RESUMO
The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant KrasG12D and mutant Trp53172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of KrasG12D/+ and Trp53172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic KrasG12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7+/- tumors have comparatively lower levels of succinate, and that cells derived from Atg7+/- tumors are also less invasive than those from Atg7+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7+/- cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy.
Assuntos
Proteína 7 Relacionada à Autofagia , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Camundongos , Mutação , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Succinatos/metabolismo , Succinatos/farmacologiaRESUMO
Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.
Assuntos
Lipídeos/análise , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Metaboloma , Proteoma/análise , Humanos , LigantesRESUMO
The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.
Assuntos
Acetanilidas/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Polifarmacologia , Sirtuína 1/antagonistas & inibidores , Tioureia/análogos & derivados , Proteína Supressora de Tumor p53/metabolismo , Autofagia , Proliferação de Células , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Tioureia/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Hepatocellular carcinoma (HCC) is driven by repeated rounds of inflammation, leading to fibrosis, cirrhosis, and, ultimately, cancer. A critical step in HCC formation is the transition from fibrosis to cirrhosis, which is associated with a change in the liver parenchyma called ductular reaction. Here, we report a genetically engineered mouse model of HCC driven by loss of macroautophagy and hemizygosity of phosphatase and tensin homolog, which develops HCC involving ductular reaction. We show through lineage tracing that, following loss of autophagy, mature hepatocytes dedifferentiate into biliary-like liver progenitor cells (ductular reaction), giving rise to HCC. Furthermore, this change is associated with deregulation of yes-associated protein and transcriptional coactivator with PDZ-binding motif transcription factors, and the combined, but not individual, deletion of these factors completely reverses the dedifferentiation capacity and tumorigenesis. These findings therefore increase our understanding of the cell of origin of HCC development and highlight new potential points for therapeutic intervention.
RESUMO
Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Indazóis/química , Indazóis/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Di-Hidro-Orotato Desidrogenase , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indazóis/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismoRESUMO
Drugging the p53 pathway has been a goal for both academics and pharmaceutical companies since the designation of p53 as the 'guardian of the genome'. Through growing understanding of p53 biology, we can see multiple routes for activation of both wild-type p53 function and restoration of mutant p53. In this review, we focus on small molecules that activate wild-type p53 and that do so in a non-genotoxic manner. In particular, we will describe potential approaches to targeting proteins that alter p53 stability and function through posttranslational modification, affect p53's subcellular localization, or target RNA synthesis or the synthesis of ribonucleotides. The plethora of pathways for exploitation of p53, as well as the wide-ranging response to p53 activation, makes it an attractive target for anti-cancer therapy.
Assuntos
Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Mutação/genética , Neoplasias/genética , Ribonucleotídeos/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfolipídeos/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Di-Hidro-Orotato Desidrogenase , Elétrons , Humanos , Ligantes , Simulação de Dinâmica Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fosfolipídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Benzamidas/farmacologia , Indóis/farmacologia , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Estrutura Molecular , Mutação , Sirtuínas/genética , Proteína Supressora de Tumor p53/genética , VemurafenibRESUMO
The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.
RESUMO
The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indazóis/farmacologia , Neoplasias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
While small-molecule inhibitors of class I/II histone deacetylases (HDAC) have been approved for cancer treatment, inhibitors of the sirtuins (a family of class III HDACs) still require further validation and optimization to enter clinical trials. Recent studies show that tenovin-6, a small-molecule inhibitor of sirtuins SirT1 and SirT2, reduces tumor growth in vivo and eliminates leukemic stem cells in a murine model for chronic myelogenous leukemia. Here, we describe a tenovin analogue, tenovin-D3, that preferentially inhibits sirtuin SirT2 and induces predicted phenotypes for SirT2 inhibition. Unlike tenovin-6 and in agreement with its weak effect on SirT1 (a p53 deacetylase), tenovin-D3 fails to increase p53 levels or transcription factor activity. However, tenovin-D3 promotes expression of the cell-cycle regulator and p53 target p21(WAF1/CIP1) (CDKN1A) in a p53-independent manner. Structure-activity relationship studies strongly support that the ability of tenovin-D3 to inhibit SirT2 contributes to this p53-independent induction of p21. The ability of tenovin-D3 to increase p21 mRNA and protein levels is shared with class I/II HDAC inhibitors currently used in the clinic and therefore suggests that SirT2 inhibition and class I/II HDAC inhibitors have similar effects on cell-cycle progression.