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[This corrects the article DOI: 10.18632/oncotarget.7685.].
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MELK is upregulated in various types of human cancer and is known to be associated with cancer progression, maintenance of stemness, and poor prognosis. OTS167, a MELK kinase inhibitor, shows potent growth-suppressive effect on human tumors in a xenograft model, but the detailed mode of action has not been fully elucidated. In this study, we demonstrate the molecular mechanism of action of MELK inhibitor OTS167 in a preclinical model. OTS167-treated cells caused morphological transformation, induced the differentiation markers, and reduced stem-cell marker expression. Furthermore, we identified DEPDC1, known as an oncogene, as an additional downstream molecule of the MELK signaling pathway. MELK enhanced DEPDC1 phosphorylation and its stability. The expression of MELK and downstream molecules was decreased in OTS167-treated xenograft tumor tissues, which revealed central necrosis and significant growth suppression. Our data should further shed light on the mechanism of action how OTS167 suppresses tumor growth through the inhibition of the MELK signaling pathway and suggest the possibility of biomarkers for the assessment of clinical efficacy.
Assuntos
Antineoplásicos/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Naftiridinas/farmacologia , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Células A549 , Animais , Biomarcadores Tumorais , Células COS , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/patologia , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC50 of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.
Assuntos
Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Terapia de Alvo Molecular , Naftiridinas/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Injeções Intravenosas , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas dos Microfilamentos/metabolismo , Estrutura Molecular , Células NIH 3T3 , Naftiridinas/química , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Domínios de Homologia de srcRESUMO
A high expression of short and immature O-glycans is one of the prominent features of breast cancer cells, which would be attributed to the upregulated expression of glycosyltransferases. Therefore, a detailed elucidation of glycosyltransferases and their substrate(s) may improve our understandings for their roles in mammary carcinogenesis. Here we report that overexpression of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), a glycosyltransferase involved in the initial step of O-glycosylation, has transformational potentials through disruptive acinar morphogenesis and cellular changes similar to epithelial-to-mesenchymal transition in normal mammary epithelial cell, MCF10A. As one of the critical O-glycan substrates, we identified fibronectin that was O-glycosylated in vivo and thereby stabilized by GALNT6. Because knockdown of fibronectin abrogated the disruptive proliferation caused by introduction of GALNT6 into epithelial cells, our findings suggest that GALNT6-fibronectin pathway should be a critical component for breast cancer development and progression.
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Fibronectinas/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Morfogênese/genética , N-Acetilgalactosaminiltransferases/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Glicosilação , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Glândulas Mamárias Humanas/anatomia & histologia , Glândulas Mamárias Humanas/patologia , Modelos Biológicos , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Estabilidade Proteica , Transfecção , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D-amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme gamma-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type gamma-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a k(cat) value of 0.0485 s(-1). Its activity has been enhanced by site directed and random mutagenesis. The k(cat)/K(m) value was increased to 3.41 s(-1) mM(-1) for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s(-1) for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype gamma-glutamyltranspeptidase of B. subtilis.
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Amidoidrolases/química , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Cefalosporinas/metabolismo , Microbiologia Industrial/métodos , Mutagênese Sítio-Dirigida/métodos , gama-Glutamiltransferase/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefalosporinas/química , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Redes e Vias Metabólicas , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismoRESUMO
The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms of such an aberrant modification have been largely unknown. We here report critical roles of a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells. Western blot and immunostaining analyses indicated that wild-type GALNT6 protein could glycosylate and stabilize an oncoprotein mucin 1 (MUC1), which was upregulated with GALNT6 in breast cancer specimens. Furthermore, knockdown of GALNT6 or MUC1 led to similar morphologic changes of cancer cells accompanied by the increase of cell adhesion molecules beta-catenin and E-cadherin. Our findings implied that overexpression of GALNT6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1 and that screening of GALNT6 inhibitors would be valuable for the development of novel therapeutic modalities against breast cancer.
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Neoplasias da Mama/metabolismo , Mucina-1/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Glicosilação , Células HeLa , Humanos , N-Acetilgalactosaminiltransferases/genética , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
Breast cancer is generated through a multistep genetic and epigenetic process including activations of oncogenes and inactivations of tumor suppressor genes. Here, we report a critical role of ubiquitin-conjugating enzyme E2T (UBE2T), an E2 ubiquitin-conjugating enzyme, in mammary carcinogenesis. Immunocytochemical staining and in vitro binding assay revealed that UBE2T interacted and colocalized with the BRCA1/BRCA1-associated RING domain protein (BARD1) complex. Knocking down of UBE2T expression with small interfering RNA drastically suppressed the growth of breast cancer cells. Interestingly, in vivo ubiquitination assay indicated BRCA1 to be polyubiquitinated by incubation with wild-type UBE2T protein, but not with C86A-UBE2T protein, an E2 activity-dead mutant, in which the 86th residue of cysteine was replaced with alanine. Furthermore, knocking down of UBE2T protein induced upregulation of BRCA1 protein in breast cancer cells, whereas its overexpression caused the decrease of the BRCA1 protein. Our data imply a critical role of UBE2T in development and/or progression of breast cancer through the interaction with and the regulation of the BRCA1/BARD1 complex.
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Neoplasias da Mama/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Northern Blotting , Western Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Through analysis of the detailed genome-wide gene expression profiles of 81 breast tumors, we identified a novel gene, G-patch domain containing 2 (GPATCH2), that was overexpressed in the great majority of breast cancer cases. Treatment of breast cancer cells MCF-7 and T47D with siRNA against GPATCH2 effectively suppressed its expression, and resulted in the growth suppression of cancer cells, suggesting its essential role in breast cancer cell growth. We found an interaction of GPATCH2 protein with hPrp43, an RNA-dependent ATPase. Their interaction could significantly enhance the ATPase activity of hPrp43 and induce a growth-promoting effect on mammalian cells. Because northern blot analyses of normal human organs implied GPATCH2 to be a novel cancer/testis antigen, targeting GPATCH2 or inhibition of the interaction between GPATCH2 and hPrp43 could be a promising novel therapeutic strategy of breast cancer.
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Adenosina Trifosfatases/metabolismo , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Helicases/metabolismo , Adenosina Trifosfatases/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Escherichia coli/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Imuno-Histoquímica , Imunoprecipitação , Rim/citologia , Masculino , Espectrometria de Massas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , RNA Helicases/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Testículo/metabolismo , TransfecçãoRESUMO
7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.
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Cefalosporinas/metabolismo , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteínas de Escherichia coli/isolamento & purificação , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Alinhamento de Sequência , gama-Glutamiltransferase/isolamento & purificaçãoRESUMO
The regulatory particle non-ATPase subunit, Nas6p, from Saccharomyces cerevisiae has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 41.43 (2), b = 61.74 (1), c = 98.09 (2) A, and contain one molecule in the asymmetric unit. A complete diffraction data set using synchrotron radiation was collected to 2.6 A resolution.