nutlin-3a and Osteosarcoma

The most prevalent form of primary osseous malignant tumor in adolescents and children is osteosarcoma (OS). A combination of surgery and neoadjuvant/post-surgery chemotherapy is currently the standard therapy. While the chemoresistance associated with OS generally leads to poor efficacy of therapeutic agents, the relevant molecular interaction is still elusive. Here, the lncRNA (long non-coding RNA) SNHG14 was found to be significantly upregulated in the nutlin3a-resistant OS cell line NR-SJSA1 and contributes to treatment resistance by suppressing ferroptosis. In NR-SJSA1 cells, knockdown of LncRNA SNHG14 resulted in a reversal of drug resistance and activation of ferroptosis, which disappeared when ferrostatin-1, a ferroptosis inhibitor, was added. Mechanistically, lncRNA SNHG14 targeted and down-regulated the expression of miR-206, further affecting the common ferroptosis inhibitor SLC7A11, and preventing NR-SJSA1 cells from undergoing ferroptosis. In conclusion, our findings highlight the involvement of lncRNA SNHG14 in ferroptosis and chemotherapy resistance of nutlin3a-resistant NR-SJSA1 cells, thus shedding new insight on how to overcome drug resistance in osteosarcoma cells and improve treatment efficacy.

Topics: Amino Acid Transport System y+; Bone Neoplasms; Drug Resistance, Neoplasm; Ferroptosis; Humans; Imidazoles; MicroRNAs; Osteosarcoma; Piperazines; RNA, Long Noncoding; Tumor Cells, Cultured

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3β (glycogen synthase kinase 3β) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3β inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3β could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3β mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.

Topics: Antineoplastic Agents; Cell Line, Tumor; Glycogen Synthase Kinase 3 beta; Humans; Imidazoles; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-mdm2

Activated p53 can promote apoptosis or cell cycle arrest. Differences in energy metabolism can influence cell fate in response to activated p53. Nutlin-3a is a preclinical drug and small molecule activator of p53. Alpha-ketoglutarate (αKG) levels were reduced in cells sensitive to Nutlin-3a-induced apoptosis and increased in cells resistant to this apoptosis. Add-back of a cell-permeable αKG analog (DMKG) rescued cells from apoptosis in response to Nutlin-3a. OGDH is a component of the αKGDH complex that converts αKG to succinate. OGDH knockdown increased endogenous αKG levels and also rescued cells from Nutlin-3a-induced apoptosis. We previously showed reduced autophagy and ATG gene expression contributes to Nutlin-3a-induced apoptosis. DMKG and OGDH knockdown restored autophagy and ATG gene expression in Nutlin-3a-treated cells. These studies indicate αKG levels, regulated by p53 and OGDH, determine autophagy and apoptosis in response to Nutlin-3a.

Topics: A549 Cells; Apoptosis; Autophagy; Bone Neoplasms; Cell Line, Tumor; Gene Knockdown Techniques; Glycolysis; Humans; Imidazoles; Ketoglutarate Dehydrogenase Complex; Ketoglutaric Acids; Lung Neoplasms; Mechanistic Target of Rapamycin Complex 1; Neoplasms; Osteosarcoma; Piperazines; Tumor Suppressor Protein p53

The human Mediator complex regulates RNA polymerase II transcription genome-wide. A general factor that regulates Mediator function is the four-subunit kinase module, which contains either cyclin-dependent kinase 8 (CDK8) or CDK19. Whereas CDK8 is linked to specific signaling cascades and oncogenesis, the cellular roles of its paralog, CDK19, are poorly studied. We discovered that osteosarcoma cells (SJSA) are naturally depleted of CDK8 protein. Whereas stable CDK19 knockdown was tolerated in SJSA cells, proliferation was reduced. Notably, proliferation defects were rescued upon the reexpression of wild-type or kinase-dead CDK19. Comparative RNA sequencing analyses showed reduced expression of mitotic genes and activation of genes associated with cholesterol metabolism and the p53 pathway in CDK19 knockdown cells. SJSA cells treated with 5-fluorouracil, which induces metabolic and genotoxic stress and activates p53, further implicated CDK19 in p53 target gene expression. To better probe the p53 response, SJSA cells (shCDK19 versus shCTRL) were treated with the p53 activator nutlin-3. Remarkably, CDK19 was required for SJSA cells to return to a proliferative state after nutlin-3 treatment, and this effect was kinase independent. These results implicate CDK19 as a regulator of p53 stress responses and suggest a role for CDK19 in cellular resistance to nutlin-3.

