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ISBN 978-952-62-1168-8 (Paperback)ISBN 978-952-62-1169-5 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1354

ACTA

Jyri Moilanen

OULU 2016

D 1354

Jyri Moilanen

FUNCTIONAL ANALYSISOF COLLAGEN XVII IN EPITHELIAL CANCERS ANDA MOUSE MODEL

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER;OULU UNIVERSITY HOSPITAL

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 3 5 4

JYRI MOILANEN

FUNCTIONAL ANALYSIS OF COLLAGEN XVII IN EPITHELIAL CANCERS AND A MOUSE MODEL

Academic Dissertation to be presented with the assentof the Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defencein Auditorium 3 of Oulu University Hospital, on 4 May2016, at 12 noon

UNIVERSITY OF OULU, OULU 2016

Copyright © 2016Acta Univ. Oul. D 1354, 2016

Supervised byProfessor Kaisa Tasanen-MäättäDocent Tiina Hurskainen

Reviewed byProfessor Juha PeltonenProfessor Malin Sund

ISBN 978-952-62-1168-8 (Paperback)ISBN 978-952-62-1169-5 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2016

OpponentProfessor Jyrki Heino

Moilanen, Jyri, Functional analysis of collagen XVII in epithelial cancers and amouse model. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center; Oulu University HospitalActa Univ. Oul. D 1354, 2016University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Basement membranes (BM) underlie epithelia and endothelia and surround many tissues. Incutaneous BM epithelial cells are attached to the stroma via multiprotein complexes calledhemidesmosomes (HD). Collagen XVII and integrin α6β4 are components of HD and they bindto laminin 332, a component of anchoring filaments, extracellularly. The main interest of thisstudy is the function of collagen XVII and its interactions with these proteins.

What is known about the function of collagen XVII is mostly derived from its role as anadhesive component in cutaneous HD. Here we demonstrate for the first time that collagen XVIIis expressed by podocytes in the human and murine glomerulus and that mutant mice lackingcollagen XVII in addition to small size, blisters and diffuse hair loss, also have deficientglomerular development and a high mortality rate.

We also show for the first time at the protein level that collagen XVII is expressed, andprobably has a functional interaction with laminin 332, in normal colon epithelia. We demonstratethat collagen XVII is expressed by the invasive cells of human colorectal carcinoma (CRC)samples and its immunostaining is increased in metastasis in CRC. The higher proportion ofcollagen XVII positive tumor cells correlates with decreased disease-free survival and cancer-specific survival times and we also suggest a functional interaction between collagen XVII andlaminin 332 in CRC.

Previous studies have suggested that collagen XVII participates in keratinocyte migration byaffecting the correlation of HD disassembly and assembly, its expression is increased in squamouscell carcinoma (SCC) and it may have a role in cell adhesion and migration in SCC carcinogenesis.Here we demonstrate upregulated collagen XVII, integrin β4 and laminin γ2 expression in actinickeratosis, Bowen’s disease and SCC. The expression of collagen XVII was increased with a highdegree of variation, especially in samples taken from areas where SCC is particularly invasive. Wealso demonstrate in the SCC-25 cell line that lack of collagen XVII or integrin β4 severely disruptsthe adhesion, migration and invasivity of these cells.

Taken together, in this study we show that collagen XVII is needed for normal glomerulardevelopment, is expressed in normal colon epithelia and participates in CRC and SCCcarcinogenesis together with laminin 332 and integrin β4.

Keywords: basement membrane, collagen, colorectal carcinoma, glomerulus, squamouscell carcinoma

Moilanen, Jyri, Kollageeni XVII toiminnan analysointi epiteliaalisissasyöpäsoluissa ja poistogeeninen hiirimalli. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical ResearchCenter; Oulun yliopistollinen sairaalaActa Univ. Oul. D 1354, 2016Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Tyvikalvot sijaitsevat epiteelin ja endoteelin alla ja ympäröivät monia kudoksia. Ihon tyvikalvos-sa epiteelisoluja alla olevaan verinahkaan kiinnittää rakenne, jota kutsutaan hemidesmosomiksi(HD). Kollageeni XVII ja integriin α6β4 ovat HD:n rakenneproteiineja. Ne kiinnittyvät solunulkopuolella laminiin 332 nimiseen proteiiniin, joka muodostaa ankkurifilamentit. KollageeniXVII ilmentyminen ja toiminta yhdessä näiden kahden proteiinin kanssa on tämän tutkimuksenkeskeisin kohde.

Valtaosa tutkimuksista, jotka käsittelevät kollageeni XVII:ää, koskevat sen toimintaa ihonkeratinosyyteissä. Tässä tutkimuksessa osoitimme ensi kertaa, että hiiren ja ihmisen munuaiske-rästen podosyyttisolut ilmentävät kollageeni XVII. Geenimanipuloidut hiiret, joilta kollageeniXVII oli poistettu, olivat pieniä, kehittivät rakkuloita ja karvattomuutta, niillä oli korkea kuollei-suus ja niiden munuaiskerästen kehitys oli häiriintynyt. Kollageeni XVII esiintymistä proteiini-tasolla, sekä mahdollista toiminnallista yhteyttä laminiin 332:een, ei aiemmin ole osoitettu pak-susuolen epiteelissä. Havaitsimme, että paksu- ja peräsuolen adenokarsinooman (CRC) invasii-vinen solukko ilmentää kollageeni XVII:ää, kollageeni XVII esiintyminen on merkittävän voi-makasta CRC:n metastasoinnin yhteydessä ja lisääntynyt kollageeni XVII esiintyminen lyhen-tää syöpävapaata aikaa ja heikentää syöpäspesifistä selviytymistä. Myös CRC:ssä kollageeniXVII toiminta voi liittyä laminiini 332:een.

Aiempien tutkimusten mukaan kollageeni XVII osallistuu keratinosyyttien migratioon vai-kuttamalla toimivien HD:ien määrään. Sen määrän on havaittu olevan korkeampi okasolu-syövässä (SCC) ja sen on ehdotettu osallistuvan syöpäsolujen adheesioon ja migraatioon SCC:nkehittyessä. Me osoitimme kohonneen kollageeni XVII, integriini β4 ja laminiini γ2 ilmenemi-sen aktiinisessa keratoosissa, Bowenin taudissa sekä SCC:ssä. Kollageeni XVII määrä oli kor-kea, mutta vaihteli paljon, sekä hiiren että ihmisen invasiivisilla SCC alueilla. Havaitsimmemyös SCC-25 solulinjalla, että kollageeni XVII tai integriini β4 puutos häiritsee vakavasti solu-jen adheesiota, migraatiota ja invaasiota.

Yhteenvetona tässä työssä osoitimme, että kollageeni XVII:ää tarvitaan munuaiskerästenkehittymisessä, sitä esiintyy paksusuolen epiteelissä, ja että kollageeni XVII osallistuu CRC:n jaSCC:n kehittymiseen yhdessä integriini β4:n ja laminiini 332:n kanssa.

Asiasanat: kollageeni, munuaiskeränen, okasolusyöpä, paksusuolen adenokarsinooma,tyvikalvo

To my family

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Acknowledgements

This study was carried out at the Department of Dermatology, Department of

Pathology, Clinical Research Center, Biocenter and Oulu Center for Cell Matrix

Research, University of Oulu from 2007 to 2016.

In particular, I owe my sincerest gratitude to my supervisor, the Head of the

Department of Dermatology, Professor Kaisa Tasanen-Määttä, MD, Ph.D, for all

the support, advice and patience you have given me during the years. It has been a

long journey and your thorough knowledge of this field of study has guided me

through the difficulties.

I wish to thank my second supervisor, Docent Tiina Hurskainen, Ph.D, who has

guided and supervised my work through the practical phases and assisted me in

each step to complete the research. You have also given me the much needed moral

support during the years.

I would like to express my deepest thanks to the former Head of the Department

of Dermatology, the chair of my follow-up group, Professor Aarne Oikarinen, MD,

Ph.D. You have always given your valuable time to provide me with the requested

information, comments and your support.

I wish to express my deepest thanks to the best lab technician there is. Anja

Mattila, without your vast expertise this project would not have been finished.

I also wish to appreciate the official pre-examinators of this dissertation,

Professor Malin Sund, MD, Ph.D, and Professor Juha Peltonen, MD, Ph.D. Your

insightful and thought-provoking comments have made a significant improvement

to this manuscript. I also thank Steve Smith, for his skilled linguistic editing of the

dissertation.

I thank all the other members of our research group, Laura Huilaja, MD, Ph.D,

Nina Kokkonen, Ph.D, Minna Kubin, MD, and Anna-Kaisa-Försti, MD, for your

intellectual support and ideas during the years. Your collegial support and advice

have given me a great deal of wisdom and strength during this project.

I also wish to thank all the collaborators that have participated in this project

and enabled its outcome. I especially would like to acknowledge: Docent Raija

Sormunen, Ph.D, Claus-Werner Franzke, PhD, Raija Soininen, PhD, Stefanie

Löffek, PhD, Docent Matti Nuutinen, MD, Ph.D, Professor Leena Bruckner-

Tuderman MD, Ph.D, Docent Helena Autio-Harmainen, MD, Ph.D, Juha Väyrynen,

MD, PhD, Erkki Syväniemi, MD, Professor Markus Mäkinen, MD, Ph.D, Kai

Klintrup, MD, PhD, Ritva Heljäsvaara, Ph.D, Professor Jyrki Mäkelä, MD, Ph.D,

10

Sirpa Salo, PhD, Docent Aki Manninen, Ph.D, Professor Tuula Salo, DDS, Ph.D,

Pilvi Riihilä, MD, PhD and Professor Veli-Matti Kähäri, MD, Ph.D.

I would also like to express my gratitude to the whole staff in the Department

of Dermatology and Clinical Research Center for your understanding and kind

assistance whenever needed.

I would also wish to show my deepest gratitude to my parents, Irma and Leo,

for their love and support. You have taught me the ethics of hard work and therefore

given me the chance to succeed in life.

I cannot go without mentioning my beloved brothers Jani and Raine, and my

sister Tanja. If there has been anything I have needed help with, work-related or

not, I have always been able to count on your help and support.

My thanks are also owed to my friends and colleagues for your endless support

and advice. You have been there to help me through some very frustrating times.

You know who you are.

Finally, above all, I wish to thank my best friend and my wife, Elisa, and our

family. You have kept me on the right path and shown me what is truly important

in life.

16.3.2016 Jyri Moilanen

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Abbreviations

BP bullous pemphigoid antigen

BP180 bullous pemphigoid antigen 180

BP230 bullous pemphigoid antigen 230

BM basement membrane

cDNA complementary DNA

CRC colorectal carcinoma

CSS cancer-specific survival

DNA deoxyribonucleic acid

DFS disease-free survival

EB epidermolysis bullosa

ECM extracellular matrix

FP foot process of podocyte

GFB glomerular filtration barrier

GFR glomerular filtration rate

HD hemidesmosome

IEM immunoelectron microscopy

IHC immunohistochemistry

KD knockdown

MAPK mitogen-activated kinase

MARCO macrophage receptor with collagenous structure

NC non-collagenous

RNA ribonucleic acid

SEM scanning electron microscopy

SCC squamous cell carcinoma

SD slit diaphragm

TEM transmission electron microscopy

TMA tissue micro array

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Original articles

This thesis is based on the following publications, which are referred throughout

the text by their Roman numerals:

I Hurskainen T, Moilanen J, Sormunen R, Franzke CW, Soininen R, Löffek S, Huilaja L, Nuutinen M, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen K (2012) Transmembrane collagen XVII is a novel component of the glomerular filtration barrier. Cell Tissue Res 348(3):579-88.

II Moilanen J, Kokkonen N, Löffek S, Väyrynen JP, Syväniemi E, Hurskainen T, Mäkinen MJ, Klintrup K, Mäkelä J, Sormunen R, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen K (2015) Collagen XVII correlates with the invasion and metastasis of colorectal cancer. Hum Pathol. 46(3):434-42.

III Moilanen J, Löffek S, Kokkonen N, Salo S, Väyrynen JP, Hurskainen T, Manninen A, Riihilä P, Heljäsvaara R, Franzke CW, Kähäri VM, Salo T, Mäkinen MJ, Tasanen K Collagen XVII and integrin β4 show similar expression in squamous cell carcinoma and their knockdown suppresses migration and invasion only of the less aggressive squamous cell carcinoma cells. Manuscript.

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Contents

Abstract

Tiivistelmä

Acknowledgements 9 Abbreviations 11 Original articles 13 Contents 15 1 Introduction 17 2 Review of the literature 19

2.1 Basement membranes and hemidesmosomes ......................................... 19 2.1.1 Cutaneous basement membrane ................................................... 21 2.1.2 Glomerular basement membrane .................................................. 23 2.1.3 Colorectal basement membrane ................................................... 24

2.2 Collagen XVII ......................................................................................... 25 2.2.1 Introduction to collagens .............................................................. 25 2.2.2 Transmembrane collagens ............................................................ 26 2.2.3 Collagen XVII .............................................................................. 27 2.2.4 Localization and function of collagen XVII in the skin ............... 28 2.2.5 Collagen XVII in other tissues ..................................................... 29 2.2.6 Collagen XVII in blistering skin diseases .................................... 29

2.3 Collagen XVII, integrin α6β4 and laminin 332 in cell migration

and invasion ............................................................................................ 30 2.3.1 The role of collagen XVII in cell migration and invation ............ 30 2.3.2 Integrin α6β4 in cell migration and invasion ............................... 31 2.3.3 Laminin 332 and cell migration and invasion .............................. 33

3 Aims of this study 35 4 Materials and methods 37

4.1 Patients and tumor samples (II-III) ......................................................... 37 4.2 Generation of Col17a1-/- mice (I) ............................................................ 37 4.3 Cell culture (I-III) .................................................................................... 38 4.4 RNA isolation, quantitative real time PCR, Western Blot (II-III) ........... 38 4.5 Immunohistochemistry and in-situ hybridisation (I-III) ......................... 38 4.6 Electron microscopical analysis (I-II) ..................................................... 40

4.6.1 Transmission electron microscopy ............................................... 40 4.6.2 Immunoelectron microscopy ........................................................ 40 4.6.3 Scanning electron microscopy ...................................................... 40

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4.7 Determing glomerular volume fraction and slit diagram count (I) ......... 40 4.8 Laser capture microdissection (II) ........................................................... 41 4.9 RNAi and overexpression of collagen XVII (II-III) ................................ 41 4.10 Cell adhesion, migration and invasion analyses (II-III) .......................... 41 4.11 Statistical analysis of collagen XVII in CRC (II) .................................... 42

5 Results 43 5.1 Collagen XVII expression and function in normal tissue ....................... 43

5.1.1 Collagen XVII is expressed in the human and murine

glomerulus and in human colon epithelia (I-II) ............................ 43 5.1.2 Lack of collagen XVII alters the phenotypes of mice and

the function of glomeruli (I) ......................................................... 44 5.2 Expression and function of collagen XVII, laminin γ2 and

integrin β4 in CRC and SCC ................................................................... 46 5.2.1 Collagen XVII and laminin γ2 are upregulated in CRC (II) ......... 46 5.2.2 Collagen XVII, laminin γ2 and integrin β4 are upregulated

in human and mouse SCC (III) ..................................................... 47 5.2.3 Collagen XVII expression is associated with TNM and

metastasis survival times in CRC patients (II) ............................. 48 5.2.4 Collagen XVII participates in adhesion, migration and

invasion (II-III) ............................................................................. 49 6 Discussion 51

6.1 Collagen XVII is a newly characterised component of the

glomerulus and affects glomerular development .................................... 51 6.2 Collagen XVII is expressed in the normal colon epithelium and

may interact with laminin 332 ................................................................. 52 6.3 Collagen XVII is expressed in CRC and its expression correlates

with tumor progression and metastasis in CRC ...................................... 53 6.4 Collagen XVII, integrin α6β4 and laminin 332 are upregulated in

SCC ......................................................................................................... 54 6.5 Collagen XVII and integrin α6β4 affect the adhesion, migration

and invasion of SCC-25 cells but has no effect on HSC-3 cells ............. 55 7 Conclusions 59 References 61 Original articles 71

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1 Introduction

Proteins are the essential building blocks of body tissue. They are large molecules

consisting of one or more long chains of amino acid residues. The structural size of

proteins ranges from tens to thousands of residues (Rodwell & Kennelly 2003,

Kannan et al. 2012).

The functional range of proteins is wide. They participate in metabolic

reactions, DNA replication, transportation, response to stimuli and serve as crucial

structural components in tissues (Rodwell & Kennelly 2003). Besides the correct

amino acid sequence, a proteins needs to be folded into a specific three-dimensonal

arrangement to function correctly. In addition, correct function also requires

posttranslational modifications such as the addition of new chemical groups or the

removal of transiently needed peptide segments (Rodwell & Kennelly 2003).

At the cellular level individual cells are dependent on proteins. The attachment

of cells to the extracellular matrix or to other cells, cell migration (pathological or

physiological) and cell invasion are all dependent on the correct function and

structure of specific proteins (Margadant et al. 2008, Kashyap et al. 2011, Buchheit

et al. 2012).

Our study concentrates on the functions and interactions of three proteins:

collagen XVII, integrin α6β4 and laminin 332, in both normal and pathological

conditions. Collagen XVII and integrin α6β4 are both best known for being

components of multiprotein structures called the type I hemidesmosome (HD)

(Margadant et al. 2008). Laminin 332 is a major component of anchoring filaments

in the skin. Collagen XVII, integrin α6β4 and laminin 332 all interact with each

other and are well known for their role as adherence molecules, attaching epithelial

cells to the underlying basement membrane (BM) (Walko et al. 2015).

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2 Review of the literature

2.1 Basement membranes and hemidesmosomes

BMs are thin, specialised extracellular matrices that surround many tissues and

which are capable of isolating a cell from, and connecting a cell to, its environment

in all metazoans. They provide mechanical stability to tissues and support

important functions, including differentiation, proliferation, migration, and

chemotaxis of cells during development.

The composition of BMs depends on the type of tissue in which they are

located. BMs are composed of diverse extracellular matrix (ECM) molecules, the

deposition and arrangement of which determines the ability of BMs to adapt to the

cell’s changing biological requirements. The complexity of BM composition

continues to be characterised in increasing detail with the discovery of previously

unknown components and isoforms. An impressive number of tissue- and site-

specific BMs are now described in the skin, kidney, colon and lung, among other

tissues (Yurchenco & Patton 2009, Kruegel & Miosge 2010, Breitkreutz et al. 2013).

In general BMs contain at least one member of laminin, type IV collagen,

nidogen, and perlecan proteins or their subtypes which determines their common

structure. Several laminins isoforms are found in BMs and they all consist of an α,

a β and a γ chain linked by disulfide bonds to form a cross-shaped structure. To

date, more than 15 isoforms with different combinations of the α1–5, β1–3 and γ1–

3 chains have been identified (Miyazaki 2006).

Tissue-specific functional diversity is determined by differential expression of

the BM’s component proteins. As the main structural components, laminin and

collagen IV form distinct networks which bond noncovalently with nidogen and

perlecan and thus form irregular polymers (Breitkreutz et al. 2013). Table 1

summarises the components of the BM, their receptors and localization sites

(Kramer 2005, Kruegel & Miosge 2010). Changes in the structure, localization or

the qualitative or quantitative composition of BM proteins are involved in the

development of different disease states (Kramer 2005, Kruegel & Miosge 2010).

BM is important in forming the epithelial barrier. In addition to the BM there

are other important structures that connect epithelial cells to each other and to the

underlying BM and mesenchyme. Adhesive epithelial cell-to-cell contacts contain

large multiprotein aggregates with structural and signaling functions. There are

three structurally prominent types of cell-to-cell junction: tight junction, adherens

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junction and desmosome (Capaldo et al. 2014). The structures and functions of

these junctions are beyond the scope of this study and thus will not be discussed in

more detail.

The attachement of epithelial cells to the underlying BM is mediated via HDs.

HDs are classified as belonging to one of two types on the basis of their components

(Figure 1). Type I (classical) HDs are found in (pseudo-) stratified epithelia, for

example, in the skin and consist of integrin α6β4, plectin, tetraspanin CD151,

collagen XVII (also referred to as bullous pemphigoid antigen 180 or BP180) and

BP230 (Borradori & Sonnenberg 1999, Margadant et al. 2008). Type II HDs are

found in simple epithelia such as that of the intestine, and consist of integrin α6β4

and plectin (Fontao et al. 1999).

Table 1. Overwiev of BM components in specialized ECMs.

BM isoform Interactive receptors/ligands Site of expression

Laminin-111 Lam α1: α-dystroglycan Development, ubiquitously in BMs, adult

articular cartilage

Laminin-211 Lam α2: α7β1, α-dystroglycan Skeletal muscle, schwann cells,

eye (limbal compartment)

Laminin-221 - Skeletal muscle, schwann cells,

neuromuscular junction

Laminin-332 Lam α3: α3β1, α6β1, α6β4 Skin, embryonic cartilage

Laminin-411 Lam α4: αvβ3 Vasculature

Laminin-421 - Vasculature, neuromuscular junction

Laminin-511 Lam α5: Lutheran Bloodgroup

glycoprotein, B-CAM, α3β1, α6β1,

α6,β4

Development, vasculature, epithelium

Perlecan Interaction with and support of

diverse growth factors

Ubiquitously in BMs, articular cartilage,

growth plate

Collagen IV α1β1, α2β1, α3β1, α6β1, α10β1,

α11β1, αvβ3, CD44, DDR-1

Development, ubiquitously in BMs, kidney,

inner ear (cochlea), eye, testis, lung, skin,

esophagus, smooth muscle cells, synovia

(knee)

Nidogen-1 αvβ3, α3β1, Laminin γ1, collagen IV,

perlecan, fibronectin, collagen I,

collagen II

Ubiquitously in BMs, limb development, rib

anlagen, adult articular cartilage

Nidogen-2 Laminin, collagen IV, perlecan,

fibronectin, collagen I, collagen II

Like nidogen-1, but more restricted in muscle,

neuromuscular junction and cartilage

Data adapted from Kruegel & Miosge 2010.

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Fig. 1. Simplified diagram of the structure and components of type I and II

hemidesmosomes. The main components of type I HD are α6β4-integrin, plectin,

tetraspanin CD151 and collagen XVII. Type II HDs consist only of α6β4-integrin and

plectin (modified from Margadant et al. 2008).

2.1.1 Cutaneous basement membrane

Although cutaneous BM is not a major target of this study, understanding its

structure is important since the basic functions of the molecules of interest

(collagen XVII, laminin 332 and integrin β4) can be better understood via their

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function in skin. The cutaneous BM zone is a complex multiprotein structure that

attaches the epidermal keratinocytes to the underlying dermis via type I HDs. The

type I HD proteins integrin α6β4 and collagen XVII bind extracellularly to laminin

332, a major component of anchoring filaments in the skin. The intracellular HD

stabilization occurs via their association with keratin intermediate filaments

through the two plakins, plectin and BP230, thus creating a stable anchoring

complex (Zillikens et al. 1999, Margadant et al. 2008, Guess & Quaranta 2009,

Rousselle & Beck 2013) (Figure 2).

The cutaneous BM also controlls cell traffic and the diffusion of bioactive

molecules in both directions and binds a variety of cytokines and growth factors,

serving as a reservoir for their controlled release (Breitkreutz et al. 2013).

The ultrastucture of cutaneous BM as observed by transmission electron

microscopy (TEM) can be divided into two segments: the lamina densa and lamina

lucida (Figure 2). The laminin and collagen IV polymers form the body of the

lamina densa below the lamina lucida. However, the lamina lucida is not detectable

in electron microscopy (EM) specimens fixed by cryopreservation, indicating that

the lamina lucida represents an artificial structure rather than the real topography

of BM (Breitkreutz et al. 2013).

Fig. 2. Schematic of the structure of the dermal-epidermal junction (modified from

McMillan et al. 2003, Masunaga 2006, Aumailley et al. 2006).

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2.1.2 Glomerular basement membrane

The glomerulus is the principal dynamic filtering unit in the kidney. It consists of

endothelial cells and podocytes, separated by a glomerular basement membrane

(GBM), which is primarily comprised of collagen IV, laminins and

glycosaminoglycans, as well as supporting mesangial cells (Miner 2005). The

three-layered structure of the endothelium, GBM, and podocytes facilitates the flow

of plasma and small solutes and restricts the flow of large plasma proteins like

albumin. The presence of high levels of albumin in the urine is considered to be

indicative of a defect in at least one of the layers of the glomerular filtration barrier

(GFB) (Miner 2011, Miner 2012). The structure of the GFB is shown in Figure 3.

Mature podocytes extend into several large projections, which divide into

smaller foot processes (FP) that interdigitate with the FPs of adjacent podocytes

(Quaggin & Kreidberg 2008). The basal surfaces of the FPs of a pair of adjacent

podocytes adhere to the GBM. The contact of two FPs form a cell-cell junction

called the slit diaphragm (SD) (Michaud & Kennedy 2007, Quaggin & Kreidberg

2008). The SD is formed by a complex of plasma membrane proteins including

nephrin, podocin, Fat1, vascular endothelial cadherin, P-cadherin, Nck proteins and

Cd2-associated protein (Patrakka & Tryggvason 2010). The SD maintains spacing

between the FPs and permits the efficient flow of water and small solutes across

the filtration barrier. Podocyte FPs are anchored to the GBM via transmembrane

cell receptors, including α3β1 integrin, α- and β- dystroglycans, and tetraspanin

CD151 (Pozzi et al. 2008, Patrakka & Tryggvason 2010).

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Fig. 3. The structure of the glomerular filtration barrier. Capillary loops are covered by

endothelial cells (red), glomerular basement membrane (green) and podocytes with

their FPs (blue). The glomerular basement membrane divides the glomerulus into two

compartments; the inner one containing the capillaries and the mesangial cells and the

outer one containing podocytes and the space into which the filtrate passess.

M = mesangial cells, C = capillary loop, BC = Bowman’s capsule, P = podocytes,

E = endothelial cells (modified from Quaggin & Kreidberg 2008).

2.1.3 Colorectal basement membrane

The BM of the colon adheres colonic epithelial cells to the underlying stroma

(Debruyne et al. 2006). The epithelium of the human colon consists of a single

sheet of columnar epithelial cells, which form finger-like invaginations into the

underlying connective tissue forming crypts, the most important functional units of

the intestine. There are millions of crypts in the intestine. The crypts are surrounded

by BM which separates the epithelial cells of the crypt from the mesenchymal cells

of the stroma. (Humphries & Wright 2008). The columnar epithelial cells are

attached to the colonic BM via type II HDs (Figure 1) (Fontao et al. 1999). The

structure of the intestinal wall including the BM zone is shown in Figure 4.

Colonocytes, mucus-secreting goblet cells, peptide hormone-secreting

endocrine cells and Paneth cells are four major terminally differentiated epithelial

cell types present in the colon. There is well founded evidence supporting the

concept that all these cells can originate from a single multipotential stem cell

through a number of committed progenitors (Cheng & Leblond 1974, Humphries

25

& Wright 2008). There is also strong evidence that these stem cells are located at

the base of the crypt. The interaction between the stem cells and underlying

mesenchymal cells of the myofibroblast lineage through the BM is considered

critical for the stem cell behaviour. It is thought that pericryptal myofibroblasts

control the stem cell habitus, cell migration and differentiation via multiple

signalling pathways, including Wnt and Notch signalling (Humphries & Wright

2008).

Fig. 4. The structure of intestinal wall (modified from www.patiencenter.com Colon and

Rectal Cancer Center).

2.2 Collagen XVII

2.2.1 Introduction to collagens

Members of the collagen family are the most abundant proteins in the extracellular

matrix. They are the major structural element of all connective tissues and are also

found in the interstitial tissue of virtually all parenchymal organs, where they

contribute to the stability of tissues and organs and maintain their structural

integrity. To date 28 types of collagen have been identified. They are composed of

three polypeptide chains, called α chains. Different α chains are numbered with

Arabic numerals (Ricard-Blum 2011). The three a chains can be identical

26

(homotrimer) or different (heterotrimer). Collagens are characterised by containing

domains with repetitions of the proline-rich tripeptide Gly-X-Y, which form

trimeric collagen triple helices. As well as triple-helical domains, collagens also

contain non triple-helical non-collagenous (NC) domains, which are used as

building blocks by other extracellular matrix proteins and are thus modular proteins

(Gelse et al. 2003, Ricard-Blum & Ruggiero 2005).

Collagens can be grouped based on their structure and supramolecular

organisation into fibril-forming collagens, fibril-associated collagens with

interrupted triple helices, network-forming collagens, anchoring fibrils, membrane

associated collagens with interrupted triple helices and others with unique functions

(Gelse et al. 2003, Jain et al. 2014).

Several diseases, such as Alport syndrome, Ehler’s Danlos syndrome and

epidermolysis bullosa (EB), arise from genetic defects that affect the molecular

structure of collagens. Thus it is crucial to understand their molecular structure,

biosynthesis, assembly and turnover (Gelse et al. 2003, Ricard-Blum & Ruggiero

2005).

2.2.2 Transmembrane collagens

The transmembrane, or membrane collagens and their distribution are summarized

in table 2. They include the homotrimeric collagens XIII, XVII, XXIII, XXV. This

subgroup also includes collagen-like membrane proteins that have, to date not been

classified as collagens, such as ectodysplasin, the macrophage receptor with

collagenous structure (MARCO) and macrophage scavenger receptors, (Tenner

1999).

Table 2. Membrane collagens.

Collagen type Distribution

XIII Endothelial cells, dermis, eye, heart

XVII Widespread: Skin, kidney, brain, intestine

XXIII Heart, retina

XXV Brain, heart, testis

Data adapted from Jain et al. 2014.

Collagens XIII, XVII, XXIII and XXV are type II transmembrane proteins with the

N-terminus located inside the cell and with a single pass hydrophobic

transmembrane domain and several extracellular collagenous domains (Franzke et

27

al. 2005) (Figure 5). The folding of the triple helix proceeds from the N- to the C-

terminus in the ectodomain of collagens XIII and XVII, in the opposite orientation

to that of the fibrillar collagens (Areida et al. 2001, Snellman et al. 2000).

Collagens XIII, XXIII and XXV, MARCO and ectodysplasin-A1 contain two

separate coiled-coil motifs, which are thought to function as independent

oligomerization domains (Latvanlehto et al. 2003, McAlinden et al. 2003,

Snellman et al. 2000). The NC4 C-terminal domain (20 amino acids) of collagens

XIII, XXIII and XXV is thought to be involved in functional interaction with the

cell surface or with extracellular matrix proteins (Banyard et al. 2003).

Membrane collagenous proteins function both as cell surface receptors and as

soluble extracellular molecules because their ectodomains are released from the

cell surface by shedding. Membrane collagens all have a triple-helical extracellular

domain. The length of the extracellular component varies between the collagens –

for example, it consists of three collagenous domains in domains in collagen XIII

(Pihlajaniemi & Rehn 1995), XXIII (Banyard et al. 2003) and XXV (Hashimoto et al. 2002) and of 15 domains in collagen XVII (Franzke et al. 2003) (Figure 5).

Fig. 5. The structure of membrane collagens (modified form Ricard-Blum 2011).

2.2.3 Collagen XVII

Collagen XVII is the largest transmembrane collagen. It consists of three 180-kDa

α1 (XVII) chains, each with an intracellular N-terminal domain of 466 amino acids,

a short transmembrane stretch of 23 amino acids, and an extracellular C-terminal

ectodomain of 1008 amino acids (Franzke et al. 2004). Its rod-like structure is

flexible and triple-helical, with pronounced thermal stability (Schacke et al. 1998,

Tasanen et al. 2000, Areida et al. 2001). The ectodomain consists of 15 collagenous

28

subdomains (COL1–COL15) with typical collagenous Gly-X-Y repeat sequences

and 16 short non-collagenous sequences (NC1–NC16A) (Franzke et al. 2003). The

NC16A sequence is located between the plasma membrane and the COL15 domain

and can therefore be considered the extracellular linker domain. It is functionally

important because it is believed to play a role in both the shedding and triple-helical

folding of collagen XVII (McLaughlin & Bulleid 1998, Myllyharju & Kivirikko

2001, Franzke et al. 2002, Franzke et al. 2004, Nishie et al. 2010).

The ectodomain can be shed from the cell surface both in vitro and in vivo to

achieve a shorter collagenous triple-helical molecule (Franzke et al. 2002, Hirako

et al. 2003, Nishie et al. 2010). The cleavage occurs at different sites within the

juxtamembranous NC16A domain (Hirako et al. 2003, Nishie et al. 2010). Under

physiological conditions, ADAM9, -10, and -17 appear to be the major sheddases,

but involvement of neutrophil elastase and serine proteinases has been suggested

in pathological settings such as BP (Franzke et al. 2009, Hofmann et al. 2009, Lin

et al. 2012, Nishie et al. 2010). The regulation mechanisms and biological functions

of collagen XVII ectodomain shedding remain uncertain, but it plays an apparent

role in binding to laminin 332, migration and differentiation of keratinocytes

(Franzke et al. 2003, Tasanen et al. 2004, Franzke et al. 2005, Qiao et al. 2009,

Nishie et al. 2011, Van den Bergh et al. 2011, Loffek et al. 2014).

2.2.4 Localization and function of collagen XVII in the skin

Collagen XVII is expressed in the HDs of the epidermal basal keratinocytes. It is a

major transmembrane constituent of the epidermal anchoring complex. The

extracellular ligands of collagen XVII are α6 integrin and laminin 332 (Fig. 1), and

its intracellular ligands include β4 integrin, plectin and BP230 within the HD

plaque (Powell et al. 2005). It has an N-terminal globular head region that localizes

to the HD plaque and a C-terminal collagenous tail that projects into the basal

lamina. The structure and location of collagen XVII mean that it acts as a core

anchor protein, connecting the intracellular and extracellular HD proteins (Van den

Bergh et al. 2011). Also strong evidence of collagen XVII involvement in directed

keratinocyte migration have been demonstrated (Qiao et al. 2009, Loffek et al. 2014)

29

2.2.5 Collagen XVII in other tissues

Most of what is currently known about collagen XVII is derived from studies of

skin and keratinocytes. However, collagen XVII is also expressed in various other

tissues (Aho & Uitto 1999, Van den Bergh & Giudice 2003, Claudepierre et al.

2005, Seppanen et al. 2006, Huilaja et al. 2008) including:

– buccal mucosa

– upper oesophagus

– small intestine

– colon

– urinary bladder

– ocular cornea, conjunctiva and retina

– heart

– central nervous system

– placenta and amniotic membrane

However, little is known of the function of collagen XVII in tissues other than the

skin.

2.2.6 Collagen XVII in blistering skin diseases

Bullous skin diseases caused by dysfunctional collagen XVII can be divided into

inherited and acquired types. In inherited diseases mutations in COL17A1 gene

lead to either the absence of collagen XVII or the expression of a structurally

altered protein. They are associated with diminished epidermal adhesion and with

skin blistering in response to minimal shearing forces in patients with junctional

EB, a genodermatosis with variable localized or generalized phenotype (McGrath

et al. 1995, Powell et al. 2005, Fine et al. 2014). Ultrastructural rudimentary HDs

and separation of the basal keratinocytes from the underlying BM can be detected.

Clinical characteristics include skin fragility and blisters, atrophic scarring, nail

dystrophy and loss, and diffuse alopecia. Deletion of the collagen XVII cytoplasmic

domain leads to a rare form of EB simplex, with mechanical fragility occurring

within the basal keratinocytes (Darling et al. 1997, Powell et al. 2005, Fine et al. 2014).

In acquired diseases autoantibodies are formed against collagen XVII (BP180).

Antibodies are found in patients with BP, pemphigoid gestationis, linear

immunoglobulin A disease, and mucous membrane pemphigoid, which are

30

clinically distinct subepidermal immunobullous diseases characterized by antibody

binding to components of the cutaneous basement membrane zone and the

development of cutaneous or mucosal blisters (Powell et al. 2005). It has been

shown with mouse models that collagen XVII is a major autoantigen in BP (Nishie

2014). BP is the most common subepidermal immunobullous disease and

autoreactivity to a 180-kDa epidermal antigen was first demonstrated in BP.

Historically autoimmunity to collagen XVII has been most studied in relation to

BP (Labib et al. 1986, Powell et al. 2005, Nishie 2014).

2.3 Collagen XVII, integrin α6β4 and laminin 332 in cell migration

and invasion

Although HDs are anchoring structures that provide stable adhesion they also

participate in the migration of epithelial cells. Components of the HDs undergo a

rapid turnover, which makes it possible for epithelial cells to change from a

stationary to a migratory state by changing the balance quickly to HD disassembly

(Kashyap & Rabinovitz 2012). HD disassembly is required for several

physiological processes, for example in cell division, differentiation and directed

migration in the wound healing process (Margadant et al. 2008, Tsuruta et al. 2011,

Loffek et al. 2014).

However, loss of attachment via HD disassembly is also a crucial criterion that

allows squamous cell carcinoma (SCC) cells to migrate and invade (Buchheit et al. 2012, Margadant et al. 2008). The roles of hemidesmosomal components and their

binding partners in SCC carcinogenesis have been studied widely, and the

importance of laminin 332 and α6β4 integrin in SCC cell migration and invasion is

well established (Beaulieu 2010, Chao et al. 1996, Giannelli & Antonaci 2000,

Guess & Quaranta 2009, Kligys et al. 2012, Miyazaki 2006). The role of collagen

XVII in SCC carcinogenesis is not as clearly characterized as those of its binding

partners, but its involvement in cancer cell migration, invasion and adhesion is

evident (Qiao et al. 2009, Nishie et al. 2011, Loffek et al. 2014).

2.3.1 The role of collagen XVII in cell migration and invation

It has been reported that collagen XVII siRNA knockdown affects keratinocyte

migration through the p38 mitogen-activated protein kinase (MAPK)-signaling

pathway. The MAPK pathway influences many cellular processes, including cell

migration via direct action on cytoskeletal components of migratory cells (Qiao et

31

al. 2009). A recent study also demonstrated a connection between collagen XVII

and the phospho-FAK/PI3K pathway in keratinocyte migration, which suggests

that collagen XVII plays a part in directed keratinocyte migration by dampening

integrin dependent PI3K activation and by stabilizing lamellipodia (Loffek et al. 2014). In collagen XVII-deficient keratinocytes, the genetic ablation of collagen

XVII leads to increased expression and increased phosphorylation of the integrin

β4 subunit and to phosphorylation of FAK, which enhance PI3K activity and induce

undirected cell migration via Rac1 activation. Phosphorylation of the collagen

XVII β4 domain is thought to inhibit the binding of the endodomain (Loffek et al. 2014). Also there has been speculation about the role of the cleaved ectodomain of

collagen XVII in adhesion and migration and there is evidence that the extodomain

may interact with laminin 332, thus independently participating in adhesion and

migration (Nishie et al. 2011). Aside from its function in migration, many studies

have been published on the participation of collagen XVII in invasion although the

information is mostly based on collagen XVII’s expression and localization.

Collagen XVII is abnormally distributed and up-regulated in solar keratosis,

Bowen’s disease, basal cell carcinomas and SCCs (Yamada et al. 1996, Parikka et al. 2003, Parikka et al. 2006, Stelkovics et al. 2008). In SCC increased collagen

XVII expression is pronounced in areas of invasive growth (Parikka et al. 2003,

Parikka et al. 2006, Stelkovics et al. 2008, Yamada et al. 1996).

2.3.2 Integrin α6β4 in cell migration and invasion

Integrins are heterodimeric cell surface glycoproteins that are composed of α and

β subunits (Dydensborg et al. 2009, Maalouf et al. 2012). To date 24 αβ

heterodimeric members has been identified (Barczyk et al. 2010). They are

transmembrane receptors for extracellular matrix proteins and transmit signals

from extracellular matrix components to the cell interior (Dydensborg et al. 2009,

Maalouf et al. 2012). Integrins play roles in several normal cellular processes that

influence the development of tumors, including the regulation of proliferation and

apoptosis, cellular motility and invasion, cell surface localization of

metalloproteinases, and angiogenesis (Hood & Cheresh 2002). Integrin interaction

with a large range of scaffold proteins results in the activation of several signalling

molecules, such as Ras and PI3K, which in turn leads to activation of other

molecules including JNK (c-JUN N-Terminal Kinase), Jun, Erk and CyclinD

(Giancotti & Tarone 2003).

32

The α chain of α6β4 integrin is very similar to the α chains of other integrins

and consists of 1073 amino acids (120 kDa), however, the β4 subunit is atypical

compared with other integrins (Mercurio et al. 2001b, Yoon et al. 2006). The β4

subunit differs those of other integrins in three ways: 1) the cytoplasmic domain is

larger (over 1000 amino acid residues and 202 kDa in size); 2) it lacks some of the

conserved cysteines in the extracellular domain; and 3) it has a unique domain

organization (Mercurio et al. 2001a, Yoon et al. 2006, Kligys et al. 2012). In intact

skin α6β4 integrin mediates the interaction of basal keratinocytes with laminin-332

located at the interface of the epidermis and dermis (Kligys et al. 2012).

The participation of α6β4 integrin in migration and invasion has been studied

widely and the evidence that α6β4 integrin facilitates the formation of some

carcinomas as well as the migration, invasion, and survival of carcinoma cells is

strong (Chao et al. 1996, Mercurio et al. 2001b, Mercurio & Rabinovitz 2001,

Chung et al. 2002, Nikolopoulos et al. 2004, Yoon et al. 2006, Beaulieu 2010,

Kligys et al. 2012, Koivisto et al. 2014). As with collagen XVII, the expression of

α6β4 integrin is often upregulated in carcinoma cells (Kligys et al. 2012). The

mechanisms by which α6β4 integrin participates in migration and carcinogenesis

of SCC are not yet fully clear but there are some theories. In cell motility and

migration it has been suggested that α6β4 integrin could be the regulator of other

integrins in keratinocytes. Regarding migration and invasion a pivotal role for α6β4

integrin has been suggested. Firstly it promotes migration and invasion directly via

variety of signalling pathways (Shaw et al. 1997, O'Connor et al. 1998, Sehgal et al. 2006, Kashyap et al. 2011, Kligys et al. 2012). These signaling pathways

include the Rac1 and cofilin pathways, regulating laminin 332 matrix assembly and

keratinocyte motility. Secondly, via phosphorylation of 4EBP1 and activation of

PI3K it determines the expression of α3β1 integrin which is shown to regulate

cellular velocity (Kligys et al. 2012). It has also been shown that α6β4 integrin is

required for tumorigenesis in a murine xenograft model of human SCC (Dajee et al. 2003). It is believed that the right correlation of HD assembly/disassembly is

the key for both migration and invasion and it has been suggested that the reduction

of HDs in SCC could be due to β4 integrin serine phosphorylation (Kashyap et al.

2011, Kashyap & Rabinovitz 2012).

33

2.3.3 Laminin 332 and cell migration and invasion

Laminins are major cell adhesion substrates in epithelial BM. They have a crucial

role in the regulation of epithelial cell adhesion and also in normal cellular

functions such as proliferation, polarity and differentiation (Miyazaki 2006).

Laminin 332 consists of an α3 (200kDa), a β3 (140kDa) and a γ2 (140kDa)

chain from which the α3 and γ2 chains undergo further processing to smaller

species of 165/145 kDa and 105 kDa, respectively (Colognato & Yurchenco 2000).

Laminin 332 is unique in both structure and activity and functions as a major

adhesion component of anchoring filaments in the epidermal BM (Miyazaki 2006).

Laminin 332 is thought to be crucial for the migration and invasion of SCC

cells, although the exact mechanisms via which laminin 332 participates in

migration and invasion remain to be clarified (Miyazaki 2006, Hamasaki et al. 2011). Like α6β4 integrin, laminin 332 or its subunits are highly expressed in

various types of human cancers. In particular, the laminin γ2 chain is expressed in

tumor cells at the invasion front or in budding tumor cells in many types of human

cancers such as adenocarcinomas of the colon, breast, pancreas and lung, and

squamous cell carcinomas and melanomas (Ono et al. 1999, Pyke et al. 1995,

Sordat et al. 1998). Overexpression of the laminin γ2 chain by tumor cells, as well

as lowered or impaired expression of the laminin α3 and/or β3 chains, may

contribute to the loss of BM structures in invasive carcinomas. Another possible

mechanism is that the laminin γ2 chain monomer itself enhances tumor invasion.

Quaranta and his group reported that the proteolytic cleavage of the 150-kDa γ2

chain of rat laminin 332 to a 80-kDa form elevates the cell migration activity of the

laminin-332 (Giannelli et al. 1997, Koshikawa et al. 2000). Another study with

recombinant laminin 332 mutants which clearly showed that the cleavage of human

laminin γ2 chain to the 105-kDa isoform increases its cell motility activity but

decreases its cell adhesion activity (Ogawa et al. 2004). In addition, it has been

shown that cleavage of the γ2 or the β3 chain impairs the ability of laminin 332 to

deposit onto, or to be assembled into, the ECM (Gagnoux-Palacios et al. 2001,

Ogawa et al. 2004, Tsubota et al. 2005). The cleavage of the β3 chain leads to a

decrease in cell adhesion activity and complete loss of the type VII collagen-

binding activity of laminin 332 (Miyazaki 2006). Cleavage of the α3 chains in the

human laminins 311 and 332 is reported to lead to an enhancement in both cell

adhesion and motility activities (Hirosaki et al. 2002, Tsubota et al. 2005). In

addition a soluble form of laminin 332 is able to stimulate cell migration by binding

to integrin α3β1 on the cell surface (Kariya & Miyazaki 2004). It has also been

34

shown that laminin 332, like α6β4 integrin, is required for tumorigenesis in a

murine xenograft model of human SCC (Dajee et al. 2003).

35

3 Aims of this study

The overall aim of this study was to broaden our knowledge concerning the

extracutaneous localization and function of collagen XVII. The specific aims were

to describe:

1. The localization and function of collagen XVII in the normal kidney

glomerulus.

2. Collagen XVII in normal colorectal epithelia.

3. The role of collagen XVII in the pathogenesis of CRC.

4. Function of collagen XVII and α6β4 integrin in SCC carcinogenesis.

36

37

4 Materials and methods

4.1 Patients and tumor samples (II-III)

The study included all patients who were operated on for CRC in Oulu University

Hospital between 2006 and 2010 (Kantola et al. 2012). The study complied with

the Declaration of Helsinki and the design was approved by the Ethical Committee

of Oulu University Hospital (58/2005, 184/2009). Tumor samples were classified

according to the TNM (tumor-node-metastasis) classification (Sobin & Wittekind

2002) and graded according to World Health Organization criteria (Hamilton et al. 2000)

Tissue collection for our SCC study was performed at the Department of

Pathology, Turku University Hospital (Kivisaari et al. 2008, Kivisaari et al. 2010,

Riihila et al. 2015). The use of archival tissue specimens and the collection of

normal skin and SCC tissues was approved by the Ethics Committee of the Hospital

District of Southwest Finland, Turku, Finland and was conducted according to the

Declaration of Helsinki.

Chemical skin carcinogenesis of wild type mice was performed using the

method described earlier (Brideau et al. 2007). The samples gathered from mice

were classified by a pathologist in a blinded manner on the basis of hematoxylin

and eosin (HE) stained sections. All animal experiments were approved by the

Animal Care and Use Committee of the University of Oulu and by the State

Provincial Office of Oulu.

4.2 Generation of Col17a1-/- mice (I)

The targeting vector contained 6.7 kb of genomic DNA with arms of 4.3 kb and 2.4

kb. Exon 18 and the surrounding intron sequences of the Col17a1 gene were

replaced with the neomycin resistance gene, driven by a phosphoglycerate kinase

promoter. Embryonic stem cell culture and the generation of chimeric mice were

performed by the Biocenter Oulu Transgenic Core Facility. Chimeric mice were

generated by blastocyst injection of embryonic stem cells carrying the targeted

mutation, and were mated with C57BL/6J OlaHsd females to produce a targeted

mouse line. F1 heterozygous mice were backcrossed for seven generations and then

intercrossed to generate Col17a1-/- mice.

38

4.3 Cell culture (I-III)

For this study we used multiple commercial cell lines including HaCaT cells

(Invitrogen, Paisley, UK), human oral squamous carcinoma cell lines SCC-15,

SCC-25 (ATCC cultures, Teddington, UK) and HSC-3 (JRCB 0623, Japan Health

Science Research Resources Bank, Osaka, Japan) cells, tumour derived primary

UT-SCC-105 cells (a generous gift from professor Reidar Grenman, University of

Turku, Finland) and the human colon adenocarcinoma cell line CaCo-2 (HTB-37,

ATCC). Cells were cultured according to the instructions provided by the

manufacturers. Primary keratinocytes from the skin of collagen XVII-deficient and

control mice were cultured in a defined serum-free keratinocyte medium

supplemented with 100 pm cholera toxin, 100 units/ml penicillin, 100 μg/ml

streptomycin, and 0.25 μg/ml amphotericin B (all from Invitrogen) on 6-well plates

coated with gelatin (2.5 mg/ml; Sigma-Aldrich). Cells were maintained at 37 °C,

5% CO2, and 95% humidity.

4.4 RNA isolation, quantitative real time PCR, Western Blot (II-III)

Total RNA was isolated from cells using QIAamp RNA Blood Mini Kit (Qiagen,

Crawley, UK) using the protocol provided by the manufacturer. cDNAs were

synthesized by standard methods (Thermo Fisher Scientific, Waltham, MA, USA).

mRNA levels were measured by quantitative RT-PCR analysis using SYBR Green

Supermix (Bio-Rad, Hercules, CA, USA) with iQ5 multicolour real-time PCR

equipment (Bio-Rad). The relative changes in collagen XVII, laminin γ2 and

integrin β4 mRNA expression were calculated by Livak’s 2-∆∆CT method (Livak &

Schmittgen 2001).

Western Blot was done using standard methods. Total protein concentration

was determined using the BioRAD DC protein Assay kit (Bio-Rad). Samples were

separated using SDS-PAGE and protein was visualized using the antibodies listed

in table 3. Images were taken with the Fusion SL imaging system (Vilber Lourmat,

Marne-la-Vallee Cedex, France).

4.5 Immunohistochemistry and in-situ hybridisation (I-III)

The antibodies used for immunohistochemical (IHC) stainings of human colon and

human and mouse kidney and SCC samples are listed in table 3. Immunoperoxidase

detection was performed using to the DAKOReal EnVision Detection System

39

(Dako Cytomation, Glostrup, Denmark). Kidney immunofluorescence (IF) staining

was viewed with an Olympus FluorView1000 confocal microscope using x60 oil

objectives and appropriate filters for the green and red channels (Alexa 488 and

Texas Red, Molecular Probes). The images were collected and saved using

Olympus software. Murine skin IF slides were mounted in VECTORSHIELD

Mounting Medium (Vector Laboratories, Peterborough, UK) containing DAPI and

recorded with Axiophot 2E microscope system (Carl Zeiss, Munchen, Germany).

Table 3. List of antibodies used in this study.

Antibody Manufacturer / reference Original article used in

Polyclonal NC14A1 (mouse) (Franzke et al. 2009) I, III

Polycolonal Endo-2 (mouse and

human)

(Franzke et al. 2002) I-II

Polyclonal tubulin (mouse) Abcam, Cambridge, MA, USA I

Polyclonal NC16A (human) (Schumann et al. 2000) I-II

Polyclonal GAPDH (human) Santa Cruz Biotechnology, Heidelberg,

Germany I

Collagen XVII Ecto-4 (human) (Huilaja et al. 2008) I

CD-34 (mouse) Novocastra, UK I

Collagen IV (mouse) Millipore, Billerica, MA, USA I

Polyclonal integrin β4 Santa Cruz III

Integrin α3 (mouse) BD Biosciences, Franklin Lakes, NJ, USA I

Integrin β1 (mouse) Gift from Dr. Aki Manninen, Biocenter Oulu,

Oulu, Finland I

Laminin α5 (mouse) Santa Cruz I

Nephrin (mouse) (Putaala et al. 2001) I

Integrin α6 (mouse) Progen, Heidelberg, Germany I

Laminin γ2 Santa Cruz I-III

Monoclonal human cytokeratin Dako, Glostrup, DK-2600, Denmark III

A tissue microarray (TMA) was constructed as previously described (Vayrynen et al. 2014). Bound antibodies were detected using the EnVisionTM system (Dako,

Copenhagen, Denmark); 3,3’-Diaminobenzidine was used as the chromogen and

hematoxylin as the counterstain. Histological images were captured with an

Olympus DP25 camera (Olympus, Center Valley, PA) attached to a Nikon Eclipse

E600 microscope (Nikon, Tokyo, Japan) using x20 and x10 objectives.

Immunoreactivity evaluation was done semi-quantitatively based on the proportion

of tumor cells found to be positive (0 to 100%).

40

Goat anti-mouse IgG-Alexa Fluor 488 and goat anti-rabbit IgG-Alexa Fluor

594 (Invitrogen, Paisley, UK) were used as secondary antibodies for IF studies of

frozen human colon tissue samples. Images were taken using an Olympus Fluor

View1000 confocal microscope and a x20 objective with appropriate filter settings.

4.6 Electron microscopical analysis (I-II)

4.6.1 Transmission electron microscopy

Standard methods were used for TEM. Thin sections were examined in a Philips

CM100 transmission electron microscope. Images were captured using a CCD

camera and analyzed with TCL-EM-Menu version 3 from Tietz Video and Image

Processing Systems GmbH (Gaunting, Germany).

4.6.2 Immunoelectron microscopy

Human kidney samples for immunoelectron microscopy (IEM) were collected

from diagnostic biopsies at the Department of Paediatrics, Oulu University

Hospital. All specimens were embedded in methylcellulose and examined using a

Philips CM100 transmission electron microscope (FEI Company, Eindhoven and

The Netherlands). Control samples were prepared by carrying out the labelling

procedure without the primary antibody. The Ethical Committee of Northern

Ostrobothnia Hospital Districts approved this element of the study, which was

performed according to the Declaration of Helsinki second revision (1983).

4.6.3 Scanning electron microscopy

Specimens were examined using a Zeiss Ultra Plus scanning electron microscope,

(Carl Zeiss MT- Nanotechnology System Division, Carl Zeiss NTS Gmbtt,

Oberkochen, Germany) with an accelerating voltage of 5 kV.

4.7 Determing glomerular volume fraction and slit diagram count

(I)

Glomerular volume fraction (VG) and glomerular surface density (SG) of the kidney

cortex were determined using a point counting method with a multipurpose test

41

grid of 100 points placed on a microscopic digital image on a monitor. Ten random

fields were counted for each case. Three Col17a1-/- mice and two control mice from

the same litter were analysed.

Three different sample pairs (wt/knock out) were chosen for counting slit

diaphragms. Statistical significance was calculated using the independent samples

t-test (SPSS, version 16).

4.8 Laser capture microdissection (II)

Two normal mucosal samples and five CRC samples were chosen for the

microdissection experiment. The PALM MicroLaser Systems (PALM

RoboSoftware 4.3 SP1, Carl Zeiss MicroImaging Gmbh, Jena, Germany) were

used to cut cancer cell islets according to manufacturers’ instructions. Total RNA

was isolated from microdissection samples using the QIAcube instrument

(QIAGEN Nordic, Sollentuna, Sweden) according to the manufacturer’s

instructions.

4.9 RNAi and overexpression of collagen XVII (II-III)

Collagen XVII knockdown (KD) by retrovirus-mediated RNAi for SCC-25 and

HSC-3 cells was generated as described in more detail previously (Schuck et al. 2004). β4 integrin knockdown was generated using the MISSION shRNA

lentivirus (Sigma, Munchen, Germany) according to the manufacturer’s protocols.

KD efficiency was evaluated by quantitative RT-PCR.

To create a stable CaCo-2 cell line overexpressing murine collagen XVII,

cDNA of full-length murine collagen XVII was cloned into the retroviral MSCV-

IRES-GFP, pMIG expression vector (Addgene, Cambridge, MA, USA). CaCo-2

cells transfected with an empty pMIG vector were used as a control. CaCo-2 cells

infected with virus particles were subsequently FACS-sorted for green fluorescent

protein to obtain stable cell lines.

4.10 Cell adhesion, migration and invasion analyses (II-III)

Cell adhesion assays were conducted using the electric cell–substrate impedance

sensing (ECIS) system (Applied Biophysics, Inc., Troy, NY, USA) according to the

manufacturer’s protocol. An elevated voltage pulse was applied with a frequency

of 40 kHz, amplitude of 5 V for 45 s.

42

The migration assay was performed using Ibidi Culture-Inserts (Ibidi,

Martinsfried, Germany) on glass-bottom 6 well plates (Mat Tek Corporation,

Ashland, MA, USA). The imaging was performed using Olympus Cell^P live-

cell/timelapse imaging system. Images were taken every 20 minutes until the

wound was closed in control cells.

Invasion analyses were done using an organotypic myoma model according to

method described earlier (Nurmenniemi et al. 2009). A MatrigelTM (BD

Biosciences, Bedford, MA) invasion analysis was done according to the

manufacturer’s protocol. Statistical significance was calculated by the independent

sample t-test using SPSS (version 22).

4.11 Statistical analysis of collagen XVII in CRC (II)

The statistical analyses for normally distributed continuous variables were

conducted using IBM SPSS Statistics 22.0 (IBM, Chicago, IL); values are

presented as mean (standard deviation), whereas other continuous variables are

presented as median (IQR). The statistical significance of the associations of

collagen XVII expression with clinicopathological factors was analyzed by the

Mann-Whitney U test (comparing two classes) or the Kruskal-Wallis test

(comparing three or more classes). A receiver operating characteristics (ROC)

analysis was used to determine an optimal cut-off score, with the shortest distance

to the coordinate (0,1), for the collagen XVII expression level to discriminate

survivors from non-survivors. A univariate survival analysis was conducted

according to the Kaplan-Meier method. Cox’s proportional hazards regression

model was used to analyze the independent prognostic significance of the

expression of collagen XVII. A two-tailed p value <0.05 was considered

statistically significant.

43

5 Results

5.1 Collagen XVII expression and function in normal tissue

5.1.1 Collagen XVII is expressed in the human and murine

glomerulus and in human colon epithelia (I-II)

Collagen XVII has not previously been studied in the kidney and only a little in the

colon. We wanted to confirm its expression in these tissues. Collagen XVII mRNA

expression was clearly detectable in the human glomerulus (Fig. 6A). In situ

hybridization showed clear expression of collagen XVII mRNA in podocytes,

endothelial cells and in the cells of the Bowman’s capsule. IHC of mouse kidney

samples showed similar localization of collagen XVII in the mouse glomerulus and

partial co-localization with nephrin in glomerular podocytes with double IF

staining. The ultrastructural localization of collagen XVII in podocytes was

investigated in normal human samples. IEM of the normal human glomerulus

showed gold particles within the pedicle and close to the cell membrane of the

pedicle soles in the lamina rara externa of the GBM (Fig. 6B). Some gold labelling

was also present in the vicinity of the slit pore between the neighbouring FPs.

The epithelial cells of the normal human colon mucosa showed collagen XVII

expression (Figure 6C). The basal cells in the anal squamous epithelium were

strongly positive for collagen XVII immunostaining. Collagen XVII was present in

the colon epithelial cells of the surface and in the crypt epithelium. Also IF staining

showed clear collagen XVII expression in normal colorectal epithelium and partial

colocalization with laminin γ2 was detected.

44

Fig. 6. Modified from original articles I-II. Collagen XVII is expressed in the human and

murine glomerulus and in human colon epithelia. IHC of mouse kidney samples showed

collagen XVII reaction in podocytes (arrows) with 40 x original magnification (A). IEM

demonstrated collagen XVII between two neighbouring foot processes and in the

glomerular basement membrane (B). IHC of colon epithelia showed clear positivity of

collagen XVII in the basal cells (arrow) in picture C. Scale bar is 100μm.

5.1.2 Lack of collagen XVII alters the phenotypes of mice and the

function of glomeruli (I)

The creation of a mouse line lacking collagen XVII enabled us to study further and

in more detailed the role and importance of collagen XVII in kidneys. These mutant

mice showed clear differences compared to normal wild type mice. Mutant mice

were smaller in size and suffered from high mortality (over 90%) within the first

weeks after birth. They developed blisters and detachment of epidermis especially

on the tail as well as both fore and hind paws. Diffuse, non-pigmented hair growth

and hair loss was also detected (Fig 7A).

The glomeruli of mutant mice appeared immature, densely packed and small

in size (Fig. 7B). Suprisingly, no major structural differences between the wild type

and mutant kidneys could be detected outside the glomerulus. Ultrastructural

analysis revealed abnormally shaped and hypertrophic podocytes and effacement

of podocyte FPs, but no major SD disruption (Fig. 7C upper pictures). In addition

endothelial cells were swollen, the capillary lumen was clogged in some places and

splitting of GBM was detected in some areas (Fig. 7C upper pictures). SEM showed

enlarged primary FPs and abnormal interdigitation of secondary pedicles

confirming the presence of disorganized podocytes (Fig. 7C lower pictures).

When analyzing functional differences in kidneys both glomerular volume

fraction (VG) and surface density (SG) were elevated in mutant mice at two weeks

45

of age, indicating that the glomerular content in mutant kidneys was increased

although the PAS staining showed no GBM thickening under light microscopy

resolution.

The impact of the absence of collagen XVII on the expression and organization

of other proteins known to be involved in GBM and FP integrity was studied by IF

stainings. No alterations in the expression and localization of collagen IV, integrins

α3, β1, laminin α5 chain or nephrin was observed compared to wild-type animals.

Fig. 7. Modified from original article I. Lack of collagen XVII affects both the structure

and function of mouce kidney and alters the fenotype of mutant mice. Mutant mice were

46

smaller in size and developed blisters and hair loss (A). Glomeruli of mutant mice were

small and immature compared to the control mice in HE staining (B). TEM (upper

pictures in C) showed abnormally shaped podocytes, foot process effacement and GBM

splitting and SEM (lower pictures in C) hypertrophic podocytes and disorganized foot

processes in mice lacking collagen XVII.

5.2 Expression and function of collagen XVII, laminin γ2 and

integrin β4 in CRC and SCC

5.2.1 Collagen XVII and laminin γ2 are upregulated in CRC (II)

Having verified the expression of collagen XVII in the normal colon, it was in our

interest to investigate whether upregulation of collagen XVII expression occurs in

CRC. 141 CRC patients were included in our study. Collagen XVII expression was

clearly detectable in most TMA patient samples. Thirty-one patients showed

negative or almost negative staining. In most positive cases the staining was both

membranous and intracellular, and more intensive than in the normal mucosa

(Figure 8A-B). Partial co-localization of collagen XVII and laminin γ2 was also

found in CRC similarly to the normal colon epithelia samples.

The localization of collagen XVII in colon epithelium and CRC was analyzed

further with immunoelectron microscopy, which revealed that the gold labeling of

collagen XVII was clearly more abundant in the cancer tissue than in the normal

colon epithelium. The localization was extracellular although some minor labelling

was also observed intracellularly (Figure 8C). In some cases concentrated

expression of collagen XVII was detected in the invasive tips of the malignant cells

(Figure 8C).

The expression of collagen XVII in CRC was quantitated by collecting normal

epithelial and colon tumor cells from patient samples by laser capture

microdissection. The collagen XVII mRNA levels of tumor samples were clearly

elevated compared to normal mucosa samples (Figure 8D).

47

Fig. 8. Modified from original article II. Collagen XVII is expressed in human CRC. In

CRC samples collagen XVII staining was strongly positive in invasive cells (arrows in

A-B), the scale bar is 100μm. In image C IEM of human CRC samples showed collagen

XVII expression intracellularly and in the extracellular matrix near basement membrane

(upper picture) and at the invasive tip of a malignant cell (lower picture). With laser

capture microdissection higher collagen XVII expression mRNA levels were

quantifically verified in CRC compared to normal epithelia (D).

5.2.2 Collagen XVII, laminin γ2 and integrin β4 are upregulated in

human and mouse SCC (III)

Due to the evident significance of collagen XVII, laminin 332 and integrin α6β4

in SCC carcinogenesis, we wanted to study their expression and localization in

precancerous, in-situ carcinoma and SCC samples compared to normal skin in a

more detailed fashion. In order to study the expression and localization of these

proteins multiple IHC stainings of human SCC samples were performed, which

showed strong expression of collagen XVII, laminin γ2 and integrin β4. Compared

to normal skin, where collagen XVII is positive in basal cells, SCC invading cells

showed membranous and cytoplasmic immunoreaction against collagen XVII. For

laminin γ2 cytoplasmic staining was limited to cells in and around the invasive

margin. A weaker immunoreaction was observed within areas of BM formation.

The staining pattern of integrin β4 was very similar to that of collagen XVII.

To investigate further, a quantitative IHC analysis of TMA samples of normal

skin, actinic keratosis, Bowen’s disease and SCC was conducted. Altogether 95

cases were included in the study (13 normal skin samples, 36 actinic keratosis

samples, 30 Bowen’s disease samples and 16 SCC samples). The results

demonstrated an increase of expression that was linear for laminin γ2 in normal to

SCC samples but not for collagen XVII or integrin β4. The highest expression

levels were found in Bowen’s disease samples, and were lower in aggressive SCC

samples. However, the positivity and intensity varied massively in Bowen’s disease

48

samples and especially in SCC samples with all three antibodies compared to

normal skin samples.

Several stainings of chemically induced mouse carcinoma samples were

performed to compare murine with human samples. The IHC of these samples

showed that the expression of collagen XVII and γ2 chain of laminin-332 were up-

regulated in the basal hyperplastic tissue of benign papillomas and in the individual

invasive cells of malignant SCCs similarly to the human samples. The signals for

both collagen XVII and laminin γ2 in the basement membrane zone were also much

stronger in all papilloma and carcinoma samples compared to normal skin samples.

To analyze whether the expression of collagen XVII, laminin γ2 and β4 integrin

was also increased in human SCC cell lines, we determined their relative expression

levels in UT-SCC-105, SCC-15, SCC-25, and HSC-3 cell lines by qRT-PCR.

Collagen XVII, laminin γ2 and β4 integrin were overexpressed in all SCC cells

compared to HaCaT keratinocytes. Collagen XVII expression was at its highest

(5,13-fold greater than in HaCaT keratinocytes) in SCC-15 cells and also elevated

in other SCC cell lines. Upregulated expression of laminin γ2 and β4 integrin was

also detected in all SCC cell lines. Moreover, an increase of collagen XVII

expression was found at the protein level.

5.2.3 Collagen XVII expression is associated with TNM and

metastasis survival times in CRC patients (II)

The upregulation of collagen XVII in CRC led to further investigation of the

correlation between clinicopathological variables in CRC patients and collagen

XVII. Analyses of the percentage of tumor cells with positive collagen XVII

staining relative to these variables revealed significant association between

increased collagen XVII expression and higher TNM stage. In particular, a higher

percentage of collagen XVII positive cells correlated with lymph node and distant

metastasis. In addition, an infiltrative growth pattern and tumor budding were

significantly associated with increased collagen XVII expression. No statistically

significant associations were found between collagen XVII staining and patient age,

gender, tumor location, WHO grade or preoperative treatments. According to the

Kaplan-Meier curve a high percentage of collagen XVII positive cancer cells

associates both with decreased disease-free survival (DFS) and cancer-specific

survival (CSS). Nevertheless, Cox regression analysis did not indicate an

independent prognostic value for collagen XVII expression.

49

5.2.4 Collagen XVII participates in adhesion, migration and invasion

(II-III)

To analyze the functional role of collagen XVII and integrin β4 in SCC, we created

KD cell lines to see how the absence of these proteins affects the adhesion,

migration and invasion of SCC cells. Both SCC-25 and HSC-3 cell lines were used

for collagen XVII KD but only SCC-25 for integrin β4 KD. The KD efficiency

varied from 78% (collagen XVII KD in SCC-25 cells) to 91% (β4 integrin KD in

SCC-25 cells). The adhesive capabilities of the cell lines were analyzed using the

ECIS analysis with either collagen XVII or integrin β4 SCC-25 KD cells. The

ability of integrin β4 SCC-25 KD cells to attach was decreased by 23% and the

ability of collagen XVII SCC-25 KD cells by 10% compared to the control cells.

KD did not affect HSC-3 cells.

The wounding assay revealed that both SCC-25 KD cell lines seemed to have

lost their ability to migrate. The KD cells were stationary and rotating; no migration

was detected. Similarly to the adhesion analysis, there was no difference in

migration between the HSC-3 control and KD cells.

The effect of KD on the ability to invade was studied using organotypic myoma

model. IHC of myoma samples using pancytokeratin antibody showed that SCC-

25 control cell lines invaded through the whole myoma section. SCC-25 cells

lacking either β4 integrin or collagen XVII did not invade to the myoma tissue at

all. However, the KD of collagen XVII did not affect the invasion of HSC-3 cells.

Since the expression of level of collagen XVII is normally very low in CaCo-

2 cells, the function of collagen XVII was studied creating CaCo-2 cells

overexpressing collagen XVII. The expression of cull-length murine collagen XVII

in the CaCo-2 cell line promoted migration of these cells in the MatrigelTM assay.

The overexpression of collagen XVII led to a 2.2-fold more effective migration in

CaCo-2-cells.

50

51

6 Discussion

6.1 Collagen XVII is a newly characterised component of the

glomerulus and affects glomerular development

Although previous studies have demonstrated collagen XVII expression in various

other tissues (Kondo et al. 1998, Aho & Uitto 1999, Van den Bergh & Giudice 2003,

Claudepierre et al. 2005, Seppanen et al. 2006) our study was the first to

demonstrate collagen XVII expression in the podocytes of the human glomerulus

and that collagen XVII is very likely needed for normal kidney development. Firstly,

the glomeruli of mutant mice lacking collagen XVII were immature and small in

size and the glomerular content of their kidneys was increased. Secondly, mutant

mice had podocyte FP effacement, which is the central cytopathological feature of

glomerular injury in the vast majority of human glomerulopathies and animal

models of glomerular diseases (D'Agati 2008, Patrakka & Tryggvason 2010). SDs

of the mutant mice were mostly normal, suggesting that collagen XVII is not an

actual component of SDs.

To understand the function of collagen XVII in the glomerulus at a molecular

level, it would be crucial to know which proteins it interacts with. However the

binding partners of collagen XVII in the glomerulus are not yet clear. In tissues

where collagen XVII functions as a component of type I HD the main intracellular

binding partners of collagen XVII are BP230, plectin and the integrin β4 subunit,

whereas the extracellular part of collagen XVII interacts with laminin 332 and the

integrin α6 subunit (Van den Bergh & Giudice 2003, Aumailley et al. 2006, Mihai

& Sitaru 2007, Nishie et al. 2011). Our hypothesis is that in the glomerulus,

collagen XVII binds components of FPs and/or the GBM, and disturbances in these

interactions result in the deficient glomerular development, podocyte effacement

and GBM splitting detected in mutant mice. Based on collagen XVII binding

partners in skin, integrins could be also be a rational possibility for collagen XVII

interaction in the kidney. Podocytes can express both α3β1- and β4-integrins

(Adler 1992, Kambham et al. 2000, Patrakka & Tryggvason 2010). Although the

exact localization and function of β4 integrin in the glomerular filtration unit is not

known, the cytoplasmic tail of β4 integrin might also be a ligand for collagen XVII

in podocytes. In keratinocytes collagen XVII also associates intracellularly with an

adherens junction protein, p120 catenin (Aho & Uitto 1999) and a microfilament

associated protein, α actinin-4 (Gonzalez et al. 2001). Both these proteins are

52

expressed in podocytes and could interact as intracellular binding partners for

collagen XVII (Michaud & Kennedy 2007). In contrast, the extracellular partners

of collagen XVII in the glomerulus are most probably different from those in the

dermo-epidermal junction, since laminin 332 is not expressed in healthy kidneys

and the integrin α6 subunit is expressed in the epithelial lining of healthy and

damaged tubules, but not in the glomerulus (Joly et al. 2006, Hahm et al. 2007).

Our finding that the expression and localization of both type IV collagen and

laminin α5 chain were unchanged in the GBM of mutant mice suggests that there

is no direct association between collagen XVII and these major GBM components.

The function of collagen XVII in podocytes is therefore still uncertain although our

findings suggest a possible role of collagen XVII in establishing cell-cell or cell-

GBM interactions of podocytes.

Interestingly, there is only one recent paper about the association between renal

lesions and BP (Hoorn et al. 2015) and no systematic analysis of kidney

involvement in this disease has yet been performed (Ross & Ahmed 1989, Plaisier

et al. 2002). Pemphigoid patients are treated with effective immunosuppressive

drugs for skin blistering, which may prevent the emergence of renal symptoms,

such as proteinuria. Based on data from the National EB Registry, renal failure may

occur in junctional EB, though mainly for secondary reasons (Fine et al. 2004,

Almaani & Mellerio 2010). Hereditary nephritis has been found in junctional EB

with pyloric atresia caused by mutations in the integrin β4 gene, although only a

minority of patients with integrin β4 mutations have renal iinvolvement (Kambham

et al. 2000, Dang et al. 2008, Almaani & Mellerio 2010). Integrin β4 null mice die

immediately after birth due to denuding of the skin (Dowling et al. 1996). The renal

consequences of integrin β4 knockout in mice have not been analysed. In the future,

the kidney function of the carriers of different type of collagen XVII mutations

must be carefully studied, since renal involvement in these cases has not yet been

analyzed in detail. However, it is also possible that patients with congenital or

autoimmune collagen XVII related skin diseases do not present any major renal

difficulties because human collagen XVII is more critical to cutaneous adhesion

than the integrity of the glomerular filtration barrier.

6.2 Collagen XVII is expressed in the normal colon epithelium and

may interact with laminin 332

We showed that collagen XVII is expressed in the colon epithelia but its role in

normal colonic epithelium is unclear. As does the kidney, the colon lacks type I

53

HDs meaning collagen XVII must have a different role in the colon than in the skin

(Fontao et al. 1999, Margadant et al. 2008). Our study demonstrated that the

expression of collagen XVII was distributed from the bottom of the crypt axis to

the tip of the normal surface colon epithelium, and thus resembled the localization

of the laminin γ2 chain and α6β4 integrin (Beaulieu 2010, Sordat et al. 1998). We

also demonstrated partial co-localization with laminin γ2. The co-localization of

collagen XVII and laminin 332 suggests a functional interaction between these

proteins in the colon epithelium.

Patients with junctional epidermolysis bullosa lacking collagen have been

found to have lower gastrointestinal tract complications such as constipation,

chronic diarrhea, anal fissures or rectal bleeding (Fine et al. 2008). These symptoms

may be explained by the fact that the epithelium of the colon is subject to milder

mechanical forces than the skin and the epithelial-mesenchymal interface of the

anorectal mucosa is very similar to that of the skin.

Based on these data it is evident that collagen XVII also has a role in colonic

epithelia. Since collagen XVII is not thought to be a component of intestinal type

II HD, its possible importance in attachment/detachment and migration could come

about via its interaction with laminin 332.

6.3 Collagen XVII is expressed in CRC and its expression

correlates with tumor progression and metastasis in CRC

The involvement of collagen XVII in CRC has not previously been described. In

our study we found out that the expression of collagen XVII in TMA patient

samples was both membranous and intracellular, and the staining was more

intensive than in normal mucosa. As in normal epithelia, partial co-localization of

collagen XVII and laminin γ2 was also detected in tumor tissue which together with

mainly extracellular localization could suggests a functional interaction between

these proteins in CRC. There were also similarities to the collagen XVII expression

in SCC since collagen XVII expression was more abundant in the tumor tissue than

in normal epithelia and there was a great deal of variation in the expression intensity

of collagen XVII within the tumor tissue (Parikka et al. 2003, Parikka et al. 2006,

Stelkovics et al. 2008, Yamada et al. 1996).

There are no previous data about the functional role of collagen XVII in CRC.

The results with TMA patient samples revealed significant correlation between

increased collagen XVII expression and advanced TNM stage and more aggressive

tumor behavior. Higher collagen XVII expression also correlates with tumor

54

budding and an infiltrative growth pattern. These are acknowledged histologic

features associated with poor outcome and are predictive of lymphovascular

invasion and lymph node involvement in CRC (Sagaert 2014). Interestingly, and

similarly to the laminin γ2 chain, increased collagen XVII immunostaining was

associated with both lymph node and distant metastasis in CRC (Shinto et al. 2005).

These similarities further strengthen our suggestion of collagen XVII and laminin

γ2 functional interaction. High expression of the laminin γ2 chain is regarded as an

independent prognostic factor in CRC (Guess & Quaranta 2009, Shinto et al. 2005).

However multivariate analysis revealed no independent significance of the

increased expression of collagen XVII, although collagen XVII positively

correlated with decreased DFS and CSS in univariate analyses. Similar to our

findings of the increased collagen XVII expression correlation to the metastasis,

another recent study had similar results in lung adenocarcinoma, where increased

collagen XVII expression had a significant correlation with brain metastasis

(Fabian et al. 2014). Our findings of increased invasion in collagen XVII-

overexpressing CaCo-2 cells strongly supports the suggestion that collagen XVII

may have an important role in CRC pathogenesis and indicates for the first time

that collagen XVII influences the migration and invasion of cancer cells derived

from non-stratified epithelia as well.

6.4 Collagen XVII, integrin α6β4 and laminin 332 are upregulated in

SCC

The upregulated expression of collagen XVII, laminin 332 and α6β4 integrin in

SCC has been reported previously (Hamasaki et al. 2011, Miyazaki 2006). Our

study was the first to quantitate and compare the expression of these binding

partners in actinic keratosis, Bowen’s disease and SCC tumours. Laminin γ2

expression increased linearly from normal skin through pre-malignant and

carcinoma in situ lesions to invasive SCC samples, which is in line with the findings

of Hamasaki and co-workers (2011) who found that the expression of laminin γ2

was higher in SCC than in Bowen’s disease (Hamasaki et al. 2011). The expression

patterns of collagen XVII and integrin β4 were alike but differed from that of

laminin γ2. In actinic keratosis and Bowen’s disease the expression of collagen

XVII and integrin β4 was up-regulated, but, surprisingly, their expression in

Bowen’s disease was higher than in SCC samples. Previous studies have

demonstrated higher expression for both collagen XVII and integrin β4 in SCC

compared to actinic keratosis or carcinoma in situ samples (Stelkovics et al. 2008,

55

Kashyap et al. 2011). However, the methods used in those studies differed from

ours. Stelkovics et al. used a different scoring system from ours and in the study by

Kashyap et al. immunostaining was performed with a phospho-specific antibody

(Stelkovics et al. 2008, Kashyap et al. 2011). It also has to be noted that as in CRC

samples, there was vast variation of the expression intensity within the tumor tissue,

both in Bowen’s disease and especially in SCC samples. We also observed that the

distribution pattern of laminin γ2 does not correspond with either collagen XVII or

integrin β4 in SCC samples which differs from what is seen in CRC. However, the

immunolocalization of collagen XVII and integrin β4 was very similar suggesting

that they interact with each other in SCC.

The elevated expression of collagen XVII and laminin γ2 in DMBA-TPA

induced cutaneous papillomas and carcinomas supports our findings in human

samples, and suggests that collagen XVII and laminin 332 are involved in both UV-

light- and chemically- induced skin carcinogenesis. The expression of collagen

XVII, laminin γ2 and integrin β4 were also clearly elevated in all investigated SCC

cell lines which was to be expected based on the findings of the aforementioned

previous studies.

6.5 Collagen XVII and integrin α6β4 affect the adhesion, migration

and invasion of SCC-25 cells but has no effect on HSC-3 cells

As previously shown, the suppression of laminin γ2 by siRNA significantly inhibits

A431 SCC cell invasion (Hamasaki et al. 2011). This prompted us to investigate

the effect of collagen XVII and integrin β4 KD by virus-mediated RNAi on the

behavior of SCC cells. The deficiency of collagen XVII or integrin β4 markedly

reduced the adhesion of SCC-25 cells, athough it was to be expected as they both

are important components of type I HDs. Thus, the consequences of collagen XVII

or integrin β4 depletion on the adhesion of SCC-25 cells are comparable to those

on keratinocytes, indicating that they also act as adhesion molecules on the surface

of malignant cells (Qiao et al. 2009, Hamill et al. 2011, Loffek et al. 2014).

Previous observations of how the lack of collagen XVII changes keratinocyte

migration are controversial: Keratinocytes derived from human or murine genetic

models show increased, but undirected motility, whereas keratinocytes with viral

collagen XVII KD have reduced migratory ability (Qiao et al. 2009, Hamill et al. 2011, Loffek et al. 2014). We observed that collagen XVII KD SCC-25 cells

exhibited stationary rotation and markedly delayed migration in the scratch wound

healing assay. Thus the effect of viral collagen XVII KD is similar in keratinocytes

56

and in SCC-25 cells. The conflicting results of previous studies may have been due

to different compensatory mechanisms of genetic or viral systems (Loffek et al. 2014) or perhaps total or partial depletion of collagen XVII has a different impact

on the migration of epithelial cells. SCC-25 cells lacking integrin β4 behaved the

same way as collagen XVII KD SCC-25 cells.

The invasion of SCC cells with collagen XVII and integrin β4 KD was

analyzed using an organotypic assay, which is based on the use of human myoma

tissue and mimics the tumor microenvironment better than collagen organotypic

models (Nurmenniemi et al. 2009, Vered et al. 2015). The effect of collagen XVII

or integrin β4 depletion on the invasion of SCC-25 cells was very clear: Instead of

invading to the myoma tissue as the control SCC-25 cells did, KD SCC-25 cells

remained proliferating at the surface of the myoma disc.

However, the results with the HSC-3 cells lacking collagen XVII were quite

the opposite compared to the SCC-25 cells. KD did not have any effect on adhesion,

migration or invasion of the very aggressive HSC-3 cells. The fact that lack of

collagen XVII does not affect aggressive HSC-3 cells but disturbs severly functions

of less aggressive SCC-25 cells, combined with our findings with IHC stainings of

TMA tumor samples, suggests that collagen XVII is involved in the early

development of malignant transformation, but it is not required anymore in highly

invasive stages of SCC. Interestingly, even though there are many similarities in

collagen XVII expression in CRC and SCC as our results clearly demonstrate, the

association of collagen XVII expression with histopathological grades, tumor

invasion or metastases in SCC has not been addressed so far.

These functional results together with expression and localization results of

collagen XVII, integrin β4 and laminin γ2, suggest that increased expression of

collagen XVII may be involved in the migration and invasion of cancer cells, and

strengthens the already strong data regarding integrin β4 and collagen XVII

participation and interaction especially in the early stages of SCC carcinogenesis.

The mechanisms via which collagen XVII and α6β4 integrin as well as their

binding partner laminin 332 participate in migration and invasion have been studied

previously in multiple studies (Borradori & Sonnenberg 1999, Franzke et al. 2003,

Franzke et al. 2005, Walko et al. 2015). It seems that the understanding of the

phosphorylation of β4 integrin would clarify the mechanisms of the attachment,

migration and invasion of SCC cells, and the role of collagen XVII is linked to this

phosphorylation process. On one hand, the absence of collagen XVII favours the

phosphorylation of the β4 integrin intracellular domain which leads to enhanced

HD disassembly and therefore to the activation of migration and invasion. But on

57

the other hand, since migration and invasion are dependant on both the creation and

disassembly of adhesive components, the total loss of collagen XVII leads to

undirected migration and noninvasive SCC cells (Kashyap & Rabinovitz 2012,

Loffek et al. 2014). Only the presence of α6β4 integrin together with collagen

XVII/BP230 and the actin cytosleleton together with laminin 332 can lead to

directed cell migration (Tsuruta et al. 2011) which is in line with our findings.

Table 4 summarizes the known expression, distribution and function of

collagen XVII in normal human tissues and collagen XVII-related pathological

conditions. Based on the current knowledge it appears clear that collagen XVII has

functional similarities regardless of its expression site and its main role seems to be

connected to cell attachment/detachment and migration together with laminin 332

and integrin α6β4.

58

Table 4. Distribution and function of collagen XVII in human.

Site of expression Localization in tissue Function in normal tissue Related pathological

conditions

Skin Basal keratinocytes, type I HD Attaches keratinocytes to the

BM and participates in

keratinocyte migration (Powell

et al. 2005)

EB, BP, PG,LAD,

MMP, skin

carcinomas (Powell et

al. 2005)

Buccal mucosa Oral keratinocytes Attaches keratinocytes to the

BM similarly to the skin

Similar to those of the

skin (Ziober et al.

2001)

Colon/intestine Epithelial cells similarly to

laminin 332

Unclear, possibly functional

interaction with laminin 332

CRC

Kidney Podocyte FPs Unclear, expressed by

podocytes and needed for

normal glomerular

development

N/A

Bladder Epithelial cells N/A N/A

Amnionic

membrane and

placenta

Syncytial trophoblasts and

cytotrophoblasts

Participation in the migration of

invasive trophoblasts (Huilaja

et al. 2008)

N/A

Nervous system Soma and proximal axons of

neurons

Participation in neuronal

migration suggested

(Seppanen et al. 2006)

N/A

Cornea,

conjunctiva and

retina

Müller cells Functional interaction with

laminin 332 suggested

(Claudepierre et al. 2005)

N/A

Heart N/A N/A, though similarly to the

skin attachment as a

component of type I HD

suggested (Kondo et al. 1998).

N/A

Abbreviations: HD: hemidesmosome; BM: basement membrane; EB: epidermolysis bullosa; PG;

pemphigoid gestationis; LAD: linear immunoglobulin A disease; MMP: mucous membrane pemphogoid;

CRC: colorectal carcinoma; FP: foot process.

59

7 Conclusions

This study expands the knowledge of the extracutaneous function of collagen XVII

and the role of collagen XVII and integrin α6β4 in epithelial cancers. It is tempting

to speculate whether in the future these findings will lead to the more careful

analyzing of the kidney function and potential defects in kidneys in patients with

collagen XVII deficiency. To speculate further it will be interesting to see whether

in the future controlling the amount of collagen XVII (soluble or membrane-bound)

could be used to control the migration and invasion of early stage SCC or CRC in

patients.

Regardless of the speculations, based on our results we were able to make

following conclusions:

1. Collagen XVII is a newly characterised component of the glomerulus

localizing in the podocyte FPs.

2. Specific deletion of collagen XVII in a gene-targeted murine model results in

severe kidney abnormalities.

3. Collagen XVII is expressed in normal the colon epithelium and there may be a

functional interaction between collagen XVII and laminin 332 in CRC.

4. The level of collagen XVII expression strongly correlates with tumor

progression and metastasis in CRC.

5. Collagen XVII and integrin α6β4 have a crucial role in the adhesion, migration

and invasion of SCC cells and there is likely a functional interaction between

collagen XVII and integrin α6β4 in SCC.

60

61

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Original articles

IV Hurskainen T, Moilanen J, Sormunen R, Franzke CW, Soininen R, Löffek S, Huilaja L, Nuutinen M, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen K (2012) Transmembrane collagen XVII is a novel component of the glomerular filtration barrier. Cell Tissue Res 348(3):579-88.

V Moilanen J, Kokkonen N, Löffek S, Väyrynen JP, Syväniemi E, Hurskainen T, Mäkinen MJ, Klintrup K, Mäkelä J, Sormunen R, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen K (2015) Collagen XVII correlates with the invasion and metastasis of colorectal cancer. Hum Pathol. 46(3):434-42.

VI Moilanen J, Löffek S, Kokkonen N, Salo S, Väyrynen JP, Hurskainen T, Manninen A, Riihilä P, Heljäsvaara R, Franzke CW, Kähäri VM, Salo T, Mäkinen MJ, Tasanen K Collagen XVII and integrin β4 show similar expression in squamous cell carcinoma and their knockdown suppresses migration and invasion only of the less aggressive squamous cell carcinoma cells. Manuscript.

Reprinted with the permissions from Springer Science and Elsevier Science.

Original publications are not included in the electronic version of the dissertation.

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