Role of Wnt11 in Kidney Ontogenesis and Development of Renal Organoid Based Models to Identify Candidate Oncogenes

D 1463 OULU 2018 D 1463

UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA

DMEDICA Qi Xu Qi Xu University Lecturer Tuomo Glumoff ROLE OF WNT11 IN KIDNEY

University Lecturer Santeri Palviainen ONTOGENESIS AND DEVELOPMENT OF RENAL Postdoctoral research fellow Sanna Taskila ORGANOID BASED MODELS Professor Olli Vuolteenaho TO IDENTIFY CANDIDATE ONCOGENES University Lecturer Veli-Matti Ulvinen

Planning Director Pertti Tikkanen

Professor Jari Juga

University Lecturer Anu Soikkeli

Professor Olli Vuolteenaho UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF BIOCHEMISTRY AND MOLECULAR MEDICINE; Publications Editor Kirsti Nurkkala BIOCENTER OULU; INFOTECH OULU ISBN 978-952-62-1910-3 (Paperback) ISBN 978-952-62-1911-0 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS D Medica 1463

QI XU

ROLE OF WNT11 IN KIDNEY ONTOGENESIS AND DEVELOPMENT OF RENAL ORGANOID BASED MODELS TO IDENTIFY CANDIDATE ONCOGENES

Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium F202 of the Faculty of Medicine (Aapistie 5 B), on 1 June 2018, at 12 noon

Supervised by Professor Seppo Vainio

Reviewed by Professor Panu Mikko Jaakkola Professor Matthias Nees

Opponent Professor Jing Zhou

ISBN 978-952-62-1910-3 (Paperback) ISBN 978-952-62-1911-0 (PDF)

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

Cover Design Raimo Ahonen

JUVENES PRINT TAMPERE 2018 Xu, Qi, Role of Wnt11 in kidney ontogenesis and development of renal organoid based models to identify candidate oncogenes. University of Oulu Graduate School; University of Oulu, Faculty of Biochemistry and Molecular Medicine; Biocenter Oulu; Infotech Oulu Acta Univ. Oul. D 1463, 2018 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract In the kidney, Wnt is involved in ureteric bud branching and nephrogenesis. Wnt mutation may lead to specific developmental dysfunctions and diseases. As part of this thesis, I show that Wnt11 is expressed in the renal tubules, except for the ureteric epitheliums, and I examine the function of Wnt11 in renal tubule organization using the new C57Bl6 Wnt11-/- mouse model. Convoluted and dilated tubules were observed in the Wnt11 mutated kidneys that may cause glomerular cysts and kidney dysfunction. More specifically, a lack of Wnt11 in the kidney reduced Six2, Hoxd1, and Hox10 expression, which may have contributed to the anomalies in the kidney tubular system. Embryogenesis and carcinogenesis share molecular characteristics. Gene expression changes take place during development to meet the demands of the tissue formation, but ectopic expression of embryonic genes by deletion, SNPs, or epigenetic modification in adult may lead to cancer. The research carried out as part of this thesis identified genes that were differentially expressed in both induced metanephric mesenchymes (MM) and human ccRCC. Gene silencing of Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr, and Itgb2 mediated by siRNA inhibited the migration, viability and invasion capacity of Renca cells (RCC). Furthermore, by using the novel 3D in vitro MM-Renca co-culture setup, the result revealed that downregulation of Bnip3, Cav1 or Gsn in Renca cells partly rescued the RCC-mediated inhibition epithelial tubules formation during nephrogenesis. Altogether, the data demonstrates that renal ontogenesis control genes play a role both in normal kidney development and in kidney cancer. The 3D RCC-MM chimera organoids developed as part of the research may also serve as a novel model for kidney cancer study.

Keywords: caveolin, gene expression, nephrogenesis, renal cell carcinoma, Wnt

Xu, Qi, Wnt11-signaalin tehtävä munuaisen kehityksessä ja uusien munuais- syöpään osallistuvien geenien identifiointi kudosteknologisten mallien avulla. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Biokemian ja molekyylilääketieteen tiedekunta; Biocenter Oulu; Infotech Oulu Acta Univ. Oul. D 1463, 2018 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä Wnt-viestit ohjaavat munuaisen kehityksen yhteydessä sekä virtsanjohtimen että munuaiskerä- sen kasvua. Virhe Wnt-proteiinia tuottavassa geenissä johtaa puolestaan vakavaan kehityshäiri- öön ja syöpään, jos geeni aktivoituu aikuisvaiheessa. Tässä väitöskirjassa osoitetaan, että Wnt11- geeni osallistuu alkion munuaisen epiteeliputkiston kehityksen säätelyyn. Wnt11-signaalin tehtä- vää tutkittiin munuaisen putkiston rakenteen synnyssä poistogeenisen C57Bl6-hiirimallin avulla. Wnt11-puutos häiritsi virtsan kokoojatiehyiden rakenteiden kehitystä. Tämä on mahdollinen syy siihen, että Wnt11-poistogeenisen hiiren munuaiseen kehittyy hiusverisuonikerästen laajen- tumia. Munuaisen toiminta on myös heikentynyt verrokkiin verrattuna. Munuaisen epiteeliputki- en kehitykseen osallistuvien geenien Six1, Hoxd1 ja Hox10 aktiivisuus oli niin ikään heikentynyt Wnt11-signaloinnin puutoksen vuoksi. Nämä seikat voivat olla puutoksen aiheuttamien kudosrakenteellisten muutosten taustalla. Solun kasvusäätely on keskeinen tekijä sekä alkion että syövän kehityksessä. Prosessien taustalla ovat myös samankaltaiset molekyylit. Wnt-solusignaalit esimerkiksi säätelevät normaalin kehityksen aikana solujen itsesäätelyä, solujen jakautumista, solujen vaeltamista ja solujen invaasiota kudoksiin ja osallistuvat syövän syntyyn niiden viestinnän häiriintyessä. Kehitystä säätelevien geenien tuotteet ohjaavat solujen jakautumista, mikä on edellytys kudosrakenteen synnylle. Jos nämä geenit syystä tai toisesta aktivoituvat uudelleen aikuisvaiheen aikana, se voi johtaa syöpäkasvaimen syntyyn. Tässä työssä verrattiin keskenään munuaisen elinkehitykseen ja ihmisen syövän kasvuun osallistuvia geenejä. siRNA-välitteinen hiljennys geeneille Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr ja Itgb2 inhiboi Renca-solujen migraatiota, elinkykyä ja invaasiota. Lisäksi tulokset paljas- tivat, että käytettäessä uutta 3D in vitro MM Renca -kasvatusmenetelmää Bnip3-, Cav1- tai Gsn- geenien hiljennys Renca-soluissa osittain pelastaa RCC-välitteisen inhibition epiteeliputkissa nefrogeneesin aikana. Kaiken kaikkiaan työssä seulottiin uusin tavoin sekä ihmisen munuaissyöpään liittyviä gee- nejä että kehitettiin lisäksi uusia niin kutsuttuja kokeellisia funktionaalisia organoidimenetel- miä. Työn tulokset tarjoavat uuden strategian, jolla geenien osuutta munuaissyöpään voidaan tut- kia seikkaperäisesti yhdessä 3D-kasvatusmenetelmien kanssa

Asiasanat: munuaissyöpä, nefrogeneesi, siRNA, tubulusmorfogeneesi, Wnt

To my family

8 Acknowledgements

This work was carried out at Biocenter Oulu, Faculty of Medical Biochemistry and Molecular Biology, University of Oulu. It was financially supported by the Biocenter Oulu Doctoral Programme and the Academy of Finland. I sincerely express my gratitude to my supervisor, Professor Seppo Vainio for providing me the opportunity to start my scientific career in his research group, and for introducing me to the beautiful world of developmental biology and cancer research. I am also very grateful for my other supervisor, Dr. Jingdong Shan, who has especially guided me the molecular biology technics during the thesis work. I would like to thank Docent Anatoliy Samoylenko, another co-supervisor, with whom we discussed and started the kidney cancer studied together, and his excellent experience in cancer inspired me in the thesis work. I would like to thank the members of my PhD follow-up group Prof. Johanna Myllyharju, Docent Aki Manninen, and Dr. Joni Mäki. They spent precious time reviewing my doctoral training plan and annual report. Their inspiring and guiding during the study made it moving smoothly. Professor Matthias Nees and Panu Jaakkola are acknowledged for their careful revision and valuable comments on this thesis. I would like to acknowledge the former head of the Center of Excellence in Cell-EMC Research Professor Taina Pihlajaniemi, head of Biocenter Oulu Professor Johanna Myllyharju, the former dean of faculty Professor Kalervo Hiltunen and the present dean of faculty Professor Peppi Karppinen, and all the other group leaders in faculty and Biocenter Oulu for providing an excellent research infrastructure and encouraging atmosphere. I wish to express my special compliments to BCO Doctoral Programme Coordinator Docent Ritva Saastamoinen for helping and encouraging me from beginning to the end. I would like to express my gratitude to all my co-authors for their valuable contribution to my research projects. I would also want to express thanks to my colleagues, former and present SV-group members for the warm and fruitful working environment during these years. Special thanks to Paula Haipus, Hannele Härkman and Johanna Kekolahti-Liias for their technical assistance in the practical lab work of the thesis. I would like to express my gratefulness to all the members of the core facility and Dr. Veli-Pekka Ponkainen for his help with the imaging, Dr. Jarkko Koivunen for his help with Incucyte and Docent Virpi Glumoff for her help with FACS.

9 I am sincerely grateful to my friends in Oulu. You bring me warmness for the long darkness winter. I wish to thanks my parents for their supporting. Finally, thanks for my husband Yongfeng and our lovely son Hang, you bring me lots of happiness.

Oulu, April 2018 Qi Xu

10 Abbreviations

ADPDK Autosomal dominant polycystic kidney disease AKT Protein kinase B APC Adenomatous polyposis coli Axin Axis Inhibition Protein Aq1 Aquaporin1 BCC Basal cell carcinoma Bnip3 BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 BMP4 Bone morphogenetic peptide 4 BMP7 Bone morphogenetic peptide 7 Ca2+ Calcium CaMKII Ca2+/calmodulin-dependent protein kinase II Cav1 Caveolin-1 ccRCC Clear Cell Renal Cell Carcinoma CDC42 Cell division control protein 42 homolog cDNA Complementary deoxyribonucleic acid c-Jun c-Jun N-terminal kinases Ck1 Casein Kinase I CKD Chronic kidney disease CM Cap mesenchyme CSC Cancer Stem Cells DAAM1 Dishevelled associated activator of morphogenesis 1 DAG Diacylglycerol DNMT3B DNA Methyltransferase 3 Beta Dvl Dishevelled segment polarity proteins Dll1 Delta like canonical Notch ligand 1 Egfr Epidermal growth factor receptor EMT Epithelial–mesenchymal transition ER Endoplasmic reticulum ERK Extracellular-signal-regulated kinase ES Embryonic stem Eya1 Eyes Absent Homolog 1 FACS Fluorescence-activated cell sorting Fgf1/2/7/10 Fibroblast Growth Factor 1/2/7/10 Fgfr2 Fibroblast Growth Factor receptor 2 FOXD3 Forkhead box D3

11 Fz Frizzled GCK Glomerulocystic kidney GDNF Glial cell-derived neurotrophic factor GFR Glomerular filtration rate Gli1/2 Glioma-associated oncogene homolog 1/2 (mouse) GSK3 Glycogen synthase kinase 3 Gsn Gelsolin HeLa Human cervical cancer cells HIF Hypoxia-inducible factor Hh Hedgehog HNF-1β Hepatocyte nuclear factor 1-beta HPRC Hereditary papillary renal carcinoma IM Intermediate mesoderm Itgb2 Integrin beta-2 IP3 Inositol trisphosphate or inositol 1,4,5-trisphosphate iPS Induced pluripotent stem cell JNK c-Jun N-terminal kinases Lgals3 Galectin-3 LRP-5/6 Lipoprotein receptor-related protein-5/6 MAML2 Mastermind-like gene family MAPK Mitogen-activated protein kinase MBF Medullary blood flow MECT1 Mucoepidermoid carcinoma translocated 1 MCKD2 Medullary cystic kidney disease 2 MET Mesenchymal-epithelial transition mK4 Induced metanephric mesenchyme undergoing epithelial conversion MM Metanephric mesenchyme mTORC1 Mammalian target of rapamycin complex 1 or mechanistic target of rapamycin complex 1 MUC1 Mucin 1 N1-ICD NOTCH1 intracellular domain NFAT Nuclear factor of activated T cells NGS Next Generation Sequence NRH1 Neurotrophin receptor homolog 1 Oct4 Octamer-binding transcription factor 4 Pax2/8 Paired box 2/8 PBRM1 Polybromo-1

12 PCP Planar cell polarity PDE Phosphodiesterase PDK1/2 Pyruvate Dehydrogenase Kinase 1/2 PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol 4,5-bisphosphate or PtdIns(4,5)P2 PLC Phospholipase C PTA Pre-tubular aggregate PTCH Protein patched homolog 1 PTK7 Tyrosine-protein kinase-like 7 / Colon carcinoma kinase 4 qRT-PCR Quantitative reverse transcription polymerase chain reaction Rac1 Ras-related C3 botulinum toxin substrate 1 Renca Renal cortical adenocarcinoma cells Ret Receptor tyrosine kinase RC Renal Corpuscle RCC Renal cell carcinoma Rho Rho family of GTPases RNA Ribonucleic acid ROCK Rho-associated protein kinase ROR2 receptor tyrosine kinase-like orphan receptor 2 RTK7 Receptor tyrosine kinase 7 RV Renal vesicle Ryk Receptor-Like Tyrosine Kinase Sal1 Supernumerary aleurone1 SETD2 SET Domain Containing 2 siRNA Small interfering RNA Six2 SIX homeobox 2 SMO Smoothened SNP Single-nucleotide polymorphism Sox2 Sex determining region Y box 2 Spry1 Sprouty RTK signaling antagonist 1 SWI/SNF Switching defective/ Sucrose nonfermenting TCF/LEF T-cell factor/lymphoid enhancer factor TGF-β Transforming growth factor beta TSC Tuberous sclerosis complex UB Ureteric bud VEGF Vascular endothelial growth factor VHL Von Hippel-Lindau

13 WD Wolffian duct Wnt4 Wingless-Type MMTV Integration Site Family, Member 4 Wnt11 Wingless-Type MMTV Integration Site Family, Member 11 Wnt9b Wingless-Type MMTV Integration Site Family, Member 9b Wt1 Wilms' tumor 1 YFP Yellow Fluorescent Protein ZO-1 Zonula occludens-1 α-SMA Alpha-Smooth Muscle Actin

14 Original publications

This thesis is based on the following publications, which are referred throughout the text by their Roman numerals:

I Nagy I#, Xu Q#, Naillat F, Ali N, Miinalainen I, Samoylenko A & Vainio S (2016). Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis. BMC Dev Biol. 16(1):30. II Xu Q, Junttila S, Scherer A, Giri KR, Kivelä O, Skovorodkin I, Röning J, Quaggin SE, Marti H, Shan J, Samoylenko A and Vainio S (2017). Renal Carcinoma/Kidney Progenitor Cell Chimera Organoid as a Novel Tumourigenesis Gene Discovery Model. Dis Model Mech. 10(12):1503-1515.

# Equal Contribution

16 Contents

Abstract Tiivistelmä Acknowledgements 9 Abbreviations 11 Original publications 15 Contents 17 1 Introduction 19 2 Review of the literature 21 2.1 Development of mammalian kidney ...... 21 2.1.1 Nephron as basic functional and structural unit ...... 21 2.1.2 Molecular regulation of nephrogenesis ...... 23 2.1.3 Branching morphogenesis and formation of the UB tree ...... 24 2.1.4 Molecular regulation of UB outgrowth and branching morphogenesis ...... 25 2.1.5 Renal cystic diseases ...... 26 2.1.6 Wnt signalling in embryonic kidney development ...... 27 2.2 Renal cancer ...... 30 2.2.1 Renal Cancer factors...... 31 2.2.2 Cancer therapies ...... 32 2.3 Similarities in embryogenesis and cancer ...... 35 2.3.1 Epithelial-to-mesenchymal transition in both embryogenesis and carcinogenesis ...... 35 2.3.2 Activation of the embryonic developmental genes in cancer ...... 37 2.3.3 Common pathways in embryogenesis and carcinogenesis ...... 38 3 Outline of the present study 43 4 Materials and methods 45 4.1 Generation of MM- YFP+ Renca 3D organoids ...... 45 5 Results 47 5.1 Hypoplastic and glomerulocystic kidney in C57Bl6 Wnt11−/− mice (I) ...... 47 5.2 Defects in the C57Bl6 Wnt11-/- collecting ducts (I) ...... 48 5.3 Involvement of Wnt11 signalling in kidney tubular organization (I) ...... 49

17 5.4 Deficiency of Wnt11 altered the cell activity during kidney development (I) ...... 50 5.5 Molecular characteristics shared between nephrogenesis and renal cell carcinoma (II) ...... 51 5.6 Embryonic gene downregulation inhibited Renca cell behaviour (II) ...... 52 5.7 Metanephric mesenchyme and YFP+ Renca 3D co-culture set up (II) ...... 53 6 Discussion 55 6.1 Wnt11 signalling is required for normal tubule system construction ...... 55 6.2 Wnt11 deficiency promotes cyst formation in mice ...... 56 6.3 Specific kidney embryonic genes could be potentially selected for gene target therapy in renal cell carcinoma ...... 57 6.4 Efficiency of in vitro 3D culture model for gene function studies ...... 59 7 Conclusions 61 References 63 Original publications 77

18 1 Introduction

Cancer may result when the developmental programs during embryogenesis are disrupted (Ma et al., 2010). The links between embryogenesis and tumorigenesis were proposed when the similarities of the cell behaviours and molecular basis were found between embryonic development and tumorigenesis. During embryogenesis the embryonic cells travel long distances to form tissues and organs, while during cancer progression the cells recapitulate the early embryonic behavioural programs to form new colonies in the distant tissues or organs (Ma et al., 2010). Previous analysis of the similarities between embryonic development and carcinogenesis has also revealed shared molecular signatures and signalling pathways (Dreesen & Brivanlou, 2007). The Wnts belong to a group of signalling proteins that are essential for embryonic development. Wnt signalling defects in the embryo may lead to abnormal organ development and even utero lethality. In the kidney, Wnts are involved in ureteric bud (UB) branching or nephrogenesis, depending on the subtypes and the signal transduced pathways. Wnt4 is expressed in the cap mesenchyme (CM), comma- and S-shaped bodies during kidney development. It is essential for the differentiation of the tubular epitheliums and their development into nephrons. A lack of Wnt4 leads to the failure of pre-tubular cell aggregates (PTA) in the kidney (Kispert, Vainio, & McMahon, 1998; Stark, Vainio, Vassileva, & McMahon, 1994). Wnt11, expressed in the UB tips, coordinates the ureteric branching by cooperation with Ret/GDNF signalling (Majumdar, Vainio, Kispert, McMahon, & McMahon, 2003). Furthermore, Wnt signalling disorder in adults has been implicated in various diseases, most notably, cancer (Duchartre, Kim, & Kahn, 2016; Polakis, 2012). Roughly about 2% of cancers are renal cell carcinomas (RCC) (Ferlay et al., 2015). Because of its resistance to chemotherapy and gene mutation characteristics, finding new gene targets may become a popular therapy strategy in the future. In the past, a number of genes have been identified as mutated in ccRCC, such as VHL, and the PI3K/Akt pathway components (Cancer Genome Atlas Research Network, 2013). These may offer new opportunities for gene therapy of renal cancer. Mouse model based in vivo studies are being used to investigate cancer, but they are costly and time-consuming. On the other hand, the monolayer cultures do not fully reflect all the characteristics of solid tumour tissues. More recently, scientists have introduced ex vivo 3D organoids cultures, which resemble tumour in the body better than cancer cell lines, and which could be used to investigate

19 tumour-host interactions (Asghar et al., 2015; Thoma, Zimmermann, Agarkova, Kelm, & Krek, 2014). This model also makes it possible to manipulate cancer cells to improve the drug development and preclinical drug tests (Weeber, Ooft, Dijkstra, & Voest, 2017). Additionally, small interfering RNAs (siRNAs) can be used to silence the expression of individual genes in cells, which may provide a promising anticancer treatment in the future (Guo, Chen, Yu, Huang, & Deng, 2013; Young, Stenzel, & Yang, 2016). This thesis focuses on clarifying the function of Wnt11 during the later stages of kidney development by using the C57Bl6 Wnt11-/- mouse model and examining the function of candidate kidney embryonic genes in kidney cancer. After comparing the expression profiles of human ccRCC and an ex vivo nephrogenesis model, the genes that had their expression pattern changed in both were selected for further functional study. Meanwhile, a novel in vitro 3D co-culture system by mixing of the cancer cells with the metanephric mesenchyme (MM) in a suitable ratio was established to help investigate the role of a specific gene in renal cancer.

20 2 Review of the literature

2.1 Development of mammalian kidney

In mice, kidney development starts during the embryonic period and continues up to about 4 weeks after birth. The postnatal renal development in mice parallels late embryonic development in humans, when most nephrons are already completed. In mammals, the pronephros and mesonephros are two transient forms of the embryonic kidneys, which subsequently degenerate. The metanephros, which develops into a functional kidney, arises from the intermediate mesoderm (IM). The mature kidney includes two parts: the collecting duct system that originated from the UB, and the nephron epithelium that is differentiated from the metanephric mesenchyme.

2.1.1 Nephron as basic functional and structural unit

The nephron forms a basic structural and functional unit in the mammalian kidney. In mice, kidney development starts at embryonic day 10.5 (E10.5) when the UB is formed as invading of the Wolffian duct (WD) into the metanephric mesenchyme. From this moment on, the ureteric epithelium will sustainably receive signals from the adjacent CM cells and undergo the branching morphogenesis to build up a mature ureteric tree. In turn, the UB tips induce the expansion and differentiation of the CM (Fig.1). Beneath each UB tip, the CM cells aggregate and form the PTA. The cells in the PTA then undergo the mesenchymal-epithelial transition (MET) and form the epithelial renal vesicle (RV), which is a precursor of the nephron. In mice, about 11,000 nephrons are generated in a mature kidney. After 24 to 36 hours of morphogenesis, the RV initiates a series of cellular rearrangements and transforms it into comma- and S-shaped bodies (Fig.1C). At this stage, the polarized distal cells of the S-shaped body will break into the lumen of the nearby collecting duct epithelium and together generate the luminal interconnection between them (Kao, Vasilyev, Miyawaki, Drummond, & McMahon, 2012). The distal and medial segments of the S-shaped body give rise to the epithelial tubule of the nephron, which further forms the proximal tubule, Loop of Henle, and distal tubule. The proximal segment separates into two distinct epithelial layers, i.e. the Bowman’s capsule and the podocytes layer, which form the Renal Corpuscle (RC) after invasion of endothelial and mesangial cells.

21 Fig. 1. Molecular crosstalk between ureteric bud and metanephric mesenchyme in the metanephros. A) Early induction of the metanephros starts E10.5 when the ureteric bud grows into the metanephric mesenchyme (MM); B) In association with ureteric branching morphogenesis, mesenchymal cells differentiate and undergo a mesenchymal-to-epithelial transformation (MET) to form the functional unit of the kidney; C) Early tubule formation with comma- and S-shaped stages of development. Modified from Themes, 2016.

The vasculature develops synchronously with the epithelial nephrogenesis in the kidney. Proper interaction between the kidney microvasculature and the nephron is crucial for kidney function such as the glomerular filtration rate (GFR), medullary blood flow (MBF), and maintenance of a medullar concentrating gradient. A group of vascular cells organizes into the nephron-associated vasculature on the glomerular cleft in the S-shape body. Meanwhile, Vegfa, which is expressed in the developing and mature podocytes, maintains the adjacent glomerular vasculature and promotes endothelial fenestration (Eremina et al., 2003). Foxd1 produced by

22 the stroma cells is also essential for differentiation and development of the renal arterial tree (Sequeira-Lopez et al., 2015). While in recent years great progress has been made to further our understanding of the kidney vasculature, the characterization of how the renal vascular system develops is still limited.

2.1.2 Molecular regulation of nephrogenesis

Cell-fate studies showed that the cell population marked as Osr1+ in the IM differentiated to form the main part of the kidney, including the nephrons, renal vasculature and the nephric duct system (Mugford, Sipilä, McMahon, & McMahon, 2008). Cells ranging from the podocytes of Bowman’s capsule to connecting segments of the nephron are all derived from the Six2+ Cited1+ progenitor population, called the cap mesenchyme (Mugford et al., 2008). A fate mapping study indicated that Cited1+ cells constitute a subpopulation of Six2+ CM that undergoes self-renewal throughout nephrogenesis (Boyle et al., 2008; Hendry, Rumballe, Moritz, & Little, 2011). However, Cited1 deficiency in mice does not disrupt nephrogenesis (Boyle, Shioda, Perantoni, & de Caestecker, 2007). Furthermore, inactivation of Six2 results in a premature and ectopic MET in the CM and loss of the progenitor pool within the metanephric mesenchyme (Self et al., 2006). All of the above indicates that Six2 regulates the fate of the CM population. A list of transcription factors, including Sal1, Wt1, Eya1, Pax2, Myc, and the Six family members are known to be important for maintaining MM progenitors. Sal1 has been identified in maintaining the Six2+Cited1+ nephron progenitor pool. In Sal1 mutant mice, overabundance of RV structures could be observed and a reduction of the progenitor cell markers was detected using qRT-PCR (Basta, Robbins, Kiefer, Dorsett, & Rauchman, 2014). Frequently, several transcription factors such as Eya1-Six1, would form a complex and regulate the fate of the progenitors (Kreidberg, 2010). Wnt signalling plays an important role in regulating nephron progenitors. According to previous studies, Wnt4 expression transiently overlaps with Six2 during the PTA stage, and induces the MET within Six2+ nephron progenitor cells to undergo nephrogenesis (Kobayashi et al., 2008; Park et al., 2012). PI3K controls self-renewal and differentiation of the nephron progenitor cells through β-catenin signalling (Lindström, Carragher, & Hohenstein, 2015). FGF and BMP signalling play important roles in maintaining the kidney progenitor pools. Bmp7 regulates the proliferation of nephron progenitors through

23 JNK signalling (Blank, Brown, Adams, Karolak, & Oxburgh, 2009). In mice, Bmp7 knockout increases apoptosis of cells in the CM and accelerates nephron maturation. The expanded expression of the RV marker Jagged1 in the mutant kidney explant indicates that Bmp7 knockout accelerates the CM differentiation (Tomita et al., 2013). Fgf9 and Fgf20 produced by the MM are required for the maintenance of the nephron progenitor pools (Barak et al., 2012). Fgf8 is necessary for developing to the S-shaped body stage. Deletion of Fgf8 eliminates Wnt4 and Lim1 expression in the nascent nephrons, both of which are required for the formation of the S- shaped bodies (Grieshammer et al., 2005). Although Notch2 is not necessary to initiate RV segmentation, it is required for the patterning of the proximal segmentation generation in the S-shaped body (podocytes and proximal convoluted tubules) (Cheng et al., 2007). Other Notch signalling components Jagged1, N1-ICD, and Dll1 all participate in the morphogenesis of the RV to the S-shaped body. Lim1 functions as a transcription factor in the patterning of the renal vesicles for nephron formation (Kobayashi et al., 2005).

2.1.3 Branching morphogenesis and formation of the UB tree

In mammals, epithelial branching morphogenesis has a vital role in the formation of various organs, such as the vasculature, mammary gland, lung and kidney. In the kidney, a “tip-trunk” is generated when the ureteric bud outgrows from the posterior end of the WD and invades into the adjacent metanephric mesenchyme (Fig.1A). During the epithelial branching process, the tip cells engage in rapid expansion, initiate the branching in the tip, and generate the epithelial tree structure by rounds of bifurcation. Generally, terminal bifurcation is the most common type of UB branching in the kidneys. In vitro kidney culture study shows most of the bifurcations are symmetrical, but terminal “trifid” branching and lateral branching were frequently observed as well (Watanabe & Costantini, 2004). The formed UB tree then develops into the urinary collecting system. The UB branching process is essential for establishing the kidney architecture, which determines the number of nephrons. A defect in UB morphogenesis leads to renal agenesis and dysplasia. Depending on the branching pattern, the UB branching can be subdivided into several developmental stages. In the early phase stage (E11.5 to E15.5), the UB undergoes rapid, iterative branching. The branching peak rate was observed at E13.5 when the mature glomeruli begin to appear (Sampogna, Schneider, & Al-Awqati, 2015). Then in the late phase, the branching

24 process becomes more random and slows down (E15.5 to E19.5), which may be due to the negative feedback from the MM-derived tubule formation (Davies, 2002). As the UB structure matures, the branching terminates, which is accompanied by completion of mesenchymal differentiation.

2.1.4 Molecular regulation of UB outgrowth and branching morphogenesis

The ureteric bud branching morphogenesis is depended on reciprocal interactions between the UB and the MM. A number of promoters and inhibitors are involved in the process. The GDNF/Ret pathway is an essential signal for the budding out of the UB from the WD and for continuing normal branching morphogenesis during kidney development (Fig.1). Expression of GDNF is restricted to the cap mesenchyme surrounding the UB tips, where Ret was detected after the branching started. Absence of GDNF, Ret, or the co-receptor Gfrα1 in mice will cause failure of UB outgrowth and renal agenesis (Costantini & Shakya, 2006; Jain, 2009). Down-regulation of GDNF by an upstream gene causes a similar phenotype in mice. Earlier it had also been shown that half of the heterozygote GDNF mice had branching defects, ranging from smaller kidney to renal aplasia (Costantini, 2006). Conversely, elevated expression of GDNF by a negative feedback loop in Spry1 mutant mice leads to multiplex ureters (Basson et al., 2006). Furthermore, the hypomorphic phenotypes caused by heterogeneity of GDNF or Spry1 could be rescued by reducing the expression of the other (Basson et al., 2006). Other molecules could together with GDNF/Ret signalling orchestrate the branching morphogenesis during development. Canonical WNT/β-catenin signalling that could regulate the cell differentiation is required for ureteric branching. Deletion of β-catenin in the epithelial cells in WD leads to UB outgrowth failure or a dramatic UB branching reduction, presumably due to the down-regulation of Ret and other downstream genes’ expression (e.g. Pax2) in the mutant mice (Bridgewater et al., 2008; Marose, Merkel, McMahon, & Carroll, 2008). Conversely, stabilization of β-catenin blocks cell differentiation of the collecting ducts leading to hypoplastic/cystic kidneys or complete kidney loss (Marose et al., 2008). Wnt11 expressed in the UB tips also participated in the UB branching through a non-canonical pathway. Deficiency of Wnt11 resulted in GDNF reduction and displayed a small kidney phenotype (Majumdar et al., 2003). In summary, Wnt signalling plays important roles in the

25 regulation of Ret expression in UB/WD epithelial cells and decide the cell fate that allows progression of morphogenesis. FGF signalling is another important receptor tyrosine kinase (RTK) pathway involved in regulating the UB branching. Loss of its receptor Fgfr2 in UB cells reduced the epithelial branching (Zhao et al., 2004). Recently, Yuri et al. demonstrated that Fgfs, including Fgf1, Fgf7 and Fgf10, are essential for the survival and proliferation of UB cells in vitro, and GDNF alone in the medium was not enough to ensure its survival (Yuri, Nishikawa, Yanagawa, Jo, & Yanagawa, 2017). Similarly, a branching reduction was also observed in Fgf7 and Fgf10 mutant mice (Ohuchi et al., 2000; Qiao et al., 1999). Retinoic Acid (RA) is also crucial for the UB branching. Blocking of the RA signalling in mouse UB cells reduced the branching number by over 50%, presumably by abolishing the Ret expression (Mendelsohn, Batourina, Fung, Gilbert, & Dodd, 1999; Rosselot et al., 2010). An in vitro branching assay showed that growth factors HGF/Met and EGF/EGFR might be involved in the branching tubulogenesis (Sakurai, Tsukamoto, Kjelsberg, Cantley, & Nigam, 1997; Santos et al., 1994). Loss of Met in the UB reduced the nephron number in adult mice and was accompanied by an upregulation of Egfr expression, which might rescue the branching defect. Consistently, mutation of both Egfr and Met markedly reduced UB branching and lead to renal failure (Ishibe et al., 2009). These results indicate that Met and EGFR signalling are complementary during normal kidney branching morphogenesis. Furthermore, programmed cell death or apoptosis is likely to be critical for the determination of the ultimate collecting system’s morphology. In vivo studies indicate that Pax2 mutation in mice increased apoptosis in collecting duct cells, leading to a lower branching rate and a reduction of nephron numbers, while application of caspase inhibitor Z-VAD-FMK rescued the terminal branch number to 152% of the untreated ones (Clark, Dziarmaga, Eccles, & Goodyer, 2004).

2.1.5 Renal cystic diseases

Renal cystic diseases can be hereditary, developmental, or acquired, but they share the characteristics of cyst formation and fibrosis in the kidney. Most of the cystic lesions originate in the collecting ducts, and only 1% of the cysts are derived from the nephron. Glomerulocystic kidney (GCK) disease is a rare human renal cystic disorder that can be inheritable or sporadic. It is more common in infants and children, but

26 occurs in adults as well (Carson, Bedi, Cavallo, & DuBose, 1987). Cystic glomeruli were reported as early as 1941, but the characterization of the cysts was done 20 years later (Bialestock, 1960; Roos, 1941). A marked dilatation of Bowman’s space was observed in GCK. In most of the cases, GCK was associated with autosomal dominant polycystic kidney disease (ADPDK), which is the most common hereditary renal cystic disease linked to the ADPDK genes (PKD1 or PKD2) (Mao, Chong, & Ong, 2016). Development of the GCK phenotype is influenced by specific genetic changes and additional stimulations at the same time. Sometimes, GCK is diagnosed simultaneously with other specific diseases. About 40% of the tuberous sclerosis complex (TSC) patients exhibited glomerulocystic kidney disease before adulthood (Bernstein, 1993). In these cases, the mTORC1 growth pathway was activated by lack of Tsc2 (Lennerz, Spence, Iskandar, Dehner, & Liapis, 2010; Siroky, Czyzyk- Krzeska, & Bissler, 2009). GCK has also been described with other genetic kidney diseases, including medullary cystic kidney, which is linked to a MCKD2 mutation, and nephronophthisis, which is linked to an NPHP3 defect (Bissler, Siroky, & Yin, 2010). Aberrant HNF-1β signalling has been reported in hypoplastic GCK (Bissler et al., 2010). HNF-1β was expressed during the early kidney developmental stage, and its target genes were co-localized with the genes participating in the formation of the primary cilium (Zhang et al., 2007). Deficiency of HNF-1β in the kidney cysts may be due to the deregulation of primary ciliary progression (Singh, Singla, Jha, Puri, & Puri, 2015). Wnt/β-catenin signalling (Wuebken & Schmidt-Ott, 2011) disorder leads to the renal cyst formation in mice as well. Our study showed that formation of the glomerular cysts in the Wnt11 mutant mice was followed by the impairment of the primary cilia (Nagy et al., 2016). These results suggest that there is a connection between cilia formation and GCK.

2.1.6 Wnt signalling in embryonic kidney development

Wnt factors as secreted lipid-modified glycoproteins are highly conserved and essential for the embryogenesis. Wnt factors regulate different cell processes, such as cell self-renewal (Merrill, 2012; Miki, Yasuda, & Kahn, 2011; Prasetyanti et al., 2013; Xu et al., 2016), proliferation (Pei et al., 2012; X. Peng et al., 2014; Tian, Benchabane, Wang, & Ahmed, 2016), migration (Bilir, Kucuk, & Moreno, 2013; De Calisto, Araya, Marchant, Riaz, & Mayor, 2005; Schambony & Wedlich, 2013) and maintenance of a variety of adult tissues (Hoppler & Moon, 2014). Depending

27 on the receptor and co-receptors used, Wnt factors transduce three different pathways: Wnt/β-catenin signalling, which is known as the canonical pathway and could determine the dynamics of cell fate in early mouse embryos and ES cells, and the two non-canonical pathways, namely planar cell polarity (PCP) and Wnt/calcium pathways (Fig.2). Different Wnt pathways orchestrate kidney organogenesis in mammalians. Up to now, a number of Wnt proteins were demonstracted to be expressed in the embryonic kidney, including Wnt2b, Wnt4, Wnt5a/b, Wnt6, Wnt7b, Wnt9b and Wnt11 (Merkel, Karner, & Carroll, 2007). Except for Wnt2b and Wnt4, which are localized in the mesenchymal cells, all other Wnt proteins are expressed in the UB during early organogenesis.

28 Fig. 2. Overview of the Wnt signalling pathways. A) Activation of canonical Wnt signalling is triggered by binding of secreted Wnt ligands to its Frizzled (Fzd) receptor and LRP co-receptors. Then β-catenin is released from the “destruction complex”, stabilized, and accumulated in the cytoplasm. This results in the translocation of β- catenin into the nucleus and the activation of target gene transcription; B) Wnt/PCP signalling is activated when Wnt ligands bind to the Fzd receptor complex to recruit the effector protein Dvl. Dvl then binds to the small GTPase Rho by de-inhibition of the cytoplasmic protein DAAM1, associated with the actin cytoskeleton morphogenesis. The small GTPase Rac1 and Dvl together trigger JNK signalling leading to transcriptional responses; C) Wnt/Ca2+ signalling regulated transcriptional responses is induced by intracellular calcium fluxes. Modified from Zhan et al., 2017.

29 The balance between expansion and differentiation of the nephron progenitor cells is a core principle in establishing a normal kidney. In addition to BMP7 and Fgf8, Wnt4 and Wnt9b have been revealed as the major factors in nephron induction. Wnt4, expressed in the kidney progenitors, is essential for kidney tubulogenesis. In Wnt4 deficient mice the PTA formation failed, but no effect was found on mesenchymal and ureteric development (Stark et al., 1994). Additionally, Wnt9b is expressed in the UB and is secreted to induce the aggregation of the progenitor cells around the UB tip. More specifically, Wnt9b was involved in the EMT process and balances the progenitor cells’ expansion and differentiation in the embryonic kidney. In the Wnt9b ablated mice, the failure of RV formation and disruption of the UB branching have been observed, both of which would result in a smaller premature kidney compared to the wild type (Carroll, Park, Hayashi, Majumdar, & McMahon, 2005; Karner et al., 2011). Examination of the genitourinary system found that Wnt11 mutation mediated the reduction of glomeruli by weakening Ret/GDNF signalling (Majumdar et al., 2003; Nagy et al., 2016). Conversely, Wnt11 was downregulated when the Ret/Gdnf signalling was absent. Our study confirmed that the C57Bl6 Wnt11 deficient mice developed glomerular cysts accompanied by the dilation of the renal tubules (Nagy et al., 2016). Another study has shown that Wnt2b expresses and controls the ureter morphogenesis but not tubulogenesis in early kidney development (Lin et al., 2001). To conclude, it appears that normal Wnt signalling regulates the kidney organogenesis order, and that the disruption of Wnt signalling might cause kidney failure and related diseases.

2.2 Renal cancer

Cancer is currently the second most important cause of death worldwide with the number of diagnosed cases rising annually. In 2015, 8.8 million people died of cancer worldwide. As a genitourinary cancer, kidney cancer is one of the most common cancers and over 90% of the kidney cancers are renal cell carcinomas (RCC). According to the statistics, cases of RCC are also rising year by year, and the incidence in men is higher than in women. However, the rate of death is showing a slight downwards trend compared to 10 years ago (Ferlay, Parkin, & Steliarova- Foucher, 2010; Ferlay et al., 2013). Renal cancer is a heterogeneous disease. Based on the phenotypes of the cancer cell, renal cancer can be grouped into several sub-types: 1) Clear cell renal cell carcinoma, which was observed in around 80% of RCC; 2) Papillary renal cell

30 carcinoma; 3) Chromophobe renal cell carcinoma; 4) Rare types of renal cell carcinoma, including collecting duct RCC, multilocular cystic RCC, medullary carcinoma, mucinous tubular and spindle cell carcinoma, and neuroblastoma- associated RCC (Muglia & Prando, 2015).

2.2.1 Renal Cancer factors

Renal cell carcinoma is a multifactorial disease. Around 3%–4% of the kidney malignancies have a family history. In these patients, genetic changes such as deficiency, mutations (e.g. SNPs, deletion) and epigenetic modifications are frequently detected. In hereditary kidney cancer, over 10 genes have been identified as high risk factors (Haas & Nathanson, 2014). There are several different types of renal cancers with identical genetic aberrations and histological phenotypes. Investigation of the genetic changes involved in the hereditary forms of renal cancer could provide us with better knowledge to develop new cancer therapeutics. Von Hippel-Lindau (VHL) disease is the most common hereditary form of ccRCC. The VHL protein is important for the regulation of the hypoxia response pathway thanks to which the cells can survive in low oxygen conditions. However, as a tumour suppressor, it is inactivated in the inherited ccRCC and most of the sporadic ccRCC through either mutation or promoter methylation (Banks et al., 2006; Nickerson et al., 2008; Yusuke Sato et al., 2013). The chromatin-remodeling complex switching defective/sucrose nonfermenting (SWI/SNF) family member Polybromo 1 (PBRM1) is the second most commonly mutated gene in ccRCC. PBRM1 encodes the protein BAF180, which regulates the position of the nucleosomes in the genome. In 2017, Gao and colleagues pointed out that loss of BAF180 may accentuate the transcriptional response to HIF in VHL deficient kidney cancer and promote the renal cancer cells’ proliferation in vivo compared to the controls (Gao, Li, Xiao, Liu, & Kaelin, 2017). Mostly, in ccRCC the gene SETD2 mutation was occurred together with a PBRM1 mutation, and BAP1 loss could lead to the formation of large tumors. Furthermore, the mutations in the components of PI3K/AKT/mTOR signalling pathway have partly been shown in ccRCC as well (Brugarolas, 2014). Except the inherited factors, unhealthy life styles, such as smoking, is a strong risk factor in urogenital tumours (Hunt, van der Hel, Olga L, McMillan, Boffetta, & Brennan, 2005; Tsivian, Moreira, Caso, Mouraviev, & Polascik, 2011). Healthy problems e.g. hypertension, obesity, and diabetes, could promote the progression of sporadic ccRCC (Chow & Devesa, 2008).

31 2.2.2 Cancer therapies

Target therapy

Genetic changes underlying clear cell renal cell carcinoma are abundant and frequent. Because of the phenomenon of substantial heterogeneity in RCC, the expression signatures can be used to identify core oncogenic phenotypes that can serve not only as biomarkers, but also as a way to identify putative therapeutic compounds (e.g. drugs and inhibitors) that might specifically target these phenotypes. Targeted therapies continue to be an integral component in the treatment of advanced and metastatic RCC. The VHL/HIF signalling pathway has been exploited as an important potential target for anticancer therapy (Fig.3). A mutation or hypermethylation of the VHL tumour suppressor gene is reported in most of the ccRCC. This allows other genes to be activated when they should not be, which can promote a cell toward becoming cancerous. Blocking of some of the hypoxia-responsive genes, such as vascular endothelial growth factor (VEGF), e.g. by the specific antibody Bevacizumab postpones the progression to an advanced RCC stage (Yang et al., 2003). Currently, multi-tyrosine kinase inhibitors (TKI) that block VEGF or PDGF signalling are used as the standard care in ccRCC, such as sunitinib, sorafenib or axitinib. pazopanib and tivozanib (Bielecka, Czarnecka, Solarek, Kornakiewicz, & Szczylik, 2014). The PI3K/Akt/mTOR signalling cascade is also important for cell survival and mitogenic signalling. In RCC, the PI3K/Akt is recurrently mutated and highly activated, suggesting a potential therapeutic target (Cancer Genome Atlas Research Network, 2013; Guo et al., 2015). Inhibition of mTOR by temsirolimus or everolimus could decrease the tumour size in one third of the RCC patients. Clinically, combination treatment with TKI plus mTOR inhibitors in advanced RCC patients could improve their survival compared to the single therapy (Leonetti, Leonardi, Bersanelli, & Buti, 2017). Additionally, the Wnt proteins transduced developmental pathways, which are known as key regulators of cell fate, proliferation, migration and differentiation in several tissues, are reactivated frequently in neoplasms, particularly in the rare subpopulation of cancer stem cells. Many kinds of drugs have been created to target the Wnt/β-catenin signalling in RCC (Fig.3). Reagents used in recent clinical trials, such as ethacrynic acid, ciclopirox olamine and piroctone olamine, which can suppress the Wnt/β-catenin signalling and trigger cell apoptosis in RCC cells, may

32 become novel treatment options for RCC patients (von Schulz-Hausmann, Schmeel, Schmeel, & Schmidt-Wolf, 2014). Also, the constitutively reactivated Hedgehog pathway may be inhibited by cyclopamine in ccRCC cells. In nude mice, inhibition of sonic hedgehog (SHH) signalling leads to decreased cell proliferation and neo- vascularization (Dormoy et al., 2009). Notch signalling, which cross-talks towards Akt/mTOR signalling in ccRCC, may present a theraputic target as well (Zhuang et al., 2017).

Immunotherapy

Immunotherapy is a treatment that boosts the body’s immune response against cancer cells, and kidney cancer seems to be one of the cancers most likely to respond to it. More than 30 years, interleukin-2 and interferon were use in the ccRCC immunotherapy (Rini, Campbell, & Escudier, 2009). However, these drugs were also systemically toxic to the patients. Anti-Programmed death-1(PD-1) can disrupt the interaction between PD-1 and its ligands have verified as an effective immumotherapy agents in RCC. Combination therapy of PD-1 antibodies and other inhibitors are going on (Weinstock & McDermott, 2015). Besides, blocking of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) could activate the anti- tumour response of the T-cells in metastatic RCC (Bailey & McDermott, 2015; Greef & Eisen, 2016).

33 Fig. 3. Potential targeting of the pathways in ccRCC development and progression. The key therapeutic points and chemicals for targeting the Wnt/β-catenin and the PI3K/AKT/mTOR signalling pathways in ccRCC are indicated.

Surgery

Due to the special characteristics of the kidney, surgery is still the main treatment for kidney cancer. Using new imaging technologies, the doctor can assess the stage and location of the tumours and decide to remove some tissue or the entire kidney. Especially for the early stage RCC, around 40-60% of patients are cured after surgical treatment (Blute, Leibovich, Lohse, Cheville, & Zincke, 2004; Margulis et al., 2007).

34 2.3 Similarities in embryogenesis and cancer

Disruption of the normal cell state and disturbance of normal developmental processes may give rise to cancer. At the end of the 1970s, when scientists found out that many embryonic genes are re-expressed in cancer cells, the hypothesis that embryonic development and tumour formation may share characteristics such as stem cell self-renewal, cell proliferation, differentiation, migration and invasion, was proposed (Ibsen & Fishman, 1979). Embryonic stem cells and cancer cells most notably share self-renewal and pluripotency. In the embryo, ES cells can maintain the same cellular state after proliferation, and pluripotency gives the cells the possibility to differentiate and develop into tissues or organs upon stimulation. In cancer, there is also a distinct cell population with long-term repopulation and self-renewal capacity known as cancer stem cells (CSC).

2.3.1 Epithelial-to-mesenchymal transition in both embryogenesis and carcinogenesis

Tumour cells can grow and spread by switching on the programs suggested to be active only during embryonic development. The epithelial-mesenchymal transition (EMT) was originally discovered as part of normal cell differentiation during early embryonic development. Loss of cell polarity and cell-cell adhesion in the EMT process helps the epithelial cells obtaining the phenotypes of mesenchymal cells and then enhances cell mobility and invasiveness. Unsurprisingly, EMT has also been detected during cancer development (Fig.4). In the embryo, as a consequence of EMT, cells dissociate from the stem cell pool and disperse to different parts of the embryo to form differentiated tissues and organs. During gastrulation, EMT participates in the generation of three germ layers (Ectoderm, Endoderm and Mesoderm) that will build all tissue types of the body. Cells of the primary mesenchyme rising from the mesoderm exhibit enhanced migratory properties via EMT. The EMT involved in the gastrulation is triggered by different molecules. Snail, as a zinc-finger transcriptional factor, represses E- cadherin and induces EMT via cell adhesion molecules during embryonic development. Other members in the Snail superfamily (E-box factors) have also been implicated in the formation of appendages, neural differentiation, cell division and cell survival (Nieto, 2002). In cancer, uncontrolled cell proliferation and angiogenesis mark the start of cancer development. The acquisition of mesenchymal characteristics by EMT

35 transition is a hallmark of tumour progression. During tumorigenesis, the EMT is activated by similar molecules that orchestrated the process during gastrulation. Subsequently, the cells eventually go through the following phases of the invasion- metastasis: intravasation, circulation through blood stream, and finally extravasation and colonization under support of the opposite process, i.e. mesenchyme-to-epithelial transition (MET) (Brabletz, 2012). Numerous studies have demonstrated that primary carcinoma cells (cancer stem cells) lose the cell polarity and cell-cell adhesion that facilitate the acquisition of a mesenchymal phenotype and the expression of mesenchymal markers such as α-SMA, Fibronectin, N-cadherin, Vimentin, and Snail (Fig.4). Once these genes are activated, each of them could trigger the EMT process together with other intracellular signalling network proteins, such as ERK, MAPK, PI3K, Akt as well as cell surface protein Integrins (Tse & Kalluri, 2007). In addition, studies have shown that Wnt/β-catenin signalling is important in the EMT process of both cancer and embryo development (Larue & Bellacosa, 2005). Accumulation of β-catenin is often followed by the loss of E-cadherin expression, which correlates with entering the EMT stage and acquisition of an invasion-metastasis phenotype in cancer (Thiery, 2002). Similarly, Wnt/β-Catenin Wnt target genes, such as Twist and Slug, can directly inhibit the E-cadherin expression and trigger the EMT process to mediate the cellular structure and behaviour in embryos (Heuberger & Birchmeier, 2010). Conversely, the MET is important for the “settling down” of migrated cells at distance areas and their proliferation for tissue construction in normal development or ectopic sarcoma formation nearby (Chaffer, Thompson, & Williams, 2007; Hugo et al., 2007). Signalling such as PI3K/Akt/mTOR and TGF-β was revealed to be involved in the MET process in certain cancers (Yang et al., 2014). Additionally, some of the microRNAs such as miR-200 may up-regulate the expression of E- cadherin and promote the MET process in specific cancers (Yang et al., 2014).

36 Fig. 4. EMT and MET take place in both cancer initiation and embryonic development. Carcinoma cells and embryonic stem cells will undergo the EMT process to acquire the properties of mesenchymal cells, which will lead the cells escaping from the original area either by seeding the nearby stroma or into the vasculature. These cells may migrate and form either a new malignancy or an organ via the cell re-epithelialization, which occurs through MET.

2.3.2 Activation of the embryonic developmental genes in cancer

Cancer cells are undifferentiated, and can be highly invasive. They may result from the disruption of the normal cell-fate determination. So genes associated with deprogramming and maintaining undifferentiated and self-renewal stem cell state may also be involved in cancer initiation and progression. The gene expression patterns shared by embryonic development and cancer may provide potential targets and clues for gene therapy. Previously, over 30 molecules including the transcription factors octamer-binding transcription factor 4 (Oct4), sex determining region Y box 2 (Sox2), and Nanog have been identified as regulatory factors in controlling ES or iPS cells self-renewal and pluripotency. Researchers who aimed to find out whether the “embryonic” state cancer cells possessed similar gene expression signatures analysed the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in various human tumour types (Ben-Porath et al., 2008; Schoenhals et al., 2009). From the gene expression map, they found that

37 the important embryonic stem cell markers Nanog, Oct4, Sox2, Nanog and c-Myc were frequently overexpressed in different cancer types (Ben-Porath et al., 2008; Schoenhals et al., 2009). FOXD3 and DNMT3B that cluster together with other genes, including SOX2, also have functions in early embryogenesis (Avilion et al., 2003). Both of them are as highly expressed in embryonal carcinoma (EC) cells as in ES cells (Sperger et al., 2003). In addition, EGFR that functions in both cell differentiation and proliferation was found to be upregulated in RCC, which may promote abnormal cell proliferation in cancer (Bayrak et al., 2014). In addition, other genes responsible for embryogenesis are activated in cancers as well. HGF/c-Met signalling is involved in the kidney branching morphogenesis, and a lack of Met in UB leads to a reduction of the number of nephrons formed (Ishibe et al., 2009). Researchers showed that Met is activated by a missense mutation or over-expression in hereditary papillary renal carcinoma (HPRC) (Salvi et al., 2008; Schmidt et al., 1997). VEGFR shows significantly lower expression in T- and B- cells, compared to ES cells. However, it is reactivated to ES cell level in leukemia by hypomethylation (Aran et al., 2016). Inactivation of the tumour suppressor genes that play roles in the inhibition of stem-cell traits have also been observed in cancers. In 2010, Mizuno et al. described that p53 silencing mutations in breast and lung cancers activate stem-cell specific patterns of gene expression and cause the tumours to exhibit stem cell-like characteristics (Mizuno, Spike, Wahl, & Levine, 2010). Retinoblastoma (Rb) gene inactivation in small cell lung cancers was accompanied by OCT4 and SOX2 amplification (Peifer et al., 2012).

