Publications by authors named "Anne Bresnick"

65 Publications

Nox4-dependent upregulation of S100A4 after peripheral nerve injury modulates neuropathic pain processing.

Free Radic Biol Med 2021 05 28;168:155-167. Epub 2021 Mar 28.

Institute of Pharmacology and Clinical Pharmacy, Goethe University, 60438 Frankfurt am Main, Germany. Electronic address:

Previous studies suggested that reactive oxygen species (ROS) produced by NADPH oxidase 4 (Nox4) affect the processing of neuropathic pain. However, mechanisms underlying Nox4-dependent pain signaling are incompletely understood. In this study, we aimed to identify novel Nox4 downstream interactors in the nociceptive system. Mice lacking Nox4 specifically in sensory neurons were generated by crossing Advillin-Cre mice with Nox4 mice. Tissue-specific deletion of Nox4 in sensory neurons considerably reduced mechanical hypersensitivity and neuronal action potential firing after peripheral nerve injury. Using a proteomic approach, we detected various proteins that are regulated in a Nox4-dependent manner after injury, including the small calcium-binding protein S100A4. Immunofluorescence staining and Western blot experiments confirmed that S100A4 expression is massively up-regulated in peripheral nerves and dorsal root ganglia after injury. Furthermore, mice lacking S100A4 showed increased mechanical hypersensitivity after peripheral nerve injury and after delivery of a ROS donor. Our findings suggest that S100A4 expression is up-regulated after peripheral nerve injury in a Nox4-dependent manner and that deletion of S100A4 leads to an increased neuropathic pain hypersensitivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2021.03.021DOI Listing
May 2021

Absence of S100A4 in the mouse lens induces an aberrant retina-specific differentiation program and cataract.

Sci Rep 2021 Jan 26;11(1):2203. Epub 2021 Jan 26.

Department of Ophthalmology, Duke University School of Medicine, Durham, NC, USA.

S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4 knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4 mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4 lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4 knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-81611-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838418PMC
January 2021

Author Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

Nature 2020 Jul;583(7814):E15

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2322-9DOI Listing
July 2020

PI3Kβ is selectively required for growth factor-stimulated macropinocytosis.

J Cell Sci 2019 08 16;132(16). Epub 2019 Aug 16.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Macropinocytosis is an actin-dependent but clathrin-independent endocytic process by which cells nonselectively take up large aliquots of extracellular material. Macropinocytosis is used for immune surveillance by dendritic cells, as a route of infection by viruses and protozoa, and as a nutrient uptake pathway in tumor cells. In this study, we explore the role of class I phosphoinositide 3-kinases (PI3Ks) during ligand-stimulated macropinocytosis. We find that macropinocytosis in response to receptor tyrosine kinase activation is strikingly dependent on a single class I PI3K isoform, namely PI3Kβ (containing the p110β catalytic subunit encoded by ). Loss of PI3Kβ expression or activity blocks macropinocytosis at early steps, before the formation of circular dorsal ruffles, but also plays a role in later steps, downstream from Rac1 activation. PI3Kβ is also required for the elevated levels of constitutive macropinocytosis found in tumor cells that are defective for the PTEN tumor suppressor. Our data shed new light on PI3K signaling during macropinocytosis, and suggest new therapeutic uses for pharmacological inhibitors of PI3Kβ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.231639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737911PMC
August 2019

PI3Kβ links integrin activation and PI(3,4)P production during invadopodial maturation.

Mol Biol Cell 2019 08 18;30(18):2367-2376. Epub 2019 Jul 18.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.

The invasion of tumor cells from the primary tumor is mediated by invadopodia, actin-rich protrusive organelles that secrete matrix metalloproteases and degrade the extracellular matrix. This coupling between protrusive activity and matrix degradation facilitates tumor invasion. We previously reported that the PI3Kβ isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and G protein-coupled receptors, is required for invasion and gelatin degradation in breast cancer cells. We have now defined the mechanism by which PI3Kβ regulates invadopodia. We find that PI3Kβ is specifically activated downstream from integrins, and is required for integrin-stimulated spreading and haptotaxis as well as integrin-stimulated invadopodia formation. Surprisingly, these integrin-stimulated and PI3Kβ-dependent responses require the production of PI(3,4)P by the phosphoinositide 5'-phosphatase SHIP2. Thus, integrin activation of PI3Kβ is coupled to the SHIP2-dependent production of PI(3,4)P which regulates the recruitment of PH domain-containing scaffolds such as lamellipodin to invadopodia. These findings provide novel mechanistic insight into the role of PI3Kβ in the regulation of invadopodia in breast cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E19-03-0182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6741064PMC
August 2019

CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release.

Elife 2019 06 7;8. Epub 2019 Jun 7.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, United States.

Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.35546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592687PMC
June 2019

A single discrete Rab5-binding site in phosphoinositide 3-kinase β is required for tumor cell invasion.

J Biol Chem 2019 03 18;294(12):4621-4633. Epub 2019 Jan 18.

From the Departments of Molecular Pharmacology and

Phosphoinositide 3-kinase β (PI3Kβ) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln and Ile) in the helical domain of the catalytic subunit (p110β) of PI3Kβ whose mutation disrupts binding to Rab5. To better define the Rab5-p110β interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an pulldown assay with GST-Rab5 Of the 35 p110β helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gβγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110β as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln and Ile residues. Analysis of the Rab5-PI3Kβ interaction by hydrogen-deuterium exchange MS identified p110β peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110β or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110β had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kβ activity , the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110β helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kβ coupling .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.006032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433078PMC
March 2019

PI3Kβ-A Versatile Transducer for GPCR, RTK, and Small GTPase Signaling.

Endocrinology 2019 03;160(3):536-555

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York.

The phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein-coupled receptors (GPCRs): the class I enzymes PI3Kβ and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kβ is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kβ by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kβ activity in vitro and in cells, and then summarizes the biology of PI3Kβ signaling in distinct tissues and in human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2018-00843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375709PMC
March 2019

S100 proteins as therapeutic targets.

Authors:
Anne R Bresnick

Biophys Rev 2018 Dec 31;10(6):1617-1629. Epub 2018 Oct 31.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.

The human genome codes for 21 S100 protein family members, which exhibit cell- and tissue-specific expression patterns. Despite sharing a high degree of sequence and structural similarity, the S100 proteins bind a diverse range of protein targets and contribute to a broad array of intracellular and extracellular functions. Consequently, the S100 proteins regulate multiple cellular processes such as proliferation, migration and/or invasion, and differentiation, and play important roles in a variety of cancers, autoimmune diseases, and chronic inflammatory disorders. This review focuses on the development of S100 neutralizing antibodies and small molecule inhibitors and their potential therapeutic use in controlling disease progression and severity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12551-018-0471-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297089PMC
December 2018

Publisher Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

Nature 2018 10;562(7725):E3

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.

Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0355-0DOI Listing
October 2018

Myosin-IIA heavy chain phosphorylation on S1943 regulates tumor metastasis.

Exp Cell Res 2018 09 25;370(2):273-282. Epub 2018 Jun 25.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, United States. Electronic address:

Nonmuscle myosin-IIA (NMHC-IIA) heavy chain phosphorylation has gained recognition as an important feature of myosin-II regulation. In previous work, we showed that phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances EGF-stimulated lamellipod extension of breast tumor cells. However, the contribution of NMHC-IIA S1943 phosphorylation to the modulation of invasive cellular behavior and metastasis has not been examined. Stable expression of phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA in breast cancer cells revealed that S1943 phosphorylation enhances invadopodia function, and is critical for matrix degradation in vitro and experimental metastasis in vivo. These studies demonstrate a novel link between NMHC-IIA S1943 phosphorylation, the regulation of extracellular matrix degradation and tumor cell invasion and metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2018.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117828PMC
September 2018

A naturally occurring antiviral ribonucleotide encoded by the human genome.

Nature 2018 06 20;558(7711):610-614. Epub 2018 Jun 20.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus and HIV. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0238-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026066PMC
June 2018

Advantages of Molecular Weight Identification during Native MS Screening.

Planta Med 2018 Nov 9;84(16):1201-1212. Epub 2018 May 9.

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Australia.

Native mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6----glucoside 8----arabinoside, sweroside, 4',5-dihydroxy-7-methoxyflavanone-6--rutinoside, loganin acid, 6--glucosylnaringenin, biochanin A 7--rutinoside and quercetin 3--rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/a-0608-4870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195442PMC
November 2018

S100A4 regulates macrophage invasion by distinct myosin-dependent and myosin-independent mechanisms.

Mol Biol Cell 2018 03 27;29(5):632-642. Epub 2017 Dec 27.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461

S100A4, a member of the S100 family of Ca-binding proteins, is a key regulator of cell migration and invasion. Our previous studies showed that bone marrow-derived macrophages from S100A4 mice exhibit defects in directional motility and chemotaxis in vitro and reduced recruitment to sites of inflammation in vivo. We now show that the loss of S100A4 produces two mechanistically distinct phenotypes with regard to macrophage invasion: a defect in matrix degradation, due to a disruption of podosome rosettes caused by myosin-IIA overassembly, and a myosin-independent increase in microtubule acetylation, which increases podosome rosette stability and is sufficient to inhibit macrophage invasion. Our studies point to S100A4 as a critical regulator of matrix degradation, whose actions converge on the dynamics and degradative functions of podosome rosettes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E17-07-0460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004585PMC
March 2018

Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ.

Biochem J 2017 11 16;474(23):3903-3914. Epub 2017 Nov 16.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, U.S.A.

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kβ, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gβγ subunits to the C2-helical domain linker of p110β, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110β. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gβγ led to an additive PI3Kβ-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kβ that cannot bind Gβγ, Gβγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kβ was blocked. Surprisingly, PI3Kβ-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110β that cannot bind Rac1. Instead of directly binding to PI3Kβ, CA-Rac1 acts by enhancing Gβγ coupling to PI3Kβ, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gβγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kβ by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gβγ coupling to PI3Kβ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BCJ20170279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858185PMC
November 2017

Deficiency in Calcium-Binding Protein S100A4 Impairs the Adjuvant Action of Cholera Toxin.

Front Immunol 2017 11;8:1119. Epub 2017 Sep 11.

Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg Vaccine Research Institute (GUVAX), Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

The calcium-binding protein S100A4 has been described to promote pathological inflammation in experimental autoimmune and inflammatory disorders and in allergy and to contribute to antigen presentation and antibody response after parenteral immunization with an alum-adjuvanted antigen. In this study, we extend these findings by demonstrating that mice lacking S100A4 have a defective humoral and cellular immune response to mucosal (sublingual) immunization with a model protein antigen [ovalbumin (OVA)] given together with the strong mucosal adjuvant cholera toxin (CT), and that this impairment is due to defective adjuvant-stimulated antigen presentation by antigen-presenting cells. In comparison to wild-type (WT) mice, mice genetically lacking S100A4 had reduced humoral and cellular immune responses after immunization with OVA plus CT, including a complete lack of detectable germinal center reaction. Further, when stimulated with OVA plus CT, S100A4 dendritic cells (DCs) showed impaired responses in several CT-stimulated immune regulatory molecules including the co-stimulatory molecule CD86, inflammasome-associated caspase-1 and IL-1β. Coculture of OVA-specific OT-II T cells with S100A4 DCs that had been pulse incubated with OVA plus CT resulted in impaired OT-II T cell proliferation and reduced production of Th1, Th2, and Th17 cytokines compared to similar cocultures with WT DCs. In accordance with these findings, transfection of WT DCs with S100A4-targeting small interfering RNA (siRNA) but not mock-siRNA resulted in significant reductions in the expression of caspase-1 and IL-1β as well as CD86 in response to CT. Importantly, also engraftment of WT DCs into S100A4 mice effectively restored the immune response to immunization in the recipients. In conclusion, our results demonstrate that deficiency in S100A4 has a strong impact on the development of both humoral and cellular immunity after mucosal immunization using CT as adjuvant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.01119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600718PMC
September 2017

GPCR Signaling Mediates Tumor Metastasis via PI3Kβ.

Cancer Res 2016 05 24;76(10):2944-53. Epub 2016 Mar 24.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York.

Inappropriate activation of PI3K signaling has been implicated strongly in human cancer. Although studies on the role of PI3K signaling in breast tumorigenesis and progression have focused most intensively on PI3Kα, a role for PI3Kβ has begun to emerge. The PI3Kβ isoform is unique among class IA PI3K enzymes in that it is activated by both receptor tyrosine kinases and G-protein-coupled receptors (GPCR). In previous work, we identified a mutation that specifically abolishes PI3Kβ binding to Gβγ (p110(526KK-DD)). Expression of this mutant in p110β-silenced breast cancer cells inhibits multiple steps of the metastatic cascade in vitro and in vivo and causes a cell autonomous defect in invadopodial matrix degradation. Our results identify a novel link between GPCRs and PI3Kβ in mediating metastasis, suggesting that disruption of this link might offer a novel therapeutic target to prevent the development of metastatic disease. Cancer Res; 76(10); 2944-53. ©2016 AACR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-15-1675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915222PMC
May 2016

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer.

J Biol Chem 2015 Dec 16;290(51):30390-405. Epub 2015 Oct 16.

From the Department of Molecular Pharmacology, Department of Biochemistry,

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.689604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683262PMC
December 2015

FLT1 signaling in metastasis-associated macrophages activates an inflammatory signature that promotes breast cancer metastasis.

J Exp Med 2015 Aug 10;212(9):1433-48. Epub 2015 Aug 10.

MRC and University of Edinburgh Centre for Reproductive Health, Queen's Medical Research Institute; and Edinburgh Cancer Research UK Centre, MRC Institute of Genetics and Molecular Medicine; University of Edinburgh, Edinburgh EH16 4TJ, Scotland, UK Department of Developmental and Molecular Biology and Department of Biochemistry, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461

Although the link between inflammation and cancer initiation is well established, its role in metastatic diseases, the primary cause of cancer deaths, has been poorly explored. Our previous studies identified a population of metastasis-associated macrophages (MAMs) recruited to the lung that promote tumor cell seeding and growth. Here we show that FMS-like tyrosine kinase 1 (Flt1, also known as VEGFR1) labels a subset of macrophages in human breast cancers that are significantly enriched in metastatic sites. In mouse models of breast cancer pulmonary metastasis, MAMs uniquely express FLT1. Using several genetic models, we show that macrophage FLT1 signaling is critical for metastasis. FLT1 inhibition does not affect MAM recruitment to metastatic lesions but regulates a set of inflammatory response genes, including colony-stimulating factor 1 (CSF1), a central regulator of macrophage biology. Using a gain-of-function approach, we show that CSF1-mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity of FLT1-inhibited MAMs. Thus, CSF1 is epistatic to FLT1, establishing a link between FLT1 and inflammatory responses within breast tumor metastases. Importantly, FLT1 inhibition reduces tumor metastatic efficiency even after initial seeding, suggesting that these pathways represent therapeutic targets in metastatic disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20141555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548055PMC
August 2015

Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling.

Mol Biol Cell 2015 Jun 15;26(12):2279-97. Epub 2015 Apr 15.

Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada

We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1-6 capped actin filaments efficiently, whereas FliI GLD 2-6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1-6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2-6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2-6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1-6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E14-11-1536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462945PMC
June 2015

Identification of the Rab5 binding site in p110β: assays for PI3Kβ binding to Rab5.

Methods Mol Biol 2015 ;1298:271-81

Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.

Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110β, and p110δ), p110β has unique properties. Unlike the other family members, p110β directly binds to Gβγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110β binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110β signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110β, and discusses methods for the analysis of p110β-Rab5 interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-2569-8_23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440688PMC
March 2016

S100 proteins in cancer.

Nat Rev Cancer 2015 Feb;15(2):96-109

Center for Biomolecular Therapeutics and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 20102, USA.

In humans, the S100 protein family is composed of 21 members that exhibit a high degree of structural similarity, but are not functionally interchangeable. This family of proteins modulates cellular responses by functioning both as intracellular Ca(2+) sensors and as extracellular factors. Dysregulated expression of multiple members of the S100 family is a common feature of human cancers, with each type of cancer showing a unique S100 protein profile or signature. Emerging in vivo evidence indicates that the biology of most S100 proteins is complex and multifactorial, and that these proteins actively contribute to tumorigenic processes such as cell proliferation, metastasis, angiogenesis and immune evasion. Drug discovery efforts have identified leads for inhibiting several S100 family members, and two of the identified inhibitors have progressed to clinical trials in patients with cancer. This Review highlights new findings regarding the role of S100 family members in cancer diagnosis and treatment, the contribution of S100 signalling to tumour biology, and the discovery and development of S100 inhibitors for treating cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrc3893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4369764PMC
February 2015

Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration.

Sci Signal 2014 Nov 25;7(353):ra112. Epub 2014 Nov 25.

Department of Pathology, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY 10467, USA.

Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)-responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding Mena(INV), an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down Mena(INV) or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that Mena(INV) and TMEM frequency are correlated prognostic markers and CSF-1 and Mena(INV) may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.2005329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266931PMC
November 2014

A generally applicable translational strategy identifies S100A4 as a candidate gene in allergy.

Sci Transl Med 2014 Jan;6(218):218ra4

The Center for Individualized Medication, Department of Clinical and Experimental Medicine, Linköping University, 581 85 Linköping, Sweden.

The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (TH2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.3007410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539009PMC
January 2014

Structure of the S100A4/myosin-IIA complex.

BMC Struct Biol 2013 Nov 20;13:31. Epub 2013 Nov 20.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Background: S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood.

Results: We determined the X-ray crystal structure of the S100A4Δ8C/MIIA(1908-1923) peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA(1908-1923) peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site.

Conclusion: The structure of the S100A4/MIIA(1908-1923) peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6807-13-31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924328PMC
November 2013

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs).

Proc Natl Acad Sci U S A 2013 Nov 4;110(47):18862-7. Epub 2013 Nov 4.

Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1304801110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839701PMC
November 2013

The heavy chain has its day: regulation of myosin-II assembly.

Bioarchitecture 2013 Jul-Aug;3(4):77-85

Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201608PMC
http://dx.doi.org/10.4161/bioa.26133DOI Listing
January 2015

Wiskott-Aldrich syndrome protein regulates leukocyte-dependent breast cancer metastasis.

Cell Rep 2013 Aug 1;4(3):429-36. Epub 2013 Aug 1.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

A paracrine interaction between epidermal growth factor (EGF)-secreting tumor-associated macrophages (TAMs) and colony-stimulating factor 1 (CSF-1)-secreting breast carcinoma cells promotes invasion and metastasis. Here, we show that mice deficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable to support TAM-dependent carcinoma cell invasion and metastasis in both orthotopic and transgenic models of mammary tumorigenesis. Motility and invasion defects of tumor cells were recapitulated ex vivo upon coculture with WASp(-/-) macrophages. Mechanistically, WASp is required for macrophages to migrate toward CSF-1-producing carcinoma cells, as well as for the release of EGF through metalloprotease-dependent shedding of EGF from the cell surface of macrophages. Our findings suggest that WASp acts to support both the migration of TAMs and the production of EGF, which in concert promote breast tumor metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2013.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777703PMC
August 2013

G protein-coupled receptor-mediated activation of p110β by Gβγ is required for cellular transformation and invasiveness.

Sci Signal 2012 Dec 4;5(253):ra89. Epub 2012 Dec 4.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β-dependent functions, we identified the site in p110β that binds to the Gβγ subunit of G proteins. Mutation of this site eliminated Gβγ-dependent activation of PI3Kβ (a dimer of p110β and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide-mediated activation. Disrupting the p110β-Gβγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kβ in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kβ could provide a therapeutic approach for tumors that depend on p110β for growth and metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.2003264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979326PMC
December 2012

Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens.

Mol Biol Cell 2012 Feb 4;23(4):533-42. Epub 2012 Jan 4.

Department of Neurology, Columbia University, New York, NY 10032, USA.

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E11-01-0039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279383PMC
February 2012
-->