RESUMO
AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to pathophysiology in cancer progression and therapeutic resistance, making it an emerging therapeutic target. The first-in-class AXL inhibitor bemcentinib (R428/BGB324) has been granted fast track designation by the U.S. Food and Drug Administration (FDA) in STK11-mutated advanced metastatic non-small cell lung cancer and was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal molecular subtype. In this study, we further explored AXL's role in mediating DNA damage responses by using OC as a disease model. AXL inhibition using R428 resulted in the increase of DNA damage with the concurrent upregulation of DNA damage response signalling molecules. Furthermore, AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress. Combinatory use of AXL and ATR inhibitors showed additive effects in OC. Through SILAC co-immunoprecipitation mass spectrometry, we identified a novel binding partner of AXL, SAM68, whose loss in OC cells harboured phenotypes in DNA damage responses similar to AXL inhibition. In addition, AXL- and SAM68-deficiency or R428 treatment induced elevated levels of cholesterol and upregulated genes in the cholesterol biosynthesis pathway. There might be a protective role of cholesterol in shielding cancer cells against DNA damage induced by AXL inhibition or SMA68 deficiency.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias Ovarianas , Humanos , Feminino , Receptor Tirosina Quinase Axl , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular Tumoral , Receptores Proteína Tirosina Quinases , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Dano ao DNARESUMO
Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.
Assuntos
Nanoporos , RNA , Adenosina/genética , Animais , Inosina/genética , Camundongos , RNA/genética , RNA/metabolismo , Edição de RNA , Análise de Sequência de RNARESUMO
Cell state transitions control the functional behavior of cancer cells. Epithelial-to-mesenchymal transition (EMT) confers cancer stem cell-like properties, enhanced tumorigenicity and drug resistance to tumor cells, while mesenchymal-epithelial transition (MET) reverses these phenotypes. Using high-throughput chemical library screens, retinoids are found to be potent promoters of MET that inhibit tumorigenicity in basal-like breast cancer. Cell state transitions are defined by reprogramming of lipid metabolism. Retinoids bind cognate nuclear receptors, which target lipid metabolism genes, thereby redirecting fatty acids for ß-oxidation in the mesenchymal cell state towards lipid storage in the epithelial cell state. Disruptions of key metabolic enzymes mediating this flux inhibit MET. Conversely, perturbations to fatty acid oxidation (FAO) rechannel fatty acid flux and promote a more epithelial cell phenotype, blocking EMT-driven breast cancer metastasis in animal models. FAO impinges on the epigenetic control of EMT through acetyl-CoA-dependent regulation of histone acetylation on EMT genes, thus determining cell states.
RESUMO
Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3 (GlcNAc)2 , and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3 (GlcNAc)2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.
Assuntos
Biomarcadores Tumorais/genética , Cistadenoma Seroso/genética , Neoplasias Ovarianas/genética , Polissacarídeos/genética , Biomarcadores Tumorais/química , Cromatografia Líquida , Cistadenoma Seroso/patologia , Feminino , Genômica/métodos , Glicosilação , Humanos , Imagem Molecular , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Interferon gama/fisiologia , Melanoma/tratamento farmacológico , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/antagonistas & inibidores , Fator 4 Ativador da Transcrição/fisiologia , Animais , Células Cultivadas , Citocinas/análise , Perfilação da Expressão Gênica , Humanos , Interferon gama/genética , Melanoma/patologia , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/fisiologia , Células-Tronco Pluripotentes/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/fisiologiaRESUMO
The walls of Nicotiana alata pollen tubes contain a linear arabinan composed of (1,5)-α-linked arabinofuranose residues. Although generally found as a side chain on the backbone of the pectic polysaccharide rhamnogalacturonan I, the arabinan in N. alata pollen tubes is considered free, as there is no detectable rhamnogalacturonan I in these walls. Carbohydrate-specific antibodies detected arabinan epitopes at the tip and along the shank of N. alata pollen tubes that are predominantly part of the primary layer of the bilayered wall. A sequence related to ARABINAN DEFICIENT1 (AtARAD1), a presumed arabinan arabinosyltransferase from Arabidopsis (Arabidopsis thaliana), was identified by searching an N alata pollen transcriptome. Transcripts for this ARAD1-like sequence, which we have named N. alata ARABINAN DEFICIENT-LIKE1 (NaARADL1), accumulate in various tissues, most abundantly in the pollen grain and tube, and encode a protein that is a type II membrane protein with its catalytic carboxyl terminus located in the Golgi lumen. The NaARADL1 protein can form homodimers when transiently expressed in Nicotiana benthamiana leaves and heterodimers when coexpressed with AtARAD1 The expression of NaARADL1 in Arabidopsis led to plants with more arabinan in their walls and that also exuded a guttation fluid rich in arabinan. Chemical and enzymatic characterization of the guttation fluid showed that a soluble, linear α-(1,5)-arabinan was the most abundant polymer present. These results are consistent with NaARADL1 having an arabinan (1,5)-α-arabinosyltransferase activity.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Glicosiltransferases/metabolismo , Nicotiana/enzimologia , Pólen/enzimologia , Polissacarídeos/metabolismo , Fluorescência , Complexo de Golgi/metabolismo , Pentosiltransferases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Multimerização Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/enzimologiaRESUMO
The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-ß-D-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.
Assuntos
Parede Celular/metabolismo , Poaceae/metabolismo , Polissacarídeos/biossíntese , beta-Glucanas/metabolismo , Domínio Catalítico , Parede Celular/ultraestrutura , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Hordeum/metabolismo , Immunoblotting , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Meristema/metabolismo , Microssomos/metabolismo , Modelos Biológicos , Peptídeo Hidrolases/metabolismo , Folhas de Planta/metabolismo , Ligação Proteica , Plântula/metabolismo , Frações Subcelulares/metabolismo , Nicotiana/metabolismo , Triticum/metabolismoRESUMO
Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.