Topics: Bone Neoplasms; Cell Proliferation; Cholesterol; Cyclin-Dependent Kinase 8; Cyclin-Dependent Kinases; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mitosis; Osteosarcoma; Piperazines; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The small-molecule inhibitors of p53-murine double minute 2 interaction, such as Nutlin-3, are effective against cancers bearing wild-type p53. However, murine double minute 2 inhibitors often are unable to completely eliminate solid tumor cells. To address this issue, we investigated the anticancer effects of Nutlin-3 in combination with Oridonin in osteosarcoma cells. We found that Oridonin at sub-toxic concentrations synergistically enhanced Nutlin-3-mediated cell viability inhibition in wild-type p53 U2OS and SJSA-1, but not in p53-mutant MNNG/HOS and in null-p53 Saos-2 osteosarcoma cell lines. Importantly, in the presence of Oridonin, Nutlin-3 could completely abolish cell viability in the wild-type p53 osteosarcoma cell lines. Western blotting analysis showed that Oridonin treatment rapidly and distinctly increased the levels of all three forms of Bim and also markedly reduced the levels of Bcl-2 and Bcl-xl in osteosarcoma cells. Western blotting analysis further showed that Oridonin considerably enhanced Nutlin-3-triggered activation of caspases-9 and -3 and poly(ADP-ribose) polymerase cleavage. Flow cytometry assay showed that Oridonin significantly enhanced Nutlin-3-mediated apoptosis in wild-type p53 osteosarcoma cells. Overall, our results suggest that the combined treatment of Nutlin-3 plus Oridonin may offer a novel therapeutic strategy for osteosarcoma.

Topics: Apoptosis; Bcl-2-Like Protein 11; bcl-X Protein; Cell Cycle; Cell Line, Tumor; Cell Survival; Diterpenes, Kaurane; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Nutlin-3a is a small molecule MDM2 antagonist and potent activator of wild-type p53. Nutlin-3a disrupts MDM2 binding to p53, thus increasing p53 levels and allowing p53 to inhibit proliferation or induce cell death. Factors that control sensitivity to Nutlin-3a-induced apoptosis are incompletely understood. In this study we isolated cisplatin-resistant clones from MHM cells, an MDM2-amplified and p53 wild-type osteosarcoma cell line. Cisplatin resistance in these clones resulted in part from heightened activation of the IGF-1R/AKT pathway. Interestingly, these cisplatin resistant clones showed hyper-sensitivity to Nutlin-3a induced apoptosis. Increased Nutlin-3a sensitivity was associated with reduced authophagy flux and a greater increase in p53 levels in response to Nutlin-3a treatment. IGF-1R and AKT inhibitors further increased apoptosis by Nutlin-3a in parental MHM cells and the cisplatin-resistant clones, confirming IGF-1R/AKT signaling promotes apoptosis resistance. However, IGF-1R and AKT inhibitors also reduced p53 accumulation in Nutlin-3a treated cells and increased autophagy flux, which we showed can promote apoptosis resistance. We conclude the IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis. First, it can inhibit apoptosis, consistent with its well-established role as a survival-signaling pathway. Second, it can enhance Nutlin-3a induced apoptosis through a combination of maintaining p53 levels and inhibiting pro-survival autophagy.

Topics: Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Drug Synergism; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Osteosarcoma; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazines; Receptor, IGF Type 1; Receptors, Somatomedin

The majority of Osteosarcoma (OS) patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. These protocols include distraction osteogenesis (DO), which is characterized by direct new bone formation. Cisplatin (CDP) is extensively used for OS chemotherapy and recent studies, using a mouse DO model, have demonstrated that CDP has profound negative effects on bone repair. Recent oncological therapeutic strategies are based on the use of standard cytotoxic drugs plus an assortment of biologic agents. Here we demonstrate that the previously reported CDP-associated inhibition of bone repair can be modulated by the administration of a small molecule p53 inducer (nutlin-3). The effects of nutlin-3 on CDP osteotoxicity were studied using both pre- and post-operative treatment models. In both cases the addition of nutlin-3, bracketing CDP exposure, demonstrated robust and significant bone sparing activity (p < 0.01-0.001). In addition the combination of nutlin-3 and CDP induced equivalent OS tumor killing in a xenograft model. Collectively, these results demonstrate that the induction of p53 peri-operatively protects bone healing from the toxic effects of CDP, while maintaining OS toxicity. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1716-1724, 2016.

Topics: Animals; Antineoplastic Agents; Bone Regeneration; Cisplatin; Female; Humans; Imidazoles; Male; Mice, Inbred C57BL; Mice, Nude; Osteogenesis, Distraction; Osteosarcoma; Piperazines; Random Allocation; Xenograft Model Antitumor Assays

Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.

Topics: Apoptosis; Cell Line, Tumor; Enzyme Activation; ets-Domain Protein Elk-1; Flow Cytometry; Humans; Imidazoles; Immunoblotting; MAP Kinase Signaling System; Minor Histocompatibility Antigens; Mitochondria; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Suppressor Protein p53

The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.

