAIL-induced apoptosis. Collectively, we identified CPI-7c as a novel small-molecule inhibitor of MDM2 with a unique two-prong mechanism of action that sensitized TRAIL-resistant cancer cells to apoptosis by modulating the MDM2-p53-DR4/DR5 pathway.

The Role of MDM2 Amplification and Overexpression in Tumorigenesis.

Mouse double minute 2 (MDM2) is a critical negative regulator of the tumor suppressor p53, playing a key role in controlling its transcriptional activity, protein stability, and nuclear localization. MDM2 expression is up-regulated in numerous cancers, resulting in a loss of p53-dependent activities, such as apoptosis and cell-cycle arrest. Genetic amplification and inheritance of MDM2 promoter single-nucleotide polymorphisms (SNPs) are the two best-studied mechanisms for up-regulating MDM2 activity. This article provides an overview of these events in human cancer, highlighting the frequent occurrence of MDM2 amplification in sarcoma and the role of SNP309 and SNP285 in regulating MDM2 expression and cancer risk. The availability of large-scale genomic profiling datasets, like those from The Cancer Genome Atlas Research Network, have provided the opportunity to evaluate the consequences of MDM2 amplification and SNP inheritance across high-quality tumor samples from diverse cancer indications.

Mouse double minute 2 (MDM2) upregulates Snail expression and induces epithelial-to-mesenchymal transition in breast cancer cells in vitro and in vivo.

The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers. In this study, we investigated the role of MDM2 in epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms in breast cancer cells in vitro and in vivo. The results showed that up-regulation of MDM2 in MCF-7 cells altered the cell morphology to a mesenchymal phenotype. Knockdown of MDM2 in MDA-MB-231 cells altered the cell morphology to the epithelial phenotype. In addition, overexpression of MDM2 increased the expression of N-cadherin and Vimentin and decreased the expression of E-cadherin, at both the mRNA and protein levels, in vitro and in vivo. Conversely, down-regulation of MDM2 decreased the expression of N-cadherin and Vimentin, and increased the expression of E-cadherin in vitro. Furthermore, MDM2 up-regulated both the mRNA and protein expression of Snail in vitro and in vivo. Knockdown of Snail almost abolished MDM2 induced EMT in vitro. Finally, we found that MDM2 expression correlated with EMT markers and Snail: Snail expression was inversely associated with E-cadherin in human breast cancer samples. Our findings demonstrated that MDM2 induces EMT by enhancing Snail expression in vitro and in vivo. Thus, MDM2 may be a potential target for therapy against human metastatic breast cancer.

Abstract A26: Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells

Abstract A multi-targeted approach will be necessary to eradicate glioblastoma multiforme (GBM) cells due to the immense genetic heterogeneity associated with GBM. Mouse double minute-2 (MDM2) regulates multiple signaling pathways and is a promising therapeutic target in GBM. In wild type (wt) p53 cells, MDM2 binds to wtp53, ubiquitinates it, and negatively regulates p53-mediated downstream events. In wtp53 and mutant (mt) p53 cells, MDM2 binds to and sequesters p73α thereby blocking p73α-mediated signaling. Our objective in the present studies was to determine if the p73α-MDM2 axis could be exploited to increase death of mtp53 GBM cells. We utilized MDM2 antagonists nutlin3a or RG7112 to block protein-protein interactions between MDM2-p53 and MDM2-p73α. In a panel of GBM cell lines, TMZ resistance was reduced in both wt53 and mt53 cells in the presence of MDM2 antagonists. In mtp53 cells, siRNA knockdown of p73α indicated that sensitivity to treatment was dependent on p73α levels. Isobologram analysis indicated that while dose-ratios of TMZ to MDM2 antagonists were additive to synergistic in inhibiting growth of wtp53 GBM cells, this was not the case in mtp53 GBM cells (SF118, GBM43, gain-of-function-mtp53 R273H U373 and MHBT32). Analysis of intracellular targets in mtp53 GBM cells exposed to TMZ and MDM2 antagonists indicated that p73α and MDM2 expression increased by 24 hours post-treatment. In addition, AKT activity was increased or sustained in mtp53 GBM cells following treatment with TMZ in the absence or presence of MDM2 antagonists. Since increased AKT activity may render cells resistant to therapy, the AKT inhibitor GDC0068 was evaluated in combination with TMZ and RG7112. As a measure of AKT-downstream target modulation, phosphorylation status of the Forkhead box O-class (FoxO) transcription factors (TFs) was determined. In the non-phosphorylated state, FoxO TFs upregulate expression of proteins involved in cell-death pathways. While phospho-FoxO1/FoxO3a TFs were increased in TMZ/RG7112-treated mtp53 GBM cells compared to controls, it was decreased in GDC0068-, TMZ/GDC0068- and TMZ/RG7112/GDC0068-treated mtp53 GBM cells which is consistent with inactivation of AKT and activation of FoxO TFs. Isobologram analysis of mtp53 GBM cell growth indicated that combination RG7112 and GDC0068 inhibited growth in a synergistic manner even in the absence of TMZ. For in vivo studies, an intermittent dosing regimen of TMZ/RG7112/GDC0068 was developed to avoid normal tissue toxicity. GBM43 flank tumor growth was significantly inhibited in mice with tumors treated with RG7112/GDC0068 and inhibited to a larger extent by the triple combination TMZ/RG7112/GDC0068 compared to vehicle and single-agent exposure (n=9-10 mice per group; single agent vs GDC0068/RG7112 or TMZ/RG7112/GDC0068, p&amp;lt;0.05). The present data indicate that targeting the p73α-MDM2 and AKT-FoxO signaling networks inhibit mtp53 GBM cell growth and with an appropriate dosing schedule can be utilized in vivo with an acceptable toxicity profile. Citation Format: Mohammad Reza Saadatzadeh, Haiyan Wang, Jixin Ding, Barbara J. Bailey, Eva Tonsing-Carter, Shanbao Cai, Nimita Dave, Harlan E. Shannon, Aaron Cohen- Gadol, Karen E. Pollok. Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A26.