2.3.3 Common pathways in embryogenesis and carcinogenesis

The molecular biological characterisation of embryonic development and carcinogenesis revealed remarkable similarities between them. The published studies have shown that several common signalling pathways are involved in both cancer and embryonic development.

Wnt signalling

Wnt signalling is one of the most intensely studied pathways both in embryogenesis and in oncogenesis. It plays a vital role in the cell-fate determination during organogenesis, such as nephrogenesis, tooth development and lung development (Xu et al., 2016). Absence of a specific Wnt factors during embryonic development

38 may cause developmental defects even embryo lethality, as described in section 2.1.6. Mis-regulation of Wnt signalling has also been associated with a number of cancers. In renal cancer for example, a certain number of Wnt proteins, Wnt receptors (Fzds) and Wnt antagonists are altered in human RCC. There are reports that advanced Wnt1/β-catenin signalling is linked to increased tumour size, tumour stage and vascular invasion in ccRCC (Kruck et al., 2013). Frequent downregulation of Wnt7a or Wnt5a contributes to kidney tumour development as well (Kondratov et al., 2012; Tamimi, Ekuere, Laughton, & Grundy, 2008). The distinct opposite expression alterations of the Wnts in RCC may be related to the pathways conducted by the Wnts in the tumorigenesis. β-catenin is a core protein in the canonical Wnt signalling pathway and its activity is inhibited by its binding partners in the absence of active Wnt. Activating mutations in β-catenin have been reported in cancers such as melanoma, ovarian cancer, colon cancer, and prostate cancer (Morin, 1999). However, β-catenin mutations are rarely detected in RCC, meaning that accumulation of β-catenin in the RCC may be regulated by other molecules (Kim et al., 2000). The expression of members of the β-catenin destruction complex APC, GSK3β, and Axin are also changed in cancer. APC is the most frequently mutated gene in human cancers. Genetic defects in APC have been associated with the accumulation of β-catenin in colorectal cancer (Clements, Lowy, & Groden, 2003). GSK3β can function as a tumour suppressor or tumour promotor, depending on its interaction with different pathways in cancers (McCubrey et al., 2014). Axin mutations in the APC and β- catenin binding domains will lead to the accumulation of β-catenin in the nuclei, and have been found in different tumours (Salahshor & Woodgett, 2005).

Hedgehog signalling

The Hedgehog (Hh) signalling pathway is essential for the process of embryonic development and for maintaining adult tissue homeostasis. The protein diverged during evolution, and controlled both cellular behaviour and stem cell maintenance in various tissues. Generally, binding of Hh protein to PTCH results in the accumulation of Smoothened (SMO), which can inhibit the proteolytic cleavage of Gli and then induce its downstream gene transcription (Fig.5). Upregulation of the Hh pathway in development results in the formation of ventral types of neurons (Pasca di Magliano & Hebrok, 2003). Unsurprisingly, in addition to developmental diseases, Hh signalling disorder contributes to cancers including RCC (Dormoy et al., 2009), basal cell carcinoma (BCC) (Gonnissen, Isebaert, & Haustermans, 2015;

39 Otsuka, Levesque, Dummer, & Kabashima, 2015), and medulloblastoma (Archer, Weeraratne, & Pomeroy, 2012; Bar, Chaudhry, Farah, & Eberhart, 2007). Ectopic Hh signalling increases cell proliferation and facilitates tumour formation. In BCCs, scientists have demonstrated that the Hh signalling pathway is activated by a PTCH or SMO mutation (Xie et al., 1998). Elevated expression of other Hh signalling components have been detected in a variety of tumours, including lung, stomach, oesophagus, pancreas, prostate, breast, liver and brain cancers (Gupta, Takebe, & Lorusso, 2010). Additionally, loss-of-function of the pathway has been linked to an increased risk of medulloblastoma and BCC formation in humans (Goodrich, Milenković, Higgins, & Scott, 1997; Taylor et al., 2002). Hh signalling has also been shown to support tumour maintenance. By inducing the expression of Oct4, Sox2 and Bmi1, Hh signalling maintains the stem cell pool in multiple cancers. Hh signalling-driven CSC maintenance was revealed in haematological malignancies and solid tumours. In leukemia, inhibition of Hh signalling may induce cellular apoptosis (Ji, Mei, Johnson, Thompson, & Cheng, 2007). Conversely, constitutive activation of Hh signalling leads to an expansion of the leukemia stem cells (Zhao et al., 2009). With the exception of hematopoietic malignancies, the CSCs in solid tumours were firstly identified and isolated in breast cancer. One study shows that the breast cancer stem cells that are able to self-renew and generate tumours in the immunodeficient mice displayed increased expression of Hh pathway components PTCH1, Gli1, and Gli2 (Liu et al., 2006). Moreover, the study demonstrates that Hh signalling has a key role in sustaining the CSC pools in other cancers, including pancreatic cancer, prostate cancer, lung cancer and melanoma (Cochrane, Szczepny, Watkins, & Cain, 2015).

40 Fig. 5. Overview of hedgehog signalling. A) Without HH, protein Patched (PTCH) inhibits smoothened (SMO). The formed complex by Glioblastoma (Gli), Costal 2 (COS2) and other kinases could be recognized and targeted for degradation by the F-box protein Slimb (SLMB). The releasing of the cleavaged form of Gli (Gli-R) then repress the expression of HH target genes. B) Binding of the HH to PTCH will results in the releasing and phosphorylation of SMO, which can enhance its interaction with COS2. Then, Gli is released from the complex after the phosphorylation of COS2. The accumulated Gli will be translocate into the nucleus and induce its target gene transcription. Modified from Ingham et al., 2011

Notch signalling

The Notch signalling pathway is functionally highly conserved from drosophila to humans. It plays a vital role in the maintenance of stem cells and the determination of cell fate during development. Notch signalling is carried out between two adjacent cells. Binding of Notch ligand to the transmembrane receptor on the signal receiving cell will subsequently induce the cleavage of Notch protein into two parts, after which the cytoplasmic part will translocate into the nucleus and regulate the expression of its target genes. The role of Notch signalling in cellular processes is based on the type of tissue or the developmental stage. Notch signalling has been evaluated in the development of the cardiovascular system, the CNS, somites and so on (Bolós, Grego-Bessa, & de la Pompa, José Luis, 2007).

41 Misregulation of Notch signalling is reportedly involved in a wide variety of malignancies including breast cancer, pancreatic cancer, and glioblastoma. Notch can work either as an oncogene or as a tumour suppressor gene, depending on the cellular context. Notch signalling can activate the expression of cancer stem cell genes, such as Survivin, Myc Nanog, Oct4, and Sox2. Notch1 chromosomal translocation as an activating mutation occurs in 50% of human T-cell acute lymphoblastic leukaemia patients (Weng et al., 2004). Similarly, activation of Notch signalling in mice may cause highly aggressive monoclonal T-cell leukaemia. Earlier studies have also demonstrated the activation of Notch signalling by a transforming MECT1-MAML2 multifunctional fusion protein, which is created via genomic translocation in epidermoid carcinoma (Bell & El-Naggar, 2013).

Transforming growth factor beta signalling

The transforming growth factor beta (TGF-β) signalling controls various cellular processes during embryonic development. It is also well known for the induction of the EMT process. Mutations in TGF-β signalling components in embryo can lead to severe defects in heart and limb (Wu & Hill, 2009). The proper functioning of the TGF-β pathway depends on its communication with other signalling pathways, such as Wnt, Hedgehog, and Notch (Guo & Wang, 2009). Generally, TGF-β and BMP together with their own receptor phosphorylate transcriptional regulator Smads (Smad-2, 3, 1, 5, 8) heterodimerise with the common partner Smad4 to form a complex, and then enter the nucleus and regulate the target genes. Dysregulation of TGF-β/BMP activity can lead to cancers. As a tumour suppressor in epithelial cells, TGF-β may inhibit cell proliferation and induce apoptosis. Decreased expression of TGF-β receptors I and II either by mutation or promoter methylation, occurs in a number of cancers. Additionally, mutations inactivating the receptor regulated Smads have also been observed in cancers (Massagué, 2008). However, at later stages of cancer progression and metastasis, TGF beta may also show tumour promoting properties. In ccRCC, TGF-β signalling was highly activated (Cardillo, Lazzereschi, Gandini, & Di Silverio, Colletta, 2001). Blocking of the TGF-β induced pathway can reduce invasive capacity of the ccRCC cells and proliferation of the RCC bone metastasis cells (Boström, Lindgren, Johansson, & Axelson, 2013; Kominsky, Doucet, Brady, & Weber, 2007; Sitaram, Mallikarjuna, Landström, & Ljungberg, 2016).

42 3 Outline of the present study

The first aim was to study the function of Wnt11 in kidney tubule formation. Wnt11 null mutation is lethal, and the mutant new-born puppies have kidney branching defects and a decreased number of nephrons (Majumdar et al., 2003). To investigate the role of Wnt11 during later kidney development, the C57Bl6 Wnt11-/- mice were selected for analysis, since some of them can survive until adulthood. The second aim was to identify new putative target genes for renal cancer therapy. Because of the similarities between embryonic and cancer development, the human ccRCC and in vitro cultured MM were compared to screen for the putative embryonic related cancer genes. The siRNA-mediated gene inhibition was used to monitor the expression levels of selected genes. In addition to the traditional in vitro cancer studies, a MM-RencaYFP ex vivo 3D culture model was established to study the functions of embryonic genes in renal cancer.

44 4 Materials and methods

Detailed description of the materials and methods used in the thesis can be found in the original publications I and II.

Table 1 Methods used in the study.

Method Original artical Cell culture II Colony-forming assay II Epithelial tubular cell counting I Glomerular number I Hematoxylin-eosin staining I Human RCC clinical samples collection II Immunohistochemistry I-II Incucyte cell proliferation, death and migration analysis II In situ hybridyzation I Microarrays and bioinformatics analysis II MM dissection and in vitro culture II Optical projection tomography I P-H3 and TUNEL assays I RNA isolation and quantitative RT-PCR I-II siRNA performing II Transgenic mouse lines I-II Trans-well cell invasion assay II Western blotting II

4.1 Generation of MM- YFP+ Renca 3D organoids

In this study, we established a 3D Renca-MM organoids co-culture protocol (Fig.6) based on the MM organoids method published before by our team (Junttila et al., 2015). The separated embryonic mice MM (E11.5 days) were incubated in 9400 U/ml collagenase Type IV for about 15 min and mechanically dissociated into single cell suspension. Then, the MM cells were mixed either with Renca-YFP cells, HeLa-YFP, or mK4-YFP cells at a 50:1 ratio. The Renca-YFP cells were pretreated with specific siRNAs twice, once 2 days before and again 3 hours before co-culture with MM onset. Afterwards, the mixed cells were re-aggregated by centrifugation at 1380rcf for 20min. The nephrogenesis continued for four days after the induction by bromoindirubin-3-oxime (BIO) for 20h in DMEM with 4.5g/L glucose and 10% FBS at 37°C.

45 The chimeric 3D organoids were fixed with 4% PFA and stained with anti-Pax2 (Abcam ab79389) and anti-Aquaporin1 (Cell Applications.INC CA0648) antibodies.

Fig. 6. 3D co-culture of E11.5 metanephric mesenchyme cells and Renca-YFP+ cells with a ratio of 50:1 ex vivo. Modified from Junttila et al., 2015.

46 5 Results

5.1 Hypoplastic and glomerulocystic kidney in C57Bl6 Wnt11−/− mice (I)

In mice, nephrogenesis continues a few weeks after birth until the fully functioning kidney is established. The postnatal renal development is similar to later kidney development in humans. Wnt11 was expressed in the ureteric epithelium during the metanephric development. Since the SV129 Wnt11-/- mice die either in utero or two days after birth, the study of kidney development after birth cannot be conducted using this model. Luckily, after crossing SV129 Wnt11-/- mice to a C57Bl6 genetic background, the symptoms of Wnt11 deficiency were relieved. Although a few of the Wnt11-/- mice still died in utero, around half of the mice could survive until adulthood. It provides us with an opportunity to investigate the function of Wnt11 in later kidney development. A hypoplastic kidney was observed in the C57Bl6 genetic background mice as well as in the SV129 upon Wnt11 deletion. Similarly, the kidneys in Wnt11-/- C57Bl6 mice are smaller than the wild type controls. Analysis of 4-5 month old kidney morphogenesis using histological inspection revealed the existence of glomeruli cysts in the Wnt11-/- kidney cortical region (Figure 1 in (I)). Statistically, one fourth of the analysed Wnt11-/- mice, including E16.5, NB and adult, had severe kidney hypoplasia accompanied with glomerular or tubular cysts. In these mice, the Bowman’s space was largely expanded as in the GCK disease compared to the wild type kidneys. From the acetylated tubulin staining, we found that the podocytes were disorganised in the Wnt11 mutant glomeruli (Figure 2 in (I)). Additionally, a reduction of primary cilia could be observed in the kidney tubules compared to the wild type (Figure S2 in (I)). The reduction of daily urine production in the Wnt11 deficient mice revealed the potential function impairment in kidney. A biochemical assay was carried out in 4-5 months old mice to evaluate the performance of the kidneys. The results indicated that creatinine clearance in Wnt11-/- mice urine was reduced by around 52% compared to the controls (Table S2 in (I)). Simultaneously, the blood urea nitrogen (BUN) in the plasma was higher in the Wnt11 mutant mice than in the controls (Table S3 in (I)). To understand the causes of the cysts in kidneys with a Wnt11 defect, we examined the expression profiles of certain genes involved in cystic kidney disease

47 (e.g. TCS2, PKD1 and HNF1β) and the core PCP pathway genes of E16.5 and NB kidneys using qRT-PCR. Our results show that the expression of certain PCP pathway associated genes in the Wnt11 mutant mice is age-dependent, while at the same time the common cystic kidney related genes, TCS2, PKD1 and HNF1β, are downregulated in Wnt11 mutant mice as observed in the other kidney cysts (Figure S3 in (I)). Moreover, the PCP pathway component Dvl2 was reduced by more than 90% due to Wnt11 deficiency in both the E16.5 and NB kidneys. Taken together, our results indicate that Wnt11 deficiency in the C57Bl6 mice may lead to a moderate degree of kidney failure. In addition, because of the increased expression of cystic genes glomerular cysts may be formed in Wnt11 deficient kidney.

5.2 Defects in the C57Bl6 Wnt11-/- collecting ducts (I)

It has previously been shown that the Wnt11 defect impairs the UB branching in kidneys by reducing the GDNF/Ret signalling in SV129 mice (Majumdar et al., 2003). In C57Bl6 Wnt11-/- kidneys, both NB and adult mice show a reduction of glomeruli compared to the wild-type controls (Figure 1 in (I)). As expected, the Ret expression is obviously downregulated at E16.5 in Wnt11-/- kidneys (n = 4, p < 0.05), while GDNF is only slightly decreased in the mutant mice (n = 4, p=0.26)). In new- born mice, the expression of Ret and GDNF was unchanged in the mutant mice when compared to the controls (Figure 2 in (I)). Similarly, the abnormal convolution of the papilla in Wnt11 mutant mice has been observed compared to the controls. Histologically, instead of being columnar, the papilla cells are squamous in appearance due to the Wnt11 deficiency (Figure 2 in (I)). To better understand the papilla cells morphology, we used immunohistochemistry staining with the collecting duct marker AQP2. In the Wnt11-/- adult mice, AQP2 expression was ectopically enriched in the CDs. It was not only present on the basolateral side of the terminal papillary tubule like in the wild type, but also on the luminal side of the CDs (Figure 3 in (I)). This data matches the loss of cell polarity in the CDs of Wnt11 mutant kidneys. Combined, the data suggests that the Wnt11 deficiency in the C57Bl6 mice kidneys leads to abnormal development of the epithelial collecting system and chronic kidney disease.

48 5.3 Involvement of Wnt11 signalling in kidney tubular organization (I)

In addition to causing branching pattern defects, Wnt11 deficiency disturbed the tubule organization in the kidneys as well. To better visualize the kidney tubule morphology, optical projection tomography (OPT) technology was used. The computer-based three-dimensional (3D) kidney reconstruction with Troma-1 staining highlighted that the tubular convolution and length were increased in the Wnt11 mutant new-born mice, compared to the wild types (1751 ± 125μm vs. 1520 ± 160μm, n = 40, p < 0.05) (Figure 4 in (I)). The lumen of the cortical tubules in the Wnt11-/- kidneys was enlarged relative to the wild types (Figure 2 in (I)). To investigate the influence of Wnt11 in the kidney tubular system formation, we analysed cross-sections of the proximal tubule (LTL) and collecting duct (AQP2) using antibody immunostaining. As shown in Fig.5 (I), the lumen of the tubules in both new-born and adult Wnt11-/- kidneys is enlarged which can be linked to increased cell numbers in a single tubule when compared to the wild types. A cell number quantification study was done to better explain the tubule organization. The results show that the cell number in a single tubular segment (PT or CD) of the Wnt11 deficient kidneys varied notably. In both Wnt11-/- new-born and adult mice, over 50% PT have more than five cells per cross- section, while the controls have only 4or 5 cells in each cross-section. Similarly, around 20% of the CDs in Wnt11-/- new-born mice have more cells in each section. More apparently, half of the CDs in Wnt11-/- kidneys have 6–8 cells/cross-section instead of 4-5 cells/section in the wild types (Fig. 5 in (I)). The 3D kidney image was generated by OPT to further investigate the organization of smaller kidney in Wnt11 mutants compared to the controls. As expected, the OPT data indicated a significant volume reduction in the whole kidney (376 ± 23μm3 vs. 595 ± 81μm3, n = 6-8, p < 0.05) and kidney pelvis (279 ± 11μm3 vs. 418 ± 32μm3, n = 6-8, p < 0.05) (Figure S4 in (I)). The characteristic shape of the kidney results from differential growth of three axes, including the cortico-medullary, dorso-ventral, and rostro-caudal axes. The 3D model clearly showed that the dorso-ventral axis in Wnt11 mutant was much thinner than in the wild type, and its area was 56% smaller compared to the controls (1455 ± 115μm vs. 1686 ± 101μm, n = 6-8, p < 0.05). The cortical-medullary axis appearance in Wnt11-/- kidney also differed from that of the controls and was about 10% smaller than in the wild type (361 ± 15μm vs. 634 ± 75μm, n = 6-8, p < 0.05) (Figure 4 in (I)).

49 Overall, these results support the hypothesis that Wnt11 signalling is involved in the organization of the kidney architecture and the tubular system.

5.4 Deficiency of Wnt11 altered the cell activity during kidney development (I)

Wnt signalling is responsible for the cell fate determination during embryonic development. To investigate whether the noted dysmorphology in Wnt11-/- kidneys is due to an alteration of cell viability, P-H3 cell proliferation and TUNEL cell apoptosis assays were performed. Our results indicate that the cells proliferate robustly and that only few cells undergo apoptosis during the development of the wild type kidney. In comparison, in the Wnt11 mutant mice only few cells proliferate at this stage, and about twice as many undergo apoptosis, mainly in the cortex region (Figure 6 in (I)). To confirm the molecular basis of the Wnt11 function, a number of metanephron-expressed markers, including Six2, Hox10 and Foxd1, were examined. Six2 plays a role in maintaining the CM progenitor pool during kidney development (Self et al., 2006). It is decreased in kidneys of E16.5 and new-born Wnt11 mutant mice compared to the controls. This may lead to less mature nephron morphology and smaller kidneys. Reduction of Foxd1 is involved in stromal cell self-renewal and Hox10, which is necessary for stromal cell differentiation during kidney development (Kobayashi et al., 2014; Yallowitz, Hrycaj, Short, Smyth, & Wellik, 2011), and may participate in the development of smaller kidney as well (Figure 2 in (I)). Previously, researchers have shown that Wnt9b is required for the regulation of secondary branching and metanephric tubule induction. A downregulation of Wnt11 and GDNF expression can be detected in the Wnt9b-/- kidney (Carroll et al., 2005). Observation of Wnt9b expression in Wnt11 targeted mutation kidneys revealed a downregulation during nephrogenesis, particularly in the early stage (Figure 7 and Figure S4 in (I)). To summarize, the data suggests that Wnt11 can regulate the process of the tubular formation during nephrogenesis.

50 5.5 Molecular characteristics shared between nephrogenesis and renal cell carcinoma (II)

Based on the cellular behaviour, in particular similarities between embryonic development and cancers, we suggested that they may also show similar molecular regulation mechanisms. To study this hypothesis, a microarray assay was performed in the ex vivo cultured metanephric mesenchyme while also reanalysing the human kidney cancer gene expression database (Eikrem et al., 2016). The results show that after 96h induction of MM with spinal cord, 1616 genes have significant differential expression in MM compared to the un-induced MM (Supplementary Table S1 in (II)). The genes that show altered expression patterns were then compared to the human ccRCC cohort (Eikrem et al., 2016). As a result, 930 genes were selected that were significantly changed in both ccRCC and nephrogenesis (Supplementary Table S2 in (II)). Closer analysis showed that around 2/3 of the genes changed expression levels in the same direction, while the rest displayed the opposite behaviour. This led us to conclude that similar genes may be involved in embryonic kidney development and renal cancer. Next, an Ingenuity Pathway Analysis was performed using the genes displaying notable expression changes (FC≥2.0 and a p<0.05) in both ccRCC and induced-MM. The diagram shows that 56 pathways are shared by the three groups (Figure 1 in (II)). As listed, atherosclerosis signalling, caveolar-mediated endocytosis signalling, dendritic cell maturation, Fcγ receptor-mediated phagocytosis in macrophages and monocytes, hepatic fibrosis/hepatic stellate cell activation, and leukocyte extravasation signalling are the pathways simultaneously changed with the highest significance levels in all three analysed groups (–log(p- value)>4). Among them, caveolar-mediated endocytosis signalling has been identified to have a role in cancer heterogeneity (Martinez-Outschoorn et al., 2015). In our study, this pathway also shows a high ratio of gene number changes in our samples compared to the total number of genes in it (Figure 1 in (II)). The genes that were associated to the caveolar-mediated endocytosis signalling and showed an increase in both ccRCC and induced-MM include galectin-3 (Lgals3), Gsn, Cav1, epidermal growth factor receptor (Egfr) and integrin beta2 (Itgb2). They were selected for further investigation. Bnip3, which had an over- expression in the ccRCC but decreased in the induced-MM and which was shown to be regulated by caveolin-1(Martinez-Outschoorn et al., 2015), was selected for the functional study as well. For comparison we also included Paired box 8 (Pax8), a gene encoding a transcription factor necessary for kidney development (Narlis,

51 Grote, Gaitan, Boualia, & Bouchard, 2007). In our microarray data, Pax8 behaved in an opposite manner to Bnip3, being downregulated in the ccRCC cohort but upregulated in the MM undergoing differentiation.

5.6 Embryonic gene downregulation inhibited Renca cell behaviour (II)

To evaluate the function of the embryonic genes in cancer, a siRNA mediated gene downregulation was performed in mice RCC cells (Renca). The efficiency of the inhibition of the gene expression by all the siRNAs tested was over 80% after 48h, 72h and 96h treatment (Figure S1 in (II)). The IncuCyte live imaging system was chosen to analyse cell proliferation, cell death and cell migration. For the proliferation assay, the percentage of the cell confluence was followed for four days after siRNA knockdown. The Renca cells with Bnip3, Gsn, and Cav1 downregulation notably reduced the cell proliferation compared to the controls, while Lgals3, Pax8, Egfr, and Itgb2 had no effect on cell growth (Figure 3 in (II)). Sytox Green nuclear acid dye (1µM) was added to the culture system to check for cell death mediated by the siRNA treatment. The percentage of dead cells at 24h after cell plating was examined. Except for siEgfr, the siRNAs promoted cell death in the Renca cells. Particularly siBnip3 and siGsn turned out to be the most effective with an induced cell death of around 2.5-fold and 2.4-fold, respectively in the Renca cells (Figure 3 in (II)). Cell migration, invasion, and colony formation are important processes in cancer metastasis. To find out whether the genes analysed for proliferation also play a role in these processes, we investigated the influence of gene downregulation on the migratory and invasive properties of Renca cells. The results suggest that all genes may be associated with cancer cell migration. However, whereas Bnip3, Cav1, Gsn, Egfr, and Lgals3 had an effect on the Renca cell invasion, Pax8 and Itgb2 did not (Figure 4 in (II)). Inhibition of these genes in the Renca cells also resulted in a poor capacity of cell colony formation compared to the controls. The most severe condition was induced by siBnip3, which showed a reduced colony count of about 70% compared to the controls (Figure 5 in (II)). Because there is evidence that changes in PI3K/Akt pathway signalling can be associated with the RCC phenotype (Cancer Genome Atlas Research Network, 2013), we investigated whether the identified functional siRNAs also have an impact on Akt signalling in cultured Renca cells. We used the degree of Akt phosphorylation as a criterion. However, among the siRNAs used, only Pax8

52 siRNA led to enhanced Akt phosphorylation, whereas the Bnip3, Cav1 and Gsn siRNAs did not have a significant influence (Figure S2B in (II)). This pointed towards a different mechanism of action for Pax8 when compared to the other genes investigated in our study.

5.7 Metanephric mesenchyme and YFP+ Renca 3D co-culture set up (II)

Generally, gene functional studies in cancers are carried out using cell based models or gene edited mouse models. The restrictions of existing tumour models may be partially compensated by the development of novel 3D in vitro models that would bridge the gap between 2D cell culture and animal setups (Rodrigues et al., 2017). Such models can better mimic the tumour microenvironment and make it possible to efficiently screen the functions of the putative oncogenes. The in vitro MM culture system which Junttila et al., developed in our laboratory provided us with new ways to study gene function. Therefore, instead of using pure MMs, we mixed a certain number of gene manipulated cancer cells with the MM and cultured the mix as 3D organoids. In order to test how the foreign cells might influence the normal nephrogenesis in the 3D in vitro culture organoids, we mixed the E11.5 MM cells with Renca-YFP cells, cervical cancer-derived HeLa-YFP cells or mK4-YPF cells derived from induced metanephric mesenchyme undergoing epithelial conversion at a 50:1 ratio (Figure 6 in (II)). Interestingly, HeLa-YFP cells and mK4-YPF cells in the chimeric organoids grew slowly and the cells formed small clusters. At the same time, the Renca-YFP cells became more robust and distributed evenly in the chimera. Interestingly, the tubular formation in the 3D culture pellet was disrupted. Compared to the pure MM organoids or other two chimeras, only few kidney tubules were formed in the MM-Renca-YFP chimeras (Figure 6 in (II)). We concluded that Renca-YFP cells in the 3D co-cultures are useful to study the role of certain embryonic genes in renal cancer. Next, we tried to answer the question whether downregulation of certain genes would influence the ability of Renca cells to grow as a part of the 3D organoids. From the cell culture assays, Bnip3, Cav1 and Gsn showed the most consistent results and piqued our interest the most for further functional investigation using the 3D co-culture system. Excitingly, downregulation of Bnip3 or Cav1 in Renca- YFP cells led to an increased number of tubular structures (Pax2+) and proximal tubes (Aq1+) in the chimeras after 4 days of culture when compared to the control

53 siRNA-treated cells. Moreover, although the downregulation of Gsn in the Renca cells disrupted the tubule structure formation in the re-aggregated pellet, this effect was less prominent than for the siRNA control-treated cells (Figure 7 and Figure 8 in (II)). We then examined whether the induction of cell death in Renca cells by siRNA treatment was the primary reason for the better formation of Pax2+ tubular structures in chimeric organoids. We chose siBnip3 for testing. The Renca cells treated with siBnip3 were mixed with the MM at a 1:20 ratio instead of 1:50, since in the proliferation assay Renca cells had the 2.5-fold increase in cell death compared to the controls. In the Pax2+ staining, the renal tubules were not as disrupted as after control siRNA treatments, suggesting that not only apoptosis but also others alterations in Renca cells caused by Bnip3 siRNA treatment decreased the Renca cells’ ability to grow in the co-culture assay (Figure S3 in (II)). Overall, we demonstrated that activation of certain embryonic genes may indeed facilitate renal cancer development. Downregulation of these embryonic genes may lead to the development of potential RCC therapies. It also appears that MM-Renca co-cultures do provide a novel model for cancer gene discovery.

54 6 Discussion

6.1 Wnt11 signalling is required for normal tubule system construction

Our results reveal the role of Wnt11 in renal tubule formation. Previous studies examining Wnt11 during kidney development suggested that Wnt11 is located in the tips of the ureteric epithelium and participates in the UB branching morphogenesis (Kispert, Vainio, Shen, Rowitch, & McMahon, 1996; Majumdar et al., 2003). However, the in utero lethality of Wnt11-/- SV129 mice restricted our study to the early stages of kidney development. Surprisingly, Wnt11-/- mice with a C57Bl6 genetic background have the potential to survive until adulthood, which provides us with an opportunity to investigate the later functions of Wnt11 in kidney maturation. In wild-type new-born mice, Wnt11 is expressed in CD, PT, and papillary cells, suggesting a potential role for Wnt11 in kidney tubulogenesis. The defects of Wnt11 altered the organization of the PT and CD in both diameter and convolution. We observed that the Wnt11-/- kidneys are smaller in size, and that their growth in three axes differed from the wild type. The question remains how Wnt11 deletion influences kidney nephrogenesis. One possibility that might explain the abnormal tubule formation in Wnt11-/- kidneys is the missing fine- tuning of the Wnt9b and Six2 expression (Karner et al., 2011; Park et al., 2012). Lack of Wnt9b decreases Wnt11 expression and the puppies die one day after birth due to kidney formation failure. The Wnt9b hypomorphic mice however survive for several days to weeks (Carroll et al., 2005; Karner et al., 2009). We observed the downregulation of Wnt9b expression in Wnt11-/- kidneys with a more severe reduction in E16.5 than in new-borns, suggesting there are reciprocal interactions between Wnt11 and Wnt9b signalling in nephrogenesis. Taken together, our results suggest that other compensatory pathways may support kidney tubulogenesis when Wnt11 activity is absent. Wnt11 may fine- tune the Wnt9b signalling to influence the kidney development. In the kidney, Six2 is expressed in the CM progenitors. Genetic inactivation of Six2 results in premature RV and ectopic differentiation of mesenchymal cells into epithelia, and a higher apoptosis has been shown before in the Six2-null kidney progenitor pool (Self et al., 2006). Similar results were also found in the Wnt11-/- kidneys. The expression of Six2 in Wnt11 mutant kidney was significantly downregulated in embryonic development as shown by in situ hybridization and

55 qRT-PCR. Therefore, Wnt11 signalling may regulate the expression of the Six2 gene in the mesenchyme to coordinate the self-renewal and commitment of the nephron progenitor cells. Wnt11 signalling may indirectly regulate the differentiation of stromal progenitor cells. The expression of Hox10 and Foxd1, involved in promoting the nephrogenesis, is altered when Wnt11 function is absent. Hox10 is critical for the proper integration of Foxd1+ cortical stromal cells into the developing kidney and further influences the distribution of nephrogenic mesenchyme progenitors (Levinson et al., 2005; Yallowitz et al., 2011). The stroma cells provide signals to activate the Ret expression and ureteric outgrowth (Batourina et al., 2001; Mendelsohn et al., 1999). Normally, the Foxd1-positive stroma comprises the renal capsule. Pelvic fused kidneys and inappropriately formed renal capsules have been observed in mice lacking Foxd1 (Levinson et al., 2005). Therefore, Wnt11 may even have a potential role in organising the nephron and stromal progenitor cell layout during metanephric kidney development. Previously, Wnt11 has been shown to positively cooperate with GDNF/Ret signalling and to regulate the ureteric branching in SV129 kidney. When Ret expression was tested in the C57Bl6 Wnt11-/- whole kidney sample, it was downregulated and it may have been weakened by the loss of UB tips in the Wnt11- /- kidneys. At the same time, GDNF expression was only slightly reduced in the mutant E16.5 and new-born kidney. These observations confirmed the Wnt11- GDNF-positive regulation loop in the ureteric epithelium branching.

6.2 Wnt11 deficiency promotes cyst formation in mice

In our study, we demonstrated that, in addition to its role in the ureteric branching, Wnt11 also has a later role in the tubule system formation. Wnt11-/- mice with a C57Bl6 genetic background develop cystic kidneys, which is similar to what occurs in human GCK diseases (Bissler et al., 2010; Lennerz et al., 2010; O'Meara et al., 2012; Woolf, Feather, & Bingham, 2002). HNF-1β loss-of-function by mutation or deletion decreased PKHD1 expression and promoted the formation of kidney cysts and renal failure in mice (Igarashi, Shao, McNally, & Hiesberger, 2005). Similarly, both PKHD1 and HNF1β within Wnt11-/- kidney were downregulated. During the early stage of kidney development, lack of HNF1β leads to severe defects in UB branching. This was characterized by decreased expression of Wnt11, Pax2 and delay of Ret expression (Desgrange et al., 2017). Based on this report combined

56 with our own study in Wnt11-/- kidney, we can conclude that Wnt11 and HNF1β synergistically regulate kidney development. Unlike in the Wnt7b-/- and Wnt9b-/- kidneys, the cysts in C57Bl6 Wnt11-/- kidneys are not the predominant phenotype (Karner et al., 2011; Yu et al., 2009), but all the survived Wnt11-/- mice had glomerular cysts and showed compromised glomerular function. The planar cell polarity is essential for convergent extension movements and oriented cell divisions. In Wnt9b mutant mice, the loss of planar cell polarity in the epithelial cells was linked to cyst formation. Furthermore, in Wnt11-/- kidney, the alteration of planar cell polarity and Wnt9b downregulation were observed, suggesting that similar mechanisms may underlie the Wnt11 related cyst development. Improper proliferation and apoptosis in cortex region may result in the expansion of PT or CD diameter as well. Impairment of primary cilia plays a role in the cystic kidney disease characteristic for Wnt11-/- kidney. Our data suggests that Wnt11 fulfils essential roles in these processes, perhaps by regulating cell orientation.

6.3 Specific kidney embryonic genes could be potentially selected for gene target therapy in renal cell carcinoma

Cancer is heterogeneous between individuals. Although the NGS helps to find numbers of alternated genes in ccRCC (Cancer Genome Atlas Research Network, 2013), there are still no clinically applicable prognostic markers for this disease. The embryonically activated kidney developmental genes associated with ccRCC were selected for our study due to the previously demonstrated similarities between embryogenesis and carcinogenesis (Kim & Orkin, 2011). Gene enrichment and pathway analyses for the genes simultaneously regulated in ccRCC and in vitro-induced MM samples gave us several pathways that were relevant both for kidney development and carcinogenesis. In all, we compared three different groups of pathways: those significantly regulated during clear cell renal carcinoma formation (marked ccRCC in Figure 1A in (II)), those induced in kidney development (marked MM (96 hr vs 0 hr)), and those found when looking at genes changed in both kidney development and renal carcinogenesis (marked MM vs ccRCC). Interestingly, analysis for the combined dataset (MM vs ccRCC) clearly gave different results compared to separate analyses of the cancer dataset and developmental dataset. Several of the pathways found in the combined dataset (such as hepatic fibrosis) require regulation of multiple genes that have very broad functions and are involved in multiple different processes, whereas others were

57 more specifically related to tumour development. For further analysis, we preferred to concentrate on a pathway already known to be involved in carcinogenesis. We decided to study the function of the genes involved in the caveolar-mediated endocytosis signalling in more detail. Caveolae, which are regulated by cavins and caveolins, are involved in endocytosis and signal transduction both under physiological conditions and in cancer (Parton & del Pozo, 2013). Among the caveolar-related embryonic genes, Cav1, Egfr, Bnip3, Gsn, Itgb2, and Lgals3 were induced in human ccRCC. Increased expression of Cav1 was observed in renal cancers (Campbell et al., 2013; Steffens et al., 2011; Waalkes et al., 2011). Caveolins are known to directly bind to EGFR and regulate EGFR signalling activity (Agelaki et al., 2009; Lajoie, Goetz, Dennis, & Nabi, 2009). Meanwhile, galectin lattices can sequester EGFR from Cav1 scaffolds, and hence promote EGFR signalling in cancer (Lajoie et al., 2007). Activation of Bnip3 by loss of CAV1 was revealed in a wide variety of cancers (Martinez-Outschoorn et al., 2015). In our study, we showed a positive correlation between over expression of developmental genes and renal cancer cells viability. The Renca cell proliferation was inhibited when the expression of selected genes was downregulated by the siRNA treatments. At the same time, cell apoptosis was induced except for in the siEGFR treated cells. Moreover, in Renca cells downregulation of these genes also suppressed the cells’ ability for invasion and migration, while previous studies had shown that the influence of cell proliferation and invasion by Cav1 downregulation is cell type dependent (Campbell et al., 2013). Also, fewer colonies were formed in the siRNA treated Renca cells, implying a lower metastasis potential for Renca cells. Using the Renca cell model we also investigated the relationship between Cav1, Bnip3, and Gsn expression and the EMT process. Cav1 expression is upregulated during EMT and influences cancer cell adhesion (Bailey & Liu, 2008). In the current study inhibition of Cav1 expression in the Renca cells did not affect the expression of epithelial marker E-cadherin and mesenchymal markers Vimentin and N-cadherin. However, Bnip3 could modulate the expression of E-cadherin in Renca cells. Moreover, silencing GSN promoted the EMT of Renca cells when verified by examining the expression of EMT markers. Similar results have previously been reported in the MDA-MB231 breast cancer cell line (Chen, Wang, Shieh, Chiu, & Liou, 2015). PI3K/Akt signalling is one of the most frequently altered pathways in ccRCC (Guo et al., 2015). It also plays a role in balancing the self-renewal and

58 differentiation of nephron progenitor cells during nephrogenesis (Lindström et al., 2015). In our study, downregulation of CAV1, Bnip3 and GSN slightly decreased the Akt phosphorylation in Renca cells. In prostate cancer cells, inactivation of CAV1 reduces the phAkt and cell viability (Li, Ren, Tahir, Ren, & Thompson, 2003). In contrast, Pax8 silencing by siRNA dramatically increased the phAkt protein in Renca cell, possibly indicating different molecular mechanism of Pax8 action. To summarize, we demonstrated that ccRCC and nephrogenesis share certain molecular characteristics and signalling pathways. The data presented here indicates that caveolar-related embryonic genes play important roles in renal cancer development. This may be taken as biomarkers to detect cancer, and the targeting of embryonic genes may well represent a future cancer therapy for the prevention and treatment of metastases.

6.4 Efficiency of in vitro 3D culture model for gene function studies

The importance of 3D cultures in modelling cell signalling, differentiation, and for drug development have been on the rise during the last ten years. More recently, the development of human patient-derived organoids has enabled more precise disease models for studying biomedical applications, translational medicine, and personalized therapy (Dutta, Heo, & Clevers, 2017). In our study, we developed a 3D co-culture system by mixing MM with kidney cancer cells to study the functions of certain genes that are relevant to both normal development and carcinogenesis. The addition of kidney-originated Renca carcinoma cells disrupted the formation of the kidney tubules, while cancer cells of different origin or immortalized non- malignant kidney cells had little effects on kidney tubule formation in the 3D chimera assay. Interestingly, this study showed that inhibition of Bnip3, GSN and Cav1 in Renca by siRNAs rescued tubule formation, which was confirmed by the positive staining of Pax2 and Aq1 in 3D organoids. It appears these genes play a role in the modulation of Renca cell behaviour. In summary, our data shows that reactivation of embryonic developmental genes may promote renal cancer development. Silencing these genes may provide us with cancer therapy options. Additionally, our ex vivo setup potentially provides a new way to study gene function, and also makes it possible to monitor cancer cell behaviour under conditions that mimic the internal tumour environment better. The siRNA-mediated inhibition of embryonic genes in kidney cancer may not only help to identify new putative oncogenes, but it may also provide new therapeutic opportunities.

60 7 Conclusions

The studies included in the thesis describe the functions of embryonic genes in kidney development and renal cancer. The novel mouse model generated by switching of Wnt11 deletion from SV129 to a C57Bl6 mice genetic background bypassed the in utero lethality of Wnt11 and provided an opportunity to study its function in the kidney maturation. In addition to the early UB branching, Wnt11 was involved in the proper organization of tubular epithelial cells, as illustrated by the dilation of renal tubules (PT and CD) and loss of planar cell polarity in Wnt11- /- kidneys. Wnt11 signalling also plays roles in regulating the differentiation of mesenchymal and stromal progenitor cells, since Six2, Foxd1, and Hox10 levels were reduced when Wnt11 was mutated. Additionally, glomerular cysts and impairment of glomerular function were observed in our Wnt11-/- model. This suggests that the C57Bl6 Wnt11-/- mouse model may be used to study the causes of human glomerulocystic kidney diseases. Disruption of the embryonic gene expression is revealed in the cancer initiation. In the thesis, we identified a list of embryonic genes that were ectopically expressed in human ccRCC, including genes related to caveolar-mediated endocytosis signalling. Cell behaviour such as proliferation, death, migration, and invasion were changed after gene silencing in the Renca cells by siRNAs. This may provide a way to find potential cancer target genes, for instance. As part of the thesis research, a novel in vitro 3D co-culture assay was established to efficiently investigate the function of the embryonic genes by mixing metanephric mesenchyme with Renca cells in a 50:1 ratio. This new 3D co-culture system may serve as a more efficient model to use for gene function studies and drug selection, which in turn may provide us with better options for cancer treatment.

62 References

Agelaki, S., Spiliotaki, M., Markomanolaki, H., Kallergi, G., Mavroudis, D., Georgoulias, V., & Stournaras, C. (2009). Caveolin-1 regulates EGFR signaling in MCF-7 breast cancer cells and enhances gefitinib-induced tumor cell inhibition. Cancer Biology & Therapy, 8(15), 1470-1477. Aran, D., Abu-Remaileh, M., Levy, R., Meron, N., Toperoff, G., Edrei, Y., Bergman, Y., & Hellman, A. (2016). Embryonic stem cell (ES)-specific enhancers specify the expression potential of ES genes in cancer. PLoS Genetics, 12(2), e1005840. Archer, T. C., Weeraratne, S. D., & Pomeroy, S. L. (2012). Hedgehog-GLI pathway in medulloblastoma. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 30(17), 2154-2156. Asghar, W., El Assal, R., Shafiee, H., Pitteri, S., Paulmurugan, R., & Demirci, U. (2015). Engineering cancer microenvironments for in vitro 3-D tumor models. Materials Today (Kidlington, England), 18(10), 539-553. Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N., & Lovell-Badge, R. (2003). Multipotent cell lineages in early mouse development depend on SOX2 function. Genes & Development, 17(1), 126-140. Bailey, A. S., & McDermott, D. F. (2015). Immune checkpoint inhibitors (anti-CTLA4, anti- PD-1) in RCC. Renal cell carcinoma (pp. 469-481) Springer, New York, NY. Bailey, K. M., & Liu, J. (2008). Caveolin-1 up-regulation during epithelial to mesenchymal transition is mediated by focal adhesion kinase. The Journal of Biological Chemistry, 283(20), 13714-13724. Banks, R. E., Tirukonda, P., Taylor, C., Hornigold, N., Astuti, D., Cohen, D., Maher, E. R., Stanley, A. J., Harnden, P., Joyce, A., Knowles, M., & Selby, P. J. (2006). Genetic and epigenetic analysis of von hippel-lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer. Cancer Research, 66(4), 2000-2011. Bar, E. E., Chaudhry, A., Farah, M. H., & Eberhart, C. G. (2007). Hedgehog signaling promotes medulloblastoma survival via BclII. American Journal of Pathology, 170(1), 347-355. Barak, H., Huh, S., Chen, S., Jeanpierre, C., Martinovic, J., Parisot, M., Bole-Feysot, C., Nitschké, P., Salomon, R., Antignac, C., Ornitz, D. M., & Kopan, R. (2012). FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man. Developmental Cell, 22(6), 1191-1207. Basson, M. A., Watson-Johnson, J., Shakya, R., Akbulut, S., Hyink, D., Costantini, F. D., Wilson, P. D., Mason, I. J., & Licht, J. D. (2006). Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1. Developmental Biology, 299(2), 466-477. Basta, J. M., Robbins, L., Kiefer, S. M., Dorsett, D., & Rauchman, M. (2014). Sall1 balances self-renewal and differentiation of renal progenitor cells. Development (Cambridge, England), 141(5), 1047-1058.

63 Batourina, E., Gim, S., Bello, N., Shy, M., Clagett-Dame, M., Srinivas, S., Costantini, F., & Mendelsohn, C. (2001). Vitamin A controls epithelial/mesenchymal interactions through ret expression. Nature Genetics, 27(1), 74-78. Bayrak, O., Sen, H., Bulut, E., Cengiz, B., Karakok, M., Erturhan, S., & Seckiner, I. (2014). Evaluation of EGFR, KRAS and BRAF gene mutations in renal cell carcinoma. Journal of Kidney Cancer and VHL, 1(4), 40-45. Bell, D., & El-Naggar, A. K. (2013). Molecular heterogeneity in mucoepidermoid carcinoma: Conceptual and practical implications. Head and Neck Pathology, 7(1), 23-27. Ben-Porath, I., Thomson, M. W., Carey, V. J., Ge, R., Bell, G. W., Regev, A., & Weinberg, R. A. (2008). An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nature Genetics, 40(5), 499-507. Bernstein, J. (1993). Renal cystic disease in the tuberous sclerosis complex. Pediatric Nephrology (Berlin, Germany), 7(4), 490-495. Bialestock, D. (1960). Anaemia of renal origin, studied by micro-dissection of the kidney. Australasian Annals of Medicine, 9, 44-52. Bielecka, Z. F., Czarnecka, A. M., Solarek, W., Kornakiewicz, A., & Szczylik, C. (2014). Mechanisms of acquired resistance to tyrosine kinase inhibitors in clear - cell renal cell carcinoma (ccRCC). Current Signal Transduction Therapy, 8(3), 218-228. Bilir, B., Kucuk, O., & Moreno, C. S. (2013). Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells. Journal of Translational Medicine, 11, 280. Bissler, J., Siroky, B., & Yin, H. (2010). Glomerulocystic kidney disease. Pediatric Nephrology, 25(10), 2049-2059. Blank, U., Brown, A., Adams, D. C., Karolak, M. J., & Oxburgh, L. (2009). BMP7 promotes proliferation of nephron progenitor cells via a JNK-dependent mechanism. Development (Cambridge, England), 136(21), 3557-3566. Blute, M. L., Leibovich, B. C., Lohse, C. M., Cheville, J. C., & Zincke, H. (2004). The mayo clinic experience with surgical management, complications and outcome for patients with renal cell carcinoma and venous tumour thrombus. BJU International, 94(1), 33- 41. Bolós, V., Grego-Bessa, J., & de la Pompa, José Luis. (2007). Notch signaling in development and cancer. Endocrine Reviews, 28(3), 339-363. Boström, A. K., Lindgren, D., Johansson, M. E., Axelson, H. (2013). Effects of TGF-β signaling in clear cell renal cell carcinoma cells. Biochemical and Biophysical Research Communications, 435(1), 126-33. Boyle, S., Misfeldt, A., Chandler, K. J., Deal, K. K., Southard-Smith, E. M., Mortlock, D. P., Baldwin, H. S., & de Caestecker, M. (2008). Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia. Developmental Biology, 313(1), 234- 245.

64 Boyle, S., Shioda, T., Perantoni, A. O., & de Caestecker, M. (2007). Cited1 and Cited2 are differentially expressed in the developing kidney but are not required for nephrogenesis. Developmental Dynamics: An Official Publication of the American Association of Anatomists, 236(8), 2321-2330. Brabletz, T. (2012). EMT and MET in metastasis: Where are the cancer stem cells? Cancer Cell, 22(6), 699-701. Bridgewater, D., Cox, B., Cain, J., Lau, A., Athaide, V., Gill, P. S., Kuure, S., Sainio, K., & Rosenblum, N. D. (2008). Canonical WNT/beta-catenin signaling is required for ureteric branching. Developmental Biology, 317(1), 83-94. Brugarolas, J. (2014). Molecular genetics of clear-cell renal cell carcinoma. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 32(18), 1968-1976. Campbell, L., Al-Jayyoussi, G., Gutteridge, R., Gumbleton, N., Griffiths, R., Gumbleton, S., Smith, M. W., Griffiths, D. F., & Gumbleton, M. (2013). Caveolin-1 in renal cell carcinoma promotes tumour cell invasion, and in co-operation with pERK predicts metastases in patients with clinically confined disease. Journal of Translational Medicine, 11, 255. Cancer Genome Atlas Research Network. (2013). Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature, 499(7456), 43-49. Cardillo, M. R., Lazzereschi, D., Gandini, O., Di Silverio, F., & Colletta, G. (2001). Transforming growth factor-beta pathway in human renal cell carcinoma and surrounding normal-appearing renal parenchyma. Analytical and Quantitative Cytology and Histology, 23(2):109-117. Carroll, T. J., Park, J., Hayashi, S., Majumdar, A., & McMahon, A. P. (2005). Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Developmental Cell, 9(2), 283-292. Carson, R. W., Bedi, D., Cavallo, T., & DuBose, T. D. (1987). Familial adult glomerulocystic kidney disease. American Journal of Kidney Diseases: The Official Journal of the National Kidney Foundation, 9(2), 154-165. Chaffer, C. L., Thompson, E. W., & Williams, E. D. (2007). Mesenchymal to epithelial transition in development and disease. Cells, Tissues, Organs, 185(1-3), 7-19. Chen, Z., Wang, P., Shieh, D., Chiu, K., & Liou, Y. (2015). Involvement of gelsolin in TGF- beta 1 induced epithelial to mesenchymal transition in breast cancer cells. Journal of Biomedical Science, 22, 90. Cheng, H., Kim, M., Valerius, M. T., Surendran, K., Schuster-Gossler, K., Gossler, A., McMahon, A. P., & Kopan, R. (2007). Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron. Development (Cambridge, England), 134(4), 801-811. Chow, W., & Devesa, S. S. (2008). Contemporary epidemiology of renal cell cancer. Cancer Journal (Sudbury, Mass.), 14(5), 288-301. Clark, P., Dziarmaga, A., Eccles, M., & Goodyer, P. (2004). Rescue of defective branching nephrogenesis in renal-coloboma syndrome by the caspase inhibitor, Z-VAD-fmk. Journal of the American Society of Nephrology: JASN, 15(2), 299-305.

65 Clements, W. M., Lowy, A. M., & Groden, J. (2003). Adenomatous polyposis coli/beta- catenin interaction and downstream targets: Altered gene expression in gastrointestinal tumors. Clinical Colorectal Cancer, 3(2), 113-120. Cochrane, C. R., Szczepny, A., Watkins, D. N., & Cain, J. E. (2015). Hedgehog signaling in the maintenance of cancer stem cells. Cancers, 7(3), 1554-1585. Costantini, F. (2006). Renal branching morphogenesis: Concepts, questions, and recent advances. Differentiation; Research in Biological Diversity, 74(7), 402-421. Costantini, F., & Shakya, R. (2006). GDNF/ret signaling and the development of the kidney. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology, 28(2), 117-127. Davies, J. A. (2002). Do different branching epithelia use a conserved developmental mechanism? BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology, 24(10), 937-948. De Calisto, J., Araya, C., Marchant, L., Riaz, C. F., & Mayor, R. (2005). Essential role of non-canonical wnt signalling in neural crest migration. Development (Cambridge, England), 132(11), 2587-2597. Desgrange, A., Heliot, C., Skovorodkin, I., Akram, S. U., Heikkilä, J., Ronkainen, V. P., Miinalainen, I., Vainio, S. J., & Cereghini, S. (2017). HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis. Development (Cambridge, England), 144(24), 4704-4719. Dormoy, V., Danilin, S., Lindner, V., Thomas, L., Rothhut, S., Coquard, C., Helwig, J. J., Jacqmin, D., Lang, H., & Massfelder, T. (2009). The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth. Molecular Cancer, 8, 123. Dreesen, O., & Brivanlou, A. H. (2007). Signaling pathways in cancer and embryonic stem cells. Stem Cell Reviews, 3(1), 7-17. Duchartre, Y., Kim, Y., & Kahn, M. (2016). The wnt signaling pathway in cancer. Critical Reviews in Oncology/Hematology, 99(Supplement C), 141-149. Dutta, D., Heo, I., & Clevers, H. (2017). Disease modeling in stem cell-derived 3D organoid systems. Trends in Molecular Medicine, 23(5), 393-410. Eikrem, O., Beisland, C., Hjelle, K., Flatberg, A., Scherer, A., Landolt, L., Skogstrand, T., Leh, S., Beisvag, V., & Marti, H. (2016). Transcriptome sequencing (RNAseq) enables utilization of formalin-fixed, paraffin-embedded biopsies with clear cell renal cell carcinoma for exploration of disease biology and biomarker development. PloS One, 11(2), e0149743. Eremina, V., Sood, M., Haigh, J., Nagy, A., Lajoie, G., Ferrara, N., Gerber, H. P., Kikkawa, Y., Miner, J. H, & Quaggin, S. E. (2003). Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. The Journal of Clinical Investigation, 111(5), 707-716. Ferlay, J., Parkin, D. M., & Steliarova-Foucher, E. (2010). Estimates of cancer incidence and mortality in europe in 2008. European Journal of Cancer (Oxford, England: 1990), 46(4), 765-781.

66 Ferlay, J., Steliarova-Foucher, E., Lortet-Tieulent, J., Rosso, S., Coebergh, J. W. W., Comber, H., Forman, D., & Bray, F. (2013). Cancer incidence and mortality patterns in europe: Estimates for 40 countries in 2012. European Journal of Cancer (Oxford, England: 1990), 49(6), 1374-1403. Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D. M., Forman, D., & Bray, F. (2015). Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer, 136(5), 359. Gao, W., Li, W., Xiao, T., Liu, X. S., & Kaelin, W. G. (2017). Inactivation of the PBRM1 tumor suppressor gene amplifies the HIF-response in VHL−/−clear cell renal carcinoma. Proceedings of the National Academy of Sciences, 114(5), 1027-1032. Gonnissen, A., Isebaert, S., & Haustermans, K. (2015). Targeting the hedgehog signaling pathway in cancer: Beyond smoothened. Oncotarget, 6(16), 13899. Goodrich, L. V., Milenković, L., Higgins, K. M., & Scott, M. P. (1997). Altered neural cell fates and medulloblastoma in mouse patched mutants. Science (New York, N.Y.), 277(5329), 1109-1113. Greef, B., & Eisen, T. (2016). Medical treatment of renal cancer: New horizons. British Journal of Cancer, 115(5), 505-516. Grieshammer, U., Cebrián, C., Ilagan, R., Meyers, E., Herzlinger, D., & Martin, G. R. (2005). FGF8 is required for cell survival at distinct stages of nephrogenesis and for regulation of gene expression in nascent nephrons. Development (Cambridge, England), 132(17), 3847-3857. Guo, H., German, P., Bai, S., Barnes, S., Guo, W., Qi, X., Lou, H., Liang, J., Jonasch, E., Mills, G. B., & Ding, Z. (2015). The PI3K/AKT pathway and renal cell carcinoma. Journal of Genetics and Genomics, 42(7), 343-353. Guo, X., & Wang, X. F. (2009). Signaling cross-talk between TGF-β/BMP and other pathways. Cell Res, 19(1), 71-88. Gupta, S., Takebe, N., & Lorusso, P. (2010). Targeting the hedgehog pathway in cancer. Therapeutic Advances in Medical Oncology, 2(4), 237. Haas, N. B., & Nathanson, K. L. (2014). Hereditary kidney cancer syndromes. Advances in Chronic Kidney Disease, 21(1), 81-90. Hendry, C., Rumballe, B., Moritz, K., & Little, M. H. (2011). Defining and redefining the nephron progenitor population. Pediatric Nephrology (Berlin, Germany), 26(9), 1395- 1406. Heuberger, J., & Birchmeier, W. (2010). Interplay of cadherin-mediated cell adhesion and canonical wnt signaling. Cold Spring Harbor Perspectives in Biology, 2(2), a002915. Mizuno, H., Spike, B. T., Wahl, G. M., & Levine, A. J. (2010). Inactivation of p53 in breast cancers correlates with stem cell transcriptional signatures. Proceedings of the National Academy of Sciences of the United States of America, 107(52), 22745-22750. Hoppler, S., & Moon, R. T. (2014). Wnt signaling in embryonic development and adult tissue homeostasis. In S. Hoppler, & R. T. Moon (Eds.), Wnt signaling in development and disease (pp. 251-252). Hoboken, NJ, USA: John Wiley & Sons, Inc.

67 Hugo, H., Ackland, M. L., Blick, T., Lawrence, M. G., Clements, J. A., Williams, E. D., & Thompson, E. W. (2007). Epithelial--mesenchymal and mesenchymal--epithelial transitions in carcinoma progression. Journal of Cellular Physiology, 213(2), 374-383. Hunt, J. D., van der Hel, Olga L, McMillan, G. P., Boffetta, P., & Brennan, P. (2005). Renal cell carcinoma in relation to cigarette smoking: Meta-analysis of 24 studies. International Journal of Cancer, 114(1), 101-108. Ibsen, K. H., & Fishman, W. H. (1979). Developmental gene expression in cancer. Biochimica Et Biophysica Acta, 560(2), 243-280. Igarashi, P., Shao, X., McNally, B. T., & Hiesberger, T. (2005). Roles of HNF-1beta in kidney development and congenital cystic diseases. Kidney International, 68(5), 1944- 1947. Ishibe, S., Karihaloo, A., Ma, H., Zhang, J., Marlier, A., Mitobe, M., Togawa, A., Schmitt, R., Czyczk, J., Kashgarian, M., Geller, D. S., Thorgeirsson, S. S., & Cantley, L. G. (2009). Met and the epidermal growth factor receptor act cooperatively to regulate final nephron number and maintain collecting duct morphology. Development (Cambridge, England), 136(2), 337-345. Jain, S. (2009). The many faces of RET dysfunction in kidney. Organogenesis, 5(4), 177- 190. Ji, Z., Mei, F. C., Johnson, B. H., Thompson, E. B., & Cheng, X. (2007). Protein kinase A, not epac, suppresses hedgehog activity and regulates glucocorticoid sensitivity in acute lymphoblastic leukemia cells. The Journal of Biological Chemistry, 282(52), 37370- 37377. Junttila, S., Saarela, U., Halt, K., Manninen, A., Pärssinen, H., Lecca, M. R., Brändli, A. W., Sims-Lucas, S., Skovorodkin, I., & Vainio, S. J. (2015). Functional genetic targeting of embryonic kidney progenitor cells ex vivo. Journal of the American Society of Nephrology: JASN, 26(5), 1126-1137. Kao, R. M., Vasilyev, A., Miyawaki, A., Drummond, I. A., & McMahon, A. P. (2012). Invasion of distal nephron precursors associates with tubular interconnection during nephrogenesis. Journal of the American Society of Nephrology: JASN, 23(10), 1682- 1690. Karner, C. M., Chirumamilla, R., Aoki, S., Igarashi, P., Wallingford, J. B., & Carroll, T. J. (2009). Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis. Nature Genetics, 41(7), 793-799. Karner, C. M., Das, A., Ma, Z., Self, M., Chen, C., Lum, L., Oliver, G., & Carroll, T. J. (2011). Canonical Wnt9b signaling balances progenitor cell expansion and differentiation during kidney development. Development (Cambridge, England), 138(7), 1247-1257. Kim, J., & Orkin, S. H. (2011). Embryonic stem cell-specific signatures in cancer: Insights into genomic regulatory networks and implications for medicine. Genome Medicine, 3(11), 75. Kim, Y. S., Kang, Y. K., Kim, J. B., Han, S. A., Kim, K. I., & Paik, S. R. (2000). Beta- catenin expression and mutational analysis in renal cell carcinomas. Pathology International, 50(9), 725-730.

68 Kispert, A., Vainio, S., & McMahon, A. P. (1998). Wnt-4 is a mesenchymal signal for epithelial transformation of metanephric mesenchyme in the developing kidney. Development (Cambridge, England), 125(21), 4225-4234. Kispert, A., Vainio, S., Shen, L., Rowitch, D. H., & McMahon, A. P. (1996). Proteoglycans are required for maintenance of wnt-11 expression in the ureter tips. Development (Cambridge, England), 122(11), 3627-3637. Kobayashi, A., Kwan, K., Carroll, T. J., McMahon, A. P., Mendelsohn, C. L., & Behringer, R. R. (2005). Distinct and sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for tubular morphogenesis during kidney development. Development (Cambridge, England), 132(12), 2809-2823. Kobayashi, A., Mugford, J. W., Krautzberger, A. M., Naiman, N., Liao, J., & McMahon, A. P. (2014). Identification of a multipotent self-renewing stromal progenitor population during mammalian kidney organogenesis. Stem Cell Reports, 3(4), 650-662. Kobayashi, A., Valerius, M. T., Mugford, J. W., Carroll, T. J., Self, M., Oliver, G., & McMahon, A. P. (2008). Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell, 3(2), 169-181. Kominsky, S. L., Doucet, M., Brady, K., Weber, K. L. (2007). TGF-beta promotes the establishment of renal cell carcinoma bone metastasis. Journal of Bone and Mineral Research, 22(1), 37-44. Kondratov, A. G., Kvasha, S. M., Stoliar, L. A., Romanenko, A. M., Zgonnyk, Y. M., Gordiyuk, V. V., Kashuba, E. V., Rynditch, A. V., Zabarovsky, E. R., & Kashuba, V. I. (2012). Alterations of the WNT7A gene in clear cell renal cell carcinomas. PloS One, 7(10), e47012. Kreidberg, J. A. (2010). WT1 and kidney progenitor cells. Organogenesis, 6(2), 61-70. Kruck, S., Eyrich, C., Scharpf, M., Sievert, K., Fend, F., Stenzl, A., & Bedke, J. (2013). Impact of an altered Wnt1/β-catenin expression on clinicopathology and prognosis in clear cell renal cell carcinoma. International Journal of Molecular Sciences, 14(6), 10944. Lajoie, P., Goetz, J. G., Dennis, J. W., & Nabi, I. R. (2009). Lattices, rafts, and scaffolds: Domain regulation of receptor signaling at the plasma membrane. The Journal of Cell Biology, 185(3), 381-385. Lajoie, P., Partridge, E. A., Guay, G., Goetz, J. G., Pawling, J., Lagana, A., Joshi, B., Dennis, J. W., & Nabi, I. R. (2007). Plasma membrane domain organization regulates EGFR signaling in tumor cells. The Journal of Cell Biology, 179(2), 341-356. Larue, L., & Bellacosa, A. (2005). Epithelial-mesenchymal transition in development and cancer: Role of phosphatidylinositol 3' kinase/AKT pathways. Oncogene, 24(50), 7443- 7454. Lennerz, J. K., Spence, D. C., Iskandar, S. S., Dehner, L. P., & Liapis, H. (2010). Glomerulocystic kidney: One hundred-year perspective. Archives of Pathology & Laboratory Medicine, 134(4), 583-605.

69 Leonetti, A., Leonardi, F., Bersanelli, M., & Buti, S. (2017). Clinical use of lenvatinib in combination with everolimus for the treatment of advanced renal cell carcinoma. Therapeutics and Clinical Risk Management, 13, 799-806. Levinson, R. S., Batourina, E., Choi, C., Vorontchikhina, M., Kitajewski, J., & Mendelsohn, C. L. (2005). Foxd1-dependent signals control cellularity in the renal capsule, a structure required for normal renal development. Development (Cambridge, England), 132(3), 529-539. Li, L., Ren, C. H., Tahir, S. A., Ren, C., & Thompson, T. C. (2003). Caveolin-1 maintains activated akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A. Molecular and Cellular Biology, 23(24), 9389-9404. Lin, Y., Liu, A., Zhang, S., Ruusunen, T., Kreidberg, J. A., Peltoketo, H., Drummond, I., & Vainio, S. (2001). Induction of ureter branching as a response to wnt-2b signaling during early kidney organogenesis. Developmental Dynamics: An Official Publication of the American Association of Anatomists, 222(1), 26-39. Lindström, N. O., Carragher, N. O., & Hohenstein, P. (2015). The PI3K pathway balances self-renewal and differentiation of nephron progenitor cells through β-catenin signaling. Stem Cell Reports, 4(4), 551-560. Liu, S., Dontu, G., Mantle, I. D., Patel, S., Ahn, N., Jackson, K. W., Suri, P., & Wicha, M. S. (2006). Hedgehog signaling and bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Research, 66(12), 6063-6071. Ma, Y., Zhang, P., Wang, F., Yang, J., Yang, Z., & Qin, H. (2010). The relationship between early embryo development and tumourigenesis. Journal of Cellular and Molecular Medicine, 14(12), 2697-2701. Majumdar, A., Vainio, S., Kispert, A., McMahon, J., & McMahon, A. P. (2003). Wnt11 and ret/gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development. Development (Cambridge, England), 130(14), 3175-3185. Mao, Z., Chong, J., & Ong, A. C. M. (2016). Autosomal dominant polycystic kidney disease: Recent advances in clinical management. F1000Research, 5, 2029. Margulis, V., Sánchez-Ortiz, R. F., Tamboli, P., Cohen, D. D., Swanson, D. A., & Wood, C. G. (2007). Renal cell carcinoma clinically involving adjacent organs: Experience with aggressive surgical management. Cancer, 109(10), 2025-2030. Marose, T. D., Merkel, C. E., McMahon, A. P., & Carroll, T. J. (2008). Beta-catenin is necessary to keep cells of ureteric bud/wolffian duct epithelium in a precursor state. Developmental Biology, 314(1), 112-126. Martinez-Outschoorn, U. E., Sotgia, F., & Lisanti, M. P. (2015). Caveolae and signalling in cancer. Nature Reviews. Cancer, 15(4), 225-237. Massagué, J. (2008). TGFβ in cancer. Cell, 134(2), 215-230. McCubrey, J. A., Steelman, L. S., Bertrand, F. E., Davis, N. M., Sokolosky, M., Abrams, S. L., Montalto, G., D'Assoro, A. B., Libra, M., Nicoletti, F., Maestro, R., Basecke, J., Rakus, D., Gizak, A., Demidenko, Z. N., Cocco, L., Martelli, A. M., & Cervello, M. (2014). GSK-3 as potential target for therapeutic intervention in cancer. Oncotarget, 5(10), 2881-2911.

70 Mendelsohn, C., Batourina, E., Fung, S., Gilbert, T., & Dodd, J. (1999). Stromal cells mediate retinoid-dependent functions essential for renal development. Development (Cambridge, England), 126(6), 1139-1148. Merkel, C., Karner, C., & Carroll, T. (2007). Molecular regulation of kidney development: Is the answer blowing in the wnt? Pediatric Nephrology, 22(11), 1825-1838. Merrill, B. J. (2012). Wnt pathway regulation of embryonic stem cell self-renewal. Cold Spring Harbor Perspectives in Biology, 4(9), a007971. Miki, T., Yasuda, S., & Kahn, M. (2011). Wnt/β-catenin signaling in embryonic stem cell self-renewal and somatic cell reprogramming. Stem Cell Reviews, 7(4), 836-846. Morin, P. J. (1999). Beta-catenin signaling and cancer. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology, 21(12), 1021-1030. Mugford, J. W., Sipilä, P., McMahon, J. A., & McMahon, A. P. (2008). Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney. Developmental Biology, 324(1), 88-98. Muglia, V. F., & Prando, A. (2015). Renal cell carcinoma: Histological classification and correlation with imaging findings. Radiologia Brasileira, 48(3), 166-174. Nagy, I. I., Xu, Q., Naillat, F., Ali, N., Miinalainen, I., Samoylenko, A., & Vainio, S. J. (2016). Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis. BMC Developmental Biology, 16(1), 30. Narlis, M., Grote, D., Gaitan, Y., Boualia, S. K., & Bouchard, M. (2007). Pax2 and pax8 regulate branching morphogenesis and nephron differentiation in the developing kidney. Journal of the American Society of Nephrology: JASN, 18(4), 1121-1129. Nickerson, M. L., Jaeger, E., Shi, Y., Durocher, J. A., Mahurkar, S., Zaridze, D., Matveev, V., Janout, V., Kollarova, H., Bencko, V., Navratilova, M., Szeszenia-Dabrowska, N., Mates, D., Mukeria, A., Holcatova, I., Schmidt, L. S., Toro, J. R., Karami, S., Hung, R., Gerard, G. F., Linehan, W. M., Merino, M., Zbar, B., Boffetta, P., Brennan, P., Rothman, N., Chow, W. H., Waldman, F. M., & Moore, L. E. (2008). Improved identification of von hippel-lindau gene alterations in clear cell renal tumors. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 14(15), 4726- 4734. Nieto, M. A. (2002). The snail superfamily of zinc-finger transcription factors. Nature Reviews. Molecular Cell Biology, 3(3), 155-166. Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S., & Itoh, N. (2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochemical and Biophysical Research Communications, 277(3), 643-649. O'Meara, C. C., Hoffman, M., Sweeney, W. E., Tsaih, S., Xiao, B., Jacob, H. J., Avner, E. D, & Moreno, C. (2012). Role of genetic modifiers in an orthologous rat model of ARPKD. Physiological Genomics, 44(15), 741- Otsuka, A., Levesque, M. P., Dummer, R., & Kabashima, K. (2015). Hedgehog signaling in basal cell carcinoma. Journal of Dermatological Science, 78(2), 95-100.

71 Park, J., Ma, W., O'Brien, L. L., Chung, E., Guo, J., Cheng, J., Valerius, M. T., McMahon, J. A., Wong, W. H., & McMahon, A. P. (2012). Six2 and wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks. Developmental Cell, 23(3), 637-651. Parton, R. G., & del Pozo, M. A. (2013). Caveolae as plasma membrane sensors, protectors and organizers. Nature Reviews. Molecular Cell Biology, 14(2), 98-112. Pasca di Magliano, M., & Hebrok, M. (2003). Hedgehog signalling in cancer formation and maintenance. Nature Reviews. Cancer, 3(12), 903-911. Pei, Y., Brun, S. N., Markant, S. L., Lento, W., Gibson, P., Taketo, M. M., Giovannini, M., Gilbertson, R. J., & Wechsler-Reya, R. J. (2012). WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum. Development (Cambridge, England), 139(10), 1724-1733. Peifer, M., Fernández-Cuesta, L., Sos, M. L., George, J., Seidel, D., Kasper, L. H., Thomas, R. K. (2012). Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature Genetics, 44(10), 1104-1110. Peng, X., Yang, L., Chang, H., Dai, G., Wang, F., Duan, X., Guo, L., Zhang, Y., & Chen, G. (2014). Wnt/β-catenin signaling regulates the proliferation and differentiation of mesenchymal progenitor cells through the p53 pathway. PloS One, 9(5), e97283. Polakis, P. (2012). Wnt signaling in cancer. Cold Spring Harbor Perspectives in Biology, 4(5), a008052. Prasetyanti, P. R., Zimberlin, C. D., Bots, M., Vermeulen, L., Melo, Felipe De Sousa E, & Medema, J. P. (2013). Regulation of stem cell self-renewal and differentiation by wnt and notch are conserved throughout the adenoma-carcinoma sequence in the colon. Molecular Cancer, 12(1), 126. Qiao, J., Uzzo, R., Obara-Ishihara, T., Degenstein, L., Fuchs, E., & Herzlinger, D. (1999). FGF-7 modulates ureteric bud growth and nephron number in the developing kidney. Development (Cambridge, England), 126(3), 547-554. Rini, B. I., Campbell, S. C., & Escudier, B. (2009). Renal cell carcinoma. The Lancet, 373(9669), 1119-1132. Rodrigues, T., Kundu, B., Silva-Correia, J., Kundu, S. C., Oliveira, J. M., Reis, R. L., & Correlo, V. M. (2017). Emerging tumor spheroids technologies for 3D in vitro cancer modeling. Pharmacology & Therapeutics, Roos, A. (1941). Polycystic kidney: Report of a case studied by reconstruction. American Journal of Diseases of Children, 61(1), 116-127. Rosselot, C., Spraggon, L., Chia, I., Batourina, E., Riccio, P., Lu, B., Niederreither, K., Dolle, P., Duester, G., Chambon, P., Costantini, F., Gilbert, T., Molotkov, A., & Mendelsohn, C. (2010). Non-cell-autonomous retinoid signaling is crucial for renal development. Development (Cambridge, England), 137(2), 283-292. S Salahshor, & J R Woodgett. (2005). The links between axin and carcinogenesis. England: British Medical Association. Sakurai, H., Tsukamoto, T., Kjelsberg, C. A., Cantley, L. G., & Nigam, S. K. (1997). EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by embryonic kidney. The American Journal of Physiology, 273(3 Pt 2), 463.

72 Salvi, A., Marchina, E., Benetti, A., Grigolato, P., De Petro, G., & Barlati, S. (2008). Germline and somatic c-met mutations in multifocal/bilateral and sporadic papillary renal carcinomas of selected patients. International Journal of Oncology, 33(2), 271- 276. Sampogna, R. V., Schneider, L., & Al-Awqati, Q. (2015). Developmental programming of branching morphogenesis in the kidney. Journal of the American Society of Nephrology: JASN, 26(10), 2414-2422. Santos, O. F., Barros, E. J., Yang, X. M., Matsumoto, K., Nakamura, T., Park, M., & Nigam, S. K. (1994). Involvement of hepatocyte growth factor in kidney development. Developmental Biology, 163(2), 525-529. Schambony, A., & Wedlich, D. (2013). Wnt signaling and cell migration Landes Bioscience. Schmidt, L., Duh, F. M., Chen, F., Kishida, T., Glenn, G., Choyke, P., … Zbar, B. (1997). Germline and somatic mutations in the tyrosine kinase domain of the MET proto- oncogene in papillary renal carcinomas. Nature Genetics, 16(1), 68-73. Schoenhals, M., Kassambara, A., De Vos, J., Hose, D., Moreaux, J., & Klein, B. (2009). Embryonic stem cell markers expression in cancers. Biochemical and Biophysical Research Communications, 383(2), 157-162. Self, M., Lagutin, O. V., Bowling, B., Hendrix, J., Cai, Y., Dressler, G. R., & Oliver, G. (2006). Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. The EMBO Journal, 25(21), 5214-5228. Sequeira-Lopez, M. L. S., Lin, E. E., Li, M., Hu, Y., Sigmund, C. D., & Gomez, R. A. (2015). The earliest metanephric arteriolar progenitors and their role in kidney vascular development. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 308(2), 138. Singh, V., Singla, S. K., Jha, V., Puri, V., & Puri, S. (2015). Hepatocyte nuclear factor-1β: A regulator of kidney development and cystogenesis. Indian Journal of Nephrology, 25(2), 70-76. Siroky, B. J., Czyzyk-Krzeska, M. F., & Bissler, J. J. (2009). Renal involvement in tuberous sclerosis complex and von hippel-lindau disease: Shared disease mechanisms? Nature Clinical Practice. Nephrology, 5(3), 143-156. Sitaram, R. T., Mallikarjuna, P., Landström, M., & Ljungberg B. (2016) Transforming growth factor-β promotes aggressiveness and invasion of clear cell renal cell carcinoma. Oncotarget. 7(24), 35917–35931. Sperger, J. M., Chen, X., Draper, J. S., Antosiewicz, J. E., Chon, C. H., Jones, S. B., Brooks, J. D., Andrews, P. W., Brown, P. O., & Thomson, J. A. (2003). Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proceedings of the National Academy of Sciences of the United States of America, 100(23), 13350- 13355. Stark, K., Vainio, S., Vassileva, G., & McMahon, A. P. (1994). Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by wnt-4. Nature, 372(6507), 679-683.

73 Steffens, S., Schrader, A. J., Blasig, H., Vetter, G., Eggers, H., Tränkenschuh, W., Kuczyk, M. A., & Serth, J. (2011). Caveolin 1 protein expression in renal cell carcinoma predicts survival. BMC Urology, 11, 25. Tamimi, Y., Ekuere, U., Laughton, N., & Grundy, P. (2008). WNT5A is regulated by PAX2 and may be involved in blastemal predominant wilms tumorigenesis. Neoplasia (New York, N.Y.), 10(12), 1470-1480. Taylor, M. D., Liu, L., Raffel, C., Hui, C., Mainprize, T. G., Zhang, X., Agatep, R., Chiappa, S., Gao, L., Lowrance, A., Hao, A., Goldstein, A. M., Stavrou, T., Scherer, S. W., Dura, W. T., Wainwright, B., Squire, J. A., Rutka, J. T., & Hogg, D. (2002). Mutations in SUFU predispose to medulloblastoma. Nature Genetics, 31(3), 306-310. Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nature Reviews. Cancer, 2(6), 442-454. Thoma, C. R., Zimmermann, M., Agarkova, I., Kelm, J. M., & Krek, W. (2014). 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Advanced Drug Delivery Reviews, 69-70, 29-41. Tian, A., Benchabane, H., Wang, Z., & Ahmed, Y. (2016). Regulation of stem cell proliferation and cell fate specification by wingless/wnt signaling gradients enriched at adult intestinal compartment boundaries. PLOS Genetics, 12(2), e1005822. Tomita, M., Asada, M., Asada, N., Nakamura, J., Oguchi, A., Higashi, A. Y., Endo, S., Robertson, E., Kimura, T., Kita, T., Economides, A. N., Kreidberg, J., & Yanagita, M. (2013). Bmp7 maintains undifferentiated kidney progenitor population and determines nephron numbers at birth. PloS One, 8(8), e73554. Tse, J. C., & Kalluri, R. (2007). Mechanisms of metastasis: Epithelial-to-mesenchymal transition and contribution of tumor microenvironment. Journal of Cellular Biochemistry, 101(4), 816-829. Tsivian, M., Moreira, D. M., Caso, J. R., Mouraviev, V., & Polascik, T. J. (2011). Cigarette smoking is associated with advanced renal cell carcinoma. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 29(15), 2027-2031. Von Schulz-Hausmann, S. A., Schmeel, L. C., Schmeel, F. C., & Schmidt-Wolf, I. G. H. (2014). Targeting the wnt/beta-catenin pathway in renal cell carcinoma. Anticancer Research, 34(8), 4101-4108. Waalkes, S., Eggers, H., Blasig, H., Atschekzei, F., Kramer, M. W., Hennenlotter, J., Tränkenschuh, W., Stenzl, A., Serth, J., Schrader, A. J., Kuczyk, M. A., & Merseburger, A. S. (2011). Caveolin 1 mRNA is overexpressed in malignant renal tissue and might serve as a novel diagnostic marker for renal cancer. Biomarkers in Medicine, 5(2), 219- 225. Watanabe, T., & Costantini, F. (2004). Real-time analysis of ureteric bud branching morphogenesis in vitro. Developmental Biology, 271(1), 98-108. Weeber, F., Ooft, S. N., Dijkstra, K. K., & Voest, E. E. (2017). Tumor organoids as a preclinical cancer model for drug discovery. Cell Chemical Biology, 24(9), 1092-1100. Guo, W., Chen, W., Yu, W., Huang, W., Deng, W. (2013). Small interfering RNA-based molecular therapy of cancers. Chin J Cancer. 32(9), 488-493.

74 Weinstock, M., & McDermott, D. (2015). Targeting PD-1/PD-L1 in the treatment of metastatic renal cell carcinoma. Therapeutic Advances in Urology, 7(6), 365-377. Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P., Silverman, L. B., Sanchez-Irizarry, C., Blacklow, S. C., Look, A. T., & Aster, J. C. (2004). Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science (New York, N.Y.), 306(5694), 269-271. Woolf, A. S., Feather, S. A., & Bingham, C. (2002). Recent insights into kidney diseases associated with glomerular cysts. Pediatric Nephrology (Berlin, Germany), 17(4), 229- 235. doi:10.1007/s00467-001-0819-5 Wu, M. Y., & Hill, C. S. (2009). Tgf-beta superfamily signaling in embryonic development and homeostasis. Developmental Cell, 16(3), 329-343. Wuebken, A., & Schmidt-Ott, K. M. (2011). WNT/β-catenin signaling in polycystic kidney disease. Kidney International, 80(2), 135-138. Xie, J., Murone, M., Luoh, S. M., Ryan, A., Gu, Q., Zhang, C., Bonifas, J. M., Lam, C. W., Hynes, M., Goddard, A., Rosenthal, A., Epstein, EH. Jr., & de Sauvage, F. J. (1998). Activating smoothened mutations in sporadic basal-cell carcinoma. Nature, 391(6662), 90-92. Xu, Z., Robitaille, A. M., Berndt, J. D., Davidson, K. C., Fischer, K. A., Mathieu, J., Potter, J. C., Ruohola-Baker, H., & Moon, R. T. (2016). Wnt/β-catenin signaling promotes self- renewal and inhibits the primed state transition in naïve human embryonic stem cells. Proceedings of the National Academy of Sciences, 113(42), E6390. Yallowitz, A. R., Hrycaj, S. M., Short, K. M., Smyth, I. M., & Wellik, D. M. (2011). Hox10 genes function in kidney development in the differentiation and integration of the cortical stroma. Plos One, 6(8), e23410. Yang, J. C., Haworth, L., Sherry, R. M., Hwu, P., Schwartzentruber, D. J., Topalian, S. L., Steinberg, S. M., Chen, H. X., & Rosenberg, S. A. (2003). A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. The New England Journal of Medicine, 349(5), 427-434. Yang, J., Du, X., Wang, G., Sun, Y., Chen, K., Zhu, X., Lazar, A. J., Hunt, K. K., Pollock, R. E., & Zhang, W. (2014). Mesenchymal to epithelial transition in sarcomas. European Journal of Cancer (Oxford, England: 1990), 50(3), 593-601. Zhang, Y., Wada, J., Yasuhara, A., Iseda, I., Eguchi, J., Fukui, K., Yang, Q., Yamagata, K., Hiesberger, T., Igarashi, P., Zhang, H., Wang, H., Akagi, S., Kanwar, Y. S., & Makino, H. (2007). The role for HNF-1b-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells. PLoS One, 2(5), e414. Young, S. W. S., Stenzel, M., & Yang, J. (2016). Nanoparticle-siRNA: A potential cancer therapy? Critical Reviews in Oncology/Hematology, 98, 159-169. Yu, J., Carroll, T. J., Rajagopal, J., Kobayashi, A., Ren, Q., & McMahon, A. P. (2009). A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney. Development (Cambridge, England), 136(1), 161-171.

75 Yuri, S., Nishikawa, M., Yanagawa, N., Jo, O. D., & Yanagawa, N. (2017). In Vitro propagation and branching morphogenesis from single ureteric Bud Cells. Stem Cell Reports, 8(2), 401-416. Sato, Y., Yoshizato, T., Shiraishi, Y., Maekawa, S., Okuno, Y., Kamura, T., Shimamura, T., Sato-Otsubo, A., Nagae, G., Suzuki, H., Nagata, Y., Yoshida, K., Kon, A., Suzuki, Y., Chiba, K., Tanaka, H., Niida, A., Fujimoto, A., Tsunoda, T., Morikawa, T., Maeda, D., Kume, H., Sugano, S., Fukayama, M., Aburatani, H., Sanada, M., Miyano, S., Homma, Y., &Ogawa, S. (2013). Integrated molecular analysis of clear-cell renal cell carcinoma. Nature Genetics, 45(8), 860. Zhao, C., Chen, A., Jamieson, C. H., Fereshteh, M., Abrahamsson, A., Blum, J., Kwon, H. Y., Kim, J., Chute, J. P., Rizzieri, D., Munchhof, M., VanArsdale, T., Beachy, P. A., & Reya, T. (2009). Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature, 458(7239), 776-779. Zhao, H., Kegg, H., Grady, S., Truong, H., Robinson, M. L., Baum, M., & Bates, C. M. (2004). Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud. Developmental Biology, 276(2), 403-415. Zhuang, Z., Lin, J., Huang, Y., Lin, T., Zheng, Z., & Ma, X. (2017). Notch 1 is a valuable therapeutic target against cell survival and proliferation in clear cell renal cell carcinoma. Oncology Letters, 14(3), 3437-3444.

76 Original publications

I Nagy I#, Xu Q#, Naillat F, Ali N, Miinalainen I, Samoylenko A & Vainio S (2016). Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis. BMC Dev Biol. 16(1):30. # Contributed equally II Xu Q, Junttila S, Scherer A, Giri KR, Kivelä O, Skovorodkin I, Röning J, Quaggin SE, Marti H, Shan J, Samoylenko A and Vainio S (2017). Renal Carcinoma/Kidney Progenitor Cell Chimera Organoid as a Novel Tumourigenesis Gene Discovery Model. Dis Model Mech. 10(12):1503-1515. Reprinted with permission from BMC developmental biology and Irina Nagy (I) and Disease models & mechanisms (II).

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1446. Sinikumpu, Suvi-Päivikki (2018) Skin diseases and their association with systemic diseases in the Northern Finland Birth Cohort 1966 1447. Nortunen, Simo (2018) Stability assessment of isolated lateral malleolar supination-external rotation-type ankle fractures 1448. Härönen, Heli (2018) Collagen XIII as a neuromuscular synapse organizer : roles of collagen XIII and its transmembrane form, and effects of shedding and over- expression in the neuromuscular system in mouse models 1449. Kajula, Outi (2018) Periytyvän rintasyöpäalttiusmutaation (BRCA1/2) kantajamiesten hypoteettinen perinnöllisyysneuvontamalli 1450. Jurvelin, Heidi (2018) Transcranial bright light : the effect on human psychophysiology 1451. Järvimäki, Voitto (2018) Lumbar spine surgery, results and factors predicting outcome in working-aged patients 1452. Capra, Janne (2018) Differentiation and malignant transformation of epithelial cells : 3D cell culture models 1453. Panjan, Peter (2018) Innovative microbioreactors and microfluidic integrated biosensors for biopharmaceutical process control 1454. Saarela, Ulla (2018) Novel culture and organoid technologies to study mammalian kidney development 1455. Virtanen, Mari (2018) The development of ubiquitous 360° learning environment and its effects on students´ satisfaction and histotechnological knowledge 1456. Vuollo, Ville (2018) 3D imaging and nonparametric function estimation methods for analysis of infant cranial shape and detection of twin zygosity 1457. Tervaskanto-Mäentausta, Tiina (2018) Interprofessional education during undergraduate medical and health care studies 1458. Peroja, Pekka (2018) Oxidative stress in diffuse large B-cell lymphoma and follicular lymphoma, and TP53 mