RESUMO
JNJ-26854165 (serdemetan) has previously been reported to inhibit the function of the E3 ligase human double minute 2, and we initially sought to characterize its activity in models of mantle cell lymphoma (MCL) and multiple myeloma (MM). Serdemetan induced a dose-dependent inhibition of proliferation in both wild-type (wt) and mutant (mut) p53 cell lines, with IC50 values from 0.25 to 3 µM/l, in association with an S phase cell cycle arrest. Caspase-3 activation was primarily seen in wtp53-bearing cells but also occurred in mutp53-bearing cells, albeit to a lesser extent. 293T cells treated with JNJ-26854165 and serdemetan-resistant fibroblasts displayed accumulation of cholesterol within endosomes, a phenotype reminiscent of that seen in the ATP-binding cassette subfamily A member-1 (ABCA1) cholesterol transport disorder, Tangiers disease. MM and MCL cells had decreased cholesterol efflux and electron microscopy demonstrated the accumulation of lipid whorls, confirming the lysosomal storage disease phenotype. JNJ-26854165 induced induction of cholesterol regulatory genes, sterol regulatory element-binding transcription factor-1 and -2, liver X receptors α and ß, along with increased expression of Niemann-Pick disease type-C1 and -C2. However, JNJ-26854165 induced enhanced ABCA1 turnover despite enhancing transcription. Finally, ABCA1 depletion resulted in enhanced sensitivity to JNJ-26854165. Overall, these findings support the hypothesis that serdemetan functions in part by inhibiting cholesterol transport and that this pathway is a potential new target for the treatment of MCL and MM.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Morte Celular/efeitos dos fármacos , Colesterol/metabolismo , Radiossensibilizantes/farmacologia , Triptaminas/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Inativação Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacosRESUMO
JNJ-26854165 was originally developed as an activator of p53 capable of inducing apoptosis in cancer cell lines. In vitro, JNJ-26854165 demonstrated cytotoxic activity. The ALL cell line panel had a significantly lower median IC(50) (0.85 µM) than the remaining cell lines. In vivo JNJ-26854165 induced significant differences in EFS distribution compared to control in 18 of 37 solid tumors and in 5 of 7 of the evaluable ALL xenografts. Objective responses were observed in 4 of 37 solid tumor xenografts, and 2 of 7 ALL xenografts achieved PR or CR. Responses were noted in xenografts with both mutant and wild-type p53.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Radiossensibilizantes/farmacologia , Sarcoma/tratamento farmacológico , Triptaminas/farmacologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Sarcoma/metabolismo , Sarcoma/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.
Assuntos
Complexo Repressor Polycomb 1 , Neoplasias da Próstata , Núcleo Celular/metabolismo , Cromatina , Histonas/metabolismo , Humanos , Masculino , Complexo Repressor Polycomb 1/genética , Neoplasias da Próstata/metabolismoRESUMO
Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Linhagem Celular , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes , Humanos , Camundongos , Mutação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA , Ratos , Proteínas Quinases Associadas a Fase S/genética , Proteínas Ligases SKP Culina F-Box/genética , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Stapled-peptides have emerged as an exciting class of molecules which can modulate protein-protein interactions. We have used a structure-guided approach to rationally develop a set of hydrocarbon stapled-peptides with high binding affinities and residence times against the oncogenic eukaryotic translation initiation factor 4E (eIF4E) protein. Crystal structures of these peptides in complex with eIF4E show that they form specific interactions with a region on the protein-binding interface of eIF4E which is distinct from the other well-established canonical interactions. This recognition element is a major molecular determinant underlying the improved binding kinetics of these peptides with eIF4E. The interactions were further exploited by designing features in the peptides to attenuate disorder and increase helicity which collectively resulted in the generation of a distinct class of hydrocarbon stapled-peptides targeting eIF4E. This study details new insights into the molecular basis of stapled-peptide: eIF4E interactions and their exploitation to enhance promising lead molecules for the development of stapled-peptide compounds for oncology.
RESUMO
Aberrant androgen signaling drives prostate cancer and is targeted by drugs that diminish androgen production or impede androgen-androgen receptor (AR) interaction. Clinical resistance arises from AR overexpression or ligand-independent constitutive activation, suggesting that complete AR elimination could be a novel therapeutic strategy in prostate cancers. IRC117539 is a new molecule that targets AR for proteasomal degradation. Exposure to IRC117539 promotes AR sumoylation and ubiquitination, reminiscent of therapy-induced PML/RARA degradation in acute promyelocytic leukemia. Critically, ex vivo, IRC117539-mediated AR degradation induces prostate cancer cell viability loss by inhibiting AR signaling, even in androgen-insensitive cells. This approach may be beneficial for castration-resistant prostate cancer, which remains a clinical issue. In xenograft models, IRC117539 is as potent as enzalutamide in impeding growth, albeit less efficient than expected from ex vivo studies. Unexpectedly, IRC117539 also behaves as a weak proteasome inhibitor, likely explaining its suboptimal efficacy in vivo. Our studies highlight the feasibility of AR targeting for degradation and off-target effects' importance in modulating drug activity in vivo.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/metabolismo , Antagonistas de Receptores de Andrógenos/metabolismo , Androgênios/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28/metabolismo , Interfase/fisiologia , Animais , Quinases relacionadas a CDC2 e CDC28/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Fase G1/fisiologia , Camundongos , Camundongos Knockout , Mitose/fisiologia , Fase S/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The ubiquitin pathway plays a central role in the regulation of cell growth and cell proliferation by controlling the abundance of key cell cycle proteins. Increasing evidence indicates that unscheduled proteolysis of many cell cycle regulators contributes significantly to tumorigenesis and is indeed found in many types of human cancers. Aberrant proteolysis with oncogenic potential is elicited by two major mechanisms: defective degradation of positive cell cycle regulators (i.e., proto-oncoproteins) and enhanced degradation of negative cell cycle regulators (i.e., tumor suppressor proteins). In many cases, increased protein stability is a result of mutations in the substrate that prevent the recognition of the protein by the ubiquitin-mediated degradation machinery. Alternatively, the specific recognition proteins mediating ubiquitination (ubiquitin ligases) are not expressed or harbor mutations rendering them inactive. In contrast, the overexpression of a ubiquitin ligase may result in the enhanced degradation of a negative cell cycle regulator. This chapter aims to review the involvement of the ubiquitin pathway in the scheduled destruction of some important cell cycle regulators and to discuss the implications of their aberrant degradation for the development of cancer.
Assuntos
Proteínas Musculares , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquitina/metabolismo , Animais , Ciclo Celular , Ciclina D , Ciclina E/fisiologia , Ciclinas/fisiologia , Humanos , Proteínas dos Microfilamentos/fisiologia , Modelos Biológicos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.
Assuntos
Azepinas/administração & dosagem , Benzamidas/administração & dosagem , Neoplasias da Mama/genética , Dano ao DNA/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Proteínas Serina-Treonina Quinases/biossíntese , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genéticaRESUMO
E2F transcription factors regulate gene expression in concert with the retinoblastoma tumor suppressor family. These transcriptional complexes are master regulators of cell cycle progression and, in addition, control the expression of genes involved in DNA repair, G 2/M checkpoint and differentiation. E2F3 has recently attracted particular attention, because it is amplified in various human tumors. Here we show that E2F3 becomes unstable as cells exit the cell cycle. E2F3 degradation is mediated by the anaphase-promoting complex/cyclosome and its activator Cdh1 (APC/C (Cdh1) ). E2F3 interacts with Cdh1 but not Cdc20, the other APC/C activator. Enforced expression of Cdh1 results in proteasome-dependent degradation of E2F3, whereas the overexpression of Cdc20 has no effect on E2F3 turnover. Finally, silencing of Cdh1 by RNA interference stabilizes E2F3 in differentiating neuroblastoma cells. These findings indicate that the APC/C (Cdh1) ubiquitin ligase targets E2F3 for proteasome-dependent degradation during cell cycle exit and neuronal differentiation.
Assuntos
Caderinas/metabolismo , Fator de Transcrição E2F3/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Antígenos CD , Caderinas/antagonistas & inibidores , Caderinas/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Neurogênese , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Serdemetan (JNJ-26854165) is a novel tryptamine compound with antiproliferative activity in various p53 wild-type (WT) tumor cell lines. We investigated its potential as radiosensitizer using four human cancer cell lines: H460, A549, p53-WT-HCT116, and p53-null-HCT116. Serdemetan inhibited clonogenic survival in all cell lines, but in a lower extent in p53-null-HCT116. In the combination studies, Serdemetan treatment at 0.25µM in H460 and at 5µM in A549 cells resulted in a sensitivity-enhancement ratio of 1.18 and 1.36, respectively. At 2Gy, surviving fractions were 0.72 and 0.97 for p53-WT HCT116 and p53-null cells exposed to 0.5µM of Serdemetan, respectively (p<0.05). Radiosensitization of H460 and A549 cells was associated with G2/M cell cycle arrest and with an increased expression of p53 and p21. In vivo, Serdemetan caused a greater than additive increase in tumor growth delay. The dose enhancement factor was 1.9 and 1.6 for H460 and A549 tumors, respectively. Serdemetan inhibited proliferation, capillary tube formation and migration of HMEC-1 cells. These effects were more marked concurrently with irradiation. These results in tumor and endothelial cells suggest that Serdemetan has potential as a radiosensitizer. Further investigations are warranted with regard to the molecular mechanisms underlying its actions and its dependency regarding p53 status.
Assuntos
Radiossensibilizantes/farmacologia , Triptaminas/farmacologia , Western Blotting , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Técnicas In Vitro , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G(0) and during the G(1) phase of the cell cycle, Skp2 is degraded via the APC/C(Cdh1) ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G(0)/G(1) state. APC/C(Cdh1) binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCF(Skp2) ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.
Assuntos
Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Serina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Linhagem Celular , Proteínas Culina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fase G1 , Humanos , Mutação , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase de Repouso do Ciclo Celular , Proteínas Quinases Associadas a Fase S/análise , Proteínas Quinases Associadas a Fase S/genética , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
By keeping the levels of Skp2 and Cks1 low during G(1) progression, APC/C(Cdh1) prevents unscheduled degradation of SCF(Skp2) substrates and premature entry into S phase. Thus, APC/C(Cdh1), a ubiquitin ligase involved in mitotic exit and maintenance of G(0)/G(1) phase, directly controls SCF(Skp2), a ubiquitin ligase involved in the regulation of S phase entry.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fase G1/fisiologia , Fase S/fisiologia , Proteínas Quinases Associadas a Fase S/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Humanos , Mitose/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Down-regulation of p27 is frequently observed in various cancers due to an enhancement of its degradation. Skp2 is required for the ubiquitination and consequent degradation of p27 protein. Another protein called Cks1 is also required for p27 ubiquitination in the SCF(Skp2) ubiquitinating machinery. In the present study, we examined Cks1 expression and its correlation with p27 in oral squamous cell carcinoma (OSCC) derived from tongue and gingiva. By immunohistochemical analysis, high expression of Cks1 was present in 62% of OSCCs in comparison with 0% of normal mucosae. In addition, 65% of samples with low p27 expression displayed high Cks1 levels. Finally, Cks1 expression was well correlated with Skp2 expression and poor prognosis. To study the role of Cks1 overexpression in p27 down-regulation, we transfected Cks1 with or without Skp2 into OSCC cells. Cks1 transfection could not induce a p27 down-regulation by itself, but both Cks1 and Skp2 transfection strongly induced. Moreover, we inhibited Cks1 expression by small interference RNA (siRNA) in OSCC. Cks1 siRNA transfection induced p27 accumulation and inhibited the growth of OSCC cells. These findings suggest that Cks1 overexpression may play an important role for OSCC development through Skp2-mediated p27 degradation, and that Cks1 siRNA can be a novel modality of gene therapy.