Role of the MDM2 promoter polymorphism (-309T>G) in acute myeloid leukemia development.

The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator of Tp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpression thereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate the possible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for the MDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCR method. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantified MDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypes and the MDM2 expression were correlated with disease free survival (DFS) rates among patients who have achieved complete remission (CR) after first induction chemotherapy. MDM2-309T>G polymorphism was significantly associated with AML development (p<0.0001). The presence of either GG genotype or G allele at MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p=0.003) increased AML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates (16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2 expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+ve cases (p=0.0036). The MDM2-309T>G polymorphism might be involved in AML development and also serve as a good prognostic indicator.

All-trans retinoic acid induces p53-depenent apoptosis in human hepatocytes by activating p14 expression via promoter hypomethylation

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, has been extensively studied for the prevention and treatment of cancer; however, the underlying mechanism of its anti-cancer potential is still unclear. Here we found that ATRA induces apoptosis in p53-positive HepG2 cells, but not in p53-negative Hep3B cells. For this effect, ATRA activated p14 expression via promoter hypomethylation, resulting in ubiquitin-dependent degradation of mouse double minute 2 (MDM2) and subsequent stabilization of p53. The potential of ATRA to stabilize p53 was almost completely abolished by knock-down of p14 in HepG2 cells and was not observed in p14-negative A549 cells. Upregulation of p14 also abolished the self-regulatory potential of p53 to repress p14 expression via DNA methylation and transcriptionally activate MDM2 expression. The accumulated p53 then activated several apoptosis-related molecules, including Bax, PUMA, caspase-9, Bid, caspase-8, caspase-3, and PARP. Ectopic expression of DNA methyltransferase 1 almost completely abolished the potential of ATRA to activate the p14-MDM2-p53 pathway and induce p53-dependent apoptosis. Therefore, we conclude that ATRA induces p14 promoter hypomethylation to trigger apoptosis.

MDM2 and CDK4 expression in periosteal osteosarcoma.

Periosteal osteosarcoma is defined by the World Health Organization as an intermediate-grade, malignant, cartilaginous, and bone-forming neoplasm arising on the surface of bone. Unlike other subtypes of osteosarcoma, no data have been published about mouse double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) expression. For this reason, we evaluated the molecular and immunohistochemical features of MDM2 and CDK4 in 27 cases relative to 20 patients with a diagnosis of periosteal osteosarcoma, surgically treated at the Rizzoli Institute between 1981 and 2014. When possible, these results were compared with the MDM2 amplification status as determined by fluorescence in situ hybridization (FISH). All but 1 case (26/27, 96.3%) were negative for MDM2 protein using immunohistochemistry both in primary and in recurrent periosteal osteosarcoma, whereas gene amplification of MDM2 was not detected in any tumor analyzed (10 cases). The positive immunohistochemical case shows a weak/moderate focal nuclear expression of MDM2 antibody in the prevalent cartilaginous component and in the spindle cells of peripheral fibroblastic areas associated with osteoid production in a primary periosteal osteosarcoma. CDK4 immunohistochemical expression was negative in all 27 cases. This retrospective analysis has demonstrated that MDM2 and CDK4 are very rarely expressed in primary and recurrent periosteal osteosarcomas and therefore do not appear to be molecules central to the control of cancer development, growth, and progression in periosteal osteosarcoma. Therefore, when compared with low-grade central and parosteal osteosarcomas, MDM2 and CDK4 markers cannot be used diagnostically to differentiate this subtype of osteosarcoma.

TSLP Induces Mast Cell Development and Aggravates Allergic Reactions through the Activation of MDM2 and STAT6

Thymic stromal lymphopoietin (TSLP) is known to promote T helper type 2 cell-associated inflammation. Mast cells are major effector cells in allergic inflammatory responses. We noted that the population and maturation of mast cells were reduced in TSLP-deficient mice (TSLP-/-). Thus, we hypothesized that TSLP might affect mast cell development. We found that TSLP induced the proliferation and differentiation of mast cells from bone marrow progenitors. TSLP-induced mast cell proliferation was abolished by depletion of mouse double minute 2 (MDM2) and signal transducers and activators of transcription 6 (STAT6), as an upstream activator of MDM2. TSLP-/-, in particular, had a considerable deficit in the expression of MDM2 and STAT6. Also, the TSLP deficiency attenuated mast cell-mediated allergic reactions through the downregulation of STAT6 and MDM2. In an antibody microarray chip analysis, MDM2 expression was increased in atopic dermatitis patients. These observations indicate that TSLP is a factor for mast cell development, and that it aggravates mast cell-mediated immune responses.