Dataset Information

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

ABSTRACT: Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

SUBMITTER: Coles GL

PROVIDER: S-EPMC7363571 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Coles Garry L GL Cristea Sandra S Webber James T JT Levin Rebecca S RS Moss Steven M SM He Andy A Sangodkar Jaya J Hwang Yeonjoo C YC Arand Julia J Drainas Alexandros P AP Mooney Nancie A NA Demeter Janos J Spradlin Jessica N JN Mauch Brandon B Le Vicky V Shue Yan Ting YT Ko Julie H JH Lee Myung Chang MC Kong Christina C Nomura Daniel K DK Ohlmeyer Michael M Swaney Danielle L DL Krogan Nevan J NJ Jackson Peter K PK Narla Goutham G Gordan John D JD Shokat Kevan M KM Sage Julien J

Cancer cell 20200611 1

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of actio ...[more]

PMID: 32531271

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Project description:BackgroundThe pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process.ResultsTo gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle.ConclusionsThe observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile environments.

Project description:Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.

Dataset Information

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Publications

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

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