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Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.
The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.
BACKGROUND & AIMS - Bile diversion to the ileum (GB-IL) has strikingly similar metabolic and satiating effects to Roux-en-Y gastric bypass (RYGB) in rodent obesity models. The metabolic benefits of these procedures are thought to be mediated by increased bile acids, although parallel changes in body weight and other confounding variables limit this interpretation.
METHODS - Global G protein-coupled bile acid receptor-1 null (Tgr5) and intestinal-specific farnesoid X receptor null (Fxr) mice on high-fat diet as well as wild-type C57BL/6 and glucagon-like polypeptide 1 receptor deficient (Glp-1r) mice on chow diet were characterized following GB-IL.
RESULTS - GB-IL induced weight loss and improved oral glucose tolerance in Tgr5, but not Fxr mice fed a high-fat diet, suggesting a role for intestinal Fxr. GB-IL in wild-type, chow-fed mice prompted weight-independent improvements in glycemia and glucose tolerance secondary to augmented insulin responsiveness. Improvements were concomitant with increased levels of lymphatic GLP-1 in the fasted state and increased levels of intestinal Akkermansia muciniphila. Improvements in fasting glycemia after GB-IL were mitigated with exendin-9, a GLP-1 receptor antagonist, or cholestyramine, a bile acid sequestrant. The glucoregulatory effects of GB-IL were lost in whole-body Glp-1r mice.
CONCLUSIONS - Bile diversion to the ileum improves glucose homeostasis via an intestinal Fxr-Glp-1 axis. Altered intestinal bile acid availability, independent of weight loss, and intestinal Akkermansia muciniphila appear to mediate the metabolic changes observed after bariatric surgery and might be manipulated for treatment of obesity and diabetes.
Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.
The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following β-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of β-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.
Blood vessel epicardial substance (BVES) is a tight-junction associated protein that was originally discovered from a cDNA screen of the developing heart. Research over the last decade has shown that not only is BVES is expressed in cardiac and skeletal tissue, but BVES is also is expressed throughout the gastrointestinal epithelium. Mice lacking BVES sustain worse intestinal injury and inflammation. Furthermore, BVES is suppressed in gastrointestinal cancers, and mouse modeling has shown that loss of BVES promotes tumor formation. Recent work from multiple laboratories has revealed that BVES can regulate several molecular pathways, including cAMP, WNT, and promoting the degradation of the oncogene, c-Myc. This review will summarize our current understanding of how BVES regulates the intestinal epithelium and discuss how BVES functions at the molecular level to preserve epithelial phenotypes and suppress tumorigenesis.
Sepsis disproportionately affects the very old and the very young. IL-1 signaling is important in innate host defense but may also play a deleterious role in acute inflammatory conditions (including sepsis) by promulgating life-threatening inflammation. IL-1 signaling is mediated by two distinct ligands: IL-1α and IL-1β, both acting on a common receptor (IL-1R1). IL-1R1 targeting has not reduced adult human sepsis mortality despite biologic plausibility. Because the specific role of IL-1α or IL-1β in sepsis survival is unknown in any age group and the role of IL-1 signaling remains unknown in neonates, we studied the role of IL-1 signaling, including the impact of IL-1α and IL-1β, on neonatal murine sepsis survival. IL-1 signaling augments the late plasma inflammatory response to sepsis. IL-1α and not IL-1β is the critical mediator of sepsis mortality, likely because of paracrine actions within the tissue. These data do not support targeting IL-1 signaling in neonates.
Copyright © 2018 by The American Association of Immunologists, Inc.
Ca/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu is present in CaMKII complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu directly interacts with purified Thr286-autophosphorylated (activated) CaMKII However, the binding of CaMKII to this CTD fragment is reduced by the addition of excess Ca/calmodulin or by additional CaMKII autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKII is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKII with full-length mGlu expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKII with mGlu in heterologous cells. First, cell-surface biotinylation studies showed that CaMKII increases the surface expression of mGlu Second, using Ca fluorimetry and single-cell Ca imaging, we found that CaMKII reduces the initial peak of mGlu-mediated Ca mobilization by about 25% while doubling the relative duration of the Ca signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
From early unicellular organisms that formed in salty water environments to complex organisms that live on land away from water, cells have had to protect a homeostatic internal environment favorable to the biochemical reactions necessary for life. In this chapter, we will outline what steps were necessary to conserve the water within our cells and how mechanisms have evolved to maintain and regulate our cellular and organismal volume. We will first examine whole body water homeostasis and the relationship between kidney function, regulation of blood pressure, and blood filtration in the process of producing urine. We will then discuss how the composition of the lipid-rich bilayer affects its permeability to water and salts, and how the cell uses this differential to drive physiological and biochemical cellular functions. The capacity to maintain cell volume is vital to epithelial transport, neurotransmission, cell cycle, apoptosis, and cell migration. Finally, we will wrap up the chapter by discussing in some detail specific channels, cotransporters, and exchangers that have evolved to facilitate the movement of cations and anions otherwise unable to cross the lipid-rich bilayer and that are involved in maintaining or regulating cell volume.
Copyright © 2018 Elsevier Inc. All rights reserved.
IMPACT STATEMENT - Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to traditional vasodilators, and inhibition of vasospasm after SAH remains a large unmet clinical need. SAH causes changes in the phosphorylation state of the small heat shock proteins (HSPs), HSP20 and HSP27, in the vasospastic vessels. In this study, the levels of HSP27 and HSP20 were manipulated using nanotechnology to mimic the intracellular phenotype of SAH-induced vasospasm, and the effect of this manipulation was tested on vasomotor responses in intact tissues. This work provides insight into potential therapeutic targets for the development of more effective treatments for SAH induced vasospasm.
Proteomics, metabolomics, and transcriptomics generate comprehensive data sets, and current biocomputational capabilities allow their efficient integration for systems biology analysis. Published multiomics studies cover methodological advances as well as applications to biological questions. However, few studies have focused on the development of a high-throughput, unified sample preparation approach to complement high-throughput omic analytics. This report details the automation, benchmarking, and application of a strategy for transcriptomic, proteomic, and metabolomic analyses from a common sample. The approach, sample preparation for multi-omics technologies (SPOT), provides equivalent performance to typical individual omic preparation methods but greatly enhances throughput and minimizes the resources required for multiomic experiments. SPOT was applied to a multiomics time course experiment for zinc-treated HL-60 cells. The data reveal Zn effects on NRF2 antioxidant and NFkappaB signaling. High-throughput approaches such as these are critical for the acquisition of temporally resolved, multicondition, large multiomic data sets such as those necessary to assess complex clinical and biological concerns. Ultimately, this type of approach will provide an expanded understanding of challenging scientific questions across many fields.