RESUMEN
The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and the presence of cancer cells with properties of normal tissue stem cells. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration. Here we show that mouse and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signalling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that these cells have features of normal tissue stem cells. Genetic perturbation of Wnt production or signalling suppressed tumour progression. Small-molecule inhibitors targeting essential posttranslational modification of Wnt reduced tumour growth and markedly decreased the proliferative potential of lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Progresión de la Enfermedad , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Nicho de Células Madre , Proteínas Wnt/biosíntesis , Vía de Señalización Wnt , Adenocarcinoma del Pulmón , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Tasa de Supervivencia , Proteínas Wnt/química , Proteínas Wnt/metabolismoRESUMEN
Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.
Asunto(s)
Proteínas de Fusión Oncogénica/metabolismo , Poliubiquitina/metabolismo , Proteolisis , Receptores de Somatomedina/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Células Cultivadas , Humanos , Proteínas de Fusión Oncogénica/genética , Fosforilación , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.
Asunto(s)
Enfermedad de Huntington/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Western Blotting , Fraccionamiento Celular , Cuerpo Estriado/metabolismo , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Proteína Huntingtina , Ratones , Proteínas del Tejido Nervioso/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.
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Mutación , Proteínas Proto-Oncogénicas c-ets/química , Receptor trkC/química , Proteínas Represoras/química , Animales , Calorimetría , Transformación Celular Neoplásica , Humanos , Lisina/química , Espectroscopía de Resonancia Magnética , Ratones , Ratones Desnudos , Células 3T3 NIH , Polímeros/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-ets/genética , Receptor trkC/genética , Proteínas Represoras/genética , Sales (Química)/química , Transducción de Señal , Electricidad Estática , Tirosina/química , Proteína ETS de Variante de Translocación 6RESUMEN
Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy, and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro, in vivo (in female mice), and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors, with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore, STINGa ADCs induce type III interferons, which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs.
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Inmunidad Innata , Inmunoconjugados , Interferones , Proteínas de la Membrana , Animales , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/inmunología , Inmunidad Innata/efectos de los fármacos , Femenino , Humanos , Ratones , Línea Celular Tumoral , Inmunoconjugados/farmacología , Inmunoconjugados/administración & dosificación , Interferones/metabolismo , Interferón lambda , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Interferón Tipo I/inmunología , Citocinas/metabolismo , Células Mieloides/inmunología , Células Mieloides/efectos de los fármacos , Inmunoterapia/métodos , Ratones Endogámicos C57BL , Receptores de IgG/agonistas , Receptores de IgG/metabolismo , Receptores de IgG/inmunologíaRESUMEN
The compounds imidazolium [trans-[RuCl(4)(1H-imidazole)(2)] (KP418) and indazolium [trans-RuCl(4)(1H-indazole)(2)] (KP1019) both show significant anticancer activity, with the latter recently having completed phase I clinical trials. An important component of this success has been associated with targeted delivery of the complexes to cancer cells by serum proteins. In this study, electron paramagnetic resonance (EPR) measurements, combined with incubation under physiological conditions, and separation of protein-bound fractions, have been used to characterize the interactions of these complexes with human serum albumin (hsA), human serum transferrin (hsTf) apoprotein, and whole human serum. The strong EPR signals observed in these experiments demonstrate that both complexes are primarily retained in the 3+ oxidation state in the presence of serum components. Rapid, noncovalent binding of KP1019 was observed in the presence of both hsA and serum, indicating that the predominant interactions occur within the hydrophobic binding sites of hsA. This sequestering process correlates with the low levels of side effects observed in clinical trials of the complex. At longer incubation times, the noncovalently bound complexes are converted slowly to a protein-coordinated form. Noncovalent interactions are not observed in the presence apo-hsTf, where only slow binding of KP1019 via ligand exchange with the protein occurs. By contrast, hydrophobic interactions of KP418 with hsA only occur with the aquated products of the complex, a process that also dominates in serum. In the presence of apo-hsTf, KP418 interacts directly with the protein through exchange of ligands, as observed with KP1019.
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Antineoplásicos/química , Apoproteínas/química , Indazoles/química , Compuestos Organometálicos/química , Compuestos de Rutenio/química , Albúmina Sérica/química , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Ligandos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This corrects the article DOI: 10.1038/nbt.3836.
RESUMEN
In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.
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Neoplasias Colorrectales/genética , Modelos Animales de Enfermedad , Edición Génica/métodos , Genes Relacionados con las Neoplasias/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Trasplante de Órganos/métodos , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Femenino , Masculino , Ratones , Ratones Transgénicos , Metástasis de la NeoplasiaRESUMEN
Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by (13)C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis.
Asunto(s)
Apoptosis/genética , Supervivencia Celular/genética , Glutamina/metabolismo , Neoplasias/metabolismo , Antioxidantes/metabolismo , Línea Celular Tumoral , Ciclo del Ácido Cítrico/genética , Glucosa/química , Glucosa/metabolismo , Glutatión/genética , Glutatión/metabolismo , Humanos , Neoplasias/patología , Oxidación-Reducción , Ácido Pirúvico/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure.
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Apoptosis/efectos de la radiación , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Inflamasomas/metabolismo , Traumatismos por Radiación/metabolismo , Animales , Médula Ósea/metabolismo , Médula Ósea/efectos de la radiación , Caspasa 1/genética , Caspasa 1/metabolismo , ADN/metabolismo , ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Inflamasomas/genética , Inflamasomas/efectos de la radiación , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Traumatismos por Radiación/genética , Radiación Ionizante , Irradiación Corporal TotalRESUMEN
O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-O-linked 2-acetamido-2-deoxy-d-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the activity of this enzyme toward a substrate bearing an excellent leaving group. Together, these results allow unambiguous assignment of Asp(174) as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp(175) as the residue that functions as the general acid/base catalyst. Therefore, the family 84 glycoside hydrolases use a DD catalytic pair to effect catalysis.