RESUMO
Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the 'fittest hit' to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.
Assuntos
Proliferação de Células , Leucemia Mieloide Aguda , Proteínas Ativadoras de ras GTPase , Humanos , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Simulação por Computador , Antineoplásicos/farmacologia , Domínios Proteicos , Animais , Proteômica/métodosRESUMO
The inactivation of tumor suppressor DOC-2/DAB2 interactive protein (DAB2IP) by epigenetic and post-transcriptional modification has been reported in multiple human malignancies. DNA methyltransferase 3A (DNMT3A) is involved in de novo establishment of DNA methylation and plays a vital role in tumorigenesis. However, whether DNMT3A can regulate colorectal cancer (CRC) progression via modulation of DAB2IP remains unclear. In this study, we revealed that DNMT3A was significantly increased in CRC, predicting a poor overall survival. Functionally, ectopic expression of DNMT3A in CRC cells enhanced cell proliferation, whereas DNMT3A knockdown had the opposite effect by inducing cell cycle arrest. Mechanistically, methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) proved that the expression of DAB2IP was epigenetically suppressed by DNMT3A-mediated promoter methylation in CRC cells. Using dual-luciferase reporter assay and ChIP-PCR assay, we further confirmed that DNMT3A restrained the transcriptional activity of DAB2IP promoter through directly binging to it. In addition, DNMT3A could activate the MEK/ERK signaling pathway via efficiently downregulating DAB2IP. Inhibition of the MEK/ERK cascade abrogated the oncogenic effects of DNMT3A on CRC cells. In conclusion, our study demonstrates that DNMT3A facilitates CRC progression by regulating DAB2IP mediated MEK/ERK activation, providing promising targets for CRC treatment.
Assuntos
Neoplasias Colorretais/patologia , DNA Metiltransferase 3A/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Metilação de DNA , DNA Metiltransferase 3A/antagonistas & inibidores , DNA Metiltransferase 3A/genética , Humanos , Prognóstico , Piridonas/farmacologia , Pirimidinonas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/imunologia , Proteínas Ativadoras de ras GTPase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Técnicas de Cultura de Células em Três Dimensões , Humanos , Imunização , Neoplasias/terapia , Ratos , Transdução de Sinais/genética , Proteínas Ativadoras de ras GTPase/antagonistas & inibidoresRESUMO
The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Vulva/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Receptores ErbB/metabolismo , Feminino , Edição de Genes , Larva/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese , Netrinas/genética , Netrinas/metabolismo , Interferência de RNA , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Vulva/citologia , Vulva/crescimento & desenvolvimento , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Safe and efficient intracellular delivery of CRISPR/Cas9 is a key step for effective therapeutic genome editing in a wide range of diseases. This remains challenging due to multiple drawbacks of the currently available vehicles. Here we report that epithelial cell -derived microvesicles (MVs) function as safe and natural carriers for efficient delivery of CRISPR/Cas9 to treat cancer. In our study, compared to epithelial cell -derived MVs, cancer -derived MVs were quickly absorbed intracellularly by recipient cancer cells in vitro and showed selective accumulation in tumors of HepG2 xenografts in vivo, due to their cancer cell tropism dependent targeting. Surprisingly, synergistic anti-tumor effect of sgIQ 1.1 loaded Cas9MVs/HEK293 + sorafenib was better than sgIQ 1.1 + Cas9MVs/HepG2 + sorafenib in vitro. In addition, qPCR results showed that miR-21 and miR-181a expression were upregulated in HepG2 cells treated with cancer cell -derived MVs that might support the cancer progression. Further, treatment of HepG2 xenografts with sgIQ 1.1 loaded Cas9MVs/HEK293 showed enhanced anti-cancer effect than sgIQ 1.1 + Cas9MVs/HepG2. Therefore, we conclude that normal cells -derived MVs can act as better and safe natural delivery systems for cancer therapeutics in the future.
Assuntos
Sistemas CRISPR-Cas , Carcinoma Hepatocelular/terapia , Micropartículas Derivadas de Células/patologia , Células Epiteliais/patologia , Neoplasias Hepáticas/terapia , Sorafenibe/farmacologia , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células , Micropartículas Derivadas de Células/metabolismo , Terapia Combinada , Células Epiteliais/metabolismo , Feminino , Edição de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
KRAS mutation-induced Ras activation plays an important role in the pathogenesis of pancreatic cancer, but the role of wild-type Ras and Ras GTPase-activating proteins remains unclear. The present study was designed to determine the expression spectra of Ras GTPase-activating proteins genes in pancreatic cancer cells, and the role of DAB2IP, a Ras GTPase-activating proteins gene, in the development and progression of pancreatic cancer. Following the analyses of the expression profiles of 16 Ras GTPase-activating proteins in 6 pancreatic cancer cell lines including Bxpc-3 (with wild-type KRAS), Capan-2, Sw1990, Aspc-1, CFPAC-1, and Panc-1 (with mutant KRAS) and 1 normal human pancreatic ductal epithelial cell line, H6C7, the expression of DAB2IP messenger RNA was further analyzed by quantitative real-time polymerase chain reaction. The role of DAB2IP in pancreatic cancer was further investigated in vitro and in vivo by upregulating DAB2IP in Bxpc-3 cells through transfection of DAB2IP into Bxpc-3 cells with recombinant lentivirus. The DAB2IP expression in pancreatic cancer cells and tissues with wild-type KRAS was significantly lower than that in cells and tissues with mutant KRAS (P < .05). In Bxpc-3 cells with wild-type KRAS, overexpression of DAB2IP decreased the expression of P-AKT and P-ERK and the Ras activity; increased the expression of P-JNK and caspase 3; inhibited cell proliferation, invasiveness, and migration; and increased the cell sensitivity to cetuximab. Overexpression of DAB2IP inhibited tumor progression in a mouse model. In conclusion, DAB2IP downregulates Ras activity in wild-type pancreatic cancer cells. Overexpression of DAB2IP decreases the Ras activity, inhibits cell proliferation, and increases sensitivity to cetuximab in wild-type pancreatic cancer cells. In conclusion, DAB2IP may serve as a potential molecular therapeutic target for the treatment of pancreatic cancer.
Assuntos
Proliferação de Células , Cetuximab/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/prevenção & controle , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Adulto , Idoso , Animais , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.
Assuntos
Actinas/metabolismo , Forminas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Forminas/antagonistas & inibidores , Forminas/genética , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transativadores/antagonistas & inibidores , Transativadores/genética , Transativadores/metabolismo , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Disruption of γ-aminobutyric acid (GABA)-ergic interneuron migration is implicated in various neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. The dopamine D1 receptor (D1R) promotes GABAergic interneuron migration, which is disrupted in various neurological disorders, some of which are also associated with mutations in the gene encoding synaptic Ras-guanosine triphosphatase-activating protein (SynGAP). Here, we explored the mechanisms underlying these associations and their possible connection. In prenatal mouse brain tissue, we found a previously unknown interaction between the D1R and SynGAP. This D1R-SynGAP interaction facilitated D1R localization to the plasma membrane and promoted D1R-mediated downstream signaling pathways, including phosphorylation of protein kinase A and p38 mitogen-activated protein kinase. These effects were blocked by a peptide (TAT-D1Rpep) that disrupted the D1R-SynGAP interaction. Furthermore, disrupting this complex in mice during embryonic development resulted in pronounced and selective deficits in the tangential migration of GABAergic interneurons, possibly due to altered actin and microtubule dynamics. Our results provide insights into the molecular mechanisms regulating interneuron development and suggest that disruption of the D1R-SynGAP interaction may underlie SYNGAP1 mutation-related neurodevelopmental disorders.
Assuntos
Actinas/metabolismo , Movimento Celular , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Microtúbulos/metabolismo , Receptores de Dopamina D1/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Neurônios GABAérgicos/citologia , Células HEK293 , Humanos , Interneurônios/citologia , Camundongos , Peptídeos/farmacologia , Proteínas Ativadoras de ras GTPase/antagonistas & inibidoresRESUMO
PURPOSE: Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. EXPERIMENTAL DESIGN: Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. RESULTS: We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. CONCLUSIONS: In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.
Assuntos
Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias Renais/patologia , Tolerância a Radiação/genética , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Animais , Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Radiação Ionizante , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Certain organs are capable of containing the replication of various types of viruses. In the liver, infection of Hepatitis B virus (HBV), the etiological factor of Hepatitis B and hepatocellular carcinoma (HCC), often remains asymptomatic and leads to a chronic carrier state. Here we investigated how hepatocytes contain HBV replication and promote their own survival by orchestrating a translational defense mechanism via the stress-sensitive SUMO-2/3-specific peptidase SENP3. We found that SENP3 expression level decreased in HBV-infected hepatocytes in various models including HepG2-NTCP cell lines and a humanized mouse model. Downregulation of SENP3 reduced HBV replication and boosted host protein translation. We also discovered that IQGAP2, a Ras GTPase-activating-like protein, is a key substrate for SENP3-mediated de-SUMOylation. Downregulation of SENP3 in HBV infected cells facilitated IQGAP2 SUMOylation and degradation, which leads to suppression of HBV gene expression and restoration of global translation of host genes via modulation of AKT phosphorylation. Thus, The SENP3-IQGAP2 de-SUMOylation axis is a host defense mechanism of hepatocytes that restores host protein translation and suppresses HBV gene expression.
Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Animais , Cisteína Endopeptidases/genética , Regulação para Baixo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Especificidade por Substrato , Sumoilação , Replicação Viral/fisiologia , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) has a poor prognosis due to recurrence and metastasis. IQ-domain GTPase-activating protein 1 (IQGAP1), a scaffold protein, plays an important role in tumorigenesis and malignant development. In this study, we aimed to explore the role of IQGAP1 in LSCC. METHODS: Expression of IQGAP1 in human LSCC specimens was assessed by immunohistochemistry. We also evaluated the roles of IQGAP1 in cell proliferation, migration and invasion and epithelial-to-mesenchymal transition (EMT) in Hep-2 cells. RESULTS: The expression of IQGAP1 protein was significantly up-regulated in LSCC tissues compared with normal laryngeal tissues (p = 0.002). Furthermore, the knockdown of IQGAP1 in Hep-2 cells inhibited cell growth, migration and invasion. Moreover, we found that IQGAP1 silencing reversed EMT. CONCLUSIONS: These results show for the first time that IQGAP1 is up-regulated in LSCC tissues and plays an important role in LSCC cell proliferation and invasiveness, which indicates that IQGAP1 could work as an oncogene and may serve as a promising molecular target for treatment of LSCC.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Laríngeas/genética , Proteínas Ativadoras de ras GTPase/genética , Carcinoma de Células Escamosas/patologia , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Neoplasias Laríngeas/patologia , Masculino , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais/genética , Proteínas Ativadoras de ras GTPase/antagonistas & inibidoresRESUMO
Dysregulation of the MAPK pathway correlates with progression of pancreatic ductal adenocarcinoma (PDAC) progression. IQ motif containing GTPase-activating protein 1 (IQGAP1) is a MAPK scaffold that directly regulates the activation of RAF, MEK, and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in gluconeogenesis, is transcriptionally downregulated in various cancers, including PDAC. Here, we demonstrate that FBP1 acts as a negative modulator of the IQGAP1-MAPK signaling axis in PDAC cells. FBP1 binding to the WW domain of IQGAP1 impeded IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity. Conversely, decreased FBP1 expression induced pERK1/2 levels in PDAC cell lines and correlated with increased pERK1/2 levels in patient specimens. Treatment with gemcitabine caused undesirable activation of ERK1/2 in PDAC cells, but cotreatment with the FBP1-derived small peptide inhibitor FBP1 E4 overcame gemcitabine-induced ERK activation, thereby increasing the anticancer efficacy of gemcitabine in PDAC. These findings identify a primary mechanism of resistance of PDAC to standard therapy and suggest that the FBP1-IQGAP1-ERK1/2 signaling axis can be targeted for effective treatment of PDAC. Cancer Res; 77(16); 4328-41. ©2017 AACR.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Desoxicitidina/análogos & derivados , Frutose-Bifosfatase/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Frutose-Bifosfatase/genética , Humanos , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Transfecção , Proteínas Ativadoras de ras GTPase/metabolismo , GencitabinaRESUMO
Obesity and type 2 diabetes are significant risk factors for malignancies, being associated with chronic inflammation and hyperinsulinemia. In this context, insulin can synergize with inflammation to promote proliferation, survival, and dissemination of cancer cells. Point mutation of p53 is a frequent event and a significant factor in cancer development and progression. Mutant p53 protein(s) (mutp53) can acquire oncogenic properties that increase metastasis, proliferation, and cell survival. We report that breast and prostate cancer cells with mutant p53 respond to insulin stimulation by increasing cell proliferation and invasivity, and that such a response depends on the presence of mutp53. Mechanistically, we find that mutp53 augments insulin-induced AKT1 activation by binding and inhibiting the tumor suppressor DAB2IP (DAB2-interacting protein) in the cytoplasm. This molecular axis reveals a specific gain of function for mutant p53 in the response to insulin stimulation, offering an additional perspective to understand the relationship between hyperinsulinemia and cancer evolution.
Assuntos
Insulina/metabolismo , Mutação , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Supressora de Tumor p53/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Complicações do Diabetes/tratamento farmacológico , Diabetes Mellitus/tratamento farmacológico , Progressão da Doença , Feminino , Humanos , Hiperinsulinismo/metabolismo , Inflamação , Masculino , Camundongos , Proteínas Mutantes/genética , Obesidade/complicações , Obesidade/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Risco , Proteínas Ativadoras de ras GTPase/antagonistas & inibidoresRESUMO
Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.
Assuntos
Melanoma/patologia , Neoplasias Bucais/patologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Técnicas de Inativação de Genes , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Camundongos , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Bucais/metabolismo , Mutação , Oncogenes , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridonas/toxicidade , Pirimidinonas/toxicidade , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
The Keap1-Nrf2 system is involved not only in biological defense but also in malignancy progression and chemoresistance. The ubiquitin-binding protein p62/Sqstm1 (p62), which is highly expressed in several cancers, competes with Nrf2 for Keap1 binding, leading to activation of Nrf2-mediated gene expression and survival of cancer cells. We had previously identified an inhibitor for the Keap1-phosphorylated-p62 (p-p62) protein-protein interaction (PPI), the acetonyl naphthalene derivative K67. In this study, we established facile synthetic routes for K67 and derivatives with various side chains on the C-2 position of naphthalene ring. K67 possessed high selectivity in the inhibition of Keap1-p-p62. Other derivatives showed potent Keap1-Nrf2 and Keap1-p-p62 PPI inhibitory activities, though the selectivity between the two activities was lower than K67.
Assuntos
1-Naftilamina/análogos & derivados , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Naftalenos/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , 1-Naftilamina/síntese química , 1-Naftilamina/química , 1-Naftilamina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Estrutura Molecular , Fator 2 Relacionado a NF-E2/química , Naftalenos/química , Ligação Proteica/efeitos dos fármacos , Proteínas de Ligação a RNA/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Proteínas Ativadoras de ras GTPase/químicaRESUMO
IQ motif-containing GTPase-activating proteins (IQGAPs) belong to a conserved family, and they are involved in various intracellular processes. IQGAP1 is expressed in all cells, while IQGAP2 and IQGAP3 are mainly expressed in hepatic cells. IQGAP1 has been suggested to be an oncogene, while IQGAP2 is considered a tumor-suppressor gene. However, the relationship between RAS family genes and IQGAP genes remains unclear. We recently demonstrated this interaction in a chemically induced mouse liver cancer. In this study, IQGAP1 expression was partially silenced in human hepatocellular carcinoma (HepG2) cells. We investigated the impact of IQGAP1 silencing on the interactions of IQGAP and RAS with several apoptotic proteins, including caspase-3 (CASP3), BCL2-associated X protein (BAX), and B-cell leukemia/lymphoma 2 (BCL2). Additionally, we investigated the effects of the interactions of these genes on cell viability, proliferation, apoptosis, and invasive capacity. IQGAP1 siRNA-treated HepG2 cells showed lower invasive capacity than the control cells, and this reduction was time- and vector concentration-dependent. In addition, IQGAP1 silencing resulted in significantly lower IQGAP1 level and subsequently higher IQGAP2 and IQGAP3 expression in HepG2 cells than in the control. Flow cytometry analyses indicated that the silencing of IQGAP1 can induce early and late apoptosis in HepG2 cells. Additionally, IQGAP2, IQGAP3, CASP3, and BAX were upregulated whereas IQGAP1 and BCL2 were downregulated in the siRNA-treated cells. Furthermore, we observed that the mRNA levels of HRAS, KRAS, NRAS, and MRAS decreased upon IQGAP1 silencing. These findings indicate that IQGAP1 potentially regulates the expression of IQGAP and RAS gene families and demonstrate its regulatory role in the apoptotic network. Taken together, our findings suggest that IQGAP1 silencing plays crucial roles in the apoptosis of HepG2 cells and lowers their proliferative and invasive capacities.
Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Movimento Celular , Neoplasias Hepáticas/patologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Biomarcadores Tumorais/genética , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Citometria de Fluxo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Despite improved outcomes in newly diagnosed multiple myeloma, virtually all patients relapse and ultimately develop drug-resistant disease. Aberrant RAS/MAPK signaling is activated in the majority of relapsed/refractory multiple myeloma patients, but its biological consequences are not fully understood. Self-renewal, as defined by the long-term maintenance of clonogenic growth, is essential for disease relapse, and we examined the role of RAS/MAPK activation on multiple myeloma self-renewal by targeting IQ motif-containing GTPase-activating protein 1 (IQGAP1), an intracellular scaffold protein required for mutant RAS signaling. We found that loss of IQGAP1 expression decreased MAPK signaling, cell-cycle progression, and tumor colony formation. Similarly, a peptide mimicking the WW domain of IQGAP1 that interacts with ERK inhibited the clonogenic growth and self-renewal of multiple myeloma cell lines and primary clinical specimens in vitro as well as tumor-initiating cell frequency in immunodeficient mice. During multiple myeloma progression, self-renewal may be enhanced by aberrant RAS/MAPK signaling and inhibited by targeting IQGAP1. Mol Cancer Ther; 15(11); 2733-9. ©2016 AACR.
Assuntos
Autorrenovação Celular , Evolução Clonal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Mimetismo Biológico , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Evolução Clonal/efeitos dos fármacos , Evolução Clonal/genética , Modelos Animais de Doenças , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Peptídeos/química , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/químicaRESUMO
IQGAP1, as a scaffold protein, integrates diverse cellular functions, including rearrangement of the actin cytoskeleton, cell adhesion, gene transcription and cell cycle regulation. IQGAP1 is elevated in a number of human cancer cells. However little is known about the expression of IQGAP in human pancreatic cancer and its association with cancer proliferation or metastasis. In the present study, we examined the expression of IQGAP1 in different pancreatic cancer cell lines and we found that IQGAP1 level is highly correlated with the degree of malignancy of pancreatic cancer cell metastasis. The proliferation, metastasis, motility and tumorigenesis in SW1990 human pancreatic cells were greatly impaired by down-regulating IQGAP1 expression with RNA interference. Mechanistic analysis indicated that Cdc42/Rac1 pathway might contribute to IQGAP1-mediated-pancreatic cell proliferation and tumorigenesis.
Assuntos
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Ativadoras de ras GTPase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Neoplasias Pancreáticas/genética , RNA Interferente Pequeno/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
OBJECTIVE: The objective of this study was to explore the effects of high glucose/high insulin on AIP1 expression in HUVECs and the possible regulation of HIF-1α signaling by AIP1. METHODS: We investigated the expression of AIP1 and HIF-1α signaling in HUVECs at the levels of mRNA and protein following exposure to 30 mmol/L glucose (high glucose), 1 nmol/L insulin (high insulin), and the combination of the two (high glucose/high insulin). We detected changes in HIF-1α and VEGF expression with AIP1 siRNA interference by real-time PCR and western blotting. The CCK8 cell proliferation assay, the scratch/wound-healing assay, and flow cytometry were used to assess cell proliferation, migration and apoptosis, respectively. Matrigel was used to perform a tubule formation assay. RESULTS: Compared with 5.5 mmol/L glucose alone (control), high glucose, high insulin, and the combination of high glucose+high insulin increased AIP1 expression at 24 h at the mRNA and protein levels. High glucose, high insulin, and high glucose+high insulin decreased HIF-1α expression at the mRNA and protein levels. AIP1 knockdown significantly increased HIF-1α and VEGF expression at both the mRNA and protein levels in HUVECs under high glucose conditions. In the presence of high insulin, the effect of high glucose on target gene expression was altered. The downregulation of AIP1 promoted cell proliferation, migration, and tubule formation, and it decreased apoptosis. CONCLUSIONS: High glucose increases AIP1 expression and decreases the expression of HIF-1α and downstream molecules. Decreased HIF-1α signaling may be regulated by increased AIP1 under high glucose.
Assuntos
Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Insulina/farmacologia , Proteínas Ativadoras de ras GTPase/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is still a major health problem, often diagnosed at an advanced stage. The multikinase inhibitor sorafenib is to date the sole approved systemic therapy. Several signalling pathways are implicated in tumour development and progression. Among these pathways, the Ras/MAPK pathway is activated in 50-100% of human HCCs and is correlated with a poor prognosis. The aim of this work was to review the main intracellular mechanisms leading to aberrant Ras pathway activation in HCC and the potential therapeutic implications. MATERIALS AND METHODS: This review is based on the material found on PubMed up to December 2014. 'Ras signaling, Ras dysregulation, Ras inhibition, MAPK pathway, cancer, hepatocarcinoma and liver cancer' alone or in combination were the main terms used for online research. RESULTS: Multiple mechanisms lead to the deregulation of the Ras pathway in liver cancer. Ras and Raf gene mutations are rare events in human hepatocarcinogenesis in contrast to experimental models in rodents. Downregulation of several Ras/MAPK pathway inhibitors such as GAPs, RASSF proteins, DUSP1, Sprouty and Spred proteins is largely implicated in the aberrant activation of this pathway in the context of wild-type Ras and Raf genes. Epigenetic or post-transcriptional mechanisms lead to the downregulation of these tumour suppressor genes. CONCLUSION: Ras/MAPK pathway effectors may be considered as potential therapeutic targets in the field of HCC. In particular after the arrival of sorafenib, more Ras/MAPK inhibitors have emerged and are still in preclinical or clinical investigation for HCC therapy.