Katrin J Svensson

All Publications

• Regulation of Energy Metabolism by Receptor Tyrosine Kinase Ligands. Frontiers in physiology Zhao, M., Jung, Y., Jiang, Z., Svensson, K. J. 2020; 11: 354

  Abstract

  Metabolic diseases, such as diabetes, obesity, and fatty liver disease, have now reached epidemic proportions. Receptor tyrosine kinases (RTKs) are a family of cell surface receptors responding to growth factors, hormones, and cytokines to mediate a diverse set of fundamental cellular and metabolic signaling pathways. These ligands signal by endocrine, paracrine, or autocrine means in peripheral organs and in the central nervous system to control cellular and tissue-specific metabolic processes. Interestingly, the expression of many RTKs and their ligands are controlled by changes in metabolic demand, for example, during starvation, feeding, or obesity. In addition, studies of RTKs and their ligands in regulating energy homeostasis have revealed unexpected diversity in the mechanisms of action and their specific metabolic functions. Our current understanding of the molecular, biochemical and genetic control of energy homeostasis by the endocrine RTK ligands insulin, FGF21 and FGF19 are now relatively well understood. In addition to these classical endocrine signals, non-endocrine ligands can govern local energy regulation, and the intriguing crosstalk between the RTK family and the TGF? receptor family demonstrates a signaling network that diversifies metabolic process between tissues. Thus, there is a need to increase our molecular and mechanistic understanding of signal diversification of RTK actions in metabolic disease. Here we review the known and emerging molecular mechanisms of RTK signaling that regulate systemic glucose and lipid metabolism, as well as highlighting unexpected roles of non-classical RTK ligands that crosstalk with other receptor pathways.

  View details for DOI 10.3389/fphys.2020.00354

  View details for PubMedID 32372975

  View details for PubMedCentralID PMC7186430

• Discovery of Hydrolysis-resistant Isoindoline N-Acyl Amino Acid Analogs that Stimulate Mitochondrial Respiration. Journal of medicinal chemistry Lin, H., Long, J. Z., Roche, A. M., Svensson, K. J., Dou, F., Chang, M. R., Strutzenberg, T., Ruiz, C., Cameron, M. D., Novick, S. J., Berdan, C. M., Louie, S., Nomura, D. K., Spiegelman, B. M., Griffin, P. R., Kamenecka, T. M. 2018

  Abstract

  N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. We found that administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure indicating that this pathway might be useful for treating obesity and associated disorders. We report the full account of the synthesis and mitochondrial uncoupling bioactivity of lipidated N-acyl amino acids and their unnatural analogs. Unsaturated fatty acid chains of medium length and neutral amino acid head groups are required for optimal uncoupling activity on mammalian cells. A class of unnatural N-acyl amino acid analogs, characterized by isoindoline-1-carboxylate head groups (37), were resistant to enzymatic degradation by PM20D1 and maintained uncoupling bioactivity in cells and in mice.

  View details for PubMedID 29533650

• Ablation of PM20D1 reveals N-acyl amino acid control of metabolism and nociception. Proceedings of the National Academy of Sciences of the United States of America Long, J. Z., Roche, A. M., Berdan, C. A., Louie, S. M., Roberts, A. J., Svensson, K. J., Dou, F. Y., Bateman, L. A., Mina, A. I., Deng, Z., Jedrychowski, M. P., Lin, H., Kamenecka, T. M., Asara, J. M., Griffin, P. R., Banks, A. S., Nomura, D. K., Spiegelman, B. M. 2018

  Abstract

  N-acyl amino acids (NAAs) are a structurally diverse class of bioactive signaling lipids whose endogenous functions have largely remained uncharacterized. To clarify the physiologic roles of NAAs, we generated mice deficient in the circulating enzyme peptidase M20 domain-containing 1 (PM20D1). Global PM20D1-KO mice have dramatically reduced NAA hydrolase/synthase activities in tissues and blood with concomitant bidirectional dysregulation of endogenous NAAs. Compared with control animals, PM20D1-KO mice exhibit a variety of metabolic and pain phenotypes, including insulin resistance, altered body temperature in cold, and antinociceptive behaviors. Guided by these phenotypes, we identify N-oleoyl-glutamine (C18:1-Gln) as a key PM20D1-regulated NAA. In addition to its mitochondrial uncoupling bioactivity, C18:1-Gln also antagonizes certain members of the transient receptor potential (TRP) calcium channels including TRPV1. Direct administration of C18:1-Gln to mice is sufficient to recapitulate a subset of phenotypes observed in PM20D1-KO animals. These data demonstrate that PM20D1 is a dominant enzymatic regulator of NAA levels in vivo and elucidate physiologic functions for NAA signaling in metabolism and nociception.

  View details for DOI 10.1073/pnas.1803389115

  View details for PubMedID 29967167

• Metastasis Stimulation by Hypoxia and Acidosis-Induced Extracellular Lipid Uptake Is Mediated by Proteoglycan-Dependent Endocytosis CANCER RESEARCH Menard, J. A., Christianson, H. C., Kucharzewska, P., Bourseau-Guilmain, E., Svensson, K. J., Lindqvist, E., Chandran, V. I., Kjellen, L., Welinder, C., Bengzon, J., Johansson, M. C., Belting, M. 2016; 76 (16): 4828-4840

  Abstract

  Hypoxia and acidosis are inherent stress factors of the tumor microenvironment and have been linked to increased tumor aggressiveness and treatment resistance. Molecules involved in the adaptive mechanisms that drive stress-induced disease progression constitute interesting candidates of therapeutic intervention. Here, we provide evidence of a novel role of heparan sulfate proteoglycans (HSPG) in the adaptive response of tumor cells to hypoxia and acidosis through increased internalization of lipoproteins, resulting in a lipid-storing phenotype and enhanced tumor-forming capacity. Patient glioblastoma tumors and cells under hypoxic and acidic stress acquired a lipid droplet (LD)-loaded phenotype, and showed an increased recruitment of all major lipoproteins, HDL, LDL, and VLDL. Stress-induced LD accumulation was associated with increased spheroid-forming capacity during reoxygenation in vitro and lung metastatic potential in vivo On a mechanistic level, we found no apparent effect of hypoxia on HSPGs, whereas lipoprotein receptors (VLDLR and SR-B1) were transiently upregulated by hypoxia. Importantly, however, using pharmacologic and genetic approaches, we show that stress-mediated lipoprotein uptake is highly dependent on intact HSPG expression. The functional relevance of HSPG in the context of tumor cell stress was evidenced by HSPG-dependent lipoprotein cell signaling activation through the ERK/MAPK pathway and by reversal of the LD-loaded phenotype by targeting of HSPGs. We conclude that HSPGs may have an important role in the adaptive response to major stress factors of the tumor microenvironment, with functional consequences on tumor cell signaling and metastatic potential. Cancer Res; 76(16); 4828-40. ©2016 AACR.

  View details for DOI 10.1158/0008-5472.CAN-15-2831

  View details for Web of Science ID 000382297700024

  View details for PubMedID 27199348

• The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria CELL Long, J. Z., Svensson, K. J., Bateman, L. A., Lin, H., Kamenecka, T., Lokurkar, I. A., Lou, J., Rao, R. R., Chang, M. R., Jedrychowski, M. P., Paulo, J. A., Gygi, S. P., Griffin, P. R., Nomura, D. K., Spiegelman, B. M. 2016; 166 (2): 424-435

  Abstract

  Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

  View details for DOI 10.1016/j.cell.2016.05.071

  View details for Web of Science ID 000380255400019

  View details for PubMedID 27374330

  View details for PubMedCentralID PMC4947008

• A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function CELL METABOLISM Svensson, K. J., Long, J. Z., Jedrychowski, M. P., Cohen, P., Lo, J. C., Serag, S., Kir, S., Shinoda, K., Tartaglia, J. A., Rao, R. R., Chedotal, A., Kajimura, S., Gygi, S. P., Spiegelman, B. M. 2016; 23 (3): 454-466

  Abstract

  Activation of brown and beige fat can reduce obesity and improve glucose homeostasis through nonshivering thermogenesis. Whether brown or beige fat also secretes paracrine or endocrine factors to promote and amplify adaptive thermogenesis is not fully explored. Here we identify Slit2, a 180 kDa member of the Slit extracellular protein family, as a PRDM16-regulated secreted factor from beige fat cells. In isolated cells and in mice, full-length Slit2 is cleaved to generate several smaller fragments, and we identify an active thermogenic moiety as the C-terminal fragment. This Slit2-C fragment of 50 kDa promotes adipose thermogenesis, augments energy expenditure, and improves glucose homeostasis in vivo. Mechanistically, Slit2 induces a robust activation of PKA signaling, which is required for its prothermogenic activity. Our findings establish a previously unknown peripheral role for Slit2 as a beige fat secreted factor that has therapeutic potential for the treatment of obesity and related metabolic disorders.

  View details for DOI 10.1016/j.cmet.2016.01.008

  View details for Web of Science ID 000373614100013

  View details for PubMedID 26876562

  View details for PubMedCentralID PMC4785066

• Exosome and microvesicle mediated phene transfer in mammalian cells SEMINARS IN CANCER BIOLOGY Christianson, H. C., Svensson, K. J., Belting, M. 2014; 28: 31-38

  Abstract

  Extracellular vesicles (EVs), e.g. exosomes and microvesicles, emerge as new signaling organelles in the exchange of information between cells at the paracrine and systemic level. It is clear that these virus like particles carry complex biological information that can elicit a pleiotropic response in recipient cells with potential relevance in physiology as well as in cancer and other pathological conditions. Numerous studies convincingly show that the molecular composition of EVs closely reflects their cell or tissue of origin. Thus, the signaling status of donor cells, more specifically their endosomal compartments, may largely determine the biological output in recipient cells, a process that we then may conceptualize as vesicle mediated phene transfer. Whereas more conventional modes of cell-cell communication mostly depend on extracellular ligand concentration and cell-surface receptor availability, the magnitude of the EV signaling response relies on the capture and uptake by target cells, allowing release of the EV content. Numerous reports point at the intriguing possibility that, among thousands of mRNAs, miRNAs, and proteins, single EV constituents effectuate the biological response, e.g. stimulation of angiogenesis and cancer cell metastasis, in recipient cells; however, we find it conceivable that strategies targeted at general mechanisms of EV function should provide more rational avenues for therapeutic intervention directed at the EV system. Such strategies include manipulation of EV formation in the endolysosomal system, EV stability in the extracellular milieu, and EV entry into target cells. Here, we provide important insights into potential mechanisms of EV transport in mammalian cells and how these may be targeted.

  View details for DOI 10.1016/j.semcancer.2014.04.007

  View details for Web of Science ID 000343019900005

  View details for PubMedID 24769057

• Meteorin-like Is a Hormone that Regulates Immune-Adipose Interactions to Increase Beige Fat Thermogenesis CELL Rao, R. R., Long, J. Z., White, J. P., Svensson, K. J., Lou, J., Lokurkar, I., Jedrychowski, M. P., Ruas, J. L., Wrann, C. D., Lo, J. C., Camera, D. M., Lachey, J., Gygi, S., Seehra, J., Hawley, J. A., Spiegelman, B. M. 2014; 157 (6): 1279-1291

  Abstract

  Exercise training benefits many organ systems and offers protection against metabolic disorders such as obesity and diabetes. Using the recently identified isoform of PGC1-? (PGC1-?4) as a discovery tool, we report the identification of meteorin-like (Metrnl), a circulating factor that is induced in muscle after exercise and in adipose tissue upon cold exposure. Increasing circulating levels of Metrnl stimulates energy expenditure and improves glucose tolerance and the expression of genes associated with beige fat thermogenesis and anti-inflammatory cytokines. Metrnl stimulates an eosinophil-dependent increase in IL-4 expression and promotes alternative activation of adipose tissue macrophages, which are required for the increased expression of the thermogenic and anti-inflammatory gene programs in fat. Importantly, blocking Metrnl actions in vivo significantly attenuates chronic cold-exposure-induced alternative macrophage activation and thermogenic gene responses. Thus, Metrnl links host-adaptive responses to the regulation of energy homeostasis and tissue inflammation and has therapeutic potential for metabolic and inflammatory diseases.

  View details for DOI 10.1016/j.cell.2014.03.065

  View details for Web of Science ID 000340881400007

  View details for PubMedID 24906147

  View details for PubMedCentralID PMC4131287

• A Smooth Muscle-Like Origin for Beige Adipocytes CELL METABOLISM Long, J. Z., Svensson, K. J., Tsai, L., Zeng, X., Roh, H. C., Kong, X., Rao, R. R., Lou, J., Lokurkar, I., Baur, W., Castellot, J. J., Rosen, E. D., Spiegelman, B. M. 2014; 19 (5): 810-820

  Abstract

  Thermogenic UCP1-positive cells, which include brown and beige adipocytes, transform chemical energy into heat and increase whole-body energy expenditure. Using a ribosomal profiling approach, we present a comprehensive molecular description of brown and beige gene expression from multiple fat depots in vivo. This UCP1-TRAP data set demonstrates striking similarities and important differences between these cell types, including a smooth muscle-like signature expressed by beige, but not classical brown, adipocytes. In vivo fate mapping using either a constitutive or an inducible Myh11-driven Cre demonstrates that at least a subset of beige cells arise from a smooth muscle-like origin. Finally, ectopic expression of PRDM16 converts bona fide vascular smooth muscle cells into Ucp1-positive adipocytes in vitro. These results establish a portrait of brown and beige adipocyte gene expression in vivo and identify a smooth muscle-like origin for beige cells.

  View details for DOI 10.1016/j.cmet.2014.03.025

  View details for Web of Science ID 000335561200010

  View details for PubMedID 24709624

  View details for PubMedCentralID PMC4052772

• Ablation of PRDM16 and Beige Adipose Causes Metabolic Dysfunction and a Subcutaneous to Visceral Fat Switch CELL Cohen, P., Levy, J. D., Zhang, Y., Frontini, A., Kolodin, D. P., Svensson, K. J., Lo, J. C., Zeng, X., Ye, L., Khandekar, M. J., Wu, J., Gunawardana, S. C., Banks, A. S., Camporez, J. P., Jurczak, M. J., Kajimura, S., Piston, D. W., Mathis, D., Cinti, S., Shulman, G. I., Seale, P., Spiegelman, B. M. 2014; 156 (1-2): 304-316

  Abstract

  A clear relationship exists between visceral obesity and type 2 diabetes, whereas subcutaneous obesity is comparatively benign. Here, we show that adipocyte-specific deletion of the coregulatory protein PRDM16 caused minimal effects on classical brown fat but markedly inhibited beige adipocyte function in subcutaneous fat following cold exposure or ?3-agonist treatment. These animals developed obesity on a high-fat diet, with severe insulin resistance and hepatic steatosis. They also showed altered fat distribution with markedly increased subcutaneous adiposity. Subcutaneous adipose tissue in mutant mice acquired many key properties of visceral fat, including decreased thermogenic and increased inflammatory gene expression and increased macrophage accumulation. Transplantation of subcutaneous fat into mice with diet-induced obesity showed a loss of metabolic benefit when tissues were derived from PRDM16 mutant animals. These findings indicate that PRDM16 and beige adipocytes are required for the "browning" of white fat and the healthful effects of subcutaneous adipose tissue.

  View details for DOI 10.1016/j.cell.2013.12.021

  View details for Web of Science ID 000329912200029

  View details for PubMedID 24439384

  View details for PubMedCentralID PMC3922400

• Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Christianson, H. C., Svensson, K. J., van Kuppevelt, T. H., Li, J., Belting, M. 2013; 110 (43): 17380-17385

  Abstract

  Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

  View details for DOI 10.1073/pnas.1304266110

  View details for Web of Science ID 000325943300052

  View details for PubMedID 24101524

  View details for PubMedCentralID PMC3808637

• Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kucharzewska, P., Christianson, H. C., Welch, J. E., Svensson, K. J., Fredlund, E., Ringner, M., Morgelin, M., Bourseau-Guilmain, E., Bengzon, J., Belting, M. 2013; 110 (18): 7312-7317

  Abstract

  Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.

  View details for DOI 10.1073/pnas.1220998110

  View details for Web of Science ID 000318682300049

  View details for PubMedID 23589885

  View details for PubMedCentralID PMC3645587

• Role of extracellular membrane vesicles in intercellular communication of the tumour microenvironment BIOCHEMICAL SOCIETY TRANSACTIONS Svensson, K. J., Belting, M. 2013; 41: 273-276

  Abstract

  Over the last few decades, extensive studies by several groups have introduced the concept of cell-derived secreted extracellular membrane vesicles as carriers of complex molecular information. Owing to their pleiotropic biological effects and involvement in a wide variety of biological processes, extracellular membrane vesicles have been implicated in physiological as well as pathological events, including tumour development and metastasis. In the present review, we discuss the role of secreted membrane vesicles in intercellular communication with a focus on tumour biology. Of particular interest is the potential role of extracellular vesicles as orchestrators of common features of the malignant tumour microenvironment, e.g. coagulation activation and angiogenesis.

  View details for DOI 10.1042/BST20120248

  View details for Web of Science ID 000314222900045

  View details for PubMedID 23356296

• Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1. J Biol Chem. Svensson, K. J., Christianson, H. C., Wittrup, A., Bourseau-Guilmain, E., Lindqvist, E., Svensson, L. M., Morgelin, M., Belting, M., et al 2013; 2013 Jun 14;288(24):17713-24.
• Standardization and Utilization of Biobank Resources in Clinical Protein Science with Examples of Emerging Applications JOURNAL OF PROTEOME RESEARCH Marko-Varga, G., Vegvari, A., Welinder, C., Lindberg, H., Rezeli, M., Edula, G., Svensson, K. J., Belting, M., Laurell, T., Fehniger, T. E. 2012; 11 (11): 5124-5134

  Abstract

  Biobanks are a major resource to access and measure biological constituents that can be used to monitor the status of health and disease, both in unique individual samples and within populations. Most "omic" activities rely on access to these collections of stored samples to provide the basis for establishing the ranges and frequencies of expression. Furthermore, information about the relative abundance and form of protein constituents found in stored samples provides an important historical index for comparative studies of inherited, epidemic, and developing disease. Standardizations of sample quality, form, and analysis are an important unmet need and requirement for gaining the full benefit from collected samples. Coupled to this standard is the provision of annotation describing clinical status and metadata of measurements of clinical phenotype that characterizes the sample. Today we have not yet achieved consensus on how to collect, manage, and build biobank archives in order to reach goals where these efforts are translated into value for the patient. Several initiatives (OBBR, ISBER, BBMRI) that disseminate best practice examples for biobanking are expected to play an important role in ensuring the need to preserve the sample integrity of biosamples stored for periods that reach one or several decades. These developments will be of great value and importance to programs such as the Chromosome Human Protein Project (C-HPP) that will associate protein expression in healthy and disease states with genetic foci along of each of the human chromosomes.

  View details for DOI 10.1021/pr300185k

  View details for Web of Science ID 000311190600002

  View details for PubMedID 22607352

• Dermatan Sulfate Is Involved in the Tumorigenic Properties of Esophagus Squamous Cell Carcinoma CANCER RESEARCH Thelin, M. A., Svensson, K. J., Shi, X., Bagher, M., Axelsson, J., Isinger-Ekstrand, A., van Kuppevelt, T. H., Johansson, J., Nilbert, M., Zaia, J., Belting, M., Maccarana, M., Malmstrom, A. 2012; 72 (8): 1943-1952

  Abstract

  Extracellular matrix, either produced by cancer cells or by cancer-associated fibroblasts, influences angiogenesis, invasion, and metastasis. Chondroitin/dermatan sulfate (CS/DS) proteoglycans, which occur both in the matrix and at the cell surface, play important roles in these processes. The unique feature that distinguishes DS from CS is the presence of iduronic acid (IdoA) in DS. Here, we report that CS/DS is increased five-fold in human biopsies of esophagus squamous cell carcinoma (ESCC), an aggressive tumor with poor prognosis, as compared with normal tissue. The main IdoA-producing enzyme, DS epimerase 1 (DS-epi1), together with the 6-O- and 4-O-sulfotransferases, were highly upregulated in ESCC biopsies. Importantly, CS/DS structure in patient tumors was significantly altered compared with normal tissue, as determined by sensitive mass spectrometry. To further understand the roles of IdoA in tumor development, DS-epi1 expression, and consequently IdoA content, was downregulated in ESCC cells. IdoA-deficient cells exhibited decreased migration and invasion capabilities in vitro, which was associated with reduced cellular binding of hepatocyte growth factor, inhibition of pERK-1/2 signaling, and deregulated actin cytoskeleton dynamics and focal adhesion formation. Our findings show that IdoA in DS influences tumorigenesis by affecting cancer cell behavior. Therefore, downregulation of IdoA by DS-epi1 inhibitors may represent a new anticancer therapy.

  View details for DOI 10.1158/0008-5472.CAN-11-1351

  View details for Web of Science ID 000302905700006

  View details for PubMedID 22350411

  View details for PubMedCentralID PMC3328612

• Chondroitin sulfate expression predicts poor outcome in breast cancer INTERNATIONAL JOURNAL OF ONCOLOGY Svensson, K. J., Christianson, H. C., Kucharzewska, P., Fagerstrom, V., Lundstedt, L., Borgquist, S., Jirstrom, K., Belting, M. 2011; 39 (6): 1421-1428

  Abstract

  Experimental studies have established that the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate act as co-receptors of cytokines and growth factors that drive the malignant cell phenotype and the remodelling of the surrounding tumor stroma. However, the clinical relevance of these studies remains ill-defined. The present study investigates the significance of chondroitin sulfate expression in malignant cells and the stroma, respectively, of tumors from two independent cohorts of breast cancer patients (cohort I: 144 patients, 130 evaluable samples; cohort II: 498 patients, 469 evaluable samples; ER-positive patients ~86% in both cohorts). Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between chondroitin sulfate and recurrence-free and overall survival. High chondroitin sulfate expression in malignant cells was shown to predict shorter recurrence-free survival (P=0.007, cohort I; P=0.024, cohort II) and overall survival (cohort I: P=0.044; cohort II: P<0.001) in both cohorts. In multivariate analysis, high chondroitin sulfate in malignant cells was shown to be an independent, predictive factor of poor overall survival (cohort I: hazard ratio 2.28: 95% confidence interval 1.08-4.81, P=0.031; cohort II: hazard ratio 1.71: 95% confidence interval 1.23-2.38, P=0.001). However, chondroitin sulfate in the stroma showed no correlation with known markers of tumor aggressiveness or with clinical outcome in either cohort. Our data suggest that high chondroitin sulfate expression in malignant cells is associated with an adverse outcome in patients with primary breast cancer, supporting the idea of a functional and potentially targetable role of chondroitin sulfate in tumor disease.

  View details for DOI 10.3892/ijo.2011.1164

  View details for Web of Science ID 000296315400010

  View details for PubMedID 21850370

• Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Svensson, K. J., Kucharzewska, P., Christianson, H. C., Skold, S., Lofstedt, T., Johansson, M. C., Morgelin, M., Bengzon, J., Ruf, W., Belting, M. 2011; 108 (32): 13147-13152

  Abstract

  Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

  View details for DOI 10.1073/pnas.1104261108

  View details for Web of Science ID 000293691400041

  View details for PubMedID 21788507

  View details for PubMedCentralID PMC3156184

• Heparan sulfate proteoglycan-mediated polyamine uptake. Methods in molecular biology (Clifton, N.J.) Welch, J., Svensson, K., Kucharzewska, P., Belting, M. 2011; 720: 327-338

  Abstract

  The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display-derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.

  View details for DOI 10.1007/978-1-61779-034-8_20

  View details for PubMedID 21318883

• Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells EXPERIMENTAL CELL RESEARCH Kucharzewska, P., Welch, J. E., Svensson, K. J., Belting, M. 2010; 316 (16): 2683-2691

  Abstract

  The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by alpha-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

  View details for DOI 10.1016/j.yexcr.2010.05.033

  View details for Web of Science ID 000281305800015

  View details for PubMedID 20594968

• Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wittrup, A., Zhang, S., Svensson, K. J., Kucharzewska, P., Johansson, M. C., Morgelin, M., Belting, M. 2010; 107 (30): 13342-13347

  Abstract

  An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intracellular vesicles of a nonclathrin-, noncaveolin-dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.

  View details for DOI 10.1073/pnas.1002622107

  View details for Web of Science ID 000280602800030

  View details for PubMedID 20624969

  View details for PubMedCentralID PMC2922147

• The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Kucharzewska, P., Welch, J. E., Svensson, K. J., Belting, M. 2009; 380 (2): 413-418

  Abstract

  Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization.

  View details for DOI 10.1016/j.bbrc.2009.01.097

  View details for Web of Science ID 000263742200039

  View details for PubMedID 19250631

• Hypoxia-Mediated Induction of the Polyamine System Provides Opportunities for Tumor Growth Inhibition by Combined Targeting of Vascular Endothelial Growth Factor and Ornithine Decarboxylase CANCER RESEARCH Svensson, K. J., Welch, J. E., Kucharzewska, P., Bengtson, P., Bjurberg, M., Pahlman, S., ten Dam, G. B., Persson, L., Belting, M. 2008; 68 (22): 9291-9301

  Abstract

  Hypoxia is a hallmark of solid tumors, which may offer opportunities for targeted therapies of cancer; however, the mechanisms that link hypoxia to malignant transformation and tumor progression are not fully understood. Here, we show that up-regulation of the polyamine system promotes cancer cell survival during hypoxic stress. Hypoxia was found to induce polyamine transport and the key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), in a variety of cancer cell lines. Increased ODC protein expression was shown in hypoxic, GLUT-1-expressing regions of tumor spheroids and experimental tumors, as well as in clinical tumor specimens. Hypoxic induction of the polyamine system was dependent on antizyme inhibitor (i.e., a key positive regulator of ODC and polyamine transport), as shown by RNA interference experiments. Interestingly, depletion of the polyamines during hypoxia resulted in increased apoptosis, which indicates an essential role of the polyamines in cancer cell adaptation to hypoxic stress. These results were supported by experiments in an in vivo glioma tumor model, showing significantly enhanced antitumor effects of the antiangiogenic, humanized anti-vascular endothelial growth factor (VEGF) antibody bevacizumab when used in combination with the well-established, irreversible inhibitor of ODC, alpha-difluoromethylornithine. Our results provide important insights into the hypoxic stress response in malignant cells and implicate combined targeting of VEGF and ODC as an alternative strategy to treat cancer disease.

  View details for DOI 10.1158/0008-5472.CAN-08-2340

  View details for Web of Science ID 000261136600025

  View details for PubMedID 19010902

• Single chain fragment anti-heparan sulfate antibody targets the polyamine transport system and attenuates polyamine-dependent cell proliferation INTERNATIONAL JOURNAL OF ONCOLOGY Welch, J. E., Bengtson, P., Svensson, K., Wittrup, A., Jenniskens, G. J., ten Dam, G. B., van Kuppevelt, T. H., Belting, M. 2008; 32 (4): 749-756

  Abstract

  The growth-promoting polyamines are polybasic compounds that efficiently enter cancer cells by as yet incompletely defined mechanisms. Strategies to inhibit their internalization may have important implications in the management of tumor disease. Here, we show that cellular binding and uptake of polyamines are inhibited by a single chain variable fragment anti-heparan sulfate (HS) antibody. Polyamine uptake was inhibited in a dose-dependent manner, and was associated with compensatory up-regulation of ornithine decarboxylase (ODC), i.e. the key enzyme of the polyamine biosynthesis pathway. Conversely, depletion of intracellular polyamines by the specific ODC-inhibitor alpha-difluoromethylornithine (DFMO) resulted in increased cellular binding of polyamine and anti-HS antibody. Importantly, anti-HS antibody also efficiently targeted DFMO-induced polyamine uptake, and combined polyamine biosynthesis inhibition by DFMO, and uptake inhibition by anti-HS antibody attenuated tumor cell proliferation in vitro. In conclusion, cell-surface HS proteoglycan is a relevant target for antibody-mediated inhibition of the uptake of polyamines, and polyamine-dependent cell proliferation.

  View details for Web of Science ID 000254357600003

  View details for PubMedID 18360702

• HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells MOLECULAR CANCER THERAPEUTICS Mani, K., Sandgren, S., Lilja, J., Cheng, F., Svensson, K., Persson, L., Belting, M. 2007; 6 (2): 782-788

  Abstract

  Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake-deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with alpha-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer.

  View details for DOI 10.1158/1535-7163.MCT-06-0370

  View details for Web of Science ID 000244262700039

  View details for PubMedID 17308074

• Synthesis and transfection efficiencies of new lipophilic polyamines JOURNAL OF MEDICINAL CHEMISTRY Gardner, R. A., Belting, M., Svensson, K., Phanstiel, O. 2007; 50 (2): 308-318

  Abstract

  A homologous series of lipophilic polyamines was synthesized and evaluated for DNA delivery and transfection efficiency. The series contained 1,4-butanediamine, 1,8-octanediamine, 2-[2-(2-amino-ethoxy)-ethoxy]-ethylamine, homospermidine, and homospermine covalently attached via their N1 terminus to a 3,4-bis(oleyloxy)-benzyl motif. In addition, homospermidine and homospermine were also attached via amide linkers. The homospermidine derivatives (i.e., benzyl tether 25 and benzamide tether 27) showed a 3-fold and 4-fold respective enhancement in delivery of AlexaFluor-488-labeled DNA over the butanediamine analogue 22. Homospermine derivative 26 was shown to inhibit 14C-spermine uptake (IC50 approximately 10 microM), which implied that 26 is able to compete effectively for polyamine recognition sites on the cell surface. This study demonstrated that the number and position of the positive charges along the polyamine scaffold plays a key role in DNA delivery and in determining the transfection efficiency.

  View details for DOI 10.1021/jm0607101

  View details for Web of Science ID 000243535600015

  View details for PubMedID 17228872