MDM2-p53 Pathway Research Articles

A Microarray-Based Gene Expression Analysis to Identify Diagnostic Biomarkers for Unknown Primary Cancer

BackgroundThe biological basis for cancer of unknown primary (CUP) at the molecular level remains largely unknown, with no evidence of whether a common biological entity exists. Here, we assessed the possibility of identifying a common diagnostic biomarker for CUP using a microarray gene expression analysis.MethodsTumor mRNA samples from 60 patients with CUP were analyzed using the Affymetrix U133A Plus 2.0 GeneChip and were normalized by asinh (hyperbolic arc sine) transformation to construct a mean gene-expression profile specific to CUP. A gene-expression profile specific to non-CUP group was constructed using publicly available raw microarray datasets. The t-tests were performed to compare the CUP with non-CUP groups and the top 59 CUP specific genes with the highest fold change were selected (p-value<0.001).ResultsAmong the 44 genes that were up-regulated in the CUP group, 6 genes for ribosomal proteins were identified. Two of these genes (RPS7 and RPL11) are known to be involved in the Mdm2–p53 pathway. We also identified several genes related to metastasis and apoptosis, suggesting a biological attribute of CUP.ConclusionsThe protein products of the up-regulated and down-regulated genes identified in this study may be clinically useful as unique biomarkers for CUP.

Abstract 3065: The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression.

Abstract MicroRNAs regulate gene expression and are differentially expressed in various tumors. While p53 is inactivated by point mutations in many tumors, melanomas rarely harbor mutations in the p53 gene. Here we report the biological role of miR-18b in melanoma via targeting the MDM2-p53 pathway. Expression of miR-18b was significantly suppressed in melanoma specimens and cell lines by virtue of hypermethylation, and was re-induced in melanoma cell lines following 5-AZA-deoxycytidine treatment. Lower levels of miR-18b expression in primary melanoma were associated with significantly reduced overall survival. MDM2 was identified as a target of miR-18b action, as miR-18b significantly suppressed the luciferase activity of a reporter plasmid containing the 3’UTR sequence of MDM2 complementary to the miR-18b seed sequence. Induction of miR-18b expression in melanoma cells resulted in reduced MDM2 expression, upregulation of p53 as well as PUMA and p21, and suppression of the anti-apoptotic BCL2 and BCL-XL genes, resulting in significant suppression of melanoma cell proliferation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell proliferation, induction of apoptosis, and reduced tumor cell growth in vivo. In addition, miR-18b overexpression suppressed melanoma cell migration and invasiveness, and reversed epithelial-to-mesenchymal transition in melanoma cell lines. Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors. Citation Format: Altaf A. Dar, Shahana Majid, David de Semir, Vladimir Bezrookove, Mehdi Nosrati, Mohammed Kashani-Sabet. The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression. [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 3065. doi:10.1158/1538-7445.AM2013-3065

The Role of miR-18b in MDM2-p53 Pathway Signaling and Melanoma Progression

BackgroundAlthough p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene. Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway.MethodsExpression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes. Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b. The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group). Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing.ResultsExpression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment. MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition.ConclusionsOur results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors.

HnRNP K: A Putative Regulator of Leukemogenesis and the p53 Pathway

Abstract 774The presence of NPM1 mutations in the absence of FLT3 mutations typically confers favorable clinical responses and long term survival in acute myeloid leukemia patients. However, a subset of NPM1MUT:FLT3WT patients have extremely poor outcomes, indicating that uncharacterized genetic alterations influence the response of these patients. Identifying the causal alterations responsible for these poor prognoses would warrant more aggressive treatment strategies for these patients. While the impact of NPM1 mutations on AML is poorly understood, in vitro studies suggest NPM1 modulates p14ARF, resulting in p53 pathway dysregulation. The importance of p53 tumor suppressive functions are highlighted in AML patients with chromosome 17p deletions (containing the p53 locus) as they respond poorly to standard therapies. However, most AML patients possess wild type p53 alleles, suggesting that alterations to regulators of p53 activities may significantly influence ablation of the p53 pathway.We currently have a limited knowledge of how p53 regulators effect AML progression and tumorigenesis in general. However, a recently described p53 and Mdm2 interacting protein, hnRNP K, has been shown to be a target of Mdm2-dependent degradation. This Mdm2 targeted degradation is thought to decrease p53-hnRNP K binding, resulting in aberrant p53-dependent transcriptional activities. Importantly, hnRNP K is aberrantly expressed in several human cancers, making it an attractive molecule for detailed examination of alterations in p53 pathway regulators. Thus, we are currently interrogating the relationship between hnRNP K and leukemic progression and are evaluating its potential as a prognostic marker and therapeutic target.Using Mdm2-transgenic (Mdm2Tg) mice, we have demonstrated that hnRNP K levels decrease in the presence of excess Mdm2, indicating an in vivo relationship between hnRNP K and the Mdm2-p53 pathway. In order to directly examine the role of hnRNP K in cancer formation and leukemia, we generated hnRNP K knockout mice. Homozygous deletion of hnRNP K results in embryo lethality prior to embryonic day 13.5. Similarly, haploinsufficiency also frequently results in a neonatal lethal phenotype. However, nearly half of the hnRNP K+/− mice survive albeit exhibiting numerous developmental defects. Surviving hnRNP K+/− mice are tumor prone and some develop hematological neoplasms.To examine the mechanisms by which hnRNP K influences the p53 pathway, we generated haploinsufficient hnRNP K mouse embryo fibroblasts (MEFs) and demonstrated that hnRNP K loss results in reduced p53 levels and diminished p53-dependent activation of p21. These results, coupled with our data from Mdm2Tg mice, establish an in vivo link between hnRNP K, the Mdm2-p53 pathway, and activation of p53 transcriptional targets.To explore the impact of hnRNP K expression in AML, we examined hnRNP K protein levels in 415 newly diagnosed AML patients. We observed that elevated hnRNP K levels associate with a significant increase in leukemic blasts and decreased survival (Figure 1A, p=0.027). Importantly, when assessed in the context of cytogenetically normal NPM1MUT:FLT3WT AML, high hnRNP K levels associate with poor outcomes within this a priori favorable prognostic group, with approximately 90% of these patients dying within 12 months (Figure 1B, p=0.049). These findings provide the first evidence that elevated hnRNP K levels associate with poor prognoses and suggest hnRNP K may represent a novel biomarker for risk assessment and a potential therapeutic target.Currently, we have generated conditional hnRNP K knockout and transgenic alleles and are crossing them with Npm1+/− and Flt3IDT mice to directly examine its impact on leukemogenesis and the p53 pathway. Additionally, we are crystallizing hnRNP K in order to examine its interaction with Mdm2 and p53 for the purpose of evaluating its potential as a therapeutic target. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study

We report a proof-of-mechanism study of RG7112, a small-molecule MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were eligible for resection. Patients with well-differentiated or dedifferentiated liposarcoma were enrolled at four centres in France. Patients received up to three 28-day neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a patient progressed at any point after the first cycle, the lesion was resected or, if unresectable, an end-of-study biopsy was done. The primary endpoint was to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation (P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and apoptosis). All analyses were per protocol. This trial is registered with EudraCT, number 2009-015522-10. Between June 3, and Dec 14, 2010, 20 patients were enrolled and completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations, assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR 4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day 8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline. The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR -12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of MIC-1 (p<0·0001) and haematological toxicity. One patient had a confirmed partial response and 14 had stable disease. All patients experienced at least one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia (nine patients), diarrhoea (nine patients), and thrombocytopenia (eight patients). There were 12 serious adverse events in eight patients, the most common of which were neutropenia (six patients) and thrombocytopenia (three patients). MDM2 inhibition activates the P53 pathway and decreases cell proliferation in MDM2-amplified liposarcoma. This study suggests that it is feasible to undertake neoadjuvant biopsy-driven biomarker studies in liposarcoma. F Hoffmann-La Roche.

Ethanol extract of paeonia suffruticosa Andrews (PSE) induced AGS human gastric cancer cell apoptosis via fas-dependent apoptosis and MDM2-p53 pathways

BackgroundThe root bark of Paeonia suffruticosa Andrews (PSE), also known as Moutan Cortex, has been widely used in Asia to treat various diseases. The molecular mechanisms by which PSE exerts its anti-oxidant and anti-inflammatory activities are well known, but its anti-cancer activity is not yet well understood. Here, we present evidence demonstrating that PSE can be used as a potent anti-cancer agent to treat gastric cancer.MethodsThe effects of the ethanol extract of PSE on cell proliferation were determined using an MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) assay. Cell cytotoxicity induced by the PSE extact is measured using an LDH leakage assay. Flow cytometry was used to analyze the cell cycle and to measure the subG0/G1 apoptotic cell fraction. Apoptosis induced by the PSE extact is also examined using a DNA fragmentation assay. Western blot analysis is used to measure the levels of apoptotic proteins such as Fas receptor, caspase-8, caspase-3, PARP, Bax, Bcl-2, MDM2, and p53.ResultsThis study demonstrated that treating AGS cells with the PSE extact significantly inhibited cell proliferation and induced cytotoxicity in a dose- and time-dependent manner. The PSE extract also induced apoptosis in AGS cells, as measured by flow cytometry and a DNA fragmentation assay. We found that the PSE extract induced apoptosis via the extrinsic Fas-mediated apoptosis pathway, which was concurrent with the activation of caspases, including caspase-8 and caspase-3, and cleavage of PARP. The MDM2-p53 pathway also played a role in the apoptosis of AGS cells that was induced by the PSE extract.ConclusionsThese results clearly demonstrate that the PSE extact displays growth-suppressive activity and induces apoptosis in AGS cells. Our data suggest that the PSE extact might be a potential anti-cancer agent for gastric cancer.

Identification of ribosomal protein S25 (RPS25)–MDM2–p53 regulatory feedback loop

There is an increasing interest in determining the role of ribosomal proteins (RPs) in the regulation of MDM2-p53 pathway in coordinating cellular response to stress. Herein, we report a novel regulatory role of ribosomal protein S25 (RPS25) in MDM2-mediated p53 degradation and a feedback regulation of S25 by p53. We demonstrated that S25 interacted with MDM2 and inhibited its E3 ligase activity, resulting in the reduction of MDM2-mediated p53 ubiquitination and the stabilization and activation of p53. S25, MDM2 and p53 formed a ternary complex following ribosomal stress. The nucleolar localization and MDM2-binding domains of S25 were critical for its role in MDM2-mediated p53 regulation. Knockdown of S25 by siRNA attenuated the induction and activation of p53 following ribosomal stress. S25 stabilized and cooperated with MDMX to regulate MDM2 E3 ligase activity. Furthermore, S25 was identified to be a transcriptional target of p53; p53 directly bound to S25 promoter and suppressed S25 expression. Our results suggest that there is a S25-MDM2-p53 regulatory feedback loop, which may have an important role in cancer development and progression.

Mdm2 RING Mutation Enhances p53 Transcriptional Activity and p53-p300 Interaction

The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A) in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the Mdm2C462A mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.

Abstract 2874: Kevetrin targets both MDM2-p53 and Rb-E2F pathways in tumor suppression

Abstract Our studies showed that Kevetrin™, a small molecule currently under development, has potent antitumor activity in several wild type and mutant p53 human tumor xenografts e.g. A549, PC-3, MDA-MB-231, HT-29, NCI-H1975, HCT-15, K-562, LNCaP. To investigate the mechanism of action of its antitumor activity in different xenograft models, we assessed Kevetrin's effects on apoptosis and cell cycle progression. Rb-E2F and MDM2-p53 pathways are crucial regulators of cell cycle progression. Remarkably, both Rb-E2F and MDM2-p53 pathways are defective in most, if not all, human tumors. There is extensive crosstalk between the Rb-E2F and MDM2-p53 pathways, especially between the transcription factors E2F1 and p53, which influence vital cellular decisions. In wild type p53 human lung carcinoma (A549), Kevetrin showed a concentration dependent increase in activation of p53 by Western blot analysis. Activated p53 triggered apoptosis by inducing the expression of PUMA. Kevetrin also increased the expression of the target gene p21, an inhibitor of cell cycle progression. Kevetrin induced apoptosis in a transcriptional independent way by altering the E3 ligase processivity of MDM2. Kevetrin strongly induced apoptosis in multiple tumor cell lines characterized by activation of caspase3 and PARP. E2F1 is a pivotal transcription factor that integrates signals from a variety of G1/S phase regulators and modulates diverse functions, such as DNA synthesis, mitosis, apoptosis and senescence. E2F1 overexpression, observed in most tumors, is associated with tumor growth. Cells which overexpress E2F1 proved to be immune to induction of senescence. A recent study showed that depletion of E2F1 hinders cell cycle progression and induces senescence. Kevetrin down regulated E2F1 expression in various p53 wild type and mutant cell lines. Kevetrin also down regulated the E2F1 target gene thymidylate synthase (TS) in various tumor cell lines. Several reports have shown E2F1 overexpression correlates with TS expression. Effective inhibition of TS by Kevetrin could play a crucial role in tackling the problem of resistance. Kevetrin acting through both major pathways of tumor suppression, Rb-E2F and MDM2-p53, has far reaching consequences. Due to the complexity of proapoptotic and antiapoptotic pathways with multiple players involved and redundant signaling networks, blocking only one antiapoptotic factor may not result in robust antitumor activity. However, Kevetrin acts via multiple targets to produce potent efficacy in various xenograft tumor models. We have also demonstrated that Kevetrin is non-genotoxic. DNA damaging drugs result in rapid phosphorylation of H2A.X at Ser 139 by PI3K-like kinases; however, Kevetrin did not induce phosphorylation of H2A.X. Since Kevetrin was well-tolerated in GLP safety pharmacology and toxicity studies, we have submitted an IND application for a Phase I clinical trial at Dana Farber / Harvard Cancer Center for 2012. 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 2874. doi:1538-7445.AM2012-2874

Abstract LB-303: Metastasis suppression by MDM2 binding protein through inhibition of ACTN4 function

Abstract MDM2 Binding Protein (MTBP) has been implicated in cancer progression. Our previous results demonstrate that MTBP haploinsufficiency in mice enhances cancer metastasis. Recently, loss of MTBP expression is shown to be associated with reduced survival of patients with head and neck carcinoma, and serves as an independent prognostic factor when p53 is mutated in tumors. Here we demonstrate one mechanism by which MTBP inhibits cancer metastasis. Overexpression of MTBP in human osteosarcoma cell lines lacking wild-type p53 did not alter primary tumor growth in mice but significantly inhibited metastases. MTBP downregulation increased the migratory potential of Mdm2-/-p53-/- mouse embryonic fibroblasts, suggesting that MTBP inhibited cell migration independently of the MDM2-p53 pathway. Co-immunoprecipitation and mass spectrometric analysis identified alpha-actinin-4 (ACTN4) as a MTBP-interacting protein. Endogenous MTBP interacted with and partially colocalized with ACTN4. MTBP overexpression inhibited cell migration and filopodia formation mediated by ACTN4. Increased cell migration by MTBP downregulation was inhibited by concomitant downregulation of ACTN4. MTBP also inhibited ACTN4-mediated F-actin bundling. We furthermore demonstrated that nuclear localization of MTBP was dispensable for inhibiting ACTN4-mediated cell migration and filopodia formation. Thus, MTBP suppresses cell migration, at least partially, by inhibiting ACTN4 function. Our study not only provides a mechanism of metastasis suppression by MTBP, but also suggests MTBP as a potential biomarker for cancer progression. 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 LB-303. doi:1538-7445.AM2012-LB-303

MTBP suppresses cell migration and filopodia formation by inhibiting ACTN4

MDM2 Binding Protein (MTBP) has been implicated in cancer progression. Here we demonstrate one mechanism by which MTBP inhibits cancer metastasis. Overexpression of MTBP in human osteosarcoma cell lines lacking wild-type p53 did not alter primary tumor growth in mice but significantly inhibited metastases. MTBP downregulation increased the migratory potential of MDM2−/−p53−/− mouse embryonic fibroblasts, suggesting that MTBP inhibited cell migration independently of the Mdm2-p53 pathway. Co-immunoprecipitation and mass spectrometric analysis identified alpha-actinin-4 (ACTN4) as a MTBP-interacting protein. Endogenous MTBP interacted with and partially colocalized with ACTN4. MTBP overexpression inhibited cell migration and filopodia formation mediated by ACTN4. Increased cell migration by MTBP downregulation was inhibited by concomitant downregulation of ACTN4. MTBP also inhibited ACTN4-mediated F-actin bundling. We furthermore demonstrated that nuclear localization of MTBP was dispensable for inhibiting ACTN4-mediated cell migration and filopodia formation. Thus, MTBP suppresses cell migration, at least partially, by inhibiting ACTN4 function. Our study not only provides a mechanism of metastasis suppression by MTBP, but also suggests MTBP as a potential biomarker for cancer progression.

Molecular mechanisms of MYCN-dependent apoptosis and the MDM2–p53 pathway: an Achille’s heel to be exploited for the therapy of MYCN-amplified neuroblastoma

The p53 oncosuppressor is very seldom mutated in neuroblastoma, but several mechanisms cooperate to its functional inactivation in this tumor. Increased MDM2 levels, due to genetic amplification or constitutive inhibition of p14 ARF, significantly contribute to this event highlighting p53 reactivation as an attractive perspective for neuroblastoma treatment. In addition to its role in tumorigenesis, MYCN sensitizes untransformed and cancer cells to apoptosis. This is associated to a fine modulation of the MDM2–p53 pathway. Indeed MYCN induces p53 and MDM2 transcription, and, by evoking a DNA damage response (DDR), it stabilizes p53 and its proapoptotic kinase Homeodomain Interacting Protein Kinase 2 (HIPK2). Through the regulation of the HIPK2-p53 inhibitor High Mobility Group protein A1 (HMGA1) and the homeobox proteins BMI-1 and TWIST-1, MYCN establishes a delicate balance between pro- and antiapoptotic molecules that might be easily perturbed by a variety of insults, leading to cell death. MDM2–p53 antagonists, such as Nutlin-3, are strikingly prone to inducing death in MYCN-amplified neuroblastoma, by further pushing on HIPK2 accumulation. Here we discuss implications and caveats of exploiting this pathway and its connections to MYCN-induced DDR for a tailored therapy of MYCN-amplified neuroblastoma.

Abstract PR9: PICT1/GLTSCR2 is a critical nucleolar binding partner of RPL11 that regulates the MDM2-p53 pathway and tumor growth

Abstract The tumor-suppressive MDM2-p53 intracellular signaling pathway is activated by cellular stress. Such stress includes DNA damage, which triggers the ATM-Chk2 and ATR-Chk1 cascades; infection or oncogene activation, which induce the p19ARF pathway; and nucleolar stress, which initiates a cascade mediated by ribosomal proteins (RPs), particularly RPL5, RPL11, RPL23, and RPS7. These RPs are usually located in the nucleolus but are rapidly released into the nucleoplasm upon nucleolar stress, activating the MDM2-p53 pathway. However, it is unknown whether there are direct connections between p53-activating RP genes and cancer development in vivo, and which mechanism these RPs employ to translocate from the nucleolus to the nucleoplasm in response to stress. PICT1/GLTSCR2 was originally identified as a candidate tumor suppressor gene located at human chromosome 19q13.32, the site of deletions in human gliomas. Subsequent in vitro studies have demonstrated that PICT1 regulates the stability of PTEN, and that low PICT1 expression caused growth advantage. Based on these findings, PICT1 has been deemed a tumor suppressor. Other evidence, however, suggests that PICT1 may not always dampen cancer progression. In oligodendroglial tumors, loss of heterozygosity (LOH) at chromosome 19q13 is associated with longer disease-free survival after chemotherapy. These contradictory observations indicate that one or more genes mapped to chromosome 19q may contribute to brain cancer development. We speculated that PICT1 might be an important chromosome 19q–mapped gene that regulates tumor progression. To clarify PICT1 function, we generated PICT1-deficient mice and ES cells and carried out extensive biochemical and molecular analyses. Our results show that PICT1 is a nucleolar protein essential for murine embryogenesis and ES cell survival. Even without DNA damage, PICT1 loss results in p53-dependent G1 arrest and apoptosis in vitro and in vivo. Strikingly, PICT1-deficient cells accumulate p53 due to impaired MDM2 function. We further show that PICT1 binds to RPL11, and that RPL11 is released from nucleoli in the absence of PICT1. In PICT1-deficient cells, increased binding of RPL11 to MDM2 blocks MDM2-mediated ubiquitination of p53, leading to p53 accumulation and repression of cell proliferation. In cases of human colon and esophageal cancer, patients with low PICT1 expression have better prognoses. Similarly, when shRNA is used to deplete PICT1 in various tumor cell lines with intact p53 signaling, the cells grow more slowly and accumulate p53. These data suggest that PICT1 is a novel key regulator of the MDM2-p53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus. Thus, PICT1 is an important cancer-related gene that, at the very least, does not behave like a typical tumor suppressor and may instead have oncogenic effects. Studies on identification of factors which influence PICT1 expression or stability, or interfering with PICT1-RPL11 binding may lead to new cancer therapeutics, especially for individuals with intact-p53 signaling. This abstract is also presented as Poster B30. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR9.

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G(1) and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.

Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53.

Maintenance of genomic integrity in hematopoietic stem cells

Hematopoietic stem cells (HSCs) maintain hematopoietic homeostasis throughout a mammal's lifespan through self-renewal and differentiation into mature blood cells. Within a bone marrow niche, HSCs adopt a quiescent state and remain in the non-dividing, G0 phase of the cell cycle. It was recently shown that maintenance of genomic integrity is crucial for the preservation of self-renewal capacity of HSCs. In this review, we focus on progress in elucidating the roles of reactive oxygen species (ROS) and DNA damage responses (DDR) in maintaining genomic integrity, and thus HSC function. Several studies have demonstrated that inappropriate ROS levels arising from disruption of the Atm, PI3K-Akt, or Mdm2-p53 pathways impair HSC function in vivo. Intriguing evidence that stem cells use specific DDR mechanisms is also accumulating. Although murine HSCs are more resistant than progenitor cells to mild DNA damage in vivo, the surviving HSCs frequently acquire genetic aberrations that can lead to leukemogenesis. Indeed, non-dividing HSCs employ the error-prone non-homologous end-joining pathway of DNA repair to fix DNA breaks, whereas progenitors undergo apoptosis; proliferating HSCs employ the high-fidelity homologous recombination mechanism. Dissection of HSC-specific mechanisms for the maintenance of genomic integrity may provide valuable insights into the biology of both HSCs and leukemia stem cells.

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

E2F1 inhibits MDM2 expression in a p53-dependent manner

MDM2 expression is down-regulated upon E2F1 over-expression, but the mechanism is not well defined. In the current study, we found that E2F1 inhibits MDM2 expression by suppressing its promoter activity. Although E2F1 binds to the MDM2 promoter, the inhibitory effect of E2F1 on the MDM2 promoter does not require the direct binding. We demonstrate that E2F1 inhibits MDM2 promoter activity in a p53-dependent manner. Knockdown of p53 in U2OS cells impairs the inhibitory effect of E2F1 on the MDM2 promoter. Consistent with this observation, E2F1 does not inhibit MDM2 promoter activity in p53-deficient H1299 cells, and the inhibition is restored when p53 is expressed exogenously. Both E2F1 and p53 are up-regulated after DNA damage stimulation. We show that such stimulation induces E2F1 to inhibit MDM2 promoter activity and promote p53 accumulation. Furthermore, inhibition of MDM2 by E2F1 promotes E2F1 induced apoptosis. These data suggest that E2F1 regulates the MDM2-p53 pathway by inhibiting p53 induced up-regulation of MDM2.

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage-induced cell-cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR-S6K1 through p38alpha MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2-mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR-S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53-dependent cell death. These findings thus establish mTOR-S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1-Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging-controlling Mdm2-p53 and mTOR-S6K pathways.

Abstract A36: Systems biology: Implications in assessing drug-drug interactions in pancreatic cancer

Abstract The era of new adjuvant targeted therapies (k-ras, EGFR, VEGF src, p53, etc.) has generated much interest in discovering better approaches for the treatment of cancers; however such approaches have met with more failure than success. The major reason for low response is related to incomplete understanding and validation of the specific molecular targets at the gene level along with complexities of genetic and epigenetic changes in cancer. This is further complicated due to redundancies and cross-talk in signaling pathways that may explain the failure of single-pathway targeted therapies. To overcome this barrier, one must fully understand the molecular interactions among key signaling pathways. This requires innovative approaches to identify genomic events to guide intelligent decision making in the design of novel therapeutic strategies for combination therapies to improve treatment outcome. An increasingly popular and scientifically robust method is to utilize integrated systems biology which combines the use of high throughput technologies with analysis and computer modeling of the potential interconnecting pathways modulated in response to drug treatments. Recently we have found that specific, orally active murine double minute 2 (MDM2) inhibitor MI-219 can synergize with chemotherapeutic drug oxaliplatin that results enhanced anti-tumor effects in pancreatic cancer. However, the mechanism behind this synergy has not been explored in greater detail. In this study we have used integrated genomic expression microarray profiling (IGEMP) coupled with pathway/genetic network modeling (PGNM) to obtain crucial information in the synergistic efficacy of MI-219-oxaliplatin in a genetically complex and by far incurable pancreatic cancer. Global gene analysis of capan-2 cell shows that MI-219 alone induces alterations in 48 genes that is expected of a targeted agent. On the other hand, oxaliplatin treatment results in alterations of more than 700 genes and is in line with the mode of action of cytotoxic drugs. However, the combination of MI-219-oxaliplatin resulted in alteration of &amp;gt;700 genes with emergence of 286 synergy unique genes. Principal component analysis revealed that each drug treatment and time point had individual global gene signatures. Further analysis of the 286 synergy unique genes revealed the presence of several local gene networks of MDM2-p53 pathway including key players such as players CREBBP (i.e., ubiquitously expressed in PC and is involved in the transcriptional coactivation of many different transcription factors including p53, CARF that is responsible for p53 stability along with NF-kB and EGR1 tumor suppressor module, all positively affecting p53 reactivation that in principle would drive cells toward increased apoptosis. Additionally, the analysis revealed a novel role of hepatocyte nuclear factor alpha (HNF4α) in aiding MI-219-oxaliplatin mediated apoptosis suggesting that this technology can also be utilized in identifying potential biomarkers. Most interestingly, all the genetic signatures could be verified and validated at the RNA and protein level. Fortified by our strong evidence we propose the use of this technology for evaluating multiple drug combinations being tested for different cancers in clinical setting. We believe that success in personalized medicine will require advances in our understanding of the true potential and concepts of this technology towards elucidating the role of most influential driver genes in drug-drug interaction that would positively impact treatment outcome. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A36.