Mutant GNAS drives pancreatic tumorigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA471090 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7153226.fastq.gz | Fastqsanger.gz | |||
| SRR7153227.fastq.gz | Fastqsanger.gz | |||
| SRR7153228.fastq.gz | Fastqsanger.gz | |||
| SRR7153229.fastq.gz | Fastqsanger.gz |
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Project description:Proteome maps were generated for mouse KGC pancreatic tumor organoid lines expressing doxycycline-inducible constitutively-active mutant GNAS (GNASR201C). The organoids were analyzed in the absence (ctrl) or presence (induced) of doxycycline. Two models, KGC Line-1 (line1) and Line-2 (line2), were analyzed in two separate experiments, 1 and 2. Proteome maps were generated using multiplexed proteomics using TMT10plex technology. Mass spectrometry was done on an Orbitrap Fusion using the SPS-MS3 method. TMT labeling was done as follows: experiment 1, line1_induced_replicate1 (126), line1_ctrl_replicate1 (127n), line2_induced_replicate1 (127c), line2_ctrl_replicate1 (128n), line1_induced_replicate2 (128c), line1_ctrl_replicate2 (129n), line2_induced_replicate2 (129c), line2_ctrl_replicate2 (130c); experiment 2, line1_induced_replicate1 (126), line1_induced_replicate2 (127n), line1_induced_replicate3 (127c), line1_ctrl_replicate1 (128n), line1_ctrl_replicate2 (128c), line1_ctrl_replicate3 (129n), line2_induced_replicate1 (129c), line2_induced_replicate2 (130n), line2_ctrl_replicate1 (130c), line2_ctrl_replicate2 (131).
2018-04-23 | MSV000082305 | MassIVE
Project description:G protein αs (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that GnasR201C cooperates with KrasG12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1-3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.
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Project description: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, inhibits 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 new PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC cancer stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover novel targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
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Project description:The GNASR201 gain-of-function mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins, driving oncogenesis in various low-grade/benign gastrointestinal and pancreatic tumors. In this study, we investigated the role of GNAS and its product Gαs in tumor progression using peritoneal models of colorectal cancer (CRC). GNAS was knocked out in multiple CRC cell lines harboring GNASR201C/H mutations (KM12, SNU175, SKCO1), leading to decreased cell-growth in 2D and 3D organoid models. Nude mice were peritoneally injected with GNAS-knockout KM12 cells, leading to a decrease in tumor growth and drastically improved survival at 7 weeks. Supporting these findings, GNAS overexpression in LS174T cells led to increased cell-growth in 2D and 3D organoid models, and increased tumor growth in PDX mouse models. GNAS knockout decreased levels of cyclic AMP in KM12 cells, and molecular profiling identified phosphorylation of β-catenin and activation of its targets as critical downstream effects of mutant GNAS signaling. Supporting these findings, chemical inhibition of both PKA and β-catenin reduced growth of GNAS mutant organoids. Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/β-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors.
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Project description:Objective: Activation of the liver glucagon receptor (GCGR) promotes amino acid catabolism, which provides substrate for glucose production. Inhibition of the receptor downregulates hepatic amino acid catabolism, leading to increases in circulating amino acid levels. Amino acids serve as a potent growth factor for pancreatic alpha cells, where glucagon is produced. Thus, GCGR inhibition-induced hyperaminoacidemia causes alpha cell hyperplasia. This liver-alpha cell feedback loop, mediated by glucagon and amino acids, has been demonstrated across species, including humans. This study was designed to delineate hepatic signaling molecules that lie downstream of GCGR and mediate the liver-alpha cell loop. Methods: We used AAV8-shRNA to knock down GCGR signaling molecules, the G-coupled protein GNAS and two GNAS downstream effectors, PKA and EPAC2 (RAPGEF4), in the liver of diet-induced obese (DIO) mice. We monitored plasma amino acid and blood glucose levels and conducted pancreas histology to derive alpha and beta cell mass. We performed liver RNA-sequencing to assess expression of glucose and amino acid metabolism genes. To examine the contribution of PKA in changes associated with GCGR inhibition, we knocked down PRKAR1A, a major inhibitory subunit of PKA, to activate PKA in liver of mice administered with GCGR blocking or control antibody. Results: Comparable suppression of hepatic amino acid catabolism gene expressions, increases in plasma amino acid levels and alpha cell hyperplasia were observed in mice with hepatic knockdown of GCGR, GNAS, and PKA. Hepatic EPAC2 knockdown did not affect amino acid metabolism or alpha cell mass in mice. Mice with hepatic PKA activation alone developed hypoaminoacidemia, hypoglucagonemia and reduced alpha cell mass. Administering GCGR blocking antibody to the mice did not alter the abnormalities. Conclusions: Hepatic PKA activation in mice fully overrides the effect of GCGR inhibition on amino acids and alpha cells. In the liver, GCGR signals through PKA to control amino acid metabolism and pancreatic alpha cell mass. Hepatic PKA plays a critical role in the liver-alpha cell loop, mediated by circulating glucagon and amino acids.
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Project description:The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms in renal epithelial cells downstream of the PTH receptor that control vitamin D metabolism remain unknown. Here we demonstrate that Salt Inducible Kinases (SIKs) control renal 1,25-vit D production downstream of PTH signaling via regulating Cyp27b1 expression. As PTH inhibits the cellular activity of SIKs by cAMP-dependent PKA phosphorylation in bone, we hypothesized that SIKs would also work as an essential mediator of PTH doownstream signaling in kidney, thus regulate vitamin D metabolism. Whole tissue and single cell transcriptomics in kidney demonstrates that both PTH and pharmacologic SIK inhibitors regulate a vitamin D gene module in specific proximal tubule cells. Moreover, small molecule SIK inhibitors directly increase 1,25-vit D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice show Cyp27b1 upregulation, elevated serum levels of 1,25-vit D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 shows PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which are also required for SIK inhibitors to increase Cyp27b1 in vivo. Lastly, in a podocyte injury mouse model of chronic kidney disease (CKD) characterized by low 1,25-vit D levels, SIK inhibitor treatment stimulates both renal Cyp27b1 expression and 1,25-vit D production.
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