P53-binding Domain Research Articles

Abstract 2765A: Co-treatment of human pancreatic cancer MiaPaCa-2 Cells with PNC-27 and Gemzar

Abstract Novel anti-cancer PNC-27 peptide, derived from the p53 binding domain of MDM2, induces necrosis of human pancreatic MiaPaCa-2 cancer cells but not normal cells in vitro, in a dose dependent fashion. Recent studies support a membranolytic mechanism of action for the peptide, initiated by binding of peptide monomers to the cells’ plasma membrane that subsequently aggregate to form transmembrane pores. Gemzar, on the other hand, is an established, non-selective chemotherapeutic used to treat pancreatic cancer. As a nucleoside analog, Gemzar inhibits DNA replication, resulting in cell cycle arrest, and ultimately apoptosis. Speculation regarding whether the two drugs, with different mechanisms, could produce a combined killing effect if administered simultaneously is investigated. Cell proliferation assays (MTT), cell cytotoxicity assays (LDH) and confocal microscopy were used to study the susceptibility of the MiaPaCa-2 cells to the co-treatment of PNC-27 and Gemzar. To determine the working concentration of PNC-27 that would result in pore formation but would not be cytotoxic to the cells, a dose response assay with serial dilutions was conducted. Results revealed that while MiaPaCa-2 cells treated with PNC-27 in DMEM have an LD50 = 150 ug/mL, the working concentration that would enable minimal pore formation without cell death was determined to be ∼10 ug/mL. Through confocal microscopy, it was then determined that propidium iodide (PI) (668.4 Da), a marker for dead cells and that is impermeable for intact cells, would nevertheless be able to enter the cell cytoplasm via PNC-27 pores. The results found PI staining in the cytoplasm but complete absence of nuclear staining. This observation not only indicates the sustained viability of the cells treated with 10 ug/mL PNC-27, but also supports the hypothesis that PI entered the cells through the PNC-27 induced pores. To explore a combined cytotoxic effect of Gemzar and PNC-27, MTT assays were carried out with Gemzar concentrations between 0 uM-100 uM for either 24 or 48 hours, in presence or absence of PNC-27. Our results demonstrate that co-treatment of MiaPaCa-2 cells with PNC-27 and Gemzar leads to a greater cytotoxic effect than with either Gemzar or PNC-27 alone. Additionally, the 48hr treatment resulted in a greater cytotoxic effect than did the 24hr condition. Our results indicate that the increased permeability of the cancer cells to Gemzar via the PNC-27-induced pores may allow for a more rapid mechanism of entry for Gemzar. Due to the selectivity of PNC-27, more Gemzar is able to target the cancer cells which have compromised membrane integrity caused by PNC-27. Therefore, co-treatment with Gemzar may allow for a dose reduction of Gemzar due to increased accessibility of the chemotherapeutic to its site of action. By reducing the dose of Gemzar required to achieve a significant cytotoxic effect, the severity of the side-effects of the drug experienced by patients may also be reduced. 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 2765A. doi:1538-7445.AM2012-2765A

Abstract 1169: Non-canonical functions of Mdm2 isoforms in estrogen influenced breast cancer cells with wild-type or mutant p53

Abstract Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in breast cancers occurred through an Mdm2 mediated pathway. We examined the effect of long-term 17β-estradiol (E2) treatment in ERα positive breast cancer cell lines that contain wild-type p53 (MCF-7) and mutant p53 (T-47D). These cell lines also contain the mdm2 SNP at position 309, which increases the expression of Mdm2 by Sp1 driven transcription. In both these cell lines, we observed that E2 treatment dramatically increased the amount of Mdm2 without decreasing the corresponding wild-type or mutant p53. Surprising the amount of p53 protein in both cell lines increased with E2 treatment. We engineered miR30-based inducible mdm2 knockdown cell lines of MCF-7 and T-47D to evaluate to impact of Mdm2 on their cancer cell phenotypes. The down-regulation of Mdm2 in MCF-7.shmdm2 and T-47D.shmdm2 inhibited cell proliferation in 2D culture and in 3D laminin-rich matri-gel. Knockdown of Mdm2 did not cause an increase in p53 protein. These data suggest a p53-independent signal transduction pathway for Mdm2-mediated activation of proliferation. Alternative spliced mdm2 transcripts have been observed in many cancers including breast, but no endogenous protein isoforms from the spliced isoforms have been described. We asked if the spliced variant Mdm2-C transcript (that lacks most of the N-terminal p53 binding domain) expressed endogenous protein in these breast cancer cell lines. We created an Mdm2-C specific antibody using a synthetic peptide to the splice junction of exons four and ten (C4-10) to detect the protein. We validated the C4-10 antibody using in vitro translated Mdm2 compared to Mdm2-C. High expression of Mdm2-C occurred in the G/G mdm2 SNP309 ER+ breast cancer cell line T-47D, but only a slight increase was detected in the presence of E2. Additionally, Mdm2-C was found in nucleolar and cytoplasmic foci before and after estrogen treatment, while the mutant p53 was predominantly nuclear. We report the first identification of endogenous tumor-associated Mdm2-C protein in breast cancer. We suggest that Mdm2 and its isoforms work through non-canonical pathways to transform estrogen influenced breast cancers through molecular pathways that remain to be elucidated. Acknowledgements: This work was supported by Breast Cancer Research Foundation to J.B. and NSF grant to JB MCB-0744316. D.O. was also partially supported by a CUNY Graduate Center Magnet Award. Special thanks to Gerard Evan for help with immunogen peptide design. 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 1169. doi:1538-7445.AM2012-1169

Interrogation of MDM2 Phosphorylation in p53 Activation Using Native Chemical Ligation: The Functional Role of Ser17 Phosphorylation in MDM2 Reexamined

The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal "lid" (residues 1-24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1-109)MDM2 and its Ser17-phosphorylated analogue (1-109)MDM2 pS17 as well as (1-109)MDM2 S17D and (25-109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1-109)MDM2 by the (15-29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation.

De-regulation of the RBBP6 isoform 3/DWNN in human cancers

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.

Identification of Three Tyrosine Phosphorylation Sites in the p53 Binding Domain of Apoptosis-Stimulating p53 Protein 2 (ASPP2) That Are Differentially Phosphorylated in Response to Chemotherapy in Patient Derived AML Blasts

Abstract 1396Acute myeloid leukemias (AML) are difficult to treat, and risk-stratification for successful chemotherapy remains a major challenge. Inactivation of the p53 tumor suppressor pathway is a frequent event in many cancers that promotes tumorigenesis and resistance to chemotherapy. However, p53 mutations are rare in AML, and thus the p53-pathway must be inactivated by other mechanisms. ASPP2 is a haploinsufficient tumor suppressor that belongs to a family of p53-binding proteins that enhance apoptosis in part by stimulation of p53-transactivation of selected pro-apoptotic target genes. High ASPP2 expression levels in the absence of p53 mutations thereby argue for proper apoptosis induction capacity and thereby for better response rates. Indeed, low ASPP2 expression levels are correlated with aggressive courses of different tumors. As we have previously shown by qPCR (Kampa-Schittenhelm et al., ASH 2010) and confirm now by intracellular immunostaining in a larger patient cohort, ASPP2 expression levels vary widely in acute leukemias. In vitro silencing of ASPP2 transcription leads to abrogation of induction of apoptosis after application of chemotherapy, arguing for inferior in vivo response rates to therapy of patients lacking ASPP2 expression. Of note, the highest expression levels we have seen was in a patient with good prognosis core binding factor leukemia lacking an autoactivating KIT mutation.The p53 core domain must interact with the ASPP2 C-terminus to fully stimulate apoptotic function. To further investigate how regulation of the p53-ASPP2 interaction may play a role in apoptosis induction in AML, we identified several highly conserved and highly predicted tyrosine phosphorylation sites at the ASPP2 C-terminus. To study whether these sites modulate the p53-ASPP2 interaction and apoptotic function, we developed phospho-specific antibodies against the three highest-scoring phosphorylation sites and confirmed. tyrosine phosphorylation at Y1029, Y1046 and Y1114 in ex vivo blasts from AML patients. Intriguingly, based on the crystal structure of the p53-ASPP2 complex, phosphorylation of all three tyrosines is predicted to disrupt p53-ASPP2 binding. Tantalizingly, we found that these phosphorylation expression patterns changed after in vitro treatment of native blasts with chemotherapy: blasts treated with daunorubicin revealed an early change of tyrosine phosphorylation patterns. Using these new phospho-specific antibodies, we are continuing to analyze changes in phosphorylation patterns in primary AML blasts (with and without ex vivo chemotherapy) and are performing univariate and multivariate analysis to correlate with available clinical data. Preliminary data suggests that altered ASPP2 tyrosine phosphorylation in AML may play an important role in modulating response to chemotherapy-induced apoptosis in the absence of inactivating p53 mutations.Ongoing work is prospectively analyzing pY-ASPP2 in patients with acute leukemia during induction chemotherapy. These results aim to evaluate ASPP2 expression as an early-on prediction marker of therapy response in acute leukemia. Further, we aim to provide new and clinically relevant insight into p53 pathway inactivation in acute leukemia – which suggests a novel potential target for therapy to increase the effectiveness of chemotherapy in these patients. Disclosures:No relevant conflicts of interest to declare.

The evolution of MDM2 family genes

MDM2 and MDM4 are proto-oncoproteins that bind to and inhibit members of the p53 protein family, p53, p73 and possibly p63. p53 is a mammalian tumor suppressor and p63 and p73 are critical for development. With the sequencing of genomes from multiple organisms there is mounting evidence for a consensus scenario of p53 gene family evolution. A single p53/p63/p73 gene is in invertebrates and required for maintenance of germline DNA. Gene duplication occurred in an ancestor in common with cartilaginous fishes, giving rise to a separate p53 gene and at least one ancestral p63/p73 gene. In bony vertebrates, all three p53 gene family paralogs, p53, p63, and p73 are distinct genes. This raises the question of how MDM2 and MDM4 genes evolved. We show evidence that MDM2 and MDM4 arose from a gene duplication event prior to the emergence of bony vertebrates more than 440 million years ago. Comparative genome studies indicate that invertebrate organisms have only one MDM homolog. In jawed vertebrates, the p53-binding domains of MDM2 and MDM4 proteins evolved at a high rate, approaching the evolution rate of the MDM2-binding domain of p53. However, the MDM2-binding domain of p73 exhibits markedly stronger conservation suggesting novel p53-independent functions. The most conserved domain within all MDM2 family members is the RING domain of the MDM2 ortholog which is responsible for ubiquitination of p53 and heterodimerization with MDM4. We suggest a model where oligomerization is an ancient function of MDM and ubiquitination activity was acquired later near the MDM gene duplication event coinciding with the time of the emergence of p53 as a distinct gene.

Abstract 613: Depletion of Mdm2 and Mdm4 by adenovirus: Molecular mechanism and anticancer therapeutic implication

Abstract Mdm2 and Mdm4 are two paralogous proteins. They contain a conserved p53-binding domain in the N-terminal region and a RING-finger domain in the C-terminal part. Both proteins physically bind p53 and prevent p53 activation. Biochemically, Mdm2 acts as an E3 ubiquitin ligase of p53 and promotes ubiquitin-dependent p53 degradation. Mdm4 does not appear to have intrinsic E3 ubiquitin ligase activity and may negatively control p53 function through suppressing p53-mediated transactivation of gene expression. Mdm2 and Mdm4 also form heterodimer, which suggests that Mdm2 and Mdm4 might cooperatively control p53 activity. In vivo, both Mdm2 and Mdm4 have an essential but non-redundant function in suppressing p53 activity to enable embryonic development. Mdm2 and Mdm4 are overexpressed in several types of human cancer such as breast cancer and osteosarcoma. In transgenic mouse models, overexpression of Mdm2 or Mdm4 leads to tumorigenesis. Thus, both Mdm2 and Mdm4 are bona fide oncogenes. Several small molecules that disrupt the interaction between p53 and Mdm2 and Mdm4 have been shown to induce p53 activation, cell cycle arrest and apoptosis. These compounds also cause tumor repression in preclinical animal models. Thus, they have clinical potentials for treating various types of human cancer. Biological agents that specifically target Mdm2 and Mdm4 have not been described. We found that adenovirus infection of diverse type of cancer cells results in dramatic downregulation of both Mdm2 and Mdm4. Adenovirus E1B-55K binds specifically both Mdm2 and Mdm4 in cells and in vitro, demonstrating specific interactions between E1B-55K and Mdm2 or Mdm4. Cotransfection experiments indicated that E1B 55K alone was sufficient to deplete Mdm2 and Mdm4. E1B-55K is known to form a functional E3 ubiquitin ligase consisting of Cul2 or Cul5. Thus, it is likely that E1B-55K targets Mdm2 and Mdm4 for ubiquitin-mediated proteosomal degradation. Based on these observations, we hypothesize that adenovirus is effective for treating cancers with overexpression of Mdm2 or Mdm4. We are currently testing whether adenoviral therapy is efficacious on treating such cancers in cell culture and animal models. (This work was supported by Bankhead-Coley Cancer Research Program of the Florida Department of Health.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 613. doi:10.1158/1538-7445.AM2011-613

Focal Adhesion Kinase as a Therapeutic Target of Bortezomib

Bortezomib, a modified dipeptidyl boronic acid, is a selective potent proteasome inhibitor that has been approved for clinical treatment of multiple myeloma and mantel cell lymphoma. Results from a growing number of basic studies and clinical trials reveal that bortezomib could be used to treat diverse types of solid tumors alone or in combination with other chemotherapeutic drugs. It has been shown that bortezomib transcriptionally suppresses focal adhesion kinase (FAK) expression by interrupting the nuclear factor kappa B (NFκB) pathway, which suggests that FAK could be a potential molecular target for bortezomib. Analysis of FAK promoter sequences revealed that FAK promoter harbors the NFκB and p53 binding domains. Further studies of FAK promoter activity, real-time PCR, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay revealed that bortezomib inhibits NFκB binding on the FAK promoter, thereby reducing FAK expression. Thus, bortezomib could inhibit cancer cell growth and migration or invasion by repressing FAK expression. Since activation and overexpression of FAK has been implicated in the progression and invasion of malignant tumors, it is likely that targeting FAK with bortezomib is a potential strategy for preventing cancer metastasis. This review focuses on the molecular regulation of FAK and the potential clinical application of bortezomib.

Turning the RING Domain Protein MdmX into an Active Ubiquitin-Protein Ligase*

The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.

Regulation of MDM4 (MDMX) function by p76MDM2: a new facet in the control of p53 activity

Under basal growth conditions, p53 function is tightly controlled by the members of MDM family, MDM2 and MDM4. The Mdm2 gene codes, in addition to the full-length p90(MDM2), for a short protein, p76(MDM2) that lacks the p53-binding domain. Despite this property and at variance with p90(MDM2), this protein acts positively toward p53, although the molecular mechanism remains elusive. Here, we report that p76(MDM2) antagonizes MDM4 inhibitory function. We show that p76(MDM2) possesses intrinsic ubiquitinating and degrading activity, and through these activities controls MDM4 levels. Furthermore, the presence of p76(MDM2) decreases the association of MDM4 with p53 and p90(MDM2), and antagonizes p53 degradation by the heterodimer MDM4/p90(MDM2). The p76(MDM2)-mediated regulation of MDM4 occurs in the cytoplasm, under basal growth conditions. Conversely, upon DNA damage, phosphorylation of MDM4Ser403 dissociates p76(MDM2) and prevents MDM4 degradation. The overall negative control of MDM4 by p76(MDM2) reflects on p53 function as p76(MDM2) impairs MDM4-mediated inhibition of p53 activity. In agreement with the positive role of p76(MDM2) toward p53, the p76(MDM2)/p90(MDM2) ratio significantly decreases in a group of thyroid tumor samples compared with normal counterparts. Overall, these findings reveal a new mechanism in the control of p53 basal activity that may account for the distinct sensitivity of tissues to stress signals depending on the balance among MDM proteins. Moreover, these data suggest an oncosuppressive function for a product of the Mdm2 gene.

D-peptide inhibitors of the p53–MDM2 interaction for targeted molecular therapy of malignant neoplasms

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed (D)PMI-alpha (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed (D)PMI-gamma (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both (D)PMI-alpha and (D)PMI-gamma failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, (D)PMI-alpha exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.

An invertebrate mdm homolog interacts with p53 and is differentially expressed together with p53 and ras in neoplastic Mytilus trossulus haemocytes

The mussel Mytilus trossulus can develop a neoplasia of the haemolymph, which occurs with high frequency (up to 40%) in nature. Associated with this disease are pro-apoptotic tumor-suppressor protein p53 isoforms, which are highly conserved between molluscs and vertebrates. The vertebrate wildtype p53 protein is maintained at low levels by the MDM2 protein in non-stressed cells to prevent undesired apoptosis. Identification of a putative invertebrate MDM-like homolog suggests early evolution of this mechanism of p53 regulation. The M. trossulus MDM homolog consists of a conserved NH(2)-terminal p53 binding domain, an acidic domain with highly conserved phosphorylation sites, and a highly conserved C-terminal RING-finger Zn-binding domain. Although BLAST queries predict this homologue to be more similar to vertebrate MDM2 than to MDM4, phylogenetic analysis suggests that it may be an ancestral form to both vertebrate MDM genes. Using yeast-two-hybrid assays and pull-down assays, we show that this molluscan MDM is able to bind to its p53 counterpart. We also show that MDM expression levels are directly correlated with p53 expression levels in healthy and in neoplastic haemocytes, but not with other p53 isoforms or with the proto-oncogene RAS. The combination of expression levels of five gene transcripts (p53, mdm, ras, Np63/73, and TAp63/73) is significantly correlated with late-stage haemic neoplasia in M. trossulus.

Abstract 1109: Identification of a novel MDM2 splice transcript that lacks nuclear transport signals but harbors the p53 binding motif in human glioma cells

Abstract MDM2 a nuclear phosphoprotein binds with E3 ubiquitin ligase activity, binds and targets p53 for proteosomal degradation, hence is a negative regulator of p53. Consequently, over-expression of MDM2 results in the loss of p53 tumor suppressor function even in the absence of inactivating mutations in the p53 gene. To date, more than 40 different alternative spliced MDM2 transcript variants have been reported in both normal and tumor cells, with most of these lacking, in part or in full, the p53 binding domain of MDM2. In this study, we examined the levels of MDM2 gene expression in a panel of glioblastoma (GBM) cell lines and in vivo primary tumor xenografts by quantitative PCR /RT-PCR and examined the full length MDM2 cDNAs for the presence of MDM2 splice transcript variants using the nested PCR technique and a combination of direct sequencing and TA vector sequencing. The results showed a significant heterogeneity in MDM2 expression and in MDM2 splice variants among the tumors. Importantly, we identified a novel splice MDM2 transcript that was consistently associated with low MDM2 transcript levels in the tumors. Sequence analysis of this MDM2 spliced transcript showed it to harbor an internal deletion of three exons, namely 7, 8, and 9, containing the nuclear localization signal (NLS), the nuclear export signal (NES) and a portion of the acidic domain (AD). The p53 binding domain was fully conserved. The deletion caused a shift in the MDM2 open reading frame that created a stop codon (TGA) at codon 316. The spliced MDM2 transcript encoded a truncated MDM2 protein that harbors the functional p53 binding motif capable of interacting with the p53 protein but without the ability to localize to the nucleolus. Overall, our results with the 15 tumors showed no strong correlation between the presence of this novel splice MDM2 transcript and the expression levels of p53in the tumors. Studies are ongoing to determine the functional consequences of this novel MDM2 spliced variant on p53 downstream function in glioma cells. Supported by grants RO1CA112519, RO1CA127872, RO1CA91438, P30CA114236 and P5OCA108786 from the National Institutes of Health (USA). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1109.

Abstract 1205: Frequent detection of MDMX isoforms in human ovarian carcinomas

Abstract The p53 tumor suppressor pathway is inactivated in many human tumors either by p53 mutations or through overexpression of negative regulators, such as MDM2 and MDMX. In analogy to MDM2, besides the full-length mRNA several transcript variants, alternatively, as well as, aberrantly spliced transcripts of MDMX have been identified in different tumor cell lines. However, so far there are no studies regarding the expression of MDMX transcript variants in ovarian cancer tissue samples. We therefore screened 34 snap frozen ovarian tumor samples, using a high-temperature First-Strand cDNA synthesis kit to avoid reverse transcriptase artefacts. We identified in addition to MDMX-S numerous new MDMX transcripts in 16 of the 34 tumor samples that were either spliced at genuine exon/intron boundaries or at cryptic splice sites within exons. Furthermore, we could detect the transcript variants MDMX-A and MDMX-XALT2 firstly in ovarian cancer tissue. The MDMX splice variants can be classified into two groups: some isoforms have only retained the p53 binding domain, such as MDMX-S; in contrast, other splice forms consist of an intact C-terminus including the RING-finger domain, such as MDMX-XALT2. In addition, we have analyzed the expression of MDMX splice forms in ovarian cancer cell lines after treatment with chemotherapeutic agent. We observed a significant increase of the MDMX-S splice form while the expression levels of full-length MDMX decreased. This suggests that MDMX splice forms may play a role in mediating chemoresistance by attenuating the p53-pathway. In ongoing studies, we are investigating the role of frequently detected MDMX splice forms regarding their impact on chemoresistance in ovarian cancer in more detail. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1205.

HDM4 is overexpressed in mantle cell lymphoma and its inhibition induces p21 expression and apoptosis

In mouse models and cell lines, murine double minute 2 (MDM2) and MDM4 have been shown to synergistically promote proteasome-mediated degradation of p21 and p53. MDM4 also inhibits p53-mediated transcriptional activation of p21. p53 expression results in increased p21 expression, a negative cell-cycle regulatory protein and an inhibitor of cyclin D1. As mantle cell lymphoma is characterized by cyclin D1 overexpression, we assessed for human homolog of MDM4 (HDM4) expression and its effect on p21 in mantle cell lymphoma. Using immunohistochemical methods, in reactive lymph nodes (n=19) germinal center cells strongly expressed HDM4 in the nucleus and the cytoplasm, but mantle zone B-cells were only dimly positive. In mantle cell lymphoma tumors, aberrant HDM4 nuclear expression was observed in 18 of 19 (95%) cases. In contrast, HDM4 in other B-cell non-Hodgkin lymphoma types retained its normal pattern of expression. To further characterize the differential upregulation of HDM4 in mantle cell lymphoma, HDM4 was assessed by quantitative real-time polymerase chain reaction in four mantle cell lymphoma cell lines (Granta 519, Z-138, SP-53, and Mino) and six mantle cell lymphoma tumors. Both the splicing variant HDM4-S, containing only the p53-binding domain, and full length HDM4 were increased compared with normal CD19+ B-cells (P<0.05). Using small interfering RNA to inhibit HDM4 in the SP53 and Mino cell lines showed increased p21 and active caspase-3, the latter indicating increased apoptosis. Our results show that HDM4 is overexpressed in mantle cell lymphoma and, at least in part, exerts its effect by suppressing p21 expression, thereby enhancing cell-cycle progression. Inhibition of HDM4 may serve as a potential approach in the design of therapy for patients with mantle cell lymphoma.

Mice Lacking Dystrophin or α Sarcoglycan Spontaneously Develop Embryonal Rhabdomyosarcoma with Cancer-Associated p53 Mutations and Alternatively Spliced or Mutant Mdm2 Transcripts

Altered expression of proteins in the dystrophin-associated glycoprotein complex results in muscular dystrophy and has more recently been implicated in a number of forms of cancer. Here we show that loss of either of two members of this complex, dystrophin in mdx mice or alpha sarcoglycan in Sgca(-/-) mice, results in the spontaneous development of muscle-derived embryonal rhabdomyosarcoma (RMS) after 1 year of age. Many mdx and Sgca(-/-) tumors showed increased expression of insulin-like growth factor 2, retinoblastoma protein, and phosphorylated Akt and decreased expression of phosphatase and tensin homolog gene, much as is found in a human RMS. Further, all mdx and Sgca(-/-) RMS analyzed had increased expression of p53 and murine double minute (mdm)2 protein and contained missense p53 mutations previously identified in human cancers. The mdx RMS also contained missense mutations in Mdm2 or alternatively spliced Mdm2 transcripts that lacked an exon encoding a portion of the p53-binding domain. No Pax3:Fkhr or Pax7:Fkhr translocation mRNA products were evident in any tumor. Expression of natively glycosylated alpha dystroglycan and alpha sarcoglycan was reduced in mdx RMS, whereas dystrophin expression was absent in almost all human RMS, both for embryonal and alveolar RMS subtypes. These studies show that absence of members of the dystrophin-associated glycoprotein complex constitutes a permissive environment for spontaneous development of embryonal RMS associated with mutation of p53 and mutation or altered splicing of Mdm2.

Runt-Related Transcription Factor RUNX3 Is a Target of MDM2-Mediated Ubiquitination

The p14(ARF)-MDM2-p53 pathway constitutes an effective mechanism for protecting cells from oncogenic stimuli such as activated Ras and Myc. Importantly, Ras activation induces p14(ARF) and often occurs earlier than p53 inactivation during cancer development. Here, we show that RUNX3, a tumor suppressor in various tumors including stomach, bladder, colon, and lung, is stabilized by Ras activation through the p14(ARF)-MDM2 signaling pathway. RUNX3 directly binds MDM2 through its Runt-related DNA-binding domain. MDM2 blocks RUNX3 transcriptional activity by interacting with RUNX3 through an acidic domain adjacent to the p53-binding domain of MDM2 and ubiquitinates RUNX3 on key lysine residues to mediate nuclear export and proteasomal degradation. Our data indicate that the lineage-specific tumor suppressor RUNX3 and the ubiquitous p53 protein are both principal responders of the p14(ARF)-MDM2 cell surveillance pathway that prevents pathologic consequences of abnormal oncogene activation.

MDM2 Controls the Timely Expression of Cyclin A to Regulate the Cell Cycle

Overexpression of MDM2 has been related to oncogenesis. In this communication, we present evidence to show that MDM2 controls the cell cycle-dependent expression of cyclin A by using a pathway that ensures its timely expression. MDM2 does not inhibit cyclin D or E expression. Silencing of endogenous MDM2 expression elevates cyclin A expression. The p53-binding domain of MDM2 harbors a SWIB region homologous to a conserved domain of a chromosome remodeling factor BRG1-associated protein. The SWIB domain of MDM2 inhibits cyclin A expression in a p53- and BRG1-dependent fashion, suggesting that MDM2 interferes with p53 binding of the BRG1 complex freeing it to repress cyclin A expression. Silencing of cyclin-dependent kinase (cdk) inhibitor p16 prevents MDM2-mediated inhibition of cyclin A expression, implicating its role in the process. MDM2-mediated repression of cyclin A expression induces G(1)-S arrest, which can be rescued by ectopic expression of cyclin A. Cancer cells lacking p53, p16, or BRG1 escape MDM2-mediated repression of cyclin A expression and growth arrest. Our data propose a novel mechanism by which MDM2 controls the cell cycle in normal cells and how cancer cells may escape this important safety barrier.

Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2.

The tumor suppressor activity of p53 is regulated by interactions with the ubiquitin ligase HDM2 and the general transcriptional coactivators CBP and p300. Using NMR spectroscopy and isothermal titration calorimetry, we have dissected the binding interactions between the N-terminal transactivation domain (TAD) of p53, the TAZ1, TAZ2, KIX, and nuclear receptor coactivator binding domains of CBP, and the p53-binding domain of HDM2. The p53 TAD contains amphipathic binding motifs within the AD1 and AD2 regions that mediate interactions with CBP and HDM2. Binding of the p53 TAD to CBP domains is dominated by interactions with AD2, although the affinity is enhanced by additional interactions with AD1. In contrast, binding of p53 TAD to HDM2 is mediated primarily by AD1. The p53 TAD can bind simultaneously to HDM2 (through AD1) and to any one of the CBP domains (through AD2) to form a ternary complex. Phosphorylation of p53 at T18 impairs binding to HDM2 and enhances affinity for the CBP KIX domain. Multisite phosphorylation of the p53 TAD at S15, T18, and S20 leads to increased affinity for the TAZ1 and KIX domains of CBP. These observations suggest a mechanism whereby HDM2 and CBP/p300 function synergistically to regulate the p53 response. In unstressed cells, CBP/p300, HDM2 and p53 form a ternary complex that promotes polyubiquitination and degradation of p53. After cellular stress and DNA damage, p53 becomes phosphorylated at T18 and other residues in the AD1 region, releases HDM2 and binds preferentially to CBP/p300, leading to stabilization and activation of p53.

Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 A resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.