RESUMO
Wnt signaling pathway plays a critical role in numerous cellular processes, including tumor initiation, proliferation, invasion/infiltration, metastasis formation and resistance to chemotherapy. In a drug discovery project aimed at the identification of inhibitors of the canonical Wnt pathway, we selected a series of quinazoline 2,4-diones as starting point for the therapeutic treatment of glioblastoma multiforme. Despite of poor physico-chemical properties of hit compound 1, our medicinal chemistry effort allowed the discovery and characterization of lead compound 33 (SEN461), with improved ADME profile, good bioavailability and active in vitro and in vivo in glioblastoma, gastric and sarcoma tumors.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Quinazolinas/metabolismo , Quinazolinas/farmacocinética , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas.
Assuntos
Antineoplásicos/farmacologia , Proteína Axina/metabolismo , Sarcoma/fisiopatologia , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Primers do DNA/genética , Feminino , Imunofluorescência , Vetores Genéticos/genética , Humanos , Immunoblotting , Lentivirus , Camundongos , Camundongos Nus , Microscopia Confocal , Plasmídeos/genética , Estabilidade Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sarcoma/tratamento farmacológicoRESUMO
Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, ß-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased ß-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Glioblastoma/patologia , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Camundongos , Camundongos Nus , Prognóstico , Transdução de Sinais , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , XenopusRESUMO
The serine/threonine kinase human Pim1 (hereafter PIM1) cooperates with human c-Myc (hereafter MYC) in cell cycle progression and tumorigenesis. However, the nature of this cooperation is still unknown. Here we show that, after stimulation with growth factor, PIM1 forms a complex with the dimer of MYC with MAX (Myc-associated factor X) via the MYC BoxII (MBII) domain. MYC recruits PIM1 to the E boxes of the MYC-target genes FOSL1 (FRA-1) and ID2, and PIM1 phosphorylates serine 10 of histone H3 (H3S10) on the nucleosome at the MYC-binding sites, contributing to their transcriptional activation. MYC and PIM1 colocalize at sites of active transcription, and expression profile analysis revealed that PIM1 contributes to the regulation of 20% of the MYC-regulated genes. Moreover, PIM1-dependent H3S10 phosphorylation contributes to MYC transforming capacity. These results establish a new function for PIM1 as a MYC cofactor that phosphorylates the chromatin at MYC-target loci and suggest that nucleosome phosphorylation, at E boxes, contributes to MYC-dependent transcriptional activation and cellular transformation.
Assuntos
Transformação Celular Neoplásica , Histonas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Serina/metabolismo , Ativação Transcricional , Células Cultivadas , Cromatina/metabolismo , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histonas/genética , Humanos , Complexos Multiproteicos , Nucleossomos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-pim-1/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The tyrosine kinase receptor fetal liver kinase 1 (Flk-1) plays a crucial role in vasculogenesis and angiogenesis, but its target genes remain elusive. Comparing Flk-1(+/+) with Flk-1(-/-) embryonic stem (ES) cells, we identified transcripts regulated by the vascular endothelial growth factor A (VEGF-A)/Flk-1 pathway at an early stage of their differentiation to endothelial and mural precursors. Further analysis of a number of these genes (Nm23-M1, Nm23-M2, Slug, Set, pp32, Cbp, Ship-1, Btk, and Pim-1) showed that their products were transiently up-regulated in vivo in endothelial cells (ECs) during angiogenesis of the ovary, and their mRNA was rapidly induced in vitro by VEGF-A in human umbilical cord vein endothelial cells (HUVECs). Functional analysis by RNA interference (RNAi) in ES cells induced to differentiate demonstrated that Pim-1 is required for their differentiation into ECs and smooth muscle cells (SMCs). In HUVECs, RNAi showed that Pim-1 is required in ECs for VEGF-A-dependent proliferation and migration. The identification of Flk-1 target genes should help in elucidating the molecular pathways that govern the vasculogenesis and angiogenesis processes.