RESUMO
Ras mutations have been frequently observed in human cancer. Although there is a high degree of similarity between Ras isomers, they display preferential coupling in specific cancer types. The binding of Ras to the plasma membrane is essential for its activation and biological functions. The present study elucidated Ras isoform-specific interactions with the membrane and their role in Ras-mediated biological activities. We investigated the role of a lipid raft protein flotillin-1 (Flot-1) in the activations of Ras. We found that Flot-1 was co-localized with H-Ras, but not with N-Ras, in lipid rafts of MDA-MB-231 human breast cells. The amino-terminal hydrophobic domain (1-38) of Flot-1 interacted with the hypervariable region of H-Ras. The epidermal growth factor-stimulated activation of H-Ras required Flot-1 which was not necessary for that of N-Ras in breast cancer cells. Flot-1 interacted with son of sevenless (SOS)-1, which promotes the conversion of Ras-bound GDP to GTP. Notably, Flot-1 was crucial for the interaction between SOS1 and H-Ras/K-Ras in breast and pancreatic cancer cells. Stable knockdown of Flot-1 reduced the in vivo metastasis in a mouse xenograft model with human breast carcinoma cells. A tissue microarray composed of 61 human pancreatic cancer samples showed higher levels of Flot-1 expression in pancreatic tumor tissues compared to normal tissues, and a correlation between K-Ras and Flot-1. Taken together, our findings suggest that Flot-1 may serve as a membrane platform for the interaction of SOS1 with H-Ras/K-Ras in human cancer cells, presenting Flot-1 as a potential target for Ras-driven cancers.
Assuntos
Proteínas de Membrana , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Neoplasias Pancreáticas/metabolismoRESUMO
Alzheimer's disease (AD) is the most prevalent type of dementia and is characterized by cognitive deficits and accumulation of pathological plaques. Owing to the complexity of AD development, paradigms for AD research and drug discovery have shifted to target factors that mediate multiple pathogenesis in AD. Increasing evidence suggests that the suppression of the Wnt/ß-catenin signaling pathway plays substantial roles in AD progression. However, the underlying mechanism for the suppression of Wnt/ß-catenin pathway associated with AD pathogenesis remains unexplored. In this study, we identified that CXXC5, a negative feedback regulator of the Wnt/ß-catenin pathway, was overexpressed in the tissues of AD patients and 5xFAD transgenic mice paired with the suppression of Wnt/ß-catenin pathway and its target genes related to AD. The level of CXXC5 was upregulated, upon aging of 5xFAD mice. AD characteristics including cognitive deficits, amyloid-ß (Aß) plaques, neuronal inflammation, and age-dependent increment of AD-related markers were rescued in Cxxc5-/-/5xFAD mice. 5-methoxyindirubin-3'-oxime (KY19334), a small molecule that restores the suppressed Wnt/ß-catenin pathway via interference of the CXXC5-Dvl interaction, significantly improved the overall pathogenic phenotypes of 5xFAD mice. Collectively, our findings revealed that CXXC5 plays a key role in AD pathogenesis and suggest inhibition of CXXC5-Dvl interaction as a new therapeutic approach for AD.
Assuntos
Doença de Alzheimer , Via de Sinalização Wnt , Animais , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , beta Catenina/metabolismo , Modelos Animais de Doenças , Proteínas de Ligação a DNA/metabolismo , Camundongos Transgênicos , Fatores de Transcrição , HumanosRESUMO
Ras protein has been considered a fascinating target for anticancer therapy because its malfunction is closely related to cancer. However, Ras has been considered undruggable because of the failure to regulate its malfunction by controlling the Ras activation mechanism. Recently, Lumakras targeting the G12C mutation was approved, and therapeutic interest in Ras for anticancer therapy has been rejuvenated. Here, we present a series of compounds that inhibit Ras via a unique mechanism of action that exploits the relationship between the Wnt/ß-catenin pathway and Ras. KYA1797K (1) binds to axin to stabilize the ß-catenin destruction complex that causes the phosphorylation and subsequent degradation of Ras, similar to canonical ß-catenin regulation. Based on the chemical structure of 1, we performed a structural optimization and identified 3-(2-hydroxyethyl)-5-((6-(4-nitrophenyl)pyridin-2-yl)methylene)thiazolidine-2,4-dione (13d) as the most potent compound. 13d displayed antitumor effects in a colorectal cancer model with enhanced inhibition activity on Ras. The results of this study suggest that the further development of 13d could contribute to the development of Ras inhibitors with novel mechanisms of action.
Assuntos
Neoplasias Colorretais , beta Catenina , Proteínas ras , Humanos , Proteína Axina/química , Proteína Axina/genética , Proteína Axina/metabolismo , beta Catenina/química , beta Catenina/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismo , Via de Sinalização WntRESUMO
The Wnt/ß-catenin signaling pathway plays important roles in the multi-phases of wound healing: homeostasis, inflammation, proliferative, and remodeling phases. However, there are no clinically available therapeutic agents targeting the Wnt/ß-catenin pathway. In this study, we tested the effect of 5, 6-dichloroindirubin-3'-methoxime (KY19382), a small molecule that activates the Wnt/ß-catenin pathway via interference with the function of the negative feedback regulator CXXC5, on cutaneous wound healing. KY19382 significantly enhanced cell migration of human keratinocytes and dermal fibroblasts with increased levels of ß-catenin, phalloidin, Keratin 14, proliferating cell nuclear antigen (PCNA), Collagen I, and alpha-smooth muscle actin (α-SMA) by activating the Wnt/ß-catenin signaling pathway without causing significant cytotoxicity. In addition, levels of Collagen I, Keratin 14, PCNA, and stem cell markers were significantly increased by KY19382 in a cutaneous murine wound healing model. Moreover, KY19382 treatment accelerated re-epithelialization and neo-epidermis formation with collagen deposition and stem cell activation at an early stage of cutaneous wound healing. Overall, KY19382 accelerates wound healing via activating the Wnt/ß-catenin pathway, and may have the potential to be used for the development of a new wound healing agent.
Assuntos
Via de Sinalização Wnt , beta Catenina , Camundongos , Humanos , Animais , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Queratina-14/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Wnt/metabolismo , Cicatrização , Colágeno/farmacologia , Proteínas de Ligação a DNA/metabolismo , Fatores de TranscriçãoRESUMO
Glycogen synthase kinase-3ß (GSK-3ß) appears to be ordinarily expressed, and functionally redundant in Wnt/ß-catenin signaling. The Wnt proteins induce transduction of a cytoplasmic protein, Dishevelled (Dvl) which negatively modulates GSK-3ß activity. CXXC5 is a negative modulator of the Wnt/ß-catenin signaling through the interaction with Dvl in the cytosol. This indicates that Wnt/ß-catenin signaling could be efficiently modulated by controlling GSK-3ß and the CXXC5-Dvl interaction. In this study, we designed a series of indirubin-3'-oxime and indirubin-3'-alkoxime derivatives containing various functional groups at the 5- or 6-position (R1) alongside alkyl or benzylic moieties at the 3'-oxime position (R2). These activate Wnt signaling through inhibitions of both GSK-3ß and the CXXC5-Dvl protein-protein interaction, in addition, the improvement of pharmacological properties. The potent activity profiles of the synthesized compounds suggested that dual inhibition of GSK-3ß and the CXXC5-Dvl interaction could be an appropriate approach towards safely and efficientlyactivating Wntsignaling. Thus, dual-targeting inhibitors are potentially better candidates for efficient activation ofWntsignaling compared to GSK-3ß inhibitors.
Assuntos
Via de Sinalização Wnt , beta Catenina , Proteínas Desgrenhadas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Indóis , Oximas/farmacologia , Regulação para Cima , beta Catenina/metabolismoRESUMO
The activation of the Wnt/ß-catenin signaling pathway plays a key role in the wound-healing process through tissue regeneration. The extract of Euodia daniellii Hemsl. (E. daniellii), a member of the Rutaceae family, activates the Wnt/ß-catenin signaling pathway. However, the function of E. daniellii in wound healing has not yet been elucidated. We performed a migration assay to determine the wound-healing effect of E. daniellii extract in vitro using human keratinocytes and dermal fibroblast. In addition, a mouse acute wound model was used to investigate the cutaneous wound-healing effect of E. daniellii extract in vivo and confirm the potential mechanism. E. daniellii extract enhanced the migration of human keratinocytes and dermal fibroblasts via the activation of the Wnt/ß-catenin pathway. Moreover, the E. daniellii extract increased the levels of keratin 14, PCNA, collagen I, and α-SMA, with nuclei accumulation of ß-catenin in vitro. E. daniellii extract also efficiently accelerated re-epithelialization and stimulated wound healing in vivo. Furthermore, we confirmed that hesperidin, one of the components of E. daniellii, efficiently accelerated the migration of human keratinocytes and dermal fibroblasts, as well as wound healing in vivo via the activation of the Wnt/ß-catenin pathway. Overall, E. daniellii extract and its active component, hesperidin, have potential to be used as therapeutic agents for wound healing.
Assuntos
Evodia , Hesperidina , Camundongos , Animais , Humanos , Via de Sinalização Wnt , beta Catenina/metabolismo , Queratina-14/metabolismo , Hesperidina/farmacologia , Evodia/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Cicatrização , Colágeno/metabolismo , Fibroblastos/metabolismoRESUMO
Phospholipase D (PLD) isoforms PLD1 and PLD2 serve as the primary nodes where diverse signaling pathways converge. However, their isoform-specific functions remain unclear. We showed that PLD1 and PLD2 selectively couple to toll-like receptor 4 (TLR4) and interleukin 4 receptor (IL-4R) and differentially regulate macrophage polarization of M1 and M2 via the LPS-MyD88 axis and the IL-4-JAK3 signaling, respectively. Lipopolysaccharide (LPS) enhanced TLR4 or MyD88 interaction with PLD1; IL-4 induced IL-4R or JAK3 association with PLD2, indicating isozyme-specific signaling events. PLD1 and PLD2 are indispensable for M1 polarization and M2 polarization, respectively. Genetic and pharmacological targeting of PLD1 conferred protection against LPS-induced sepsis, cardiotoxin-induced muscle injury, and skin injury by promoting the shift toward M2; PLD2 ablation intensified disease severity by promoting the shift toward M1. Enhanced Foxp3+ regulatory T cell recruitment also influenced the anti-inflammatory phenotype of Pld1LyzCre macrophages. We reveal a previously uncharacterized role of PLD isoforms in macrophage polarization, signifying potential pharmacological interventions for macrophage modulation.
Assuntos
Macrófagos/fisiologia , Fosfolipase D/metabolismo , Cicatrização/fisiologia , Ferimentos e Lesões/prevenção & controle , Animais , Polaridade Celular/fisiologia , Inflamação/patologia , Inflamação/prevenção & controle , Janus Quinase 3/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos/lesões , Fator 88 de Diferenciação Mieloide/metabolismo , Fosfolipase D/genética , Receptores de Interleucina-4/metabolismo , Sepse/imunologia , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/metabolismo , Ferimentos e Lesões/patologiaRESUMO
BACKGROUND: Most cancer cells employ the Warburg effect to support anabolic growth and tumorigenesis. Here, we discovered a key link between Warburg effect and aberrantly activated Wnt/ß-catenin signalling, especially by pathologically significant APC loss, in CRC. METHODS: Proteomic analyses were performed to evaluate the global effects of KYA1797K, Wnt/ß-catenin signalling inhibitor, on cellular proteins in CRC. The effects of APC-loss or Wnt ligand on the identified enzymes, PKM2 and LDHA, as well as Warburg effects were investigated. A linkage between activation of Wnt/ß-catenin signalling and cancer metabolism was analysed in tumour of Apcmin/+ mice and CRC patients. The roles of PKM2 in cancer metabolism, which depends on Wnt/ß-catenin signalling, were assessed in xenograft-tumours. RESULTS: By proteomic analysis, PKM2 and LDHA were identified as key molecules regulated by Wnt/ß-catenin signalling. APC-loss caused the increased expression of metabolic genes including PKM2 and LDHA, and increased glucose consumption and lactate secretion. Pathological significance of this linkage was indicated by increased expression of glycolytic genes with Wnt target genes in tumour of Apcmin/+ mice and CRC patients. Warburg effect and growth of xenografted tumours-induced by APC-mutated-CRC cells were suppressed by PKM2-depletion. CONCLUSIONS: The ß-catenin-PKM2 regulatory axis induced by APC loss activates the Warburg effect in CRC.
Assuntos
Proteínas de Transporte/metabolismo , Neoplasias Colorretais/metabolismo , Genes APC , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Hormônios Tireóideos/metabolismo , Efeito Warburg em Oncologia , Via de Sinalização Wnt , Animais , Proteínas de Transporte/genética , Neoplasias Colorretais/genética , Xenoenxertos , Humanos , L-Lactato Desidrogenase/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Neoplasias/genética , Proteômica , Tiazolidinas/farmacologia , Hormônios Tireóideos/genética , Análise Serial de Tecidos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Pancreatic cancer is a major malignant tumor without an effective treatment. KRAS mutations occur in 90% of the pancreatic cancer patients and are a major obstacle for treatment of pancreatic cancer. Pancreatic cancer patients have been treated with limited chemotherapeutic agents such as gemcitabine. However, patients often develop resistance to gemcitabine that is attributed to KRAS mutations. Gemcitabine treatment activates both the Wnt/ß-catenin and RAS/ERK pathways. These signaling pathways are also activated in the gemcitabine-resistant pancreatic cancer cell lines, suggesting that they play an important role in gemcitabine resistance in pancreatic cancer. The gemcitabine-resistant cell lines show enhanced migratory and invasive capabilities than their parental lines. Therefore, we investigated the effects of a small molecule, KYA1797K that degrades both ß-catenin and RAS, on pancreatic cancer. KYA1797K decreased the expression level of both ß-catenin and KRAS in pancreatic cancer cell lines expressing either wild-type or mutant KRAS. It also suppressed migration and invasion of gemcitabine-resistant and parental pancreatic cancer cells. Overall, we demonstrated that inhibiting the Wnt/ß-catenin and RAS/ERK pathways by destabilizing ß-catenin and RAS could be a therapeutic approach to overcome gemcitabine resistance in pancreatic cancer.
Assuntos
Desoxicitidina/análogos & derivados , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas ras/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Humanos , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tiazolidinas/farmacologia , beta Catenina/metabolismo , GencitabinaRESUMO
Mutant KRAS provides a driving force for enhancement of cancer stem cells (CSCs) characteristics contributing transformation of colorectal cancer (CRC) cells harboring adenomatous polyposis coli (APC) mutations. Here, we identified the factors mediating the promotion of CSCs properties induced by KRAS mutation through microarray analyses of genes specifically induced in CRC spheroids harboring both KRAS and APC mutations. Among them, REG4 was identified as a key factor since CRISPR/Cas9-mediated knockout of REG4 most significantly affected the stem cell characteristics in which CSCs markers were effectively suppressed. We show that REG4 mediates promotion of CSCs properties via Wnt/ß-catenin signaling in various in vitro studies including tumor organoid systems. Furthermore, expression patterns of CSCs markers and REG4 correlated in intestinal tumors from Apcmin/+ /KrasG12D LA2 mice and in CRC patient tissues harboring both KRAS and APC mutations. The role of REG4 in the tumor-initiating capacity accompanied by enhancement of CSCs characteristics was also revealed by NSG mice xenograft system. Collectively, our study highlights the importance of REG4 in promoting CSCs properties induced by KRAS mutation, and provides a new therapeutic strategy for CRC harboring both APC and KRAS mutations.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Associadas a Pancreatite/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Transformação Celular Neoplásica/genética , Xenoenxertos , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: Cancer stem cells (CSCs), the major driver of tumorigenesis, is a sub-population of tumor cells responsible for poor clinical outcomes. However, molecular mechanism to identify targets for controlling CSCs is poorly understood. METHODS: Gene Set Enrichment Analyses (GSEA) of Wnt/ß-catenin and RAS signaling pathways in stem-like subtype of colorectal cancer (CRC) patients were performed using two gene expression data set. The therapeutic effects of destabilization of ß-catenin and RAS were tested by treatment of small molecule KYA1797K using CRC patient derived cells. RESULTS: Treatment with KYA1797K, a small molecule that destabilizes both ß-catenin and RAS via Axin binding, effectively suppresses the stemness of CSCs as shown in CRC spheroids and small intestinal tumors of ApcMin/+/K-RasG12DLA2 mice. Moreover, KYA1797K also suppresses the stemness of cells in CRC patient avatar model systems, such as patient-derived tumor organoids (PDTOs) and patient-derived tumor xenograft (PDTX). CONCLUSION: Our results suggest that destabilization of both ß-catenin and RAS is a potential therapeutic strategy for controlling stemness of CRC cells. Video abstract.
Assuntos
Antineoplásicos , Carcinogênese/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tiazolidinas , beta Catenina/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas , Organoides , Cultura Primária de Células , Tiazolidinas/administração & dosagem , Tiazolidinas/farmacologiaRESUMO
RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HEK293 , Humanos , Camundongos Nus , Modelos Biológicos , Modelos Moleculares , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/química , beta Catenina/genéticaRESUMO
BACKGROUND: Stabilization of RAS is a key event for the hyper-activation of Wnt/ß-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC. METHODS: We generated mice with deletion of Wdr76 (Wdr76-/-) and crosses of Wdr76-/- with ApcMin/+ (Wdr76-/-; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture. RESULTS: Wdr76-/- mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76-/-; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and ß-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival. CONCLUSIONS: In analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Neoplásicas/patologia , Proteólise , Proteínas ras/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Citosol/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , Via de Sinalização WntRESUMO
Ras signaling is tightly regulated during neural stem cell (NSC) differentiation, and defects in this pathway result in aberrant brain development. However, the mechanism regulating Ras signaling during NSC differentiation was unknown. Here, we show that stabilized HRas specifically induces neuronal differentiation of NSCs. Lentivirus-mediated HRas overexpression and knockdown resulted in stimulation and inhibition, respectively, of NSC differentiation into neuron in the ex vivo embryo. Retinoic acid, an active metabolite of vitamin A, promoted neuronal differentiation of NSCs by stabilizing HRas, and HRas knockdown blocked the retinoic acid effect. Vitamin-A-deficient mice displayed abnormal brain development with reduced HRas levels and a reduced thickness of the postmitotic region containing differentiated neurons. All of these abnormal phenotypes were rescued with the restoration of HRas protein levels achieved upon feeding with a retinoic-acid-supplemented diet. In summary, this study shows that retinoic acid stabilizes HRas protein during neurogenesis, and that this is required for NSC differentiation into neurons and murine brain development.
Assuntos
Encéfalo/embriologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Neurais/citologia , Neurônios/citologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tretinoína/farmacologia , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Deficiência de Vitamina A/patologiaRESUMO
Both the Wnt/ß-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both ß-catenin and Ras, via targeting the Wnt/ß-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating ß-catenin and Ras degradation through enhancement of the ß-catenin destruction complex activating GSK3ß. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both ß-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/ß-catenin and Ras pathways.
Assuntos
Proteína Axina/química , Proteína Axina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas RGS/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tioidantoínas/farmacologia , beta Catenina/metabolismo , Animais , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genes APC , Genes ras , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas RGS/metabolismo , Tioidantoínas/síntese química , Tioidantoínas/química , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/químicaRESUMO
The Wnt/ß-catenin signaling pathway plays a significant role in the control of osteoblastogenesis and bone formation. CXXC finger protein 5 (CXXC5) has been recently identified as a negative feedback regulator of osteoblast differentiation through a specific interaction with Dishevelled (Dvl) protein. It was reported that targeting the Dvl-CXXC5 interaction could be a novel anabolic therapeutic target for osteoporosis. In this study, complex structure of Dvl PDZ domain and CXXC5 peptide was simulated with molecular dynamics (MD). Based on the structural analysis of binding modes of MD-simulated Dvl PDZ domain with CXXC5 peptide and crystal Dvl PDZ domain with synthetic peptide-ligands, we generated two different pharmacophore models and applied pharmacophore-based virtual screening to discover potent inhibitors of the Dvl-CXXC5 interaction for the anabolic therapy of osteoporosis. Analysis of 16 compounds selected by means of a virtual screening protocol yielded four compounds that effectively disrupted the Dvl-CXXC5 interaction in the fluorescence polarization assay. Potential compounds were validated by fluorescence spectroscopy and nuclear magnetic resonance. We successfully identified a highly potent inhibitor, BMD4722, which directly binds to the Dvl PDZ domain and disrupts the Dvl-CXXC5 interaction. Overall, CXXC5-Dvl PDZ domain complex based pharmacophore combined with various traditional and simple computational methods is a promising approach for the development of modulators targeting the Dvl-CXXC5 interaction, and the potent inhibitor BMD4722 could serve as a starting point to discover or design more potent and specific the Dvl-CXXC5 interaction disruptors.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas Desgrenhadas/antagonistas & inibidores , Simulação de Dinâmica Molecular , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Ligação a DNA , Bases de Dados de Compostos Químicos , Proteínas Desgrenhadas/química , Humanos , Ligantes , Domínios PDZ , Ligação Proteica , Fatores de Transcrição , Via de Sinalização WntRESUMO
Infection with Helicobacter pylori is closely linked to an increased risk of gastric cancer. Although cytotoxin-associated gene A (CagA), a major virulence factor of H. pylori, is known to be a causal factor for gastric carcinogenesis, the molecular link between CagA and gastric cancer-initiating cell (CIC)-like properties remains elusive. Here, we demonstrate that CagA is required for increased expression of ß-catenin and its target CIC markers via downregulation of microRNA (miR)-320a and miR-4496. CagA promoted gastric CIC properties and was responsible for chemoresistance. miR-320a and miR-4496 attenuated the in vitro self-renewal and tumour-initiating capacity of CagA-expressing CICs by targeting ß-catenin. Moreover, miR-320a and miR-4496 decreased CagA-induced chemoresistance by targeting ATP-binding cassette, subfamily G, member 2 (ABCG2) at the transcriptional and post-transcriptional levels, respectively. Combination therapy with 5-fluorouracil and miR-320a/miR-4496 suppressed gastric tumourigenesis and metastatic potential in an orthotopic mouse model, probably via suppression of CagA-induced CIC properties and chemoresistance. Our results provide novel evidence that CIC properties, chemoresistance and tumourigenesis associated with H. pylori are linked to CagA-induced upregulation of ß-catenin and ABCG2. These data provide novel insights into the molecular mechanisms of CagA-induced carcinogenisis and the therapeutic potential of of miR-320a and miR-4496. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Infecções por Helicobacter/genética , Helicobacter pylori/patogenicidade , MicroRNAs/genética , Neoplasias Gástricas/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Carcinogênese , Autorrenovação Celular , Transformação Celular Neoplásica , Citotoxinas/genética , Citotoxinas/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Regulação para Cima , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Dishevelled (Dvl) plays a crucial role in Wnt signaling by interacting with membrane-bound receptors and downstream molecules through its PDZ domain. CXXC5 is one of the key molecules that interacts with Dvl and negatively regulates the Wnt/ß-catenin pathway in osteoblast differentiation. Recently, the Dvl-CXXC5 interaction has been identified as an excellent target for osteoporosis treatment. Therefore, it is desirable to have detailed structural information for the Dvl-CXXC5 interaction. Although solution structures of the Dvl1 PDZ domain have been reported, a high-resolution crystal structure would provide detailed sidechain information that is essential for drug development. Here, we determined the first crystal structure of the Dvl-1 PDZ domain at a resolution of 1.76 Å, and compared it with its previously reported solution structure. The Dvl1 PDZ domain crystal belonged to the space group H32 with unit-cell parameters a = b = 72.837, c = 120.616, α = ß = 90.00, γ = 120.00. The crystal structure of Dvl1 PDZ shared its topology with the previously reported structure determined by nuclear magnetic resonance (NMR); however, the crystal structure was quite different from the solution structure in both the secondary structural region and the ligand-binding pocket. Molecular modeling based on NMR and X-ray crystallographic data yielded detailed information about the Dvl1/CXXC5 interaction, which will be useful for designing inhibitors.
Assuntos
Proteínas Desgrenhadas/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Domínios PDZ , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Soluções , Fatores de Transcrição , Via de Sinalização WntRESUMO
Myelination in corpus callosum plays important role for normal brain functions by transferring neurological information between various brain regions. However, the factors controlling expression of myelin genes in myelination are poorly understood. Here, CXXC5, a recently identified protein with CXXC-type zinc finger DNA binding motif, was characterized as a transcriptional activator of major myelin genes. We identified expression of CXXC5 expression was increased by Wnt/ß-catenin signaling. CXXC5 specifically expressed in the white matter induced expression of myelin genes through the direct binding of CXXC DNA-binding motif of CXXC5 on the MBP promoter. During the differentiation of neural stem cells (NSCs) of CXXC5(-/-) mice, the expressions of myelin genes were simultaneously reduced. The CXXC5(-/-) mice exhibited severely reduction of myelin genes expression in corpus callosum as well as abnormalities in myelin structure. The disrupted structural integrity of myelin in the CXXC5(-/-) mice resulted in reduced electrical conduction amplitudes at corpus callosum. These findings indicate that the regulation of myelin genes expression by CXXC5 is important for forming myelin structure involved with axonal electrical signal transfer in the corpus callosum.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/fisiologia , Potenciais de Ação/genética , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/ultraestrutura , Células Cultivadas , Corpo Caloso/crescimento & desenvolvimento , Corpo Caloso/metabolismo , Proteínas de Ligação a DNA , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/genética , Condução Nervosa/genética , Células-Tronco Neurais , Oligodendroglia/ultraestrutura , Fatores de Transcrição , Via de Sinalização Wnt/genética , Proteína Wnt3A/farmacologia , beta Catenina/metabolismoRESUMO
The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/ß-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2µM affinity and had a 0.186µM KD value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural-kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.