Topics: Antineoplastic Agents; Binding Sites; Consensus Sequence; Dimethyl Sulfoxide; DNA, Neoplasm; Doxorubicin; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genes, p53; HCT116 Cells; Histones; Humans; Imidazoles; Methylation; Nucleotide Motifs; Osteosarcoma; Piperazines; Promoter Regions, Genetic; Protein Binding; Transcriptional Activation; Tumor Suppressor Protein p53

Nutlin-3 is a small-molecule antagonist of murine double minute 2 (MDM2) that blocks its binding to p53, leading to an increase in p53 protein levels. The tumor suppressor p53 induces growth arrest or apoptosis in response to genotoxic stress. Along with its growth-suppressive effect, it has been reported that p53 stimulates the mitogen-activated protein kinase (MAPK) pathway via the upregulation of heparin- binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFR) ligand, and discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, at the transcription level. In this study, we propose a novel mechanism involved in the p53-induced MAPK activation. Nutlin-3 induced the phosphorylation of EGFR, MAPK/ERK kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 in U2OS human osteosarcoma cells harboring wild-type p53. This phosphorylation was completely inhibited by p53 siRNA, but not by pifithrin (PFT)-α, an inhibitor of the trans-criptional activity of p53. While the nutlin-3-induced EGFR phosphorylation was prevented by the inactivation of ERK1/2, the nutlin-3-induced MEK1/2-ERK1/2 phosphorylation was still observed in the cells in which EGFR phosphorylation was inhibited using EGFR siRNA and AG1478, an inhibitor of EGFR tyrosine kinase. Of note, nutlin-3 caused the accumulation of mitochondrial reactive oxygen species (ROS) and this correlated with the mitochondrial translocation of p53. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), a ROS scavenger, prevented the phosphorylation of ERK1/2. PFT-μ, which prevented the mitochondrial localization of p53, suppressed ERK1/2 phosphorylation, as well as ROS accumulation. Finally, we analyzed the effect of ERK1/2 activation on nutlin-3-induced apoptosis. The knockdown of MEK1/2 and ERK1/2 activity using U0126, a MEK inhibitor, or siRNAs, resulted in the enhancement of nutlin-3-induced apoptosis. In addition, TEMPO and PFT-μ also promoted nutlin-3-induced apoptosis. Taking the above findings into account, it can be concluded that nutlin-3 activates ERK1/2 prior to EGFR phosphorylation via ROS generation following the mitochondrial translocation of p53 and that nutlin-3-induced ERK1/2 activation may play a role in protecting U2OS cells from p53-dependent apoptosis, constituting a negative feedback loop for p53-induced apoptosis.

Topics: Antioxidants; Apoptosis; Benzothiazoles; Butadienes; Cell Line, Tumor; Cyclic N-Oxides; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; Imidazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Osteosarcoma; Phosphorylation; Piperazines; Protein Transport; Quinazolines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Toluene; Tumor Suppressor Protein p53; Tyrphostins

Topics: Apoptosis; Biomarkers, Tumor; Bone Neoplasms; Cell Cycle Proteins; Gene Amplification; Gene Dosage; Humans; Imidazoles; Mutation; Nuclear Proteins; Osteosarcoma; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Retrospective Studies; Tumor Suppressor Protein p53

Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy. Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. In this study, we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro. Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53, resulting in a pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo. p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA. These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Mice, SCID; Neoplasm Transplantation; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Tumor Suppressor Protein p53

Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC(50) did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually "inactive" in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Biological Transport; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Fluorescent Dyes; Humans; Imidazoles; Mitoxantrone; Neoplasm Proteins; Osteosarcoma; Piperazines

Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (for example, ionizing and ultraviolet radiation or chemotherapeutic drug treatment) can activate p53, but also induce mutations in the P53 gene, and thus select for p53-mutated cells. Nutlin-3a (Nutlin) is pre-clinical drug that activates p53 in a non-genotoxic manner. Nutlin occupies the p53-binding pocket of murine double minute 2 (MDM2), activating p53 by blocking the p53-MDM2 interaction. Because Nutlin neither binds p53 directly nor introduces DNA damage, we hypothesized Nutlin would not induce P53 mutations, and, therefore, not select for p53-mutated cells. To test this, populations of SJSA-1 (p53 wild-type) cancer cells were expanded that survived repeated Nutlin exposures, and individual clones were isolated. Group 1 clones were resistant to Nutlin-induced apoptosis, but still underwent growth arrest. Surprisingly, while some Group 1 clones retained wild-type p53, others acquired a heterozygous p53 mutation. Apoptosis resistance in Group 1 clones was associated with decreased PUMA induction and decreased caspase 3/7 activation. Group 2 clones were resistant to both apoptosis and growth arrest induced by Nutlin. Group 2 clones had acquired mutations in the p53-DNA-binding domain and expressed only mutant p53s that were induced by Nutlin treatment, but were unable to bind the P21 and PUMA gene promoters, and unable to activate transcription. These results demonstrate that non-genotoxic p53 activation (for example, by Nutlin treatment) can lead to the acquisition of somatic mutations in p53 and select for p53-mutated cells. These findings have implications for the potential clinical use of Nutlin and other small molecule MDM2 antagonists.

