RESUMO
BACKGROUND: Activating mutations of the KRAS occurs in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, direct pharmacological targeting of the activated KRAS protein has been challenging. We previously reported that KR12, a DNA-alkylating pyrrole-imidazole polyamide designed to recognize the KRAS G12D/V mutation, showed an anti-tumor effect in colorectal cancer. In this study, we evaluated the anti-tumor effect of KR12 in PDAC. METHODS: KR12 was synthesized by an automated peptide synthesizer PSSM-8 and tested for anti-tumor effect in PDAC mouse models. RESULT: KR12 inhibited tumor growth in a spontaneous PDAC mouse model, although the anti-tumor activity appeared to be limited in a human PDAC xenograft model. We developed a pyrrole-imidazole polyamide screening process based on the hypothesis that genetic elements otherwise unaffected by KR12 could exert attenuating effects on KRAS-suppression-resistant PDAC. We identified RAD51 as a potential therapeutic target in human PDAC cells. A RAD51 inhibitor showed an inhibitory effect on cell growth and affected the cytotoxic activity of KR12 in PDAC cells. CONCLUSION: These data suggested that the simultaneous inhibition of RAD51 and mutant KRAS blockage would be an important therapeutic strategy for PDAC.
Assuntos
Antineoplásicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Humanos , Nylons/farmacologia , Nylons/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , DNA/uso terapêutico , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Neoplasias PancreáticasRESUMO
INTRODUCTION: Hepatoblastoma (HB) is the most common paediatric liver tumour, and epigenetic aberrations may be important in HB development. Recently, the Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) developed risk stratification based on clinicopathological factors. This study aimed to construct a more accurate model by integrating CHIC-HS with molecular factors based on DNA methylation. METHODS: HB tumour specimens (N = 132) from patients treated with the Japanese Pediatric Liver Tumors Group-2 protocol were collected and subjected to methylation analysis by bisulfite pyrosequencing. Associations between methylation status and clinicopathological factors, overall survival (OS), and event-free survival (EFS) were retrospectively analysed. We investigated the effectiveness of the evaluation of methylation status in each CHIC-HS risk group and generated a new risk stratification model. RESULTS: Most specimens (82%) were from post-chemotherapy tissue. Hypermethylation in ≥2 of the four genes (RASSF1A, PARP6, OCIAD2, and MST1R) was significantly associated with poorer OS and EFS. Multivariate analysis indicated that ≥2 methylated genes was an independent prognostic factor (hazard ratios of 6.014 and 3.684 for OS and EFS, respectively). Two or more methylated genes was also associated with poorer OS in the CHIC-very low (VL)-/low (L)-risk and CHIC-intermediate (I) risk groups (3-year OS rates were 83% vs. 98% and 50% vs. 95%, respectively). The 3-year OS rates of the VL/L, I, and high-risk groups in the new stratification model were 98%, 90%, and 62% (vs. CHIC-HS [96%, 82%, and 65%, respectively]), optimising CHIC-HS. CONCLUSIONS: Our proposed stratification system considers individual risk in HB and may improve patient clinical management.
Assuntos
Hepatoblastoma , Neoplasias Hepáticas , ADP Ribose Transferases/genética , ADP Ribose Transferases/uso terapêutico , Criança , DNA , Metilação de DNA , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Neoplasias/genética , Estudos Retrospectivos , Medição de RiscoRESUMO
Anaplastic lymphoma kinase (ALK) aberration is related to high-risk neuroblastomas and is an important therapeutic target. As acquired resistance to ALK tyrosine kinase inhibitors is inevitable, novel anti-ALK drug development is necessary in order to overcome potential drug resistance against ATP-competitive kinase inhibitors. In this study, to overcome ALK inhibitor resistance, we examined the growth inhibition effects of newly developed ALK-targeting pyrrole-imidazole polyamide CCC-003, which was designed to directly bind and alkylate DNA within the F1174L-mutated ALK gene. CCC-003 suppressed cell proliferation in ALK-mutated neuroblastoma cells. The expression of total and phosphorylated ALK was downregulated by CCC-003 treatment but not by treatment with a mismatch polyamide without any binding motif within the ALK gene region. CCC-003 preferentially bound to the DNA sequence with the F1174L mutation and significantly suppressed tumor progression in a human neuroblastoma xenograft mouse model. Our data suggest that the specific binding of CCC-003 to mutated DNA within the ALK gene exerts its anti-tumor activity through a mode of action that is distinct from those of other ALK inhibitors. In summary, our current study provides evidence for the potential of pyrrole-imidazole polyamide ALK inhibitor CCC-003 for the treatment of neuroblastoma thus offering a possible solution to the problem of tyrosine kinase inhibitor resistance.
Assuntos
Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Antineoplásicos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/administração & dosagem , Neuroblastoma/tratamento farmacológico , Pirróis/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Nylons/síntese química , Nylons/química , Fosforilação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Receptor tyrosine kinases (RTKs) receive different modulation before transmitting proliferative signals. We previously identified neuronal leucine-rich repeat 1 (NLRR1) as a positive regulator of EGF and IGF-1 signals in high-risk neuroblastoma cells. Here, we show that NLRR1 is up-regulated in various adult cancers and acts as a key regulator of tumor cell proliferation. In the extracellular domains of NLRR1, fibronectin type III (FNIII) domain is responsible for its function to promote cell proliferation. We generated monoclonal antibodies against the extracellular domains of NLRR1 (N1mAb) and screened the positive N1mAbs for growth inhibitory effect. The treatment of N1mAbs reduces tumor cell proliferation in vitro and in vivo, and sensitizes the cells to EGFR inhibitor, suggesting that NLRR1 is a novel regulatory molecule of RTK function. Importantly, epitope mapping analysis has revealed that N1mAbs with growth inhibitory effect recognize immunoglobulin-like and FNIII domains of NLRR1, which also indicates the importance of FNIII domain in the function of NLRR1. Thus, the present study provides a new insight into the development of a cancer therapy by targeting NLRR1 as a modulator of proliferative signals on cellular membrane of tumor cells.
RESUMO
PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Antineoplásicos Alquilantes/síntese química , Antineoplásicos Alquilantes/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Mutação com Ganho de Função , Humanos , Imidazóis/síntese química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Camundongos , Nylons/síntese química , Nylons/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/síntese química , Pirróis/farmacologia , Pirróis/uso terapêutico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Neuroblastoma/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína GAP-43/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para CimaRESUMO
In the search for new pharmaceutical leads, especially with DNA-binding molecules or genome editing methods, the issue of side and off-target effects have always been thorny in nature. A particular case is the investigation into the off-target effects of N-methylpyrrole-N-methylimidazole polyamides, a naturally inspired class of DNA binders with strong affinity to the minor-groove and sequence specificity, but at < 20 bases, their relatively short motifs also insinuate the possibility of non-unique genomic binding. Binding at non-intended loci potentially lead to the rise of off-target effects, issues that very few approaches are able to address to-date. We here report an analytical method to infer off-target binding, via expression profiling, based on probing the relative impact to various biochemical pathways; we also proposed an accompanying side effect prediction engine for the systematic screening of candidate polyamides. This method marks the first attempt in PI polyamide research to identify elements in biochemical pathways that are sensitive to the treatment of a candidate polyamide as an approach to infer possible off-target effects. Expression changes were then considered to assess possible outward phenotypic changes, manifested as side effects, should the same PI polyamide candidate be administered clinically. We validated some of these effects with a series of animal experiments, and found agreeable corroboration in certain side effects, such as changes in aspartate transaminase levels in ICR and nude mice post-administration.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Nylons/metabolismo , Nylons/farmacologia , Algoritmos , Animais , Sítios de Ligação/genética , Fenômenos Bioquímicos , Linhagem Celular , DNA/genética , DNA/metabolismo , Descoberta de Drogas , Feminino , Edição de Genes/métodos , Perfilação da Expressão Gênica , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Pirróis/metabolismo , Pirróis/farmacologiaRESUMO
Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein inspired us to develop a new gene-level-inhibitory strategy using a sequence-specific gene regulator. Here, we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within the MYCN transcript. Pharmacologic suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN nonamplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, treatment with MYCN-A3, but not MYCN nontargeting PI polyamide, precipitated a copy number reduction of MYCN in neuroblastoma cells with MYCN amplification. These findings suggest that directly targeting MYCN with MYCN-A3 is a novel therapeutic approach to reduce copy number of the MYCN gene for MYCN-amplified neuroblastoma. SIGNIFICANCE: This study presents a novel approach to drugging an amplified oncogene by showing that targeting gene amplification of MYCN suppresses MYCN expression and neuroblastoma growth.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/prevenção & controle , Nylons/farmacologia , Alquilação , Animais , Antineoplásicos Alquilantes/química , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Imidazóis/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/patologia , Nylons/química , Pirróis/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To examine the hydrophobic structure of PI polyamides on tumor accumulation in vivo, PI polyamide-fluorescein conjugates 1-5 with the distinct number of N-methylimidazole (Im) units were synthesized. There existed an inverse relationship between the Im unit number of the compounds and their hydrophobicity. Compound 1 with one Im unit and 3 with three Im units accumulated and retained preferentially in tumor tissues compared to 5 with five Im units. These results suggest the importance of a PI polyamide's primary structure, which partly contributes to its hydrophobic property, on its accumulation and/or retention in tumor tissues in vivo.
Assuntos
Imidazóis/metabolismo , Neoplasias/metabolismo , Nylons/metabolismo , Pirróis/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Feminino , Fluoresceínas/síntese química , Fluoresceínas/química , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/síntese química , Imidazóis/química , Camundongos Endogâmicos BALB C , Estrutura Molecular , Nylons/síntese química , Nylons/química , Pirróis/síntese química , Pirróis/química , Distribuição TecidualRESUMO
Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as seco-CBI. These molecules also provide an alternative for oncogenes deemed "undruggable" at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide's possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.
Assuntos
Antineoplásicos Alquilantes/metabolismo , Sítios de Ligação , Neoplasias Colorretais/genética , Genoma Humano , Imidazóis/metabolismo , Nylons/metabolismo , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Neoplasias Colorretais/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Humanos , Imidazóis/química , Imidazóis/farmacologia , Mutação , Nucleossomos/metabolismo , Motivos de Nucleotídeos , Nylons/química , Nylons/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNARESUMO
In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1-/- mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Proliferação de Células , Espaço Extracelular/metabolismo , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , Ligação ProteicaRESUMO
The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Sistema de Sinalização das MAP Quinases , Neuroblastoma/genética , Neuroblastoma/metabolismo , Ativação Transcricional , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Interferente Pequeno/genética , Estresse Fisiológico/genética , Tretinoína/farmacologiaRESUMO
Brain-derived neurotrophic factor (BDNF) and its high affinity receptor tyrosine kinase receptor B (TrkB) are involved in neuronal survival, maintenance, differentiation and synaptic plasticity. Deficiency of BDNF was reported to be associated with psychological disorders such as depression. Hence we examined proliferative effect of 11 candidate TrkB agonistic compounds in TrkB-expressing SH-SY5Y cells, via a hypothesis that some candidate compounds identified in our previous in silico screening for a small molecule targeting the BDNF binding domain of TrkB should activate TrkB signaling. In the present study, two promising compounds, 48 and 56, were identified and subsequently assessed for their ability to induce TrkB phosphorylation in vitro and in vivo. Likewise those seen in BDNF, the compounds mediated TrkB phosphorylation was blocked by the Trk inhibitor, K252a. Since BDNF-TrkB signaling deficiency is associated with the pathogenesis of depression and reactivation of this signaling by antidepressants is a cause of the pathogenic state recovery, the compounds were subjected to the assessment for forced swim test, which is a mouse model of depression. We found that compound 48 significantly reduced mouse immobility time compared with the control vehicle injection, suggesting the confirmation of hypothetical antidepressant-like efficacy of 48 compound in vivo. Thus, our present study demonstrated that compound 48, selected through in silico screening, is a novel activator of TrkB signaling and a potential antidepressant molecule.
Assuntos
Antidepressivos/administração & dosagem , Depressão/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Receptor trkB/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Depressão/tratamento farmacológico , Depressão/psicologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Resultado do TratamentoRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor that originates from multipotent neural crest cells. NB cell populations that express embryonic stem cell-associated genes have been identified and shown to retain a multipotent phenotype. However, whether somatic reprogramming of NB cells can produce similar stem-cell like populations is unknown. Here, we sought to reprogram NB cell lines using an integration-free Sendai virus vector system. Of four NB cell lines examined, only SH-IN cells formed induced pluripotent stem cell-like colonies (SH-IN 4F colonies) at approximately 6 weeks following transduction. These SH-IN 4F colonies were alkaline phosphatase-positive. Array comparative genomic hybridization analysis indicated identical genomic aberrations in the SH-IN 4F cells as in the parental cells. SH-IN 4F cells had the ability to differentiate into the three embryonic germ layers in vitro, but rather formed NBs in vivo. Furthermore, SH-IN 4F cells exhibited resistance to cisplatin treatment and differentiated into endothelial-like cells expressing CD31 in the presence of vascular endothelial growth factor. These results suggest that SH-IN 4F cells are partially reprogrammed NB cells, and could be a suitable model for investigating the plasticity of aggressive tumors.
Assuntos
Plasticidade Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Neuroblastoma/genética , Neuroblastoma/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Hibridização Genômica Comparativa , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/citologia , Vetores Genéticos/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Vírus SendaiRESUMO
Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Imidazóis/síntese química , Imidazóis/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Nylons/síntese química , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células CACO-2 , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HT29 , Humanos , Imidazóis/farmacologia , Camundongos Nus , Mutação , Nylons/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Tretinoína/farmacologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neuroblastoma/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteólise , Ensaio Tumoral de Célula-TroncoRESUMO
The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3ß, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3ß, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3ß inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3ß activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.
Assuntos
Elementos Antissenso (Genética)/genética , Quinase 3 da Glicogênio Sintase/genética , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Animais , Linhagem Celular Tumoral , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/etiologia , Neuroblastoma/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genéticaRESUMO
Neuroblastoma (NB) is one of the most frequent solid tumors in children and its prognosis is still poor. The neurotrophin receptor TrkB and its ligand brain-derived neurotrophic factor (BDNF) are expressed at high levels in high-risk NBs and are involved in defining the poor prognosis of the patients. However, the TrkB targeting therapy has never been realized in the clinic. We performed an in silico screening procedure utilizing an AutoDock/grid computing technology in order to identify novel small chemical compounds targeting the BDNF-binding domain of TrkB. For the first screening, a library of three million synthetic compounds was screened in silico and was ranked according to the Docking energy. The top-ranked 37 compounds were further functionally screened for cytotoxicity by using NB cell lines. We have finally identified seven compounds that kill NB cells with the IC50 values of 0.07-4.6 µmol/L. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that these molecules induce apoptosis accompanied by p53 activation in NB cell lines. The candidate compounds and BDNF demonstrated an antagonistic effect on cell growth, invasion, and colony formation, possibly suggesting competition at the BDNF-binding site of TrkB. The candidate compounds had tumor-suppressive activity in xenograft and in vivo toxicity tests (oral and intravenous administrations) using mice, and did not show any abnormal signs. Using in silico Docking screening we have found new candidate TrkB inhibitors against high-risk NBs, which could lead to new anti-cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Neuroblastoma/tratamento farmacológico , Proteínas Quinases/genética , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Glicoproteínas de Membrana , Camundongos , Simulação de Acoplamento Molecular , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Proteínas Tirosina Quinases , Receptor trkB , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas PequenasRESUMO
Human anaplastic lymphoma kinase (ALK) has been identified as an oncogene that is mutated or amplified in NBLs. To obtain a better understanding of the molecular events associated with ALK in the pathogenesis of NBL, it is necessary to clarify how ALK gene contributes to NBL progression. In the present study, we found that ALK expression was significantly high in NBL clinical samples with amplified MYCN (n = 126, P < 0.01) and in developing tumors of MYCN-transgenic mice. Indeed, promoter analysis revealed that ALK is a direct transcriptional target of MYCN. Overexpression and knockdown of ALK demonstrated its function in cell proliferation, migration and invasion. Moreover, treatment with an ALK inhibitor, TAE-684, efficiently suppressed such biological effects in MYCN amplified cells and tumor growth of the xenograft in mice. Our present findings explore the fundamental understanding of ALK in order to develop novel therapeutic tools by targeting ALK for aggressive NBL treatment.