Hyaluronan synthases in development and cancer : immunohistochemical studies

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DISSERTATIONS | KARI TÖRRÖNEN | HYALURONAN SYNTHASES IN DEVELOPMENT AND CANCER | No 371

uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2226-7 ISSN 1798-5706

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

KARI TÖRRÖNEN

HYALURONAN SYNTHASES IN DEVELOPMENT AND CANCER

Functions of hyaluronan, hyaluronan

synthases and hyaluronan receptors during embryonic development and cancer progression are dependent on their spatial

and temporal distribution in cells and tissues. This thesis gives new information on these molecules and confirms the importance

of hyaluronan during embryogenesis and cancer development, and establishes basis for future studies aiming to generate novel

treatments.

KARI TÖRRÖNEN

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Hyaluronan Synthases in Development and Cancer

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KARI TÖRRÖNEN

Hyaluronan Synthases in Development and Cancer

Immunohistochemical studies

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in lecture hall SN200, Kuopio, on Friday 14th, October 2016, at 12 noon

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 371

Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland

Kuopio 2016

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Grano Oy Joensuu, 2016

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical physiology and isotope medicine Faculty of Health Sciences

Professor Hannele Turunen, Ph.D.

Department of Nursing Science Faculty of Health Sciences

Assosiate professor Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Professor Kai Kaarniranta, M.D., PhD Institute of Clinical Medicine, Opthalmology

Faculty of Health Science

University lecturer Veli-‐‑Pekka Ranta, Ph.D.

School of Pharmacy Faculty of Health Science

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-‐‑70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-‐‑952-‐‑61-‐‑2226-‐‑7 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2227-‐‑4

ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714

ISSN-‐‑L: 1798-‐‑5706

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Author’s address: Institute of Biomedicine/School of Medicine University of Eastern Finland

KUOPIO FINLAND

Supervisors: Docent Kirsi Rilla, Ph.D.

Institute of Biomedicine/School of Medicine University of Eastern Finland

KUOPIO FINLAND

Professor Raija Tammi, M.D., Ph.D.

KUOPIO FINLAND

Professor Markku Tammi, M.D., Ph.D.

KUOPIO FINLAND

Reviewers: Professor Juha Tuukkanen, Ph.D Institute of Biomedicine

University of Oulu OULU

FINLAND

Docent Lauri Eklund, Ph.D

Faculty of Biochemistry and Molecular Medicine Biocenter Oulu, University of Oulu

Oulu

Finland

Opponent: Docent Sirkku Peltonen, M.D., Ph.D.

Dermatology Outpatient Clinic Turku University Hospital Turku

Finland

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Törrönen, Kari

Hyaluronan Synthases in Development and Cancer – Immunohistochemical Studies University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences 371. 2016. 51 p.

ISBN (print): 978-‐‑952-‐‑61-‐‑2226-‐‑7 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2227-‐‑4 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706

ABSTRACT

Hyaluronan is a common glycosaminoglycan of our body essential for fetal development and tissue regeneration throughout life. It is an important component of the extracellular matrix and regulates many homeostatic processes in various tissues like differentiation of keratinocytes and moisture balance in the skin. Disturbance in the regulation of homeostasis may lead to emergence of malignant cells, which develop into tumors.

Hyaluronan accumulation on the cell surface and surrounding extracellular matrix is suggested to participate in the creation of a favorable growth environment for the cancer cells. Hyaluronan is an effective cell growth promoter both in health and disease.

Hyaluronan is synthesized by enzymes, called hyaluronan synthases. Therefore, these enzymes and the regulation of their activities have an important role in the normal functioning of the body and in the development of therapies for diseases in which hyaluronan is produced in excess. The genes encoding hyaluronan synthases (HAS1-‐‑3) have been identified more than twenty years ago, and novel information on their regulation has been obtained in recent studies. However, the exact functions and subcellular localizations of the hyaluronan synthase proteins in cells and tissues are still not known.

The aim of this study was to determine the location of these three enzymes in cells and tissues, utilizing both immunohistochemical stainings and fusion proteins. We also wanted to find out for the first time at protein level the spatial and temporal distribution of these enzymes during development, with mouse fetuses as a research model.

We used mouse model to study changes in hyaluronan, HASes and CD44 during ultraviolet radiation (UVR) induced skin squamous cell carcinoma. In addition, we studied one of the most lethal types of cancer, mesothelioma, which originates from the actively hyaluronan synthesizing mesothelial cells. We compared the incidence of hyaluronan synthases, hyaluronan, and the main receptor for hyaluronan, CD44, in mesotheliomas with the other general lung cancer type, adenocarcinoma. The aim was to find out whether hyaluronan and associated proteins could be utilized to identify these two cancer types.

It was found that the presence of different hyaluronan synthases during fetal development is partly overlapping both spatially and temporally, but some differences were observed. In particular, the connective tissue of the heart, skin and cartilage were highly positive for all isoforms. Long-‐‑term exposure to UVR increased the amount of hyaluronan, CD44 and hyaluronan synthases in mouse epidermis, which correlated with development of epidermal hyperplasia and formation of carcinomas.

This thesis gives novel information on the importance of hyaluronan in fetal development and tumorigenesis. Careful studies on intracellular location of hyaluronan synthases and their differences provide novel cell biological data and support the development of therapy.

National Library of Medicine Classification: QS 604, QU 83, QU 141, QZ 365

Medical Subject Headings:Glycosaminoglycans; Hyaluronic Acid; Glucuronosyltransferase; Antigens, CD44;

Embryonic Development; Fetal Development; Neoplasms; Ultraviolet Rays; Carcinoma, Squamous Cell;

Mesothelioma; Adenocarcinoma; Disease Models, Animal; Mice

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Törrönen, Kari

Hyaluronan Synthases in Development and Cancer – Immunohistochemical Studies Itä-‐‑Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences 371. 2016. 51 s.

ISBN (print): 978-‐‑952-‐‑61-‐‑2226-‐‑7 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2227-‐‑4 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706

TIIVISTELMÄ

Hyaluronaani on hyvin yleisenä elimistössämme esiintyvä glykosaminoglykaaneihin kuuluva polysakkaridi, joka on välttämätön sikiönkehitykselle ja lisäksi edistää kudosten uusiutumista läpi elämän. Se on tärkeä soluväliaineen komponentti ja säätelee mm. ihon kosteustasapainoa ja ihon pintasolujen eli keratinosyyttien erilaistumista. Elimistön säätelyjärjestelmien pettäessä syntyy pahanlaatuisia soluja, joista kehittyy kasvaimia.

Hyaluronaanin ajatellaan osallistuvan syöpäsolujen kasvulle suotuisan kasvuympäristön luomiseen kertymällä solujen pinnalle ja niitä ympäröivään soluväliaineeseen.

Hyaluronaani on siis solujen kasvun edistäjä sekä hyvässä että pahassa.

Hyaluronaanin valmistus tapahtuu entsyymien, hyaluronaanisyntaasien (HAS1-‐‑3) avulla. Siksi nämä entsyymit ja niiden toiminnan säätely ovat tärkeässä asemassa, kun pyritään ymmärtämään normaalin elimistön toimintaa ja suunnittelemaan mahdollisia hoitoja sairauksiin, joissa hyaluronaania tuotetaan liikaa. Hyaluronaanisyntaaseja 1-‐‑3 koodaavat geenit on tunnistettu jo parikymmentä vuotta sitten, ja niiden toiminnasta on saatu viimeaikaisissa tutkimuksissa paljon tietoa. Kuitenkin itse proteiinien säätelystä ja sijannista soluissa ja kudoksissa tiedetään varsin vähän. Tämän väitöskirjatyön tarkoituksena oli selvittää ja osoittaa näiden kolmen entsyymin sijainti soluissa ja kudoksissa sekä immunohistokemiallisia värjäyksiä että fuusioproteiineja käyttäen.

Halusimme myös selvittää ensimmäistä kertaa proteiinitasolla näiden entsyymien esiintymisen eri sikiönkehitysvaiheessa, käyttäen eri-‐‑ikäisiä hiiren sikiöitä tutkimusmallina.

Tutkimme hiirimallin avulla ultraviolettisäteilyn (UVR) vaikutusta hyaluronaanin ja siihen liittyviin proteiineihin sekä levyepiteelisyövän kehitykseen. Lisäksi käytimme esimerkkinä yhtä tappavimmista syöpätyypeistä, mesotelioomaa, joka saa alkunsa runsaasti hyaluronaania tuottavista keuhkopussia verhoavista mesoteelisoluista.

Vertasimme hyaluronaanisyntaasien, hyaluronaanin ja tärkeimmän hyaluronaaniresep-‐‑

torin, CD44:n esiintyvyyttä mesotelioomissa toiseen yleiseen keuhkoissa esiintyvään syöpätyyppiin, adenokarsinoomaan. Tarkoituksena oli selvittää, voisiko hyaluronaania ja siihen liittyviä proteiineja hyödyntää näiden kahden toisistaan hankalasti erotettavan syöpätyypin erottamisessa.

Totesimme että eri hyaluronanisyntaasien esiintyminen sikiönkehityksen aikana on osittain samanaikaista, mutta tietyissä elimissä eroja kuitenkin havaittiin. Erityisesti sidekudoksessa, sydämessä, ihossa ja rustossa niiden esiintyminen oli merkittävää.

Isoentsyymien solunsisäinen lokalisaatio poikkesi myös toisistaan. Lisäksi pitkäaikainen altistus ultraviolettisäteilylle lisäsi sekä hyaluronaanin, CD44:n että hyaluronaanisyntaasien määrää hiiren epidermiksessä ja korreloi hyperplasian ja karsinoomien kanssa.

Tämä väitöskirjatyön tulokset tukevat hyaluronaanin merkitystä sikiönkehityksessä ja syövässä. Hyaluronaanisyntaasien solunsisäisen sijainnin ja niiden erojen perinpohjainen selvittäminen antavat tärkeää perussolubiologista tietoa ja tukevat hoitojen kehittämistä.

Luokitus: QS 604, QU 83, QU 141, QZ 365

Yleinen Suomalainen asiasanasto: hyaluronaani; alkionkehitys; ultraviolettisäteily; kasvaimet; syöpätaudit;

karsinoomat; adenokarsinooma; mesoteliooma; koe-‐‑eläinmallit; hiiret

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To Vanessa, Oliver, Nastja and Dasha

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Acknowledgements

There is a long journey behind of this doctoral thesis work. It started two decades ago in Department of Anatomy, Faculty of Medicine, University of Kuopio and finished in Institute of Biomedicine, School of Medicine, University of Eastern Finland.

First of all, I want to thank my principal supervisor Docent Kirsi Rilla, Ph.D.. She has almost endless patience, great knowledge of microscopy and cell biology. I know you were sometimes quite desperate and exhausted with my thesis work, but still you get somewhere strength to continue pushing ahead me with writing. We have had many philosophical discussions about life and sometimes also about science. We have laught and cried during this process and finally finished it.

I am grateful to my second supervisor Professor Emerita Raija Tammi. You have teached me the meaning of accuracy and importance to repeat experiments. You have a great sense of humour.

I wish to express my sincere gratitude to my third supervisor Professor Markku Tammi.

You introduced me the world of hyaluronan. It has been a honour to work in the HA research group. I have also enjoyed our floorball games.

I owe my warm tanks to present and former colleagues in HA-‐‑group, Sanna Pasonen-‐‑

Seppänen, Ph.D., Sanna Oikari, Ph.D., Hanna Siiskonen, M.D., Ph.D., Anne Kultti, Ph.D., Tiina Jokela, Ph.D., Katri Makkonen, Ph.D, Leena Rauhala, M.Sc., Piia Takabe, M.Sc., Lasse Hämäläinen, M.Sc., Susanna Hartmann-‐‑Petersen, M.D. Ph.D., Ashik Jawahar Deen, Ph.D., Uma Thanigai Arasu, M.Sc., Ritva Tumelius, Ph.D., Juha-‐‑Pekka Pienimäki, M.D., Ville Koistinen, M.D., Kai Härkönen, M.Sc., Raquel Melero, Ph.D., and Genevieve Bart, PhD.

I am grateful to the official reviewers, professor Juha Tuukkanen and Docent Lauri Eklund, Ph.D., for reviewing this thesis work. I thank Eeva Nukarinen, M.Ed., for her revision of the language in this thesis.

I want to thank proficient laboratory professionals attended in this thesis work. Riikka Kärnä, M.Sc., has done numerous laboratory experiments and sheered up everyone with her laught. Ms. Eija Rahunen and Mr. Kari Kotikumpu have done most of the histology and have been hard competirors in floorball. Ms. Arja Venäläinen and Ms. Eija Kettunen have done precious work in laboratory.

My special thanks go to the former head of Anatomy Professor Emiritus Heikki Helminen. He is an old style gentleman and great scientist in cartilage research area. He has been like a godfather for me and many other “young” scientists. I also want to thank Anitta Mahonen, the head of Institute of Biomedicine, for providing some extra time for this thesis work. Rita Sorvari, D.D.S., is always in my heart. She has mentored and engouraged me in the field of teaching. I thank Petteri Nieminen, M.D., Ph.D., and Virpi Tiitu, Ph.D., for their valuable help with teaching of anatomy.

During these years I have got help from several “office” persons. I am grateful to Ms. Eija Vartianen, Ms. Arja Hoffren, Ms. Karoliina Tenkanen, Ms. Outi Tikkanen, Ms. Irma Pääkkönen, and Ms. Kati Bolodin.

I have been fortunate to have great collegues and friends from the cartilage and cancer research areas. Specially, I like to thank Docent Reijo Sironen, M.D., Ph.D., Professor Mikko Lammi, Hannu Karjalainen, Ph.D., Janne Sahlman, M.D, Ph.D, Mika Elo, M.D., Ph.D., Professor Juha Töyräs, and Associated Professor Rami Korhonen for collaboration during work and free time. I also want to thank other members of Cargo-‐‑ and BBC-‐‑groups.

I would like to thank to the whole personel of former Department of Anatomy and present Institute of Biomedicine. I want to thank the personel of SibLabs and former Department of Electron microscopy, for their help with microscopy. Additionally, during these two decades I have worked with many other people. I want to thank all of you collectively.

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I am grateful of financial support of North Savo Cultural Foundation and Paavo Koistinen’s foundation.

I want to thank my sister Anu Törrönen and my brothers Kalle, Ville and Eero Törrönen for helping and supporting me during my life.

My loving thanks belong to my family. Dasha you are the light in my life. Our dear children Oliver, Nastja and little Vanessa have teached me a lot of about the true of meaning life.

Kuopio, August 2016

Kari Törrönen

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List of the original publications

This dissertation is based on the following original publications, which are referred to in the text by their Roman numerals:

I Törrönen K, Nikunen K, Kärnä R, Tammi M, Tammi R and Rilla K. Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes.

Histochem Cell Biol. 1414(1):17-‐‑31, 2014

II Siiskonen H, Törrönen K, Kumlin T, Rilla K, Tammi MI and Tammi RH. Chronic UVR causes increased immunostaining of CD44 and accumulation of hyaluronan in mouse epidermis. J Histochem Cytochem. 59(10):908-‐‑17, 2011

III Törrönen K, Soini Y, Pääkkö P, Parkkinen J, Sironen R and Rilla K.

Mesotheliomas show higher hyaluronan positivity around tumor cells than metastatic pulmonary adenocarcinomas. Histol Histopathol, 31(10):1113-‐‑1122, 2016

The publications were adapted with the permission of the copyright owners.

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Abbreviations

bHABC biotinylated hyaluronan binding complex

CD44 cluster of differentiation 44 EGFP enhanced green fluorescent

protein

ECM extracellular matrix

EGFR epidermal growth factor receptor GlcNAc N-‐‑acetylglucosamine

GlcUA glucuronic acid

HABC hyaluronan binding complex HAS hyaluronan synthase

HexNac N-‐‑acetylhexosamine HYAL hyaluronidase

ICAM-‐‑1 intercellular adhesion molecule 1 LYVE-‐‑1 lymph vessel endothelium

receptor 1

RHAMM receptor for hyaluronan-‐‑

mediated motility

SHAP serum-‐‑derived hyaluronan-‐‑

associated protein

TGF-‐‑β transforming growth factor beta TSG-‐‑6 tumor necrosis stimulated gene 6 TNFIP6 tumor necrosis factor-‐‑induced

protein-‐‑6

UDP uridine diphosphate UVR ultraviolet radiation

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1 Introduction

Hyaluronan is the simplest of glycosaminoglycans and a major constituent of the extracellular matrix (ECM), acting as a space filler, moisturizer and lubricator in most tissues of our body. However, it has special properties and is synthesized by a special mechanism on the plasma membrane, which makes it unique among other ECM molecules.

Hyaluronan content is high in rapidly renewing and growing tissues, like in tumorigenesis and embryonic development. It is unclear whether this accumulation of hyaluronan is a cause or consequence of the high cellular growth rate that occurs during cancer progression and development. Furthermore, it is not clear how crucial the role of hyaluronan synthases and their activation is in hyaluronan accumulation. Detailed information about hyaluronan synthases and hyaluronan binding receptors would help to solve these questions.

Hyaluronan is produced by specific enzymes, hyaluronan synthases (HAS1-‐‑3) at the inner face of the plasma membrane, and this localization is highly dependent on HAS activity. However, only little information on the subcellular localization of the three synthase proteins is available. The aim of this thesis work was to study the distribution and localization of hyaluronan, its main receptor CD44 and hyaluronan synthases in cultured cells and in rapidly growing tissues like embryonic tissues, tumors, and UV radiated skin.

The results of this thesis work provide new data on the properties and functions of hyaluronan and proteins related to it and support the hypothesis that hyaluronan is an important regulator of growth of cells and homeostasis of tissues. These properties make hyaluronan and proteins associated with it as potential therapeutic targets in many clinical conditions, like cancer and inflammation.

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2 Review of the literature

2.1 HYALURONAN

2.1.1 Hyaluronan overview and history

Hyaluronan, a nonsulfated glycosaminoglycan, is one of the major components of the extracellular matrix. The importance of hyaluronan in normal biological and pathological processes has been noticed during last decades (Table 1). Hyaluronan has a significant role in embryogenesis (Camenisch et al. 2000, Tien and Spicer 2005), angiogenesis (Slevin et al.

2007), immunoregulation (Jackson 2009), and cancer progression and cancer therapeutics (Toole et al. 2008). Hyaluronan has several functions including space filling, matrix formation and lubrication in joints. During wound healing and tissue repair hyaluronan synthesis is elevated (Slevin et al. 2007).

Table 1. Historical milestones of hyaluronan research

Year Event Reference

1934 Isolated from the vitreous

humor of bovine eyes (Meyer and Palmer 1934) 1930-‐‑40s Isolated from synovial fluid,

skin, umbilical cord, tumors and rooster combs

(Kabat 1939, Meyer and Palmer 1936, Meyer and Chaffee 1941, Ogston et al. 1939, Ragan and Meyer 1949)

1951 Chemical structure (Meyer et al. 1951) 1970 Clinical use (Butler et al. 1970) 1972 Interaction with cartilage

proteoglycans (Hardingham and Muir 1972) 1979 Hyaluronan receptor CD44 (Underhill and Toole 1979) 1993 Group A Streptococcus

synthase (DeAngelis et al. 1993)

1996 Molecular cloning of

mammalian hyaluronan

synthase

(Itano and Kimata 1996)

1998 Hyaluronan is an independent

prognostic factor in cancer (Ropponen et al. 1998) 1999 Active hyaluronan synthase (Tlapak-‐‑Simmons et al. 1999)

1999 EGFP-‐‑tagged HASs (Müllegger et al. 2001, Rilla et al. 2005, Spicer and Nguyen 1999)

2000 HAS2 knockout is lethal in mouse

(Camenisch et al. 2000)

2012 Target of cancer therapy (Jiang et al. 2012a)

Hyaluronan is found in many species from some simple bacteria (Lowther and Rogers 1955) to vertebrates (Prehm 1990). High concentrations of hyaluronan exist e.g. in the vitreous body of the eye (Meyer and Palmer 1934), the umbilical cord (Hadidian and Pirie 1948), skin (Mier and Wood 1969) and synovial fluid (Ragan and Meyer 1949).

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3 2.1.2 Structure of hyaluronan

Hyaluronan is composed of repeating disaccharides, D-‐‑glucuronic acid and N-‐‑acetyl-‐‑

glucosamine (Figure 1). There are alternating β(1-‐‑3) and β(1-‐‑4)glucuronidic bonds between the sugars (Weissmann et al. 1954). Hyaluronan chain may contain up to 25 000 disaccharides, and molecular mass of one hyaluronan molecule can be as high as 10⁷ Da and length 25 µμm (Toole 2004). At physiological pH hyaluronan is highly hydrophilic due to negatively charged glucuronic acid groups. In aqueous solutions linear hyaluronan chain forms a random coiled structure. Unlike the other glycosaminoglycans, hyaluronan does not covalently attach to core protein to form proteoglycans.

Figure 1. The general chemical structure of the disaccharide unit of hyaluronan. Hyaluronan is composed of alternating residues of β-‐‑D-‐‑(1→3) glucuronic acid (GlcA) and β-‐‑D-‐‑(1→4)-‐‑N-‐‑

acetylglucosamine (GlcNAc).

2.2 HYALURONAN SYNTHESIS

2.2.1 Hyaluronan is synthesized on the plasma membrane

Hyaluronan is synthesized by hyaluronan synthases (HAS), integral transmembrane proteins that act on the inner face of the plasma membrane and extrude the growing hyaluronan chain through the plasma membrane into the extracellular space (Prehm 1984).

Mammals have three hyaluronan synthase isoenzymes, HAS1, HAS2 and HAS3 (Toole 2004). These enzymes utilize two precursors, UDP-‐‑N-‐‑acetylglucosamine and UDP-‐‑

glucuronic acid for hyaluronan synthesis. The new sugar units are added into the reducing end of the growing chain by the native vertebrate enzyme (Weigel et al. 1997). Studies with a recombinant enzyme and the enzyme from Pasteurella multocida show chain growth in the non-‐‑reducing end (Bodevin-‐‑Authelet et al. 2005, DeAngelis 1999).

The amino acid sequences of HAS isoenzymes are quite homologous between different species. The predicted structure of the enzyme consists of 4-‐‑6 transmembrane domains and 1-‐‑2 membrane associated domains (Figure 2). A large cytoplasmic domain is suggested to contain the enzymatically active area. The transmembrane domains that span the lipid

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bilayer are suggested to create a pore in the plasma membrane for the protruding hyaluronan chain (Weigel et al. 1997). There has been speculation on the structure of the pore. A recent review by Weigel concludes that the HAS enzyme itself forms the pore (Weigel 2015), while previous studies using inhibition of multidrug resistance transporters had suggested that they are involved in the export of hyaluronan from the inner to the outer surface of plasmamembrane (Schulz et al. 2007).

Figure 2. A schematic structure of vertebrate hyaluronan synthase hyaluronan binding proteins and degradation enzymes. The synthase is composed of 7 transmembrane or membrane-‐‑associated domains and a large cytoplasmic domain, latter assumed to contain the enzymatic activity. Aggrecan is an example of extracellular hyaluronan binding protein.

CD44 is the main cell surface receptor for hyaluronan. Hyaluronidase 2 is a cell surface degradation enzyme of hyaluronan. Modified from Itano and Kimata (Itano and Kimata 2002), Anderegg et al. (Anderegg et al. 2014) and Chowdhury et al. (Chowdhury et al.

2016).

The human HAS1 gene is localized in chromosome 19 and the mouse gene in chromosome 17 (Spicer and McDonald 1998). HAS1 is the isoenzyme with lowest activity (Itano et al.

1999), (Rilla et al. 2013a), and HAS1 knockout mice have no apparent phenotype (Kobayashi et al. 2010). The hyaluronan chains synthesized by HAS1 are suggested to be smaller as compared to those of HAS2 (Itano et al. 1999). Hyaluronan production by HAS1 is highly dependent of the intracellular UDP-‐‑sugar concentration and high concentrations are necessary for full enzymatic activity (Rilla et al. 2013a). TGF-‐‑β stimulated synoviocytes

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have elevated HAS1 expression and increased hyaluronan production (Stuhlmeier and Pollaschek 2004a). HAS1 promoter contains SP3 and SMAD3 elements and these elements regulate the level of its expression(Chen et al. 2012).

High expression of HAS1 is typical for some diseases like rheumatoid arthritis (Stuhlmeier and Pollaschek 2004b), osteoarthritis (Lambert et al. 2014) and infectious lung disease (Chang et al. 2014). Bone marrow mesenchymal progenitor cells isolated from myeloma patients have higher HAS1 mRNA expression as compared to cells collected from healthy people, with a corresponding elevation in HA production (Calabro et al. 2002).

The location of the human HAS2 gene is in chromosome 8 and that of mouse in chromosome 15 (Spicer and McDonald 1998). HAS2 deletion has lethal effects during embryonic development. HAS2 produces large hyaluronan polymers with an average molecular mass of >2 MDalton (Itano et al. 1999). The availability of UDP-‐‑HexNac in the cytosol limits the synthesis rate of hyaluronan and feedback regulates the expression of HAS2 (Jokela et al. 2011). On the other hand extracellularly applied UDP-‐‑Glucose activates HAS2 expression by binding to the P2Y14-‐‑ plasma membrane receptor, leading to the phosphorylation of STAT3 in tyrosine 705 and binding to the promoter of HAS2 (Jokela et al. 2014).

Interestingly, HAS2 is overexpressed in the hereditary cutaneous mucinosis of Shar Pei dogs (Zanna et al. 2009), and fibroblasts from Shar Pei dogs have higher numbers of plasma membrane protrusions (Docampo et al. 2011). The lifespan of naked mole rat is the longest among rodents, even exceeding 30 years (Buffenstein and Jarvis 2002). Skin fibroblasts of naked mole rat have high expression levels for HAS2 and produce extremely high molecular weight hyaluronan, which accumulates in the subcutaneous tissue (Tian et al.

2013).

HAS2 is overexpressed in fibroblasts isolated from patients suffering idiopathic pulmonary fibrosis. These fibroblasts are more invasive compared to fibroblasts from healthy people. This invasion capacity is regulated by CD44 (Li et al. 2011).

Human and mouse HAS3 genes are localized in chromosomes 16 and 8, respectively (Spicer and McDonald 1998). HAS3 knockout mice are viable and have no specific morphological phenotype (Bai et al. 2005), but they have epileptic phenotype (Arranz et al.

2014). Hyaluronan produced by HAS3 is usually shorter than hyaluronan made by HAS1 and by HAS2 (Brinck and Heldin 1999, Itano et al. 1999). The promoter area of HAS3 has been recently characterized. There are binding sites for C/EBP and NFκB and Sp1, which seem to be essential for promoter activity (Wang et al. 2015). HAS3 is abundant on the plasma membrane (Rilla et al. 2005), and a specific feature of HAS3 is its accumulation into plasma membrane protrusions that collapse after hyaluronidase digestion or inhibition of hyaluronan synthesis (Kultti et al. 2006). This suggests that the protrusions are dependent on HAS3 activity.

2.2.5 Regulation of hyaluronan synthesis

Hyaluronan synthesis is stimulated in many physiological and pathological states, like in inflammation, after tissue injury and during tumor progression (Cyphert et al. 2015). The HAS expression is regulated by numerous local and systemic stimuli, like growth factors, cytokines and hormones.

The effects of growth factors on HAS activity are mainly mediated at the transcriptional level, since they induce rapid changes in HAS mRNA levels, usually associated with a simultaneous increase in hyaluronan synthesis (Jacobson et al. 2000, Karvinen et al. 2003b, Pienimäki et al. 2001, Yamada et al. 2004). For example, keratinocyte growth factor (KGF) (Karvinen et al. 2003b) and epidermal growth factor (EGF) (Pienimäki et al. 2001) stimulate the expression of HAS2 and HAS3 in keratinocytes. In fact, HAS2 is one of the direct target genes for EGF signaling (Saavalainen et al. 2005). Examples of hormones that induce HAS

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expression are estrogen (Tellbach et al. 2002) and progesterone (Uchiyama et al. 2005). Also several cytokines, like interleukin-‐‑ 1β (IL-‐‑1β) upregulate HAS expression and hyaluronan synthesis in many cell types, like fibroblasts (Yamada et al. 2004), endothelial cells (Vigetti et al. 2010) and lung adenocarcinoma cells (Chow et al. 2010). Hydrocortisone inhibits hyaluronan synthesis in human epidermis (Ågren et al. 1995). Glucocorticoids almost totally block HAS2 expression in dermal fibroblasts (Zhang et al. 2000)

4-‐‑methylumbelliferone (4-‐‑MU) has been reported to specifically inhibit hyaluronan synthesis in cultured mammalian cells (Kosaki et al. 1999a, Kultti et al. 2009, Nakamura et al. 1995, Nakamura et al. 1997, Sohara et al. 2001) and in Streptococcus equi FM100 cells (Kakizaki et al. 2002). It inhibits melanoma cell adhesion and locomotion (Kudo et al. 2004), and metastasis (Yoshihara et al. 2005). 4-‐‑MU also reverses the effect of HAS2 transfection on hyaluronan synthesis and colony formation of tumor cells (Kosaki et al. 1999a). Nowadays 4-‐‑MU is widely utilized as a research tool to inhibit hyaluronan synthesis.

At post-‐‑transcriptional level the availability of UDP-‐‑sugar precursors is a potential regulator of HAS activity (Itano et al. 1999). This hypothesis is supported with a finding that depletion of the UDP-‐‑glucuronic acid pool through glucuronidation of 4-‐‑MU (Kakizaki et al. 2004) reduces hyaluronan synthesis rate. Also reduction of UDP-‐‑GlcNA by mannose reduces hyaluronan synthesis rate (Jokela et al. 2011). In general, the availability of both UDP-‐‑sugars regulates the activity of hyaluronan synthesis (Deen et al. 2016, Jokela et al.

2011, Kakizaki et al. 2004, Kultti et al. 2009, Rilla et al. 2013a, Vigetti et al. 2009).

It has been suggested that hyaluronan synthases can form homo-‐‑ and heteromers in plasma membrane, which offers one more possible way of regulation for HAS activity (Bart et al. 2015, Karousou et al. 2010). Bart and coworkers showed reduced hyaluronan synthesis in HAS2 and HAS3 overexpressing cells cotransfected with Has1 (Bart et al. 2015). Other putative factors regulating HAS activity are post-‐‑transcriptional modifications in HAS2 enzyme, including phosphorylation (Goentzel et al. 2006, Vigetti et al. 2011), ubiquitination (Karousou et al. 2010) and O-‐‑GlcNAcylation (Deen et al. 2016, Vigetti et al. 2012).

Because HASs are known to be active only in the plasma membrane (Rilla et al. 2005), their traffic is potentially an important post-‐‑transcriptional factor regulating hyaluronan synthesis (Deen et al. 2014). All HAS isoenzymes follow the normal route for transmembrane proteins, travelling from ER to Golgi apparatus and further to the plasma membrane (Müllegger et al. 2003). Regulation of HAS trafficking is not fully understood, but posttranslational modifications of HAS and intracellular UDP-‐‑sugar levels affect it (Müllegger et al. 2003, Siiskonen et al. 2014). HAS1 seems to be less present on the plasma membrane (Siiskonen et al. 2014) than HAS2 and HAS3 (Rilla et al. 2005, Siiskonen et al.

2014). Accordingly, HAS2 and especially HAS3 are more active in inducing plasma membrane protrusions (Kultti et al. 2006, Rilla et al. 2005) and secretion of extracellular vesicles (Rilla et al. 2013b, Rilla et al. 2014).

2.3 HYALURONAN DEGRADATION

Hyaluronan is degraded by hyaluronidases. Vertebrate hyaluronidases produce a large size range of hyaluronan oligomers (Stern and Jedrzejas 2006). They can also break down chondroitin, chondroitin-‐‑4 sulfate, chondroitin-‐‑6 sulfate and to some extent dermatan sulfate (Kreil 1995). There are six hyaluronidase genes in humans, hyaluronidases 1-‐‑4, PH-‐‑

20 and HYALP1 (Stern and Jedrzejas 2006). Free radicals and reactive oxygen species can fragment hyaluronan without hyaluronidases (Monzon et al. 2010, Ågren et al. 1997b).

The most studied hyaluronidases are HYAL1 and HYAL2. In humans HYAL1 is found in many organs, like liver, kidney, spleen and heart (Csoka et al. 2001). This enzyme is

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mainly located in lysosomes and requires a low pH for activity (Csoka et al. 2001). HYAL1 is also detected in serum (Csoka et al. 2001) and urine (Csoka et al. 1997). High serum levels of HYAL1 are explained by its continuous secretion and uptake in cells (Gasingirwa et al.

2010).

HYAL2 is a glycosylphosphatidylinositol-‐‑linked (GPI-‐‑linked) protein on cell surface (Fig.

2). Its expression has been detected in all mouse tissues except brain (Rai et al. 2001).

HYAL2 has a relatively low hyaluronidase activity and it hydrolyses only high molecular weight hyaluronan (Lepperdinger et al. 1998). A recent study has suggested possible HYAL2 binding to CD44. Shedding of HYAL2 from cell surface is correlated with CD44 shedding (Hida et al. 2015, Tian et al. 2013).

2.4 CD44 AND OTHER HYALURONAN RECEPTORS/BINDING PROTEINS There are several hyaluronan-‐‑binding proteins including CD44, LYVE1, HARE/Stabilin2, RHAMM, TSG-‐‑6/TNFIP6, aggrecan, brevican, versican and SHAP (Knudson and Knudson 1993). Some of these proteins like CD44, LYVE, HARE, are membrane associated proteins and some like aggrecan, brevican, versican and TSG-‐‑6 are found in the extracellular space.

RHAMM is found intracellularly and on cell suface (Day and Prestwich 2002). Most of these proteins have a constitutive hyaluronan binding protein motif, so called link module (Kohda et al. 1996) while in some of them like RHAMM B(X⁷)B motif (B is arginine or lysine, X is any non-‐‑acidic amino acid) is responsible for hyaluronan binding (Yang et al.

1994).

2.4.1 CD44

The main cell surface hyaluronan binding protein is CD44 (Fig.2) (Aruffo et al. 1990). CD44 is a glycosylated transmembrane protein containing an N-‐‑terminal link-‐‑module. The structure of the extracellular domain and its glycosylations are regulated via alternative splicing (Lesley et al. 1993). Ten variable exons can be inserted in the extracellular part of CD44 and there are two alternative exons for the intracellular domain (Thorne et al. 2004).

The intracellular part of CD44 is rather short (Thorne et al. 2004). CD44 has several N-‐‑ and O-‐‑glycolysationsites (Lesley and Hyman 1998). Substitution of CD44 by chondroitin sulfate, heparan sulfate and keratan sulfate chains is possible (Piepkorn et al. 1997, Piepkorn et al.

1999, Takahashi et al. 1996, Tuhkanen et al. 1997). These glycosaminoglycan side chains make CD44 capable of binding growth factors, like hepatocyte growth factor (Orian-‐‑

Rousseau et al. 2002). There are three phosphorylation sites in the standard CD44 molecule.

Serine 325 relays CD44 association with the ezrin–radixin-‐‑moesin complex, and through them to actin cytoskeleton (Thorne et al. 2004). CD44-‐‑actin cytoskeleton interaction is stabilized by lipid rafts in the plasma membrane (Oliferenko et al. 1999). Palmitoylation of the transmembrane domain of CD44 might regulate its association in lipid rafts (Thankamony and Knudson 2006). CD44 is expressed in most human and mouse tissues (Sherman et al. 1997). The capacity of CD44 to bind hyaluronan varies. It can be totally inactive, activatableby certain signals and constitutively active (Lesley and Hyman 1992).

CD44 affinity to HA is quite low. Clustering of CD44 molecules increases affinity to hyaluronan (Sleeman et al. 1996). Specific de-‐‑N-‐‑glycosylation at the hyaluronan binding site of CD44 increases its affinity to HA (English et al. 1998). CD44-‐‑HA interaction has several roles in tissues. CD44 binding to hyaluronan links cells to the pericellular matrix. CD44 also mediates the uptake of hyaluronan in many cell types, like macrophages and chondrocytes (Culty et al. 1992). Hyaluronan binding to CD44 has been shown to recruit signaling molecules including ErbB2 and epidermal growth factor receptor (Toole 2009). The

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interactions between hyaluronan and CD44 have an important role in the development, inflammation, T-‐‑cell recruitment, tumor growth and metastasis (Lesley et al. 1993).

Overexpression of CD44 variants has a role in many tumors such as ovarian (Bourguignon et al. 2001), gastric (Carvalho et al. 2006) and head and neck cancers (Wang et al. 2007). A phenotype with CD44 overexpression is generally more malignant. In squamous cell carcinomas CD44 is decreased in high malignant cases (Karvinen et al.

2003a, Kosunen et al. 2004). There are also reports suggesting suppression of metastasis by CD44 (Gao et al. 1997, Kuo et al. 2006). CD44 is one of the markers for cancer stem cells for example in head and neck (Bourguignon et al. 2012), and lung (Wang et al. 2013) cancers.

2.4.2 Other hyaldherins

LYVE1 (lymphatic vessel endothelial hyaluronan receptor 1) is found in human (Banerji et al. 1999) and mice (Prevo et al. 2001) lymphatic vessels. It is a homolog of CD44 and has a link module (Banerji et al. 1999). LYVE1 is present in hepatic sinusoidal endothelial cells (Mouta Carreira et al. 2001) and in the reticular cells of lymph nodes (Wrobel et al. 2005).

HARE/stabilin 2 is a hyaluronan receptor for endosytosis. It was first found in liver endothelial cells (Zhou et al. 2000). Different splice variants have been found in tissues like spleen, lymph node and bone marrow (Hare and Harris 2015). HARE is a plasma membrane protein that binds to hyaluronan with high affinity and the bound hyaluronan is rapidly endocytosed. There are several other ligands for HARE, like chondroitin sulfate, dermatan sulfate and heparin. Stabilin 2 knockout mouse has an otherwise normal phenotype, except that the serum level of hyaluronan is high (Schledzewski et al. 2011).

RHAMM (receptor for hyaluronan mediated motility) is expressed in many tissue types (Hardwick et al. 1992). Hyaluronan interaction with RHAMM has an important role during tissue injury and repair (Zaman et al. 2005). For example, upregulation of RHAMM expression has been detected in bovine aortic smooth muscle cells after injury (Savani et al.

1995).

Brevican is a brain-‐‑specific proteoglycan, which exists both as secreted and cell surface glycosylphosphatidytinositol-‐‑anchored isoforms (Jaworski et al. 1999). Neurocan is a proteoglycan specific for brain and nervous tissues. During brain remodelling processes neurocan and hyaluronan distributions and functions are associated (Rauch et al. 2004).

Neurocan expression is increased following brain (Asher et al. 2000) and spinal cord (Jones et al. 2003) injuries. Aggrecan is a cartilage-‐‑specific high molecular weight proteoglycan. It has a long core protein with a large number of glycosaminoglycan side chains (Fig.2). In cartilage about 100 aggrecan molecules bind to a hyaluronan chain and make very large aggregates. These aggregates are negatively charged and bind water and positive ions. The aggregates are trapped in the collagen network. This helps to create the high osmotic pressure of cartilage (Chandran and Horkay 2012). Versican is another large chondroitin sulfate proteoglycan. Some versican is present in most tissues. It has four splice variants.

Versican is involved in cell adhesion, proliferation, extracellular matrix modeling, and migration (Wight et al. 2014). Versican can regulate inflammation (Wight et al. 2014) and has a role in cancer development and metastasis (Ricciardelli et al. 2009).

Hyaluronan binding protein-‐‑2 (HABP2) was originally isolated from plasma (Choi-‐‑

Miura et al. 1996). This extracellular protease takes part in blood coagulation and has been linked to atherosclerosis (Kanse et al. 2008). Low molecular weight hyaluronan activates HAPB2 and high molecular weight hyaluronan inhibits. This activation results in endothelial barrier dysfunction (Mambetsariev et al. 2010). HABP2 is an important in human lung carcinoma (Mirzapoiazova et al. 2015).

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9 2.5 HYALURONAN AND CANCER

2.5.1 Hyaluronan in cancer cell cultures

The rate of hyaluronan secretion is typically high in cancer cells compared to non-‐‑

malignant cells, and often correlates with the malignant properties of the cells.

Overexpression of HAS2 promotes anchorage-‐‑independent growth of cultured fibrosarcoma cells (Kosaki et al. 1999b) and mesothelioma cells (Li and Heldin 2001), while inhibition of HAS2 leads to decreased anchorage-‐‑independent growth in breast cancer cells (Li et al. 2007a). Moreover, growth of colon carcinoma cells is dependent on HAS3 expression (Bullard et al. 2003). Increased production of hyaluronan enhances multidrug resistance of many cultured cancer cell lines (Misra et al. 2003). Hyaluronan and hyaluronan binding proteins have been shown to take part in epithelial-‐‑mesenchymal transition (Cho et al. 2012, Itano and Kimata 2008). HAS3 overexpression and hyaluronan accumulation disrupted epithelial cell polarity. This failure of tissue organization is a possible premalignant feature that can lead to malignant growth (Rilla et al. 2012).

2.5.2 Hyaluronan in animal tumor models

The importance of hyaluronan in cancer progression has been shown in several animal models. Hyaluronan overproduction by HAS2 overexpression or HYAL1 inhibition promotes tumorigenicity of rat colon carcinoma cells (Jacobson et al. 2002). Transgenic mice with HAS2 overexpression in the mammary gland show increased tumor growth and angiogenesis (Koyama et al. 2007). In a tumor xenocraft model intratumoral lymphangiogenesis is promoted by a hyaluronan-‐‑rich tumor stroma (Koyama et al. 2008).

Furthermore, depletion of hyaluronan by a clinically formulated hyaluronidase increased the tumor delivery of chemotherapeutic agents in a mouse model of pancreatic cancer (Jacobetz et al. 2013).

2.5.3 Hyaluronan in human cancer

Hyaluronan is a strong indicator of malignant growth and acts as an independent unfavourable prognostic factor in many tumors of epithelial origin. Poor prognosis of cancer is associated with hyaluronan accumulation in breast, colorectal, prostate, lung, ovarian, and gastric cancers (Tammi et al. 2008).

Hyaluronan accumulates in the stroma of several tumors, like breast (Auvinen et al.

2000), ovarian (Anttila et al. 2000), prostate (Lipponen et al. 2001), and thyroid (Böhm et al.

2002) cancers. Strong hyaluronan staining in the tumor stroma is linked to invasion and is usually correlated with poor prognosis. Cell associated hyaluronan is also increased in tumors like bladder (Hautmann et al. 2001), ovarian (Anttila et al. 2000), and colorectal cancers (Ropponen et al. 1998) and even nuclear positivity has been detected in some cases, like in basal cell carcinoma (Karvinen et al. 2003a) and breast cancer (Auvinen et al. 2000).

In squamous cell carcinomas originating from mouth (Kosunen et al. 2004), lung (Pirinen et al. 1998), larynx (Hirvikoski et al. 1999) skin (Karvinen et al. 2003a), and melanoma (Karjalainen et al. 2000) show reducing levels of hyaluronan during the progression of cancer. Poor prognosis of mouth carcinoma is associated with reduced hyaluronan level (Kosunen et al. 2004).

Hyaluronan synthases in development and cancer : immunohistochemical studies

uef.fi

Dissertations in Health Sciences

KARI TÖRRÖNEN

HYALURONAN SYNTHASES IN DEVELOPMENT AND CANCER

KARI TÖRRÖNEN

Hyaluronan Synthases in Development and Cancer

Hyaluronan Synthases in Development and Cancer

Immunohistochemical studies

To Vanessa, Oliver, Nastja and Dasha

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the literature