ABSTRACT
Diagnostic markers are desperately needed for the early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity and the differential expression of PDA markers were identified by screening single cell (sc) RNAseq from tumor samples of a mouse model for PDA (KIC) at early and late stages of PDA progression compared to that of a normal pancreas. Candidate genes were identified from three sources: (1) an unsupervised screening of the genes preferentially expressed in mouse PDA tumors; (2) signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes, or genes encoding proteins that were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines; and (3) genes whose expression is associated with poor or better prognoses (PAAD, oncolnc.org). The developmental progression of PDA was detected in the temporal order of gene expression in the cancer cells of the KIC mice. The earliest diagnostic markers were expressed in epithelial cancer cells in early-stage, but not late-stage, PDA tumors. Other early markers were expressed in the epithelium of both early- and late-state PDA tumors. Markers that were expressed somewhat later were first elevated in the epithelial cancer cells of the late-stage tumors, then in both epithelial and mesenchymal cells, or only in mesenchymal cells. Stromal markers were differentially expressed in early- and/or late-stage PDA neoplasia in fibroblast and hematopoietic cells (lymphocytes and/or macrophages) or broadly expressed in cancer and many stromal cell types. Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and the acinar-specific homozygous deletion of differentiation transcription factors (dTFs), were screened for the early expression of all PDA markers identified in the KIC neoplasia. Prognostic markers associated with a more rapid decline were identified and showed differential and cell-type-specific expression in PDA, predominately in late-stage epithelial and/or mesenchymal cancer cells. Select markers were validated by immunohistochemistry in mouse and human samples of a normal pancreas and those with early- and late-stage PDA. In total, we present 2165 individual diagnostic and prognostic markers for disease progression to be tested in humans from pancreatitis to late-stage PDA.
Subject(s)
Biomarkers, Tumor , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Pancreatitis , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/diagnosis , Carcinoma, Pancreatic Ductal/pathology , Pancreatitis/metabolism , Pancreatitis/genetics , Pancreatitis/pathology , Pancreatitis/diagnosis , Mice , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/pathology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Humans , Prognosis , Gene Expression Regulation, Neoplastic , Disease Models, Animal , Cell Line, Tumor , Disease ProgressionABSTRACT
G protein-coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gß, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gß subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2-/- mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2-/- macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gß1) deletion was lethal, but mice lacking Gnb2 (Gß2) were viable. Gnb2-/- macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gß2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.
Subject(s)
Calcium Signaling , Chemotaxis , Heterotrimeric GTP-Binding Proteins/metabolism , Macrophages/metabolism , Models, Biological , Receptor, Anaphylatoxin C5a/metabolism , Animals , Heterotrimeric GTP-Binding Proteins/genetics , Mice, Knockout , Receptor, Anaphylatoxin C5a/geneticsABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer that resists efforts to identify better chemotherapeutics. PDA is associated with chronic pancreatitis and acinar cell dedifferentiation. This reduces enzyme production by the exocrine pancreas, resulting in digestive insufficiencies. Malabsorption of partially digested food causes bloating, overfilled intestines, abdominal pain, excessive feces, steatorrhea, and malnutrition. These maladies affect quality of life and restrict treatment options for pancreatitis and PDA. Here, we characterize health benefits and risks of dietary pancreatic enzymes in three mouse models of PDA-KC, KCR8-16, and KIC. KC expresses oncogenic KrasG12D in pancreatic tissue whereas KCR8-16 also has deletions of the Rgs8 and Rgs16 genes. Rgs proteins inhibit the release of digestive enzymes evoked by G-protein-coupled-receptor agonists. KC and KCR8-16 mice developed dedifferentiated exocrine pancreata within 2 months of age and became malnourished, underweight, hypoglycemic, and hypothermic. KC mice adapted but KCR8-16 mice rapidly transitioned to starvation after mild metabolic challenges. Dietary pancreatic enzyme supplements reversed these symptoms in KC and KCR8-16 animals, and extended survival. Therefore, we tested the benefits of pancreatic enzymes in an aggressive mouse model of PDA (KIC). Median survival improved with dietary pancreatic enzyme supplements and was extended further when combined with warfarin and gemcitabine chemotherapy. However, dietary pancreatic enzymes stimulated tumor growth in the terminal stages of disease progression in KIC mice.
Subject(s)
Carcinoma, Pancreatic Ductal/complications , Malnutrition/drug therapy , Pancreatic Neoplasms/complications , Animals , Blood Glucose , Carcinoma, Pancreatic Ductal/pathology , Disease Models, Animal , Disease Progression , Eating , Female , Insulin/blood , Male , Malnutrition/etiology , Malnutrition/pathology , Mice , Pancreatic Neoplasms/pathologyABSTRACT
BACKGROUND: Mutations activating the α subunit of heterotrimeric Gs protein are associated with a number of highly specific pathological molecular phenotypes. One of the best characterized is the McCune Albright syndrome. The disease presents with an increased incidence of neoplasias in specific tissues. MAIN BODY: A similar repertoire of neoplasms can develop whether mutations occur spontaneously in somatic tissues during fetal development or after birth. Glands are the most "permissive" tissues, recently found to include the entire gastrointestinal tract. High frequency of activating Gαs mutations is associated with precise diagnoses (e.g., IPMN, Pyloric gland adenoma, pituitary toxic adenoma). Typically, most neoplastic lesions, from thyroid to pancreas, remain well differentiated but may be a precursor to aggressive cancer. CONCLUSIONS: Here we propose the possibility that gain-of-function mutations of Gαs interfere with signals in the microenvironment of permissive tissues and lead to a transversal neoplastic phenotype.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs/genetics , Gain of Function Mutation , Neoplasms/pathology , Humans , Neoplasms/genetics , PhenotypeABSTRACT
Proper maintenance of mature cellular phenotypes is essential for stable physiology, suppression of disease states, and resistance to oncogenic transformation. We describe the transcriptional regulatory roles of four key DNA-binding transcription factors (Ptf1a, Nr5a2, Foxa2 and Gata4) that sit at the top of a regulatory hierarchy controlling all aspects of a highly differentiated cell-type-the mature pancreatic acinar cell (PAC). Selective inactivation of Ptf1a, Nr5a2, Foxa2 and Gata4 individually in mouse adult PACs rapidly altered the transcriptome and differentiation status of PACs. The changes most emphatically included transcription of the genes for the secretory digestive enzymes (which conscript more than 90% of acinar cell protein synthesis), a potent anabolic metabolism that provides the energy and materials for protein synthesis, suppressed and properly balanced cellular replication, and susceptibility to transformation by oncogenic KrasG12D. The simultaneous inactivation of Foxa2 and Gata4 caused a greater-than-additive disruption of gene expression and uncovered their collaboration to maintain Ptf1a expression and control PAC replication. A measure of PAC dedifferentiation ranked the effects of the conditional knockouts as Foxa2+Gata4 > Ptf1a > Nr5a2 > Foxa2 > Gata4. Whereas the loss of Ptf1a or Nr5a2 greatly accelerated Kras-mediated transformation of mature acinar cells in vivo, the absence of Foxa2, Gata4, or Foxa2+Gata4 together blocked transformation completely, despite extensive dedifferentiation. A lack of correlation between PAC dedifferentiation and sensitivity to oncogenic KrasG12D negates the simple proposition that the level of differentiation determines acinar cell resistance to transformation.
Subject(s)
Pancreas, Exocrine , Pancreatic Neoplasms , Mice , Animals , Acinar Cells/metabolism , Epithelium/metabolism , Transcription Factors/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Phenotype , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolismABSTRACT
G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ß-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.
Subject(s)
Fatty Acids/metabolism , Nuclear Proteins/physiology , RGS Proteins/physiology , Transcription Factors/physiology , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Fatty Acids/biosynthesis , Fatty Acids/genetics , Gluconeogenesis , Glucose/biosynthesis , Glucose/physiology , Hepatocytes/metabolism , Liver/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Oxidation-Reduction , Receptors, G-Protein-Coupled/metabolism , Transcription, GeneticABSTRACT
The recent paper by Pfeil et al., "Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gßγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs", opens another path from biochemical in vitro reconstitution to understanding the complex regulation of calcium signaling inside the cell.
Subject(s)
Calcium , GTP-Binding Protein alpha Subunits, Gq-G11 , Calcium/metabolism , Calcium Signaling , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Phospholipase C beta/metabolismABSTRACT
Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA. IMPLICATIONS: These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Plasticity/physiology , Neoplasm Metastasis/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Cell Line, Tumor , Cell Plasticity/drug effects , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/physiology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/drug therapy , Tumor Microenvironment/drug effects , Tumor Microenvironment/physiology , Gemcitabine , Axl Receptor Tyrosine KinaseABSTRACT
The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5' GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Gene Expression Regulation, Neoplastic/genetics , Pancreatic Neoplasms/genetics , CRISPR-Cas Systems , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Movement/genetics , GTP-Binding Proteins/metabolism , Gene Expression/genetics , Humans , Methylation , Neoplasm Invasiveness/genetics , Pancreatic Neoplasms/pathology , Prognosis , Promoter Regions, Genetic/genetics , RNA, Messenger , RNA, Small Interfering , Signal TransductionABSTRACT
PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Acinar Cells/drug effects , Acinar Cells/metabolism , Acinar Cells/pathology , Adenocarcinoma/metabolism , Animals , Calcium/metabolism , Carcinogenesis/drug effects , Carcinogenesis/metabolism , Carcinogenesis/pathology , Carcinoma, Pancreatic Ductal/metabolism , Cell Dedifferentiation/drug effects , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , Ceruletide/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Disease Progression , GTP-Binding Proteins/metabolism , Histone Deacetylase Inhibitors/pharmacology , Mice , Pancreatic Ducts/drug effects , Pancreatic Ducts/metabolism , Pancreatic Neoplasms/metabolism , Pancreatitis/drug therapy , Pancreatitis/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , RGS Proteins/metabolism , Signal Transduction/drug effects , Gemcitabine , Pancreatic NeoplasmsABSTRACT
Large G protein alpha subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Galpha subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein-protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Galpha subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Cell Division/physiology , GTP-Binding Protein alpha Subunits/metabolism , Models, Biological , Nuclear Proteins/metabolism , RGS Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Caenorhabditis elegans Proteins/genetics , Cell Division/genetics , GTP-Binding Protein alpha Subunits/genetics , Guanine Nucleotide Exchange Factors , Hormones/metabolism , Microtubules/physiology , Nuclear Proteins/genetics , Phylogeny , RGS Proteins/genetics , Receptors, G-Protein-Coupled/genetics , Signal Transduction/geneticsABSTRACT
BACKGROUND: Heterotrimeric G protein signaling in liver helps maintain carbohydrate and lipid homeostasis. G protein signaling is activated by binding of extracellular ligands to G protein coupled receptors and inhibited inside cells by regulators of G protein signaling (RGS) proteins. RGS proteins are GTPase activating proteins, and thereby regulate Gi and/or Gq class G proteins. RGS gene expression can be induced by the ligands they feedback regulate, and RGS gene expression can be used to mark tissues and cell-types when and where Gi/q signaling occurs. We characterized the expression of mouse RGS genes in liver during fasting and refeeding to identify novel signaling pathways controlling changes in liver metabolism. RESULTS: Rgs16 is the only RGS gene that is diurnally regulated in liver of ad libitum fed mice. Rgs16 transcription, mRNA and protein are up regulated during fasting and rapidly down regulated after refeeding. Rgs16 is expressed in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominates. Restricting feeding to 4 hr of the light phase entrained Rgs16 expression in liver but did not affect circadian regulation of Rgs16 expression in the suprachiasmatic nuclei (SCN). CONCLUSION: Rgs16 is one of a subset of genes that is circadian regulated both in SCN and liver. Rgs16 mRNA expression in liver responds rapidly to changes in feeding schedule, coincident with key transcription factors controlling the circadian clock. Rgs16 expression can be used as a marker to identify and investigate novel G-protein mediated metabolic and circadian pathways, in specific zones within the liver.
ABSTRACT
Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.
Subject(s)
Antineoplastic Agents/pharmacology , Lactams, Macrocyclic/pharmacology , Microtubules/drug effects , Neurons/drug effects , Oxazoles/pharmacology , Vinca Alkaloids/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Genomics , Humans , Lactams, Macrocyclic/chemistry , Mice , Microscopy, Electron , Mitosis , Neoplasm Transplantation , Oxazoles/chemistry , Tubulin/chemistry , Vinblastine/analogs & derivatives , Vinblastine/chemistry , Vinblastine/pharmacology , Vinca Alkaloids/chemistry , VinorelbineABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States, and is projected to be second by 2025. It has the worst survival rate among all major cancers. Two pressing needs for extending life expectancy of affected individuals are the development of new approaches to identify improved therapeutics, addressed herein, and the identification of early markers. PDA advances through a complex series of intercellular and physiological interactions that drive cancer progression in response to organ stress, organ failure, malnutrition, and infiltrating immune and stromal cells. Candidate drugs identified in organ culture or cell-based screens must be validated in preclinical models such as KIC (p48(Cre);LSL-Kras(G12D);Cdkn2a(f/f)) mice, a genetically engineered model of PDA in which large aggressive tumors develop by 4 weeks of age. We report a rapid, systematic and robust in vivo screen for effective drug combinations to treat Kras-dependent PDA. Kras mutations occur early in tumor progression in over 90% of human PDA cases. Protein kinase and G-protein coupled receptor (GPCR) signaling activates Kras. Regulators of G-protein signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback-regulate GPCR signaling. We crossed Rgs16::GFP bacterial artificial chromosome (BAC) transgenic mice with KIC mice and show that the Rgs16::GFP transgene is a Kras(G12D)-dependent marker of all stages of PDA, and increases proportionally to tumor burden in KIC mice. RNA sequencing (RNA-Seq) analysis of cultured primary PDA cells reveals characteristics of embryonic progenitors of pancreatic ducts and endocrine cells, and extraordinarily high expression of the receptor tyrosine kinase Axl, an emerging cancer drug target. In proof-of-principle drug screens, we find that weanling KIC mice with PDA treated for 2 weeks with gemcitabine (with or without Abraxane) plus inhibitors of Axl signaling (warfarin and BGB324) have fewer tumor initiation sites and reduced tumor size compared with the standard-of-care treatment. Rgs16::GFP is therefore an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens such as Axl-targeted agents. This screening strategy can potentially be applied to identify improved therapeutics for other cancers.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , Drug Evaluation, Preclinical , Pancreatic Neoplasms/drug therapy , Albumin-Bound Paclitaxel/pharmacology , Albumin-Bound Paclitaxel/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Biological Assay , Carcinogenesis/pathology , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Genes, Reporter , Green Fluorescent Proteins/metabolism , Humans , Mice , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , RGS Proteins/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Gemcitabine , Axl Receptor Tyrosine Kinase , Pancreatic NeoplasmsABSTRACT
Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) for alpha subunits of the Gi and/or Gq class of heterotrimeric G proteins. RGS GAP activity is inhibited by phosphatidic acid (PA), lysophosphatidic acid (LPA), and phosphatidylinositol 3,4,5-trisphosphate (PIP3) but not by other phospholipids, phosphoinositides, or diacylglycerol. Both PA and PIP3 can inhibit RGS4 GAP activity and their inhibition is additive, suggesting that PA and PIP3 interact with different domains of RGS4. The N terminus of RGS4 (1-57 amino acids) is required for PA binding and inhibition. Mutation at Lys20, far from the RGS domain of RGS4, decreases PA-mediated inhibition of RGS4 by more than 85%. Amino acid substitutions in helix 5 within the RGS domain of RGS4, opposite to the RGS/Galpha protein contact face, reduce binding affinity and inhibition by PIP3. Calmodulin binds all RGS proteins tested in a Ca(2+)-dependent manner at two sites, one in the N-terminal 33 amino acids and another in the RGS domain. Ca2+/calmodulin does not directly affect GAP activity of RGS4 but reverses PA and PIP3-mediated inhibition. In summary, these results demonstrate that phospholipids such as PA and PIP3 act as allosteric inhibitors of RGS proteins, and Ca2+/calmodulin competition with PA and PIP3 may provide an intracellular mechanism for feedback regulation of Ca2+ signaling evoked by G-protein-coupled agonists.
Subject(s)
Allosteric Regulation , GTP-Binding Protein Regulators/metabolism , GTPase-Activating Proteins/antagonists & inhibitors , Phospholipids/metabolism , RGS Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Amino Acid Substitution , Animals , Calcium/metabolism , Calmodulin/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go , GTPase-Activating Proteins/chemistry , Humans , Point Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , RGS Proteins/chemistry , RGS Proteins/geneticsABSTRACT
Regulators of G-protein signaling (RGS) play a critical role in G-protein-coupled receptor signaling in mammalian cells. RGS proteins are GTPase-accelerating proteins (GAPs) for alpha subunits of heterotrimeric G proteins of the Gi and Gq class. RGS GAPs can modulate the frequency and duration of G-protein signaling and may constitute a new family of therapeutic targets. Identifying the tissue distribution and cellular localization of RGS proteins has been hindered by the lack of effective antibodies for immunodetection. The measurement of mRNA levels for RGS proteins, however, can provide insight into their tissue specificity and regulation. This article describes the use of a highly sensitive and rapid method for measuring RGS mRNA in mouse tissues. This quantitative real-time polymerase chain reaction method is established for the 19 reported mouse RGS genes and is used to study the tissue distribution of the R4 family of RGS genes and the diurnal regulation of RGS16 in mouse liver.
Subject(s)
Mice/metabolism , RGS Proteins/physiology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Signal Transduction , Animals , Forecasting , Gene Expression Regulation , Humans , Male , Mice, Inbred C57BL , RGS Proteins/analysis , RGS Proteins/chemistry , RGS Proteins/genetics , Sensitivity and Specificity , Tissue DistributionABSTRACT
AIMS/HYPOTHESIS: Pathological cardiac hypertrophy is an early phenotype in both types 1 and 2 diabetes. The primary stimulus for hypertrophic growth in diabetes is yet unknown and may involve neurohumoral stimulation of Gq-coupled receptors as well as direct glucose-dependent mechanisms. To discriminate between these hypertrophic stimuli we analyzed hypertrophic signalling pathways in wildtype and Gα11-knockout mice. METHODS: Experimental diabetes was induced in wildtype and knockout mice by intraperitoneal injection of streptozotocin. 8 weeks after induction of diabetes myocardial function and structure was assessed by echocardiography before sacrifice. To identify prohypertrophic signalling pathways expression and translocation of protein kinase C isoforms α, ßII, δ, ε and ζ were analyzed by immunohistochemical staining and immunoblot analysis after tissue fractionation. Changes in calcineurin signalling were identified by immunoblot analysis and functional assays. Expression levels of transcription factors GATA4 and NF-κB were quantified by real-time RT-PCR. RESULTS: Diabetic wildtype mice developed myocardial hypertrophy with preserved cardiac function. Calcineurin signalling was not different between the two groups. However, diabetic wildtype mice showed increased protein levels of PKC-α and PKC-ζ, translocation of PKC-α, -δ and -ε to cellular membranes and higher levels of NF-κB expression. In contrast, diabetic Gα11-knockout mice showed no altered phenotype and no changes in NF-κB or PKC expression, although translocation of PKC-ε occurred as in wildtypes. CONCLUSIONS: Gα11 is essential for the development of cardiac hypertrophy in type 1-diabetes. Stimulation of hypertrophic signalling through PKC-α, PKC-δ, PKC-ζ, and NF-κB appears to be receptor-dependent, whereas PKC-ε is activated by hyperglycemia, independent of Gα11.
Subject(s)
Cardiomegaly/metabolism , Diabetes Mellitus, Experimental/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/deficiency , Myocardium/metabolism , Signal Transduction/physiology , Animals , Cardiomegaly/pathology , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/pathology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/pathologyABSTRACT
Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic beta-cells. Although beta-cells can replicate in younger adults, the available diabetes therapies do not specifically target beta-cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and beta-cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates beta-cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal-associated cells and islet beta-cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated beta-cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and beta-cell expansion in embryos and metabolically stressed adults.
Subject(s)
Diabetes Mellitus, Type 1/embryology , Diabetes Mellitus, Type 1/pathology , Islets of Langerhans/embryology , Islets of Langerhans/pathology , RGS Proteins/metabolism , Aging/drug effects , Aging/pathology , Animals , Animals, Newborn , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/genetics , Disease Models, Animal , Exenatide , Female , Gene Expression Regulation, Developmental/drug effects , Green Fluorescent Proteins/metabolism , Hyperglycemia/complications , Hyperglycemia/pathology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Mice , Mice, Obese , Peptides/pharmacology , Pregnancy , RGS Proteins/genetics , Recombinant Fusion Proteins/metabolism , Regeneration/drug effects , Stem Cells/drug effects , Stem Cells/metabolism , Venoms/pharmacologySubject(s)
GTPase-Activating Proteins/metabolism , Guanosine Triphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Oligopeptides/chemistry , RGS Proteins/chemistry , GTPase-Activating Proteins/chemistry , Guanosine Diphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/chemistry , Kinetics , Protein Subunits , RGS Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Endothelin-A (ET(A)) is a G-protein-coupled receptor expressed in the neural crest-derived mesenchyme of the pharyngeal arches during craniofacial development. Targeted deletion of the ET(A) receptor or its ligand endothelin-1 (ET-1) causes cleft palate and hypoplasia of the mandible, otic cup, and tympanic ring. Previously we showed that Galpha(q)/Galpha(11)-null mice die around E11.0, whereas Galpha(q)((-/-))Galpha(11)((+/-)) mice survive to birth with hypomorphic phenotypes similar to, but less severe than, ET(A) or ET-1-null mice. To determine whether ET-1 signaling is transduced by Galpha(q)/Galpha(11) proteins, we examined the expression patterns of several ET-1 dependent and independent transcription factors in Galpha(q)/Galpha(11)-deficient embryos. Expression of genes encoding the ET-1-dependent transcription factors Dlx3, Dlx6, dHAND, and eHAND was specifically downregulated in the pharyngeal arches of Galpha(q)/Galpha(11)-deficient mice. In contrast, pharyngeal arch expression of the homeobox gene Msx1, which is not regulated by ET-1 signaling, was maintained in these embryos. We conclude that the Galpha(q) and Galpha(11) proteins serve as the intracellular mediators of ET-1 signaling in the pharyngeal arch mesenchyme.