Topics: Apoptosis; Bone Neoplasms; Cell Line, Tumor; Clone Cells; Humans; Imidazoles; Mutation; Osteosarcoma; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Promyelocytic leukemia nuclear bodies (PML-NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML-NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53-dependent growth inhibition. ALT-associated PML bodies (APBs) are specialized PML-NBs that include telomere-repeat binding-factor TRF1 and are exclusively in telomerase-negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell-cycle and p53 responses in ALT-positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin-3a. Both IR and Nutlin-3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin-3a. p53 was recruited to PML-NBs 3-4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML-NBs marked sites of apparently unrepaired DNA double-strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin-3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin-3a-treated cells. These data indicate (1) p53 is recruited to PML-NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53-p21 pathway activation increases the percentage of APB-positive cells, (3) p21 and p53 are recruited to ALT-associated PML-NBs after Nutlin-3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance.

Topics: Cell Line, Tumor; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; DNA Repair; Humans; Imidazoles; Multiprotein Complexes; Nuclear Proteins; Osteosarcoma; Piperazines; Promyelocytic Leukemia Protein; Protein Transport; Proto-Oncogene Proteins c-mdm2; Telomere; Transcription Factors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

We report herein the design of potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. Compound 5 binds to MDM2 with a K(i) of 0.6 nM, activates p53 at concentrations as low as 40 nM, and potently and selectively inhibits cell growth in tumor cells with wild-type p53 over tumor cells with mutated/deleted p53. Compound 5 has a good oral bioavailability and effectively inhibits tumor growth in the SJSA-1 xenograft model.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Biological Availability; Bone Neoplasms; Cell Line, Tumor; Drug Design; HCT116 Cells; Humans; Indoles; Mice; Morpholines; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Rats; Spiro Compounds; Structure-Activity Relationship; Tumor Suppressor Protein p53

microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.

Topics: Bone Neoplasms; Cell Adhesion; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Genes, p53; HCT116 Cells; HT29 Cells; Humans; Imidazoles; MicroRNAs; Neoplasms; Oligonucleotide Array Sequence Analysis; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Transfection; Tumor Suppressor Protein p53; Up-Regulation

Nutlin-3, a small molecule inhibitor, activates p53 by disrupting p53-HDM2 association. In this study, we found that Nutlin-3 suppressed cell growth and induced apoptosis in the absence of wild-type p53, suggesting a p53-independent mechanism for Nutlin-3-induced cell death. Like p53, its homolog p73 transactivates proapoptotic genes and induces cell death. Since HDM2, a key negative regulator of p53, also binds to and inhibits p73, we asked whether p73 could mediate Nutlin-3-induced apoptosis. We demonstrate that Nutlin-3 inhibits endogenous binding between the proapoptotic p73 isoform TAp73alpha and HDM2 in p53-null cells. Dissociation of p73 and HDM2 leads to increased p73 transcriptional activity with upregulation of p73 target genes noxa, puma and p21, as well as enhanced apoptosis. p73 knockdown by siRNA results in rescue of Nutlin-3-treated cells, indicating that Nutlin-3-induced apoptosis is, at least in part, p73 dependent. In addition, Nutlin-3 treatment increases TAp73alpha protein levels with prolongation of p73 half-life. These results provide the first evidence that Nutlin-3 disrupts endogenous p73-HDM2 interaction and enhances the stability and proapoptotic activities of p73 and thus, provides a rationale for the use of Nutlin-3 in the large number of human tumors in which p53 is inactivated.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bone Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; HCT116 Cells; Humans; Imidazoles; Immunoblotting; Immunoprecipitation; Nuclear Proteins; Osteosarcoma; Piperazines; Protein Binding; Protein Biosynthesis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Transcription, Genetic; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time.. The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied.. Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1beta to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA.. Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells.. These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.

Topics: Cell Adhesion; Cell Differentiation; Cell Lineage; Cell Proliferation; Cells, Cultured; Coculture Techniques; Humans; Imidazoles; Macrophage Colony-Stimulating Factor; Osteoclasts; Osteosarcoma; Piperazines; Proto-Oncogene Proteins c-mdm2; RANK Ligand; Time Factors; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation