MDM2 Protein Expression Research Articles

MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas.

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.

Fabrication and toxicity characterization of a hybrid material based on oxidized and aminated MWCNT loaded with carboplatin

This study focused on the fabrication and toxicity characterization of a hybrid material-based on the multiple functionalizations of multiwalled carbon nanotubes (MWCNTs) with carboxyl or amino groups and the anti-tumor drug carboplatin (CP). The functionalization was evidenced by Fourier transformed infrared spectroscopy (FTIR) and high performance liquid chromatography (HPLC). The amount of platinum ions released in the simulated body fluid (SBF) was assessed by inductively coupled plasma mass spectrometry (ICP-MS). Cell viability, nanotubes cellular uptake, cell proliferation, superoxide anion production, SOD activity, intracellular glutathione and protein expression of several molecules involved in breast tumor cell survival and death were investigated after 24h exposure. Exposure to the aminated carbon nanotubes loaded with carboplatin resulted in a greater decrease of viability compared to oxidized carbon nanotubes loaded with the same drug, which was in an inversely proportional relationship with the production of superoxide anions in breast cancer cells. The inhibition of Hsp60, Hsp90, p53 and Mdm2 protein expression was induced as a consequence of the cytoprotection mechanism failure. Overexpression of Beclin1 and the reduction of Bcl2 expression were also observed, suggesting that functionalized MWCNT loaded with CP trigger cell death via autophagy in breast cancer cells.

Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells.

Abstract 3703: MDM2 regulation of MYC and MYCN in pediatric neural cancers

Abstract Background MYC and MYCN have critical roles in a wide range of cancers and can be deregulated through diverse transcriptional and post-transcriptional mechanisms. Recent studies have shown that the high-level MYCN expression needed to drive retinoblastoma and MYCN-amplified neuroblastoma proliferation depends upon efficient MYCN translation mediated by highly expressed MDM2 and acting via a p53-independent mechanism. Here, we assessed MDM2-mediated regulation of MYC as well as MYCN in other pediatric neural tumors, specifically in Group 3 medulloblastoma and in MYC-overexpressing neuroblastoma. We also assessed MDM2-mediated regulation of MYCN in small cell lung cancer (SCLC), which has genetic and morphologic features in common with retinoblastoma. Methods MDM2, MYCN, and MYC expression were defined in Group 3 medulloblastoma lines D283, D425, D341; in MYC-overexpressing neuroblastoma lines CHLA 255 and SY-5Y; and in p53-wild type and p53-mutant SCLC lines H69 and H526 by western blotting. Cells were infected with shMDM2 and scrambled control lentivirus and effects on MDM2, MYC, and MYCN protein expression defined using western blot and effects on cell growth defined using Cell Titer-Glo. In neuroblastoma cells, MDM2 knockdown effects were defined with or without p53 co-knockdown. Results Group 3 medulloblastoma lines D283, D341, D425 highly expressed MYC but not MYCN. MDM2 knockdown did not decrease MYC expression in these cells. MDM2 knockdown decreased MYC expression In CHLA 255 and SY5Y neuroblastoma cells, along with p53 induction and cell death, and MYC down-regulation and cell death were mitigated by co-knockdown of p53. MDM2 knockdown decreased MYCN expression and impaired viability in p53-wild type H69 SCLC cells and in p53-mutant H526 SCLC cells. Conclusions MDM2 is not needed to sustain high-level MYC expression in three Group 3 medulloblastoma cell lines. MDM2 sustains high-level MYC in two MYC-overexpressing neuroblastoma cell lines via a p53-dependent mechanism distinct from the p53-independent MYCN regulation in MYCN-amplified neuroblastomas. In SCLC, MDM2 sustains high-level MYCN expression and viability in a p53-independent manner, similar to MDM2 effects in retinoblastoma and in MYCN-amplified neuroblastoma. These findings suggest that MDM2 selectively sustains MYCN but not MYC expression through a p53-independent mechanism and raise the possibility that cis-acting elements specific to MYCN-RNA mediate MDM2 regulation. Citation Format: Hung N. Tran, Donglai Qi, David Cobrinik. MDM2 regulation of MYC and MYCN in pediatric neural cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3703.

The miR-367-3p Increases Sorafenib Chemotherapy Efficacy to Suppress Hepatocellular Carcinoma Metastasis through Altering the Androgen Receptor Signals

The androgen receptor (AR) was found to suppress hepatocellular carcinoma (HCC) metastasis at late stages. Due to this discovery, we searched for some AR enhancers to increase the efficacy of Sorafenib chemotherapy, and identified the microRNA (miR)-367-3p, whose expression is positively correlated with AR expression in advanced HCC, as an HCC metastasis suppressor. Combining miR-367-3p with Sorafenib showed better efficacy to suppress HCC cell invasion in vitro and in vivo. Mechanism dissection revealed that miR-367-3p could increase AR expression via directly targeting the 3′UTR of MDM2 to decrease MDM2 protein expression. The resultant increase of AR expression might then promote the expression of FKBP5 and PHLPP, thus dephosphorylating and inactivating AKT and ERK, to suppress the HCC cell invasion. Interestingly, the suppression of pAKT by miR-367-3p could subsequently attenuate the phosphorylation of AR and MDM2, giving rise to additional enhancement of AR protein expression, effectively forming a positive feedback loop. Together, these results suggest that miR-367-3p may function as an AR enhancer to increase Sorafenib chemotherapy efficacy via altering the MDM2/AR/FKBP5/PHLPP/(pAKT and pERK) signals to better suppress HCC metastasis. Successful development of this newly combined chemotherapy in the future may help us to better suppress the HCC metastasis at late stages.

Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene

The objective of the present study was to explore the invitro and invivo anticancer effects of PlatycodinD (PD), derived from Platycodin grandiflorum, on highly metastatic MDA-MB-231 breast cancer cells. Using the MTT assay, we found that PD inhibited MDA-MB-231 cell growth in a concentration-dependent manner, with an IC50 value of 7.77±1.86µM. Further studies showed that PD had anti-proliferative effects and induced cell cycle arrest in the G0/G1 phase. To explore the detailed mechanism(s) by which PD suppressed MDA-MB-231 cell growth, western blot analyses were used to detect the expression levels of proteins related to cell proliferation and survival. The data showed that PD decreased the expression of proteins related to the G0/G1 phases, downregulated the protein expression of MDM2, MDMX, and mutant p53, and increased the expression levels of p21 and p27 invitro. We verified the effects of PD on the expression of MDM2, MDMX, mutant p53, p21 and p27 using a pcDNA3-Flag-MDM2 plasmid and MDM2 siRNA transfection, and found that PD inhibited MDA-MB-231 cell viability by targeting MDM2 and mutant p53. Compared with the corresponding parental cells, the cells with siRNA-MDM2 transfection had a greater decrease in cell viability and proliferation, while those with pcDNA3-MDM2 plasmid transfection did not show any increase in the effects of PD. We also established a MDA-MB-231 xenograft model in BALB/c nude mice, and found that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in these mice. The expression levels of various proteins in the tumor tissue exhibited changes similar to those observed invitro. These findings indicate that PD exerted invitro and invivo anticancer effects against MDA-MB-231 breast cancer cells, that PD is a potential MDM2/MDMX inhibitor, and that the anticancer effects of PD were likely associated with its inhibition of these proteins. Our observations help to identify a mechanism by which PD functions as an anti-breast cancer agent.

Histone deacetylase inhibitor sodium butyrate suppresses proliferation and promotes apoptosis in osteosarcoma cells by regulation of the MDM2-p53 signaling.

Histone deacetylase inhibitors have been reported to induce tumor cell growth arrest, differentiation, and apoptosis. This study aimed to investigate the effects of one histone deacetylase inhibitor – sodium butyrate (SB) – on osteosarcoma (OS) cell proliferation and apoptosis and also the molecular mechanisms by which SB exerts regulatory effects on OS cells. U2OS and MG63 cells were treated with SB at various concentrations. Then, cell proliferation and apoptosis were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and flow cytometry assays, respectively; the expression of Ki67, Bax, Bcl-2, MDM2, and p53 proteins was determined by using Western blot assay. The results showed that SB suppressed proliferation in a concentration-dependent manner and promoted apoptosis of OS cells. In addition, SB enhanced p53 expression and decreased MDM2 expression, indicating that SB can regulate MDM2–p53 feedback loop. p53 inhibited proliferation and promoted apoptosis, whereas MDM2 promoted proliferation and suppressed apoptosis, which indicated that functional effect of SB on OS cell lines at least in part depended on the MDM2–p53 signaling. We also explored the effect of SB on OS cells in vivo and found that SB suppressed the growth of OS cells with no noticeable effect on activity and body weight of mice in vivo. These findings will offer new clues for OS development and progression and offer SB as a potent targeted agent for OS treatment.

Intravenous leiomyomatosis: an unusual intermediate between benign and malignant uterine smooth muscle tumors

Intravenous leiomyomatosis is an unusual smooth muscle neoplasm with quasi-malignant intravascular growth but a histologically banal appearance. Herein, we report expression and molecular cytogenetic analyses of a series of 12 intravenous leiomyomatosis cases to better understand the pathogenesis of intravenous leiomyomatosis. All cases were analyzed for the expression of HMGA2, MDM2, and CDK4 proteins by immunohistochemistry based on our previous finding of der(14)t(12;14)(q14.3;q24) in intravenous leiomyomatosis. Seven of 12 (58%) intravenous leiomyomatosis cases expressed HMGA2, and none expressed MDM2 or CDK4. Colocalization of hybridization signals for probes from the HMGA2 locus (12q14.3) and from 14q24 by interphase fluorescence in situ hybridization (FISH) was detected in a mean of 89.2% of nuclei in HMGA2-positive cases by immunohistochemistry, but in only 12.4% of nuclei in negative cases, indicating an association of HMGA2 expression and this chromosomal rearrangement (P=8.24 × 10−10). Four HMGA2-positive cases had greater than two HMGA2 hybridization signals per cell. No cases showed loss of a hybridization signal by interphase FISH for the frequently deleted region of 7q22 in uterine leiomyomata. One intravenous leiomyomatosis case analyzed by array comparative genomic hybridization revealed complex copy number variations. Finally, expression profiling was performed on three intravenous leiomyomatosis cases. Interestingly, hierarchical cluster analysis of the expression profiles revealed segregation of the intravenous leiomyomatosis cases with leiomyosarcoma rather than with myometrium, uterine leiomyoma of the usual histological type, or plexiform leiomyoma. These findings suggest that intravenous leiomyomatosis cases share some molecular cytogenetic characteristics with uterine leiomyoma, and expression profiles similar to that of leiomyosarcoma cases, further supporting their intermediate, quasi-malignant behavior.

NORE1A Regulates MDM2 Via β-TrCP.

Mouse Double Minute 2 Homolog (MDM2) is a key negative regulator of the master tumor suppressor p53. MDM2 regulates p53 on multiple levels, including acting as an ubiquitin ligase for the protein, thereby promoting its degradation by the proteasome. MDM2 is oncogenic and is frequently found to be over-expressed in human tumors, suggesting its dysregulation plays an important role in human cancers. We have recently found that the Ras effector and RASSF (Ras Association Domain Family) family member RASSF5/NORE1A enhances the levels of nuclear p53. We have also found that NORE1A (Novel Ras Effector 1A) binds the substrate recognition component of the SCF-ubiquitin ligase complex β-TrCP. Here, we now show that NORE1A regulates MDM2 protein levels by targeting it for ubiquitination by SCF-β-TrCP. We also show the suppression of NORE1A protein levels enhances MDM2 protein expression. Finally, we show that MDM2 can suppress the potent senescence phenotype induced by NORE1A over-expression. Thus, we identify a mechanism by which Ras/NORE1A can modulate p53 protein levels. As MDM2 has several important targets in addition to p53, this finding has broad implications for cancer biology in tumor cells that have lost expression of NORE1A due to promoter methylation.

Alterations in PTEN, MDM2, TP53 and AR protein and gene expression are associated with canine prostate carcinogenesis

The PTEN, AR, MDM2 and p53 protein network plays a central role in the development of many human cancers, thus eliciting the development of targeted cancer therapeutics. Dogs spontaneously develop tumours, and they are considered a good model for comparative oncology initiatives. Due to the limited information on these proteins in canine tumours, this study aimed to investigate gene and protein alterations in PTEN, AR, MDM2 and p53 in canine prostate cancer (PC). Protein expression was evaluated by immunohistochemistry (15 normal, 22 proliferative inflammatory atrophy (PIA) and 19 PC samples) and Western blotting (2 normal prostate tissue, 2 BPH, 2 PIA samples and 2 PC samples) and gene expression by RT-qPCR (10 normal, 10 PIA and 15 PC samples) of formalin-fixed tissue. We identified nuclear and cytoplasmic expression of PTEN and p53 in all samples, with only nuclear staining found for MDM2 and AR. Our results revealed high expression of MDM2 in PC and PIA samples compared to normal samples, whereas PTEN, P53 and AR expression was down-regulated in PC compared to normal tissue. All tumour samples (n=19) showed loss of nuclear PTEN expression, and all cancer mimickers showed positive nuclear staining. Therefore, nuclear PTEN staining could be a good diagnostic marker for differentiating between malignant lesions and mimickers. Canine prostate carcinogenesis involves increased expression of MDM2 in association with decreased expression of PTEN, p53 and AR, such as occurs in hormone refractory PC in men. Thus, dogs may be an important model for studying advanced stage PC.

The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans.

The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53-Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway.

P53 causes butein‑mediated apoptosis of chronic myeloid leukemia cells.

Progression of chronic myeloid leukemia, marked by the oncogenic Bcr-Abl mutation, is tightly associated with an alteration of the p53 pathway. It is known that butein extracted from various plants represses cancer growth. Although the anticancer effects of butein are widely accepted, the mechanisms by which butein induces apoptosis of chronic myeloid leukemia cells remains to be elucidated. The present study demonstrated that butein-induced apoptosis was mediated by p53. KBM5 chronic myeloid leukemia (CML) cells expressing wild-type p53 were more sensitive to butein compared with p53-null K562 CML cells in terms of apoptotic cell death. In addition, butein arrested KBM5 cells at S-phase and altered the expression levels of certain cyclins and the p53-downstream targets, MDM2 and p21. In addition, while butein reduced the protein expression of MDM2 in the KBM5 and K562 cells, it resulted in proteasome-independent MDM2 degradation in p53-expressing KBM5 cells, however, not in p53-null K562 cells. Therefore, the present study suggested that p53 causes the butein-mediated apoptosis of leukemic cells.

Clinical Response to Idasanutlin (RG7388) in Acute Myeloid Leukemia Patients Is Associated with Pre-Treatment MDM2 Protein Expression in Leukemic Blasts and Leukemic Stem Cells

Introduction. Despite years of translational research and discovery, outcomes in Acute Myeloid Leukemia remain poor. The MDM2 antagonist idasanutlin has shown promising clinical activity in solid tumors and acute leukemias. Idasanutlin enhances the activity of the tumor suppressor, p53, through antagonism of MDM2:p53 interaction. Such protein:protein disruption abolishes MDM2 targeting of p53 for ubiquitination and degradation. Subsequent stabilization of the p53 protein allows it to exert tumor suppressor transcriptional regulation and induction of apoptotic pathways. Identification of patients in whom functional p53 activation drives efficacy may translate into improved outcomes for patients treated with idasanutlin.Patients and Methods. Trial NP28679 (NCT01773408) is a Phase 1/1b study evaluating idasanutlin (as monotherapy or in combination with cytarabine) in relapsed or refractory AML patients, with clinical response as the primary endpoint. To identify biomarkers of response, patients' pre-treatment peripheral blood specimens were evaluated for MDM2 protein expression levels in leukemic blasts and leukemic stem cells. Association of MDM2 percent cell positivity with clinical outcomes was evaluated using intracellular flow cytometry gated on CD45dim leukemic blasts and CD45dim/CD117+/CD34+ leukemic stem cells.Results. We observed in a proof-of-principle training data set that MDM2 expression in leukemic blasts was associated with patients more likely to exhibit Complete Remission (CR, CRp, CRi) versus Progressive Disease (PD, HI, PR) [n=61; Wilcoxon p = .0041 (AUC = .74; 95%CI[.57, .91])] TP53 mutational status alone was not tightly associated with patient response (Fisher's Exact Test p = .19 [AUC = .60; 95%CI [.52,.67.])] When MDM2 protein expression and TP53 mutational status were analyzed as co-variates, an enhanced association with patient CR was observed (AUC = .76; 95% CI [.59, .93.]) A separate group of patients treated with an optimized idasanutlin formulation was utilized as a validation set. Association of CR with MDM2 expression in blasts was also shown in this separate patient population [n=24; Wilcoxon p = .0052 (AUC = .82; 95% CI [.64, 1.00.]) Comparable results were observed for proof-of-principle and validation set analyses of response association with MDM2 protein expression in AML patients CD45dim/CD117+/CD34+ stem cell subpopulations.Conclusions. In summary, training and validation sets reveal that MDM2 protein expression in leukemic blasts and stem cells are associated with idasanutlin-induced CR in patients with AML. We will continue to monitor this potentially predictive biomarker in future randomized clinical studies of idasanutlin. On a broader level, the data presented here support the concept that leukemic blasts may be "oncogene-addicted" to MDM2 in AML, i.e. that AML tumor cells may rely on a dominant oncogene for growth and survival, such that inhibition of the function of this specific oncogene is sufficient to halt the neoplastic phenotype. Thus, the concept of oncogene addiction may apply not only to mutated kinases but also to ubiquitin ligases such as MDM2. DisclosuresReis:Roche Pharma: Employment, Equity Ownership. Jukofsky:Roche Pharma: Employment, Equity Ownership. Chen:Roche Pharma: Employment, Equity Ownership. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Zhong:Roche Pharma: Employment, Equity Ownership. So:Roche Pharma: Employment, Equity Ownership. Drummond:Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees. Assouline:Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy. Hashemyan:Roche Pharma: Employment, Equity Ownership. Theron:Roche Pharma: Employment, Equity Ownership. Blotner:Roche Pharma: Employment, Equity Ownership. Rueger:Roche Pharma: Employment, Equity Ownership. Middleton:Roche Pharma: Employment, Equity Ownership. Vey:Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria. Nichols:Roche Pharma: Employment, Equity Ownership. Chen:Roche Pharma: Employment, Equity Ownership. Pierceall:Roche Pharma: Employment, Equity Ownership.

Sodium glycididazole enhances the radiosensitivity of laryngeal cancer cells through downregulation of ATM signaling pathway.

The purpose of this study was to evaluate the radiation-enhancing effect of sodium glycididazole, and the corresponding mechanisms of action on laryngeal cancer cells. Two laryngeal cancer cell lines (Hep-2 and UT-SCC-19A) were irradiated with X-rays in the presence or absence of sodium glycididazole. Cell survival, DNA damage and repair, cell apoptosis, cell cycle distribution, expression of proteins related to cell cycle checkpoint, and apoptosis were measured. Significantly increased DNA damages, decreased cells in the G1 phase, arrested cells at G2/M phase, decreased DNA repair protein XRCC1 foci formation, and enhanced cell apoptosis were observed in laryngeal cell lines treated by sodium glycididazole combined with irradiation compared with the irradiation alone. The combined treatment downregulated the protein expressions of ataxia-telangiectasia mutated (ATM), p-ATM, CHK2, and P53 but upregulated the protein expressions of MDM2 and Cdk2. This study indicates that sodium glycididazole enhances the radiosensitivity of laryngeal cancer cells through downregulation of ATM signaling pathway in vitro and in vivo.

GLI1 expression is an important prognostic factor that contributes to the poor prognosis of rhabdomyosarcoma.

The GLI1 and MDM2 genes are amplified or exhibit copy number gains in rhabdomyosarcoma (RMS). Here, we used immunohistochemistry to determine the relationships between GLI1 and MDM2 protein expression and several clinicopathological variables of RMS. GLI1 and MDM2-positivity rates were 61.36% and 13.64%, respectively. GLI1 expression correlated with presence of the PAX3-FOXO1 fusion gene (P=0.040) and lymph node metastasis (P=0.034), and a significant association was found between GLI1 expression and overall survival (OS) (P=0.008). However, there was no association between MDM2 expression and any of the clinicopathological parameters or OS. Thus, GLI1 may be a biomarker of poor prognosis in RMS patients, and could itself be a therapeutic target. This contrasts with the apparent lack of clinical importance of MDM2 in RMS pathology, at least in the cohorts we examined.

Dual inhibition of nuclear factor kappa-B and Mdm2 enhance the antitumor effect of radiation therapy for pancreatic cancer

IntroductionRadiation therapy, alone or in combination with chemotherapy, is effective for patients with locally advanced and recurrent pancreatic cancer. Ionizing radiation induces cell cycle arrest and cell apoptosis through enhancement several signals such as p53, p21Waf1/Cip1, and caspase. However, the therapeutic efficacy is attenuated by radiation-induced activation of NF-κB. Nafamostat mesilate, a synthetic serine protease inhibitor, inhibits NF-κB activation in pancreatic cancer. Therefore, we hypothesized that nafamostat mesilate inhibited radiation-induced activation of NF-κB and improves therapeutic outcome. ResultsIn combination group, NF-κB activation was significantly inhibited in comparison with that of radiation group. Nafamostat mesilate obviously down-regulated the expression levels of Mdm2 compared with control cells or irradiated cells. Consequently, p53 expression was stabilized inversely in correlation with Mdm2 protein expression level. The expression levels of p53, p21Waf1/Cip1, cleaved caspase-3 and -8 were the highest in the combination group. Nafamostat mesilate enhanced ionizing radiation-induced cell apoptosis and G2/M cell cycle arrest. In combination group, cell proliferation and tumor growth were significantly slower than those in other groups. ConclusionCombination therapy of radiation with nafamostat mesilate exerts enhanced anti-tumor effect against human pancreatic cancer.

Expression and Clinical Significance of MDM2 and p21WAF1 Proteins in Human Glioma

Expression and Clinical Significance of MDM2 and p21WAF1 Proteins in Human Glioma

Abstract 1785: Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner

Abstract The mechanisms that determine p53 cell fate decision after its non-genotoxic activation by MDM2 inhibitors are poorly understood. Wild-type p53-inducible phosphatase-1 (WIP1/PPM1D) forms an autoregulatory loop with p53 and is involved in the homeostasis of the p53 stress response network. WIP1 is activated, gained/amplified and overexpressed in a range of p53 wild-type malignancies. We investigated cellular growth/proliferation of p53 wild-type (wt) and matched p53 mutant (mut)/null cell line pairs, differing in WIP1 genetic status, in response to Nutlin-3 + GSK2830371 (WIP1i)/WIP1 siRNA-mediated knockdown. We also assessed the effects of WIP1i or knockdown in combination with Nutlin-3 on p53 post-translational modifications, p53 downstream transcriptional targets and apoptosis. Phosphorylation of p53S15 (pp53S15) was used as a marker of WIP1 catalytic activity. WIP1-amplified MCF-7p53wt cells were very sensitive to the WIP1i, with a 50% growth inhibitory concentration (GI50) of 2.65μM + 0.54 (SEM). The three p53 wild-type and mutant/null cell line pairs showed relatively little sensitivity to single agent WIP1i (GI50>10μM). However the WIP1i, at a non-growth inhibitory dose (2.5μM) in vitro, potentiated their response to Nutlin-3 in a p53-dependent manner. HCT116p53+/+; WIP1L450fs*1/+ (L450fs*1 is a WIP1 activating mutation), NGPp53wt and SJSA-1p53wt cell lines showed a 2.4-fold (p = 0.007), 2.1-fold (p = 0.04) and 1.3-fold (p = 0.02) decrease in their Nutlin-3 GI50 values respectively in the presence of WIP1i at 2.5μM. In contrast Nutlin-3 GI50 did not change in HCT116p53-/−, N20R1p53Mut and SN40R2p53Mut, NGP and SJSA-1 Nutlin-3 resistant daughter cell lines respectively. WIP1 transient siRNA knockdown also resulted in potentiation of cells to Nutlin-3. In WIP1-non-amplified (WNA) cells treated with either Nutlin-3 at the GI50 dose or 2.5μM WIP1i, pp53S15 was not detected, however in combination, these drugs resulted in dramatic levels of pp53S15. Interestingly protein expression of p53 targets MDM2 and p21 were not affected in WNA cells by the increased p53S15 phosphorylation; whereas BAX expression was elevated. The Nutlin-3 + WIP1i/siRNA knockdown combination also resulted in a significant increase in cell death markers between 24-48Hrs post-treatment in a p53-dependent manner. Although the strongest WIP1i potentiation of Nutlin-3 was observed in HCT116p53+/+; WIP1L450fs*1/+ cells which possess an activating WIP1 mutation, the NGPp53wt and SJSA-1p53wt cell lines with wild-type WIP1 also showed potentiation. These findings highlight the mechanistic importance of the p53-WIP1 autoregulatory loop in cell fate determination after non-genotoxic p53 activation by Nutlin-3. Potent inhibitors of WIP1 may potentiate the response to other therapeutics aiming to activate p53 mediated tumour suppression. Citation Format: Arman Esfandiari, Nicola J. Curtin, John Lunec. Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1785. doi:10.1158/1538-7445.AM2015-1785

Study of Immunohistochemical Expression of Mdm2 and Bax Proteins in Oral Squamous Cell Carcinoma

Study of Immunohistochemical Expression of Mdm2 and Bax Proteins in Oral Squamous Cell Carcinoma

Plumbagin alters telomere dynamics, induces DNA damage and cell death in human brain tumour cells

Natural plant products may possess much potential in palliative therapy and supportive strategies of current cancer treatments with lesser cytotoxicity to normal cells compared to conventional chemotherapy. In the current study, anti-cancer properties of plumbagin, a plant-derived naphthoquinone, on brain cancer cells were determined. Plumbagin treatment resulted in the induction of DNA damage, cell cycle arrest and apoptosis, followed by suppression of the colony forming ability of the brain tumour cells. These effects were substantiated by upregulation of PTEN, TNFRSF1A and downregulation of E2F1 genes, along with a drop in MDM2, cyclin B1, survivin and BCL2 protein expression. Plumbagin induced elevated levels of caspase-3/7 activity as well. For the first time, we show here that plumbagin inhibits telomerase in brain tumour cells and results in telomere shortening following chronic long-term treatment. This observation implies considerable cytotoxicity of plumbagin towards cancer cells with higher telomerase activity. Collectively, our findings suggest plumbagin as a potential chemotherapeutic phytochemical in brain tumour treatment modalities.