MDM2 Levels Research Articles

Quinuclidinone derivative 6 induced apoptosis in human breast cancer cells via sphingomyelinase and JNK signaling

Novel quinuclidinone derivatives have been previously reported by our laboratory. In this study, we investigated the impact of two novel quinuclidinone derivatives 4 and 6 on apoptotic signaling in breast cancer cells (MCF-7) and their normal counterparts (MCF-12a). Our data revealed that derivatives 4 and 6 reduced proliferation and induced apoptosis in breast cancer cells. However, derivative 6 was less cytotoxic to normal breast epithelial cells than breast cancer cells; therefore, we focused on derivative 6 for further investigation. Flow cytometric analysis showed that quinuclidinone derivative 6 reduced the percentage of MCF-7 cells in G2/M which is confirmed by increased expression levels of cyclin B, while it arrests MCF12a in G1 phase judging from increased p21. Quinuclidinone derivative 6 increased expression levels of p53 and Bax at both protein and mRNA levels and reduced expression level of Mdm2, Bcl2, Akt and Bcl-XL It also increased mitochondrial apoptotic pathways by activating release of cytochrome c which is consistent with activation of caspase-9 as confirmed by caspase-9 inhibitor LEHD-CHO. Finally, it increased sphingomyelinase signaling and ceramide formation as well as its downstream targets ERK1/2, p38, and JNK. Inhibition of ERK1/2 with PD98059 exerted little effect on the derivative 6-induced apoptosis and p38 inhibition with SB203580 slightly lessened apoptosis, whereas inhibition of JNK with SP600125 markedly suppressed derivative 6-induced apoptosis. These results indicate that derivative-6 induced the activation of sphingomyelinase signaling and that JNK played a pivotal role in induction of apoptosis in human breast cancer cells. In vivo studies and molecular docking experiments are now in progress for further anticancer investigations.

Decreased Mdm2 Expression Inhibits Tumor Development and Extends Survival Independent of Arf and Dependent on p53

Inactivation of the Arf-Mdm2-p53 tumor suppressor pathway is a necessary event for tumorigenesis. Arf controls Mdm2, which in turn regulates p53, but Arf and Mdm2 also have p53-independent functions that affect tumor development. Moreover, inhibition of oncogene-induced tumorigenesis relies on Arf and p53, but the requirements of Arf and p53 in tumor development initiated in the absence of overt oncogene overexpression and the role of Mdm2 in this process remain unclear. In a series of genetic experiments in mice with defined deficiencies in Arf, Mdm2 and/or p53, we show Mdm2 haploinsufficiency significantly delayed tumorigenesis in mice deficient in Arf and p53. Mdm2 heterozygosity significantly inhibited tumor development in the absence of Arf, and in contrast to Myc oncogene-driven cancer, this delay in tumorigenesis could not be rescued with the presence of one allele of Arf. Notably, Mdm2 haploinsufficieny blocked the accelerated tumor development in Arf deficient mice caused by p53 heterozygosity. However, tumorigenesis was not inhibited in Mdm2 heterozygous mice lacking both alleles of p53 regardless of Arf status. Surprisingly, loss of Arf accelerated tumor development in p53-null mice. Tumor spectrum was largely dictated by Arf and p53 status with Mdm2 haploinsufficiency only modestly altering the tumor type in some of the genotypes and not the number of primary tumors that arose. Therefore, the significant effects of Mdm2 haploinsufficiency on tumor latency were independent of Arf and required at least one allele of p53, and an Mdm2 deficiency had minor effects on the types of tumors that developed. These data also demonstrate that decreased levels of Mdm2 are protective in the presence of multiple genetic events in Arf and p53 genes that normally accelerate tumorigenesis.

MDM2 regulates a novel form of incomplete neoplastic transformation of Theileria parva infected lymphocytes

Our efforts are concerned with identifying features of incomplete malignant transformation caused by non viral pathogens. Theileria parva (T. parva) is a tick-transmitted protozoan parasite that can cause a fatal lymphoproliferative disease in cattle. The T. parva-infected lymphocytes display a transformed phenotype and proliferate in culture media like the other tumor cells, however those cells will return to normal after antiprotozoal treatment reflecting the incomplete nature of transformation. To identify signaling pathways involved in this form of transformation of T. parva-infected cells, we screened a library of anticancer compounds. Among these, TIBC, a specific inhibitor of MDM2, markedly inhibited proliferation of T. parva-infected lymphocytes and promoted apoptosis. Therefore we analyzed MDM2 function in T. parva-infected cells. Several T. parva-infected cell lines showed increased expression level of MDM2 with alternatively spliced isoforms compared to the lymphoma cells or ConA blasts. In addition, buparvaquone affected MDM2 expression in T. parva transformed cells. Moreover, p53 protein accumulation and function were impaired in T. parva-infected cells after cisplatin induced DNA damage despite the increased p53 transcription level. Finally, the treatment of T. parva-infected cells with boronic-chalcone derivatives TIBC restored p53 protein accumulation and induced Bax expression. These results suggest that the overexpression of MDM2 is closely linked to the inhibition of p53-dependent apoptosis of T. parva-infected lymphocytes. Aberrant expression of host lymphocyte MDM2 induced by cytoplasmic existence of T. parva, directly and/or indirectly, is associated with aspects of this type of transformation of T. parva-infected lymphocytes. This form of transformation shares features of oncogene induced malignant phenotype acquisition.

A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Inflammation‐mediated abrogation of androgen signaling: An in vitro model of prostate cell inflammation

As a link between inflammation and cancer has been reported in many studies, we established an in vitro model of prostatic inflammation to investigate the loss of androgen receptor (AR)-mediated signaling in androgen responsive prostate cell lines. First, the U937 monocyte cell line was differentiated into macrophages using phorbol acetate (PMA), and cells were induced with lipopolysaccharide (LPS) for cytokine secretion. Next, the cytokine levels (TNFα, IL-6, and IL1β) in conditioned media (CM) were analyzed. Prostate cells were then fed with CM containing varying concentrations of TNFα, and IkB degradation, nuclear factor kappa B (NFκB) translocation and transactivation, and the expression of matrix metalloproteinase-8 (MMP8) and matrix metalloproteinase-9 (MMP9) were then assessed. As a result of CM treatment, ubiquitin-mediated AR degradation, which was restored using anti-TNFα antibody neutralization, led to both a decrease in KLK4, PSA, and NKX3.1 expression levels and the upregulation of GPX2. In addition to the loss of AR, acute and chronic CM exposure resulted in p53 degradation and consequent p21 downregulation, which was also restored by either androgen administration or ectopic NKX3.1 expression via the stabilization of MDM2 levels in LNCaP cells. Additionally, CM treatment enhanced H2AX((S139)) phosphorylation (a hallmark of DNA damage) and genetic heterogeneity in the absence of androgens in prostate cells without activating mitochondrial apoptosis. Thus, the data suggest that inflammatory cytokine exposure results in the loss of AR and p53 signaling in prostate cells and facilitates genetic heterogeneity via ROS accumulation to promote prostate carcinogenesis.

Transcription Factor NFAT1 Activates the mdm2 Oncogene Independent of p53

Although the MDM2-p53 interaction has been well documented, MDM2 overexpression is observed in human cancers with little or no functional p53, suggesting that mdm2 expression is regulated by mechanisms independent of p53. Dysregulation of NFAT signaling is associated with malignant transformation and cancer development and progression. In this study, we demonstrate that the human mdm2 P2 promoter contains a consensus binding site for the NFAT1 transcription factor. NFAT1 directly binds the mdm2 P2 promoter in vitro and in vivo, resulting in the up-regulation of mdm2 transcription. Enforced expression of NFAT1 results in an elevated MDM2 protein level and reduces p53 activation and function in response to DNA damage. Both NFAT1 and MDM2 are highly expressed in human hepatocellular carcinoma tissues, compared with adjacent normal liver tissues. There is a positive correlation between the NFAT1 and MDM2 levels in tumor tissues. The novel function of NFAT1 in the control of MDM2 expression provides a basis for future investigations of the role of NFAT1 in cancer development, progression, and therapy.

Murine double minute‐2 expression is required for capillary maintenance and exercise‐induced angiogenesis in skeletal muscle

Exercise-induced angiogenesis is a key determinant of skeletal muscle function. Here, we investigated whether the E3 ubiquitin ligase murine double minute-2 (Mdm2) exerts a proangiogenic function in exercised skeletal muscle. Mdm2 hypomorphic (Mdm2(Puro/Δ7-9)) mice have a 60% reduction in Mdm2 expression compared with that in wild-type animals. Capillary staining on muscle sections from Mdm2(Puro/Δ7-9) sedentary mice with a wild-type or knockout background for p53 revealed that deficiency in Mdm2 resulted in 20% capillary regression independently of p53 status. In response to one bout of exercise, protein expression of the proangiogenic vascular endothelial growth factor-A (VEGF-A) was increased by 64% in muscle from wild-type animals, and endothelial cell outgrowth from exercised muscle biopsy samples cultured in a 3-dimensional collagen gel was enhanced by 37%. These proangiogenic responses to exercise were impaired in exercised Mdm2(Puro/Δ7-9) mice. Prolonged exercise training resulted in increased Mdm2 protein expression (+49%) and capillarization (+24%) in wild-type muscles. However, exercise training-induced angiogenesis was abolished in Mdm2(Puro/Δ7-9) mice. Finally, exercise training restored Mdm2, VEGF-A, and capillarization levels in skeletal muscles from obese Zucker diabetic fatty rats compared with those in healthy animals. Our results define Mdm2 as a crucial regulator of capillary maintenance and exercise-induced angiogenesis in skeletal muscle.

Identification of FDA‐approved Drugs that Computationally Bind to MDM2

The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers p53 is inactivated by abnormally high levels of MDM2--an E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft, and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present 15 FDA-approved drugs predicted to inhibit p53/MDM2 interaction.

Reactivation of p53 mutant protein by PRIMA-1 and induction of apoptosis in pancreatic cancer cells.

e13546 Background: Inactivating TP53 mutations are common events in different types of cancers, including pancreatic adenocarcinoma, where it occurs in as much as 70% of cases. Accumulation of mutant p53 provides a molecular target that may be reactivated into a conformation capable of arresting tumor growth. The small molecule PRIMA-1 (p53 reactivation and induction of massive apoptosis) has been shown to selectively induce apoptosis in tumor cells by reactivating some p53 mutants, but no previous studies have investigated its effects in pancreatic cancer. Methods: PANC-1 (mutant TP53 R273H) and CAPAN-2 (wild-type TP53) pancreatic cell lines were used as in vitro models. We tested the effects of PRIMA-1 on cell viability (MTT assay), apoptosis (morphology, AnnexinV/FITC-FACS), cell cycle (BrdU incorporation) and expression of p53 regulated proteins by western blotting. As control of p53-dependent effects, PANC-1 cell lines were transfected with siRNA against TP53 (sip53). Results: PRIMA-1 selectively induced apoptosis in PANC-1 cells compared to CAPAN-2 cells and this effect was concomitant with an increase in the levels of MDM2, Bax and cleaved caspase-3 as detected by western blot analysis. Treatment with PRIMA-1 for 24h induced a 50% reduction in DNA synthesis whereas G2/M arrest was detected after 12h of treatment. p53 silencing in PANC-1 decreased the cytotoxicity of PRIMA-1, characterizing a p53-dependent effect. Finally, N-acetylcysteine completely blocked PRIMA-1-induced growth suppression and apoptosis, suggesting that PRIMA-1 exerts its effect at least in part via restoring redox-dependent effects to mutant p53. Conclusions: Our data indicate that PRIMA-1 induces apoptosis in TP53 mutant pancreatic cancer cells by promoting the re-activation of p53 and subsequent of proapoptotic signaling pathways, suggesting a possible mechanism for effective targeting of pancreatic cancer.

Association of p53 mutations with progression-free survival (PFS) and overall survival (OS) in EGFR-mutated non-small cell lung cancer (NSCLC) patients (p) treated with erlotinib.

e18143 Background: Advanced NSCLC p with EGFR mutations have a median PFS of 14 months (m) and OS of 27 m when treated with erlotinib. In NSCLC cell lines, tyrosine kinase inhibitors (TKIs) induce p53 translocation from the cytoplasm to the nucleus and subsequent upregulation of Fas and caspase activation leading to apoptosis, but this mechanism was defective in p53-null cells. We tested whether TP53 mutations influence outcome to erlotinib in EGFR-mutated p. Expression levels of the p53 repressor MDM2 were also examined. Methods: We assessed p53 status in pretreatment paraffin-embedded tumor samples from 93 erlotinib-treated, EGFR-mutated advanced NSCLC p. Mutations in exons 5, 6, 7 and 8 were screened by High Resolution Melting analysis followed by sequencing of the amplified products with non-wild-type (wt) melt curves. All mutant samples were re-confirmed by standard PCR and sequencing. Expression levels of MDM2 mRNA were determined by quantitative RT-PCR. Results: Mutations in exons 5-8 of TP53 were detected in 26 of 93 p (28%). We found an unusually high frequency of in-frame and frameshift deletions (23% of mutations), indicating that the spectrum of p53 mutations might be different in EGFR-mutated NSCLC. Mutations were less frequent in p with ECOG PS >2 and more frequent in p with the T790M mutation. OS was 15 m in the 16 p with missense mutations 31 m in p with wt p53 and not reached in p with non-missense mutations (P=0.04). PFS was 9 m for 14 p with mutations in one of the p53 DNA binding motifs (DBMs), compared to 19 m for wt p and 27 m for p with non-DBM mutations. MDM2 mRNA levels were significantly lower in tumors with p53 mutations, especially in DBM mutations. In the case of wt p, high MDM2 expression correlated with longer PFS and OS in p with wt p53. Conclusions: TP53 mutations co-exist with EGFR mutations in a significant number of p; missense mutations correlate with shorter OS and mutations in DBMs correlate with shorter PFS. This finding paves the way for the possibility of combining erlotinib with a drug restoring p53 function in those p harboring certain types of mutations in the TP53 gene.

Pharmacological activation of the p53 pathway by nutlin-3 exerts anti-tumoral effects in medulloblastomas

Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%-70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.

Evaluation of MDM2, p16, and p53 staining levels as biomarkers of biochemical recurrence following salvage radiation therapy for recurrent prostate cancer

The selection of appropriate candidates for salvage radiation therapy (SRT) to address a rising PSA following radical prostatectomy remains challenging. Herein, we provide the first evaluation of the ability of staining levels of the tumor based biomarkers MDM2, p16, and p53 to aid in prediction of biochemical recurrence (BCR) among men undergoing SRT for recurrent prostate cancer. We identified 152 patients who were treated with SRT between July 1987 and July 2003. Staining levels of MDM2, p16, and p53 in primary tumor samples removed during prostatectomy were detected using monoclonal antibodies and quantified by use of a computer-assisted method. Associations of staining levels with BCR were evaluated using Cox proportional hazards regression models; relative risks (RRs) and 95% confidence intervals (CIs) were estimated. Compared to patients with low staining (≤median) as measured by percentage of cells with nuclear staining, there was no significant difference in risk of BCR for patients with high MDM2 staining (RR: 0.90, 95% CI: 0.57-1.45, P = 0.67), high p16 staining (RR: 0.88, 95% CI: 0.54-1.44, P = 0.62), or high p53 staining (RR: 1.33, 95% CI: 0.84-2.11, P = 0.23) in multivariable analysis. These results were consistent when considering alternate percentile cutpoints and alternate quantifications of biomarker staining. Our results provide evidence that MDM2, p16, and p53 staining levels are not useful in the prediction of BCR after SRT. As such, these biomarkers are of little clinical use in the selection of appropriate candidates for SRT.

Abstract 5213: MDM2 at the crossroads of cell fate decisions in cerebellar development and medulloblastoma

Abstract During post-natal cerebellar development, Granule Neuron Precursors (GNPs) undergo a proliferative expansion in response to Sonic hedgehog (Shh) signaling, prior to differentiation and migration to their final niche. Aberrations in this tightly controlled process can tip the balance toward persistent proliferation leading to Medulloblastoma (MB), or toward apoptosis or premature differentiation leading to neurodegeneratiion. Here, we report that MDM2, the principal inhibitor of the p53 tumor suppressor protein, is required for cerebellum development as well as for MB tumorigenesis. Our studies employed a novel hypomorphic allele of Mdm2 (Mdm2puro) in combination with the Mdm2del7-9 null allele. Mdm2puro/del7-9 mice express ∼30% of the wild-type level of MDM2. Mdm2puro/del7-9 cerebella exhibit decreased foliation, reduction in GNP numbers, and laminar disorganization. These phenotypes are attributed, in part, to high levels of p53-dependent apoptosis. In addition, we have recently determined through fate mapping studies that loss of MDM2 also promotes the premature migration of GNPs. Global transcriptome analyses of the Mdm2puro/del7-9 cerebellum supports dysregulation of the transcriptional programs that regulate GNP differentiation. Further, Western blot analyses reveal an increase in the pro-differentiation protein, Numb, and the neuronal maturation marker, βIII-Tubulin, in GNPs. A concomitant decrease in Gli1 and Gli2, two effectors of Shh signaling known to be degraded by Numb, suggests an increase in Numb activity. These experiments suggest that loss of MDM2 and subsequent increase in p53 activity accelerates GNPs migration and possibly favors GNP differentiation over proliferation. In complementary experiments, we employed Shh-responsive primary GNP cultures from wild-type mice. Shh stimulation of GNPs results in an increase in the steady state level of MDM2 and an increase in MDM2 phosphorylation at Ser166, a modification known to enhance the anti-p53 function of MDM2. These findings suggest that Shh signaling may upregulate MDM2 to prevent p53 activation during GNP proliferation. In keeping with the concept that GNP proliferation and MB tumor formation are governed by similar mechanisms, we also observed increased levels of p-MDM2Ser166 in Shh-induced preneoplastic lesions (PNLs) and MB tumors in a Ptch1+/- mice, a model of human MB. Notably, a 70% reduction in the level of MDM2 in these mice abrogates the formation of PNLs, supporting a key role for MDM2 in the initiation of some types of MB. Overall, our results suggest that MDM2 mediated inhibition of p53 plays in important role in survival and migration of GNPs cerebellum development. This role of MDM2 may be high-jacked by deregulated Shh signaling in certain MB tumors thereby limiting the tumor suppressive function of p53 and obviating a need for p53 gene mutations. 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 5213. doi:1538-7445.AM2012-5213

Abstract 977: Elevated MDM2 protein contributes to activity of MDM2 antagonists by enabling p53 induction of antitumor microRNAs

Abstract Small-molecule inhibitors of p53-MDM2 binding, the nutlins, can free p53 from MDM2 control and effectively activate its growth suppressive and pro-apoptotic activity in cancer cells (Vassilev et al., Science, 2004, 303, 844). Optimized members of this family of molecules are currently under clinical investigation as novel therapeutic modality for cancers expressing wild-type p53. We have shown previously that MDM2 antagonists are most effective in cancer cells that overproduce MDM2 as a result of mdm2 gene amplification (Tovar et al., PNAS, 2006, 103, 1888). Here, we show that reduction of MDM2 protein by RNA interference decreases the effectiveness of nutlin-3a in osteosarcoma cells with mdm2 gene amplification, suggesting that MDM2 protein contributes to antitumor activity of MDM2 antagonists. In search of the mechanism behind this phenomenon, we found that nutlin-induced p53 activates the transcription of a specific subset of microRNAs in cancer cells with mdm2 amplification but not in cells expressing normal levels of MDM2 protein. Five specific members of the class (miR1, miR133, miR141, miR184, miR375) were studied and showed that their p53-dependent transcription is also dependent on the presence of high levels of MDM2 protein, generated by continuous p53 activation of MDM2 in the presence of nutlin. Weak p53 response elements were found in the promoter regions of all five miRs and p53 binding was conformed by ChIP analysis of cell lysates and p53 reporters in experiments with miR375. Transcriptional cofactor p300 was found elevated in cancer cells with mdm2 gene amplification and dependent on the presence of high MDM2 protein levels. Our experimental data suggests that in the presence of high p300, and high MDM2, generated by nutlin treatment, p53 acquires the ability to activate weak p53-responsive miR promoters in MDM2-amplified cells. Interestingly, miR mimics of two of the five p53-induced miRs showed growth suppressive effects in cancer cells and enhanced the activity of low dose nutlin-3a. These findings reveal a novel mechanism for modulation of p53 transcriptional activity and indicate that the high levels of MDM2, generated in mdm2-amplified cancer cells in the presence of nutlin, contribute to its antitumor. 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 977. doi:1538-7445.AM2012-977

Abstract 4596: Missense mutations in the p53 DNA-binding motifs (DBMs) are associated with shorter time to progression (TTP) in erlotinib-treated, EGFR-mutated patients

Abstract Background: Advanced non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations has an average time to progression (TTP) of 14 months (mo) and overall survival of 27 mo when treated with erlotinib. However, EGFR mutations can only imperfectly predict outcome. In NSCLC cell lines, tyrosine kinase inhibitors (TKIs) induce p53 translocation from the cytoplasm to the nucleus and subsequent up-regulation of Fas and caspase activation leading to apoptosis. This mechanism was defective in p53-null cells. We tested whether mutations in the TP53 gene influence outcome to erlotinib in EGFR-mutated patients (p). Expression levels of the p53 repressor MDM2 were also examined. Methods: We assesed p53 status in pretreatment paraffin-embedded tumor samples from 93 erlotinib-treated, EGFR mutated advanced NSCLC p. Mutations in exons 5, 6, 7 and 8 were screened by High Resolution Melting analysis (HRM) followed by sequencing of the amplified products with non-wild type melt curves. All mutant samples were re-confirmed by standard PCR and sequencing. Expression levels of MDM2 mRNA were determined by quantitative RT-PCR. Results: Mutations in exons 5-8 of the TP53 gene were detected in 26 of 93 p (28%). We found an unusually high frequency of in-frame and frameshift deletions (23% of mutations), indicating that the spectrum of p53 mutations might be different in EGFR-mutated NSCLC. Mutations in the TP53 gene were not associated with sex, age, smoking status, histology, exon 19 vs. 21 mutation, or response, but, within the study population, were significantly less frequent in p with ECOG 2 or above. The mutations were also associated with the presence of the pretreatment T790M mutation. 14 p had mutations in one of the p53 DNA binding motifs (DBMs), and showed TTP to erlotinib of only 9 mo, compared to 19 months for wt p and 27 mo for those carrying a non-DBM mutation. Survival was 24 mo vs. 31 mo, and not-reached, respectively. Finally, MDM2 mRNA levels were significantly lower in tumors with p53 mutations, especially when these affected DBMs. In the case of wt p, high MDM2 expression correlated with a better TTP and survival. Conclusions: TP53 mutations co-exist with EGFR mutations in a significant number of p; and those in the DBMs are associated with poorer response to erlotinib. This finding paves the way for the possibility of combining erlotinib with a drug restoring p53 function in those p harboring DBM mutations in the TP53 gene. 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 4596. doi:1538-7445.AM2012-4596

Abstract 5026: Loss of microRNA-143/145 disturbs cell growth of cancers by impairing the MDM2-p53 feedback loop

Abstract BACKGROUND: Dysregulated microRNAs (miRNAs) play an important role in many malignant tumors, including epithelial cancers, but little is known about the molecular mechanisms and clinical significances of miRNAs dysregulation. The aim is to identify and determine the miR-143 and miR-145 how to specifically be involved in controlling MDM2 expression in a tumor tissue-specific manner. METHODS: Using real-time PCR, the expression of miR-143, miR-145 and MDM2 were evaluated and analyzed in epithelial cancer cell lines and oral cancer specimens. The different PCR-derived fragments and mutagenesis from the MDM2 mRNA 3′ UTR were inserted into the pGL3 vector to generate the luciferase reporters and the reporter activities were assayed by luciferase assay kits. The effects of overexpression of miR-143 and miR-145 on MDM2 and p53 levels were analyzed by western blot. The effects of miR-143 and miR-145 on cell proliferation and cell cycle were determined by the EdU assay and flow cytometry analysis. The effects of miR-143 and miR-145 on tumorigenicity in vivo were investigated with HNSCC cell xenograft models and the cell apoptotic rate as well as mechanism were tested by TUNEL and apoptosis-protein expression in the tumor tissues. RESULTS: The results showed that both miR-143 and miR-145, which belong to the same miRNA cluster, can negatively modulate expression of their target gene, MDM2. The miR-143 and miR-145 is post-transcriptionally activated by up-regulated p53, thereby generating a short miRNAs-MDM2-p53 feedback loop. Re-expression of these miRNAs suppresses cellular growth and triggers the apoptosis of epithelial cancer, in vitro and in vivo, by enhancing p53 activity via MDM2 turnover. Moreover, the miRNA-dependent MDM2 turnover contributes to the equilibrium of repeated p53 pulses in response to DNA damage stress. CONCLUSIONS: These findings suggest that MDM2 dysregulation caused by down-regulation of miR-143 and miR-145 contributes to epithelial cancer development and palys a key role in regulating cellular proliferation and apoptosis. Recruitment of miR-143 and miR-145 may be a reasonable strategy for treatment of epithelial cancers. KEYWORDS: miRNA-143, miRNA-145, MDM2, p53, epithelial cancers. ACKNOWLEDGMENTS: This study was supported by the National Natural Science Foundation of China (Grant No. 30973343 and 81101515), Projects of the Shanghai Science and Technology Committee (Grant No. 08JC1414400 and 10XD1402500), the Shanghai Leading Academic Discipline Project (S30206). *The first two authors contributed equally to this work. 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 5026. doi:1538-7445.AM2012-5026

Abstract 1810: Mechanistic studies of the dual Akt and JNK inhibitor Chaetoglobosin K: Effect on downstream pathways and AP-1 proteins in tumorigenic cells

Abstract We have previously shown that the anti-tumorigenic indolylcytochalasin Chaetoglobosin K (ChK) concomitantly down-regulates Akt and JNK activation in ras-transformed cells. This dual inhibition was found not be regulated by Rac1, a Rho protein that is involved in both signaling pathways, or MKK4, an upstream kinase of JNK. The purpose of this study was to determine: 1) the effect of ChK on the activation of downstream mediators of the PI3K/Akt (including mdm2), and JNK (including the activator protein-1 (AP-1) proteins and stat3) signaling pathways; 2) whether the upstream mitogen-activated protein kinase kinase 7 (MKK7) is involved in ChK's effect on JNK. The AP-1 transcription factor complex, composed of proteins of the JUN, FOS, and ATF families, has become a promising therapeutic target due to the proposed role of AP-1 activation in tumorigenesis. In these studies, ras-transformed rat liver epithelial cells were grown to confluence and treated with either ChK (5μM or 10μM) or vehicle (DMSO) for 24 hours. Cell proteins were extracted and separated using SDS-PAGE. Western blot analysis was performed using phosphorylation site-specific antibodies to monitor changes in MKK7, c-JUN, ATF-2, mdm2, and stat3 protein activation levels. The results of these studies indicate that ChK treatment decreased c-JUN and ATF-2 phosphorylation, but had no effect on stat3 phosphorylation. Additionally, ChK treatment decreased mdm2 phosphorylation. ChK did not alter MKK7 activation levels and, taking into consideration its lack of effect on MKK4, suggest ChK does not inhibit JNK via its archetypical MAPK signaling pathway. These studies indicate ChK may serve as a small molecule inhibitor of the AP-1 pathway proteins, as well as the Akt/mdm2 pathway. This data further supports ChK's potential as a unique targeted tumor therapy. This project was funded by NIH grant 1R15CA135415 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 1810. doi:1538-7445.AM2012-1810

Abstract 2786: Potent inhibition of human liposarcoma cell growth and survival by novel modulators of the MDM2-p53 interaction

Abstract The tumor suppressor p53 is commonly inactivated in human malignancies. One mechanism is by genomic amplification and consequent overexpression of the ubiquitin E3 ligase MDM2, which targets p53 for proteosome-mediated degradation. The MDM2 gene is amplified in >95% of human liposarcomas, a disease for which the efficacy of systemic therapies is limited. Here, we compared the effects of two novel highly potent and selective inhibitors of the MDM2-p53 interaction, with the classic MDM2 inhibitor Nutlin-3 on the expression and activity of p53 and the resulting changes in human liposarcoma cell proliferation and survival. We determined MDM2 copy number, p53 mutational status, and relative expression levels of MDM2 and p53 proteins in 8 human liposarcoma cell lines and normal human preadipocytes/adipocytes. TP53 status was wild type in 7 of 8 liposarcoma cell lines, which also had genomic amplification of MDM2. MDM2 protein expression correlated with the degree of amplification and was highly expressed in comparison to normal preadipocytes/adipocytes. The two spiro-oxindole derivatives - Compounds A and B- (1) and Nutlin-3 were applied to liposarcoma cell lines with or without p53 siRNA and the biological consequences were determined by immunoblot, cell viability, cell cycle and apoptosis assays. Treatment with MDM2 inhibitors led to increased p53 protein levels and induction of its transcriptional targets MDM2 and p21. Cell viability was markedly decreased in a dose-dependent manner in liposarcoma cells with wild-type p53, with IC50 values of 0.40-4.23 μM, 0.37-1.88 μM and 2.72-9.83 μM for Compound A, Compound B, and Nutlin-3, respectively. These effects were markedly abrogated by siRNA-mediated p53 knockdown and in a cell line harboring a de novo p53 mutation. Compound A and Compound B also induced cell cycle arrest and apoptosis at approximately 5 fold lower concentrations than Nutlin-3. At low concentrations, G0/G1 cell arrest occurred, whereas higher concentrations resulted in G2/M arrest and induction of apoptosis. Cell line and tumor xenograft experiments are ongoing. These data demonstrate that Compounds A and B are highly potent and specific inhibitors of MDM2 with activity in genetically characterized liposarcoma cells. Further study and clinical development in this disease is warranted. 1. Wang S. et al., AACR, Orlando FL, 2011, Abstract LB-204 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 2786. doi:1538-7445.AM2012-2786

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

Overexpression of SKI Oncoprotein Leads to p53 Degradation through Regulation of MDM2 Protein Sumoylation

Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer.