RESUMO
Transcription factor NF-κB potently activates anti-apoptotic genes, and its inactivation significantly reduces tumor cell survival following genotoxic stresses. We identified two structurally distinct lead compounds that selectively inhibit NF-κB activation by DNA double-strand breaks, but not by other stimuli, such as TNFα. Our compounds do not directly inhibit previously identified regulators of this pathway, most critically including IκB kinase (IKK), but inhibit signal transmission in-between ATM, PARP1, and IKKγ. Deconvolution strategies, including derivatization and in vitro testing in multi-kinase panels, yielded shared targets, cdc-like kinase (CLK) 2 and 4, as essential regulators of DNA damage-induced IKK and NF-κB activity. Both leads sensitize to DNA damaging agents by increasing p53-induced apoptosis, thereby reducing cancer cell viability. We propose that our lead compounds and derivatives can be used in context of genotoxic therapy-induced or ongoing DNA damage to increase tumor cell apoptosis, which may be beneficial in cancer treatment.
Assuntos
NF-kappa B , Transdução de Sinais , NF-kappa B/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , DNARESUMO
DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here, we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, the depletion of both DHX30 isoforms leads to higher global translation but slower proliferation and lower mitochondrial energy metabolism. Isoform-specific silencing supports a role for cytoplasmic DHX30 in modulating global translation. The impact on translation and proliferation was confirmed in U2OS and MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified fourteen mitoribosome transcripts we propose as direct DHX30 targets that can be used to explore the prognostic value of this mechanism in cancer. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.
RESUMO
Impairment of the p53 pathway is a critical event in cancer. Therefore, reestablishing p53 activity has become one of the most appealing anticancer therapeutic strategies. Here, we disclose the p53-activating anticancer drug (3S)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (MANIO). MANIO demonstrates a notable selectivity to the p53 pathway, activating wild-type (WT)p53 and restoring WT-like function to mutant (mut)p53 in human cancer cells. MANIO directly binds to the WT/mutp53 DNA-binding domain, enhancing the protein thermal stability, DNA-binding ability, and transcriptional activity. The high efficacy of MANIO as an anticancer agent toward cancers harboring WT/mutp53 is further demonstrated in patient-derived cells and xenograft mouse models of colorectal cancer (CRC), with no signs of undesirable side effects. MANIO synergizes with conventional chemotherapeutic drugs, and in vitro and in vivo studies predict its adequate drug-likeness and pharmacokinetic properties for a clinical candidate. As a single agent or in combination, MANIO will advance anticancer-targeted therapy, particularly benefiting CRC patients harboring distinct p53 status.
Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Pirróis/farmacologia , Tiazóis/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/síntese química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Doxorrubicina/farmacologia , Descoberta de Drogas , Sinergismo Farmacológico , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Ligação Proteica , Pirróis/síntese química , Tiazóis/síntese química , Proteína Supressora de Tumor p53/agonistas , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC.
RESUMO
The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.
Assuntos
Genes p53/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ativação Transcricional/genética , Leveduras/crescimento & desenvolvimento , HumanosRESUMO
Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe three reversine-related molecules, designed by docking calculation, that present structural modifications in the diamino units at positions 2 and 6. We investigated the conformations of the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas phase, water and chloroform, the last solvent considered to give insights into the detection of broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the HN(7) form, but the most stable conformations changed in different solvents. Molecules 1â»3 were evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy. Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell lines used, similar concentrations of 1â»3 were effective only in cells where p53 was deleted or down-regulated. Therefore, our findings support a potential selective role of these structurally simplified, reversine-related molecules in p53-defective cancer cells.