Increase In MDM2 Research Articles

KLF11 promotes the proliferation of breast cancer cells by inhibiting p53-MDM2 signaling

Abnormal Krüppel-like factor 11 (KLF11) expression is frequently found in tumor tissues and is associated with cancer prognosis, but its biological functions and corresponding mechanisms remain elusive. Here, we demonstrated that KLF11 functions as an oncoprotein to promote tumor proliferation in breast cancer cells. Mechanistically, at the transcription level, KLF11 decreased TP53 mRNA expression. Notably, KLF11 also interacted with and stabilized MDM2 through inhibiting MDM2 ubiquitination and subsequent degradation. This increase in MDM2 in turn accelerated the ubiquitin-mediated proteolysis of p53, leading to the reduced expression of p53 and its target genes, including CDKN1A, BAX, and NOXA1. Accordingly, data from animals further confirmed that KLF11 significantly upregulated the growth of breast cancer cells and was inversely correlated with p53 expression. Taken together, our findings reveal a novel mechanism for breast cancer progression in which the function of the tumor suppressor p53 is dramatically weakened.

Hyperprogression of a mismatch repair-deficient colon cancer in a humanized mouse model following administration of immune checkpoint inhibitor pembrolizumab.

Immunotherapy is an established treatment modality in oncology. However, in addition to primary or acquired therapy resistance with immune checkpoint blockade (ICB), hyperprogressive disease (HPD) or hyperprogression (HP) with acceleration of tumor growth occurs in a subset of patients receiving ICB therapy. A validated and predictive animal model would help investigate HPD/HP to develop new approaches for this challenging clinical entity. Using human cytotoxic T-cell line TALL-104 injected intraperitoneally into immunodeficient NCRU-nude athymic mice bearing mismatch repair-deficient (MMR-d) human colon carcinoma HCT116 p53-null (but not wild-type p53) tumor xenograft, we observed accelerated tumor growth after PD-1 blockade with pembrolizumab administration. There was increased colon tumor cell proliferation as determined by immunohistochemical Ki67 staining of tumor sections. There was no increase in MDM2 or MDM4/MDMX in the p53-null HCT116 cells versus the wild-type p53-expressing isogenic tumor cells, suggesting the effects in this model may be MDM2 or MDM4/MDMX-independent. Human cytokine profiling revealed changes in IFN-γ, TRAIL-R2/TNFRSF10B, TRANCE/TNFSF11/RANK L, CCL2/JE/MCP-1, Chitinase 3-like 1, IL-4 and TNF-α. This represents a novel humanized HPD mouse model with a link to deficiency of the p53 pathway of tumor suppression in the setting of MMR-d. Our novel humanized preclinical TALL-104/p53-null HCT116 mouse model implicates p53-deficiency in an MMR-d tumor as a possible contributor to HPD/HP and may help with evaluating therapeutic strategies in cancer immunotherapy to extend clinical benefits of ICB’s in a broader patient population.

Abscopal Gene Expression in Response to Synchrotron Radiation Indicates a Role for Immunological and DNA Damage Response Genes

Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.

Contemplations on MDMX (MDM4) driving triple negative breast cancer circulating tumor cells and metastasis.

MDMX (MDM4) is emerging as an important breast cancer (BC) biomarker, and oncoprotein, that can be targeted in combination with its well-known family member MDM2. While MDM2 has previously been implicated in driving BC metastasis, information about the role of MDMX in driving circulating tumor cells (CTCs) and BC metastasis is lacking. BCs often have alterations of MDM2, MDMX, and mutant p53 (mtp53). Therefore, the role of MDM2 and MDMX in the context of mtp53 in BCs requires further clarification. Our group has recently reported that triple negative breast cancer (TNBC) metastasis is dependent on both MDM2 and MDMX, and depleting MDM2 results in increased MDMX, but depleting MDMX does not cause an increase in MDM2. In the context of human TNBC expressing mtp53 in an orthotopic mouse model the down-regulation of MDMX virtually cleared CTCs from the blood. Contemplations, using the available literature, suggest that disrupting the stability and/or function of MDMX protein (and its downstream targets), in the context of mtp53 expressing BCs, might be beneficial for patient survival. It remains to be determined if blocking mtp53-MDMX pathways can inhibit early stage TNBC and eliminate CTCs that have the potential to form metastatic lesions.

Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells

Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2SNP309G contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2SNP309 T/G. lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 ± 0.2 fold increase in Mdm2 and a 2.0 ± 0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 ± 0.2 fold), survival against apoptosis (2.2 ± 0.2 fold), migration (10 ± 1.5%) and contraction (112.7 ± 14.1 mm2) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 – such as proliferative vitreoretinopathy.

NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway.

Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFβ) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.

Functional Activation of Mutant p53 by Platinum Analogues in Cisplatin-Resistant Cells Is Dependent on Phosphorylation.

Dysfunctionality of the p53 tumor suppressor is a major cause of therapeutic drug resistance in cancer. Recently, we reported that mutant, but otherwise functional, p53v172F was inactivated in cisplatin-resistant 2780CP/Cl-16 and 2780CP/Cl-24 human ovarian tumor cells by increased recruitment of the inhibitor MDM4. The current study demonstrates that, unlike cisplatin, platinum analogues oxaliplatin and DACH-diacetato-dichloro-Pt(IV) (DAP) strongly stabilize and activate p53v172F in resistant cells, as indicated by prolonged p53 half-life and transactivation of targets p21 (CDKN1A) and MDM2. This increase in MDM2 reduced MDM4 levels in cell lysates as well as the p53 immunocomplex and prevented reversion of p53 to the inactive p53-MDM2-MDM4-bound state. Phosphorylation of p53 at Ser15 was demonstrated by all three drugs in sensitive A2780 and corresponding resistant 2780CP/Cl-16 and 2780CP/Cl-24 cell lines. However, cisplatin induced Ser20 phosphorylation in A2780 cells only, but not in resistant cells; in contrast, both DAP and oxaliplatin induced this phosphorylation in all three cell lines. The inference that Ser20 phosphorylation is more important for p53 activation was confirmed by ectopic expression of a phosphomimetic (S20D) mutant p53 that displayed reduced binding, relative to wild-type p53, to both MDM2 and MDM4 in p53-knockout A2780 cells. In consonance, temporal studies demonstrated drug-induced Ser15 phosphorylation coincided with p53 stabilization, whereas Ser20 phosphorylation coincided with p53 transactivation.Implications: Cisplatin fails to activate the pathway involved in phosphorylating mutant p53v172F at Ser20 in resistant cells, but this phosphorylation is restored by oxaliplatin and DAP that reactivates p53 function and circumvents cisplatin resistance. Mol Cancer Res; 15(3); 328-39. ©2016 AACR.

Role of Six Trans Membrane Protein of Prostate (STAMP) Proteins in Prostate Cancer- Relation with Survival Genes

Prostate cancer studies focus on identification of androgen receptor (AR) regulated genes that are also highly expressed in the prostate. As a promising candidate, STAMP family genes STAMP1/STEAP2, STAMP2/STEAP4 and STEAP3 are involved in apoptosisand the cell cycle in metastatic prostate cancer. Vascular NADPH oxidase generates superoxide and other ROS, which stimulates IkappaB degradation and NF-kB activation by subunits of NADPH oxidases, namely p47phox and p67phox induced by different stimuli such as hydrogen peroxide. Hydrogen peroxide increased the expression levels of p67phox. They also have a role in redox-sensitive genes such as STAMP gene family. Flow cytometry analysis of LNCaP cells was performed using Annexin V staining and apoptotic index charts were drawn. STAMP1 and STAMP2 showed total anti-oxidant capacity versus control with hydrogen peroxide incubation. Using siRNA technology in LNCaP cells expressing mutant p53 silencing of p53 showed significant increase in MDM2 and decrease of caspase 9 mRNA levels at RT-PCR. Silencing of STAMP2, a significant decrease in p47phox was shown but STAMP1 silencing counteracted this effect on Cu/ZnSOD expression.As a conclusion, STAMP proteins have effects on oxidative stress-induced genes with significant and opposite changes.

The p53-MDM2 Antagonist RG7388 Activates p53 and Induces a Predominantly Pro-Apoptotic Gene Expression Signature in Chronic Lymphocytic Leukemia

Introduction. Current treatment protocols for chronic lymphocytic leukemia (CLL), including FCR (fludarabine, cyclophosphamide and rituximab), have dramatically improved response rates. However, since none of these therapies is curative, continued preclinical studies on innovative therapeutic strategies are warranted. In particular, the identification of new agents that interfere with the survival of CLL cells by promoting their apoptosis is one approach to improve therapeutic outcome. Taking into account that p53 deletions and/or mutations in CLL are restricted to 10%-15% of patients at diagnosis, we have investigated the efficacy of the second-generation MDM2 antagonist RG7388, in primary CLL samples, examining its ability to disrupt the p53-MDM2 interaction, resulting in expression of p53 transcriptional targets that trigger apoptosis.Methods. Heparinized peripheral blood samples were obtained from CLL patients (n=42) with informed consent, in accordance with institutional guidelines and the Declaration of Helsinki. Mononuclear cells were purified by Lymphoprep density-gradient centrifugation. Cells were resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were treated with increasing concentrations of RG7388 and incubated at 37°C, 5% CO2. Ex vivo cytotoxicity was assessed in all samples 48 hours after treatment using an XTT assay. A pilot time-course experiment identified 6 hours after treatment as the most informative time point for gene expression analysis. Therefore, in 8 samples cells were treated for 6 hours and RNA was extracted using standard methods. qRT-PCR on a panel of nine p53 transcriptional targets (CDKN1A, MDM2, PUMA, BAX, FAS, FDXR, ZMAT3, TP53INP1, DR5) was performed using SYBR Green chemistry. The functional integrity of p53 was assessed by western blot following treatment with MDM2 antagonists in 20 samples. Apoptosis was assessed using a Caspase 3/7 luminescent assay.Results. Treatment with RG7388 significantly decreased cell viability (LC50 < 1µM) in 28/42 CLL samples (mean LC50 = 0.40 ± 0.05 µM). As expected, the 8 CLL samples that displayed non functional p53 on western blot and/or mutated TP53 by sequencing were more drug-resistant (mean LC50 = 5.22 ± 1.13 µM) than those sensitive responders (LC50 < 1µM) with a functional p53 (mean LC50 = 0.39 ± 0.05 µM, n=12). Interestingly, among p53 functional samples, we identified a small subset that showed intermediate response (1 µM <LC50< 10 µM, n=2) or resistance (LC50 >10 µM, n=3) to RG7388 treatment.RG7388 significantly upregulated the mRNA expression of MDM2 and of p53 transcriptional targets involved in the intrinsic pathway of apoptosis (PUMA and BAX), and the extrinsic pathway of apoptosis (death receptor 5 (DR5)) (Figure 1A, n=5 samples). Interestingly, only a slight upregulation of CDKN1A was observed and upregulation of pro-apoptotic genes dominated. Overall, in p53 functional CLL samples, exposure to 3 µM RG7388 for 6 hours led to a 7.8-fold increase in MDM2, a 7-fold increase in PUMA, a 4.9-fold increase in DR5, a 4.5-fold increase in BAX, and a 3.3-fold increase in CDKN1A (Figure 1A). Conversely, no significant upregulation of p53 target genes was seen in p53 non-functionalCLL samples (Figure 1B, n=3 samples).Treatment with RG7388 led to a concentration-dependent increase in caspase 3/7 activity in p53 functional CLL samples (Figure 1C). No significant difference in caspase 3/7 activity was observed in p53 non-functional samples after RG7388 treatment (Figure 1D).Interestingly, when we analysed samples taken two years apart from a fludarabine- and chlorambucil-resistant CLL patient, we found that treatment with RG7388 decreased cell viability, induced p53 accumulation and upregulation of p53-target genes, suggesting that inhibiting the p53-MDM2 interaction might be a promising treatment strategy in chemo-resistant CLL patients that show a functional p53.Conclusion. Taken together, our data indicate that RG7388 induces a characteristic dominantly pro-apoptotic gene expression profile of p53-target genes in primary p53-functional CLL samples. RG7388 showed a consequent potent apoptotic effect on CLL cells, which was dependent on the presence of a functional p53. Identification of patients in whom functional p53 activation drives apoptosis may translate into improved outcomes for patients treated with non-genotoxic MDM2 antagonists. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells.

The Mdm2 oncogene is a negative regulator of the p53 tumor suppressor and recently identified inhibitor of DNA break repair. Nutlin-3 is a small-molecule inhibitor of Mdm2-p53 interaction that can induce apoptosis in cancer cells through activation of p53. Although this is a promising therapy for those cancers with wild-type p53, half of all human cancers have inactivated p53. Here, we reveal that a previously unappreciated effect of Nutlin is inhibition of DNA break repair, stemming from its ability to increase Mdm2 protein levels. The Nutlin-induced increase in Mdm2 inhibited DNA double-strand break repair and prolonged DNA damage response signaling independent of p53. Mechanistically, this effect of Nutlin required Mdm2 and acted through Nbs1 of the Mre11-Rad50-Nbs1 DNA repair complex. In ovarian cancer cells, where >90% have inactivated p53, Nutlin combined with the genotoxic agents, cisplatin or etoposide, had a cooperative lethal effect resulting in increased DNA damage and apoptosis. Therefore, these data demonstrate an unexpected consequence of pharmacologically increasing Mdm2 levels that when used in combination with genotoxic agents induces synthetic lethality in ovarian cancer cells, and likely other malignant cell types, that have inactivated p53. Data reveal a therapeutically beneficial effect of pharmacologically increasing Mdm2 levels combined with chemotherapeutic agents for malignancies that have lost functional p53.

Abstract 42: The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells

Abstract Introduction: The signaling pathways for activin and TGFβ are frequently disrupted in colorectal cancers (CRC) and these mutations are associated with metastatic disease. Understanding the cellular consequences wrought by mutations to these signaling pathways may provide opportunities for the development of new therapeutic approaches. Our lab has previously shown opposing effects on a downstream target of TGFβ and activin, namely the cyclin-dependent kinase (CDK) inhibitor p21. TGFβ induces SMAD4-dependent upregulation of p21, while activin downregulates p21 in a SMAD4-independent mechanism via ubiquitination and degradation allowing for enhanced cell migration. However, the mode of regulation leading to p21 degradation is not completely known. We hypothesize that regulation of the E3 ubiquitin ligase MDM2 by NF-κB is involved in p21 degradation. Here we explore the role of MDM2 in the regulation of p21 in response to activin and TGFβ treatment in FET CRC cells. Methods: Colon cancer cell lines were assessed for ligand-induced PI3K/NF-κB/MDM2/p21 pathway engagement. FET cells were transfected with increasing concentrations of either wild type p65 or a dominant-negative mutant of p65 and treated with activin or TGFβ prior to immunoblot analysis for IκB-alpha, MDM2 and p21. DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). The requirement for NF-κB activation was determined through use of the selective inhibitor peptide NBD (NEMO-binding domain), and PI3 kinase activity was confirmed by inhibition with LY294002. Results: Because activin induces ubiquitination of p21, we investigated the potential involvement of MDM2 and further the regulation of MDM2 by NF-κB. NF-κB has been reported to regulate MDM2 through direct binding to an NF-κB binding site in the promoter of MDM2. Activin induced activation of NF-κB as measured by increased DNA binding activity to an NF-κB consensus-binding site as well as by a reduction in IκB-alpha protein. Furthermore, we found this regulation to be dependent on activation of PI3 kinase. Overexpression of wild type p65 plasmid but not dominant negative p65 was found to increase MDM2 expression while the NF-κB specific inhibitor NBD blocked the activin-induced increase in MDM2. By contrast, TGFβ treatment did not contribute to an increase in MDM2 levels via NF-κB pathway. Conclusions: Here, we provide evidence to indicate that in colon cancer cells, activin may be working through NF-κB to promote MDM2 activity leading to the degradation of p21 in a PI3 kinase dependent mechanism. Future experiments will explore if pharmaceutical intervention in this pathway can inhibit the metastatic activity of activin as a therapeutic approach for advanced colon cancer. Citation Format: Arundhati Jana, Seung Hyua Baik, Timothy Carroll, Ozkan Ozden, Nancy L. Krett, Barbara Jung. The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells. [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 42. doi:10.1158/1538-7445.AM2015-42

Association of p53 and mdm2 in the development and progression of non-small cell lung cancer.

MDM2 protein is an important regulator of the p53 pathway and has a large effect on the anti-tumorigenic activity of the p53. Presently, we aimed to analyze the possible association of p53 and mdm2 in the development and progression of non-small cell lung cancer. In addition, impact of an important gene promoter polymorphism of MDM2 (T309G, rs 2279744) on its gene expression and ultimately of the TP53 was investigated in non-small cell lung cancer patients. A case-control study using peripheral blood samples of 100 non-small cell lung cancer patients and 100 cancer free healthy controls was conducted. Expression profile of MDM2 and TP53 gene were evaluated by using quantitative real time polymerase chain reaction assay, and MDM2 promoter polymorphism were analyzed by amplification refractory mutation system polymerase chain reaction. Non-small cell lung cancer patients expressed more than 6-fold increased mdm2 and about 7-folds decreased in p53 expression levels compared to healthy controls. Higher fold change increase of mdm2 and/or decrease of p53 were associated with advanced status and poor clinical outcome of the patients. A significant increase in mdm2 of about 14-folds and decrease in p53 of about 16.5-fold were observed among patients with MDM2 (309GG) genotype vs just 2.2-fold increase in mdm2 and 1.9-fold decrease in p53 among patients with MDM2 (309TT) genotype. In conclusion, present study demonstrated that MDM2 (309 T > G) polymorphism may be one of the important factors for the increased expression mdm2, which was associated with down-regulation of p53 at messenger RNA (mRNA) level and ultimately may contribute in the poor clinical outcome of the non-small cell lung cancer patients, thus may prove as a promising target for the treatment of non-small cell lung cancer at molecular level.

TP53 and MDM2 polymorphisms and the risk of endometrial cancer in postmenopausal women.

The aim of the study was to determine an association of TP53 codon 72 (Arg72Pro, G>C transversion, rs1042522) and MDM2 SNP309 (T>G change, rs2279744) polymorphisms in endometrial cancer (EC) of postmenopausal women, regarding grading and staging of EC. In the study, endometrial samples from 202 postmenopausal female patients (the study group, n = 152, was women with EC; the control group, n = 50, cancer-free patients) were taken for the evaluation of two gene polymorphisms: TP53 codon 72 and MDM2 SNP309, respectively. Genotypic analyses were performed using the PCR–RFLP technique. There were significant differences in the frequency of TP53 and MDM2 genotypes in EC patients—increased EC occurrence was observed with the presence of MDM2 G/G and TP53 Arg/Arg genotypes, while allele Pro of TP53 decreased cancer risk. Analysis of combined MDM2/TP53 polymorphisms revealed that T/T-Pro/Arg genotype decreased EC risk, whereas G/G-Arg/Arg genotype increased it. Association of these genetic polymorphisms with histological grading showed increased MDM2 G/G homozygote and TP53 Arg/Arg homozygote frequencies in grading 2 as well as allele G overrepresentation in G1 and G3 EC patients. Finally, with clinical FIGO staging under evaluation, an increase in MDM2 G/G and TP53 Arg/Arg homozygote frequencies in staging I and TP53 Arg/Arg homozygote frequencies in staging II were observed. Co-occurrence of some MDM2 SNP309 and TP53 codon 72 polymorphisms seems to influence EC risk, involving grading and staging of this neoplasm at the same time.

Abstract 3056: miR-125b suppresses p14ARF and modulates p53-dependent and p53-independent apoptosis in prostate cancer cells.

Abstract Increasing evidence has revealed that miR-125b functions as a potential oncogene in different types of human cancer. Our previous studies demonstrated that miR-125b directly targets p53, Puma and Bak1 in human prostate cancer (CaP). To further elucidate the role of miR-125b in modulating the p53 network, we utilized a bioinformatics approach to identify miR-125b-regulated, CaP-relevant tumor suppressor gene, and identified p14ARF gene as a miR-125b target. We first evaluated the effect of miR-125b on the regulation of p14ARF expression, and found that miR-125b treatment induced reduction of p14ARF by 80% in LNCaP CaP cells and 60% in 22Rv1 CaP cells. Conversely, anti-miR-125b increased the p14ARF level by 40% in LNCaP and 30% in 22Rv1. We then performed luciferase assay to validate that the miR-125b binding site in the 3’UTR of p14ARF mRNA is responsible for the regulation of p14ARF by miR-125b. Since p14ARF accelerates Mdm2 degradation, resulting in p53 up-regulation, we thus assessed the effect of miR-125b on the expression of Mdm2 and p53. Compared with miRNA control, miR-125b treatment induced a dramatic increase in Mdm2 and a significant reduction of p53 in CaP cell lines, as well as in miR-125b-overexpressed xenograft tumor. Additionally, miR-125b treatment resulted in a reduction of interaction between p14ARF and Mdm2. Next, we examined the effect of miR-125b on CaP cell proliferation. Both LNCaP and 22Rv1 cells were transfected with miR-125b and cell proliferation was determined by clone formation assay. When compared with the control, miR-125b treatment increased clonogenic survival of CaP cells, while anti-miR-125b caused a dramatic reduction in the number of colonies. We evaluated the effect of downregulation of miR-125b on apoptosis in p53-positive 22Rv1 CaP cells. Data from TUNEL assay demonstrated that anti-miR-125 caused 63% of cells to undergo apoptosis. Similarly, in p53-negative PC3 CaP cells, miR-125b treatment induced a 2-fold increase in survival, while anti-miR-125b caused 50% of cells to undergo apoptosis. In summary, we observed that overexpression of miR-125b negatively regulates the expression of p14ARF, resulting in promotion of cell proliferation and inhibition of apoptosis. Inactivation of miR-125b using anti-miR-125b affects apoptosis involving both p53-dependent and p53-independent pathways. Therefore, our data suggest that miR-125b has a great potential to be used as a therapeutic target for CaP treatment. Citation Format: Xu-Bao Shi, Sumaira Amir, Ai-Hong Ma, Lingru Xue, Ralph W. deVere White. miR-125b suppresses p14ARF and modulates p53-dependent and p53-independent apoptosis in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3056. doi:10.1158/1538-7445.AM2013-3056

Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent

In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin sensitivity in the human TC setting using a p53 short interfering (si)RNA approach. The cisplatin-sensitive TC cell line (Tera), the subline with acquired cisplatin resistance (Tera-CP) and a panel of intrinsically resistant TC cell lines (Scha and 2102EP), all expressing wild-type p53, were used. p53 and p53 transcriptional targets MDM2 and p21Waf1/Cip1 (p21) were expressed in a p53 transactivation-dependent way in all TC cell lines. Following cisplatin exposure, expression levels of p53 increased, with a subsequent increase in MDM2 and p21 mRNA and protein levels and Fas cell membrane levels. Downregulation of p53 with siRNA lowered cisplatin-induced apoptosis in Tera and Tera-CP, which was associated with a diminished Fas membrane expression. In contrast, p53 suppression augmented cisplatin-induced apoptosis in Scha and 2102EP and concomitantly strongly suppressed MDM2 and p21 mRNA and protein expression. Our results indicate that p53 is involved in transactivation of pro- and anti-apoptotic genes in untreated and cisplatin-treated TC cells, but subtle differences are present between TC cell lines. The opposite role of p53 in cisplatin-induced apoptosis among TC cell lines demonstrates the importance of the cellular context for the p53 transactivation phenotype in TC cells.

A phase I study of MDM2 antagonist RG7112 in patients (pts) with relapsed/refractory solid tumors.

e13600^ Background: RG7112 is a small molecule MDM2 antagonist, designed to non-genotoxically activate p53. A phase I dose escalation (DE) was performed, followed by a sarcoma biomarker extension (EXT) in pts with wild type TP53. Methods: 106 pts (58M, 48 F), median age 57.9 yrs (range 22-84) participated; 76 in 9 cohorts (DE) from 20 to 1800 mg/m2, orally QD x 10 q28 days. 30 pts with sarcoma were treated at MTD (2500 mg) (EXT) with pre-and on dose (d5+/-2) biopsies. Analyses included TP53 mutation (AmpliChip), MDM2 amplification (ISH), p53 and p21 IHC, MDM2 RT-PCR, Ki-67, TUNEL and [18F]-FLT-PET. Blood was obtained for PK and MIC-1, a PD marker of p53 activation. Results: DE: MTD was1440 mg/m2/d (2500 mg flat dose). PK was ~dose linear (t½ 1-1.5 d) with high variability (CV~70%) in AUC and Cmax . Adverse events included nausea/GI and exposure-related neutropenia/thrombocytopenia. 3 DLTs: diarrhea, pancytopenia, hyponatremia occurred (at ≥ 640 mg/m2). Evidence of activity included: 1) concentration dependent increase in plasma MIC-1 (% baseline), 2) decrease in [18F]-FLT PET and PR each in liposarcoma pts at 1800 mg/m2 and 1440 mg/m2, respectively. EXT: Grade3/ 4 cytopenias at MTD precluded subsequent cycles in 6/8 pts (10 day schedule). Dosing was changed to 5 days, and only 3 patients had Gr3/4 cytopenias. 8/22 pts remained on study for &gt;4 cycles, including 2 pts with SD for 7 and 9 cycles respectively. 3 of 4 EXT pts had decreased [18F]-FLT-PET activity. Biopsies (pre- and on treatment) demonstrated: 1) increase in p53 (median 1.5 fold change (X) by IHC, n=15); 2) increase in p21 (median 2.7X by IHC, n=14); 3) increase in MDM2 (median 2.5X by RT-PCR, n = 27); 4) decrease in % Ki-67(+) cells (median % change from baseline -65.4%, range -91% to +275%, n=17 ); 5) increase in TUNEL(+) cells of 9.0 (density of + cells/mm2, range -26.2 to +45.5, n=22). These results were seen both in tumors with and without MDM2 gene amplification, and in multiple sarcoma subtypes. Conclusions: RG7112 has manageable AEs (GI ) and cytopenias correlating with AUC. Single agent disease control, and biomarker activity was seen in both MDM2 amplified and non-amplified, heavily pretreated soft tissue sarcoma pts, with changes reflecting activation of p53-related pathways.

Abstract 4422: Activation of beta-catenin/TCF-4 signaling leading to intestinal tumorigenesis in APCMin/+ mice is radiation quality dependent

Abstract Purpose: Radiation quality depends on loss of energy in tissues per unit distance travelled and could be low-linear energy transfer (low-LET) or high-linear energy transfer (high-LET). The low-LET radiation like gamma-rays and x-rays loose less and hence deposits less energy in tissues and are sparsely ionizing. However, the high-LET radiation like α-particle, neutron, and heavy ions loose more and so deposits more energy in tissues traversed. Epidemiological studies showed strong correlation between radiation exposure and colorectal cancer (CRC) and CRC is the second leading cause of cancer mortality in the USA. However, it is not known if activation of molecular pathways involved in CRC and consequent intestinal tumorigenesis is radiation quality dependent. Methods: Female APCMin/+ mice (6-8 weeks) were irradiated with 5 Gy of gamma radiation, a tumorigenic dose in these mice. After 90 days mice were euthanized, intestinal tumors counted and tumor samples were flash frozen. Tumor samples were subjected to histology, western blot and immunohistochemistry (IHC) to dissect the APC/β-catenin/TCF4 pathway, commonly altered in human CRC. Results of gamma radiation were compared to two qualitatively different types of radiation - high-energy proton (energy: 1 giga electron volt (GeV)) and iron (56-Fe) ion (energy: 1 GeV/nucleon). We used 4 Gy of 56-Fe and 4.7 Gy of proton, which are isotoxic to the gamma radiation dose of 5 Gy determined using a quality factor 1.25 and 1.06 respectively. Quantitation of IHC and western blot were performed using ImageJ v2.45 softwere. Results: Quantitative observations showed enhanced intestinal tumorigenesis after exposure to proton and 56-Fe radiation. Marked increases in β-catenin and TCF4 were associated with a decreased phospho-β-catenin and increased phospho-GSK3β in proton and 56-Fe mice. Increased β-catenin was also associated with higher levels of cyclin-D1 and c-myc in these samples. Furthermore, compared to gamma radiation, strikingly lower level of p53 along with higher MDM2 was observed in proton and 56-Fe samples. Interestingly, however, the decrease in p53 and increase in MDM2 was higher in 56-Fe. These observations were further supported by IHC staining of tumor sections. Statistical analyses to support these observations in proton and 56-Fe compared to ≤ ray or spontaneous tumors will be presented. Conclusions: Considering higher active β-catenin and inactivated GSK3β, we speculate that exposure to high-LET radiation caused stabilization of oncogenic β-catenin and upregulation of c-myc and cyclin-D1 leading to higher tumorigencity. Enhanced potential of high-LET radiation for carcinogenesis is further supported by downregulation of p53. In conclusion, this is the first evidence showing greater potential of high-LET radiation for intestinal carcinogenesis mediated via activation of β-catenin/TCF4 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4422. doi:1538-7445.AM2012-4422

Abstract 5432: Inhibition of Hif Prolyl 4-hydroxylases Protect Against Reoxygenation Injury in Endothelial Cells

Background: Hypoxia inducible factor-1 (Hif-1 α ) modulates expression of genes essential for cell survival under hypoxia. The oxygen-sensing enzyme prolyl 4-hydroxylase2 (PHD2) targets Hif-1 α for destruction under normoxia. Hypoxia/Reoxygenation is associated with p53 activation, a master regulator of apoptosis in many type of cells. We hypothesized that preservation of Hif-1 α beyond the period of hypoxia protects endothelial cells from reperfusion injury. To analyze the protective effect of Hif-1 α , human umbilical veins endothelial cells were exposed to 1 h of hypoxia (Po2&lt;5mmHg) followed by 24 hrs of reoxygenation (Reox.; Po2=140mmHg). Exposure to hypoxia causes a increase in Hif-1 α , p53, and Mdm2 (E3 ubiqitin ligase) by 3.1±0.2, 2±0.3, and 1.9±0.2-fold compared to control, respectively. During Reox. Hif-1 α and Mdm2 declined towards basal level, while p53 remained unaltered. Under the same conditions endothelial apoptosis was increased to 47±3% (annexin V staining). Silencing of PHD2, (53% reduction of PHD2 content) lead to an increase of the Hif-1 α content during hypoxia and maintained it at that level during Reox. Hif-1 α stabilization was accompany by an increase in the Mdm2 content and phosphorylation, whereas expression of p53 was reduced. PHD2 silencing reduced apoptosis to half. Additon of DMOG (1mM dimethyloxalylglycine), a PHD inhibitor, at the onset of Reox had the same effect. Reduction of p53 content was restored when the proteosome inhibitor MG-132 was added. Interaction of Mdm2 and P53 (co-immunoprecipitation) was increased compare to Reox. Downregulation of Hif-1 α by siRNA (64% reduction of Hif-1 α content) increased apoptosis to 66±5% and abrogate Mdm2-p53 complex formation. Downregulation of Mdm2 by siRNA (38% reduction of content) had no effect on Hif-1 α but increased p53 level. Conclusions: siRNA downregulation as well as pharmacological inhibition of PDH2, manoeuvre which inhibit Hif-1 α degradation and maintained it at that level during Reox. protect against reperfusion injury. This effect of Hif-1 α involves stimulation of Mdm2 induced degradation of the proapoptotic p53.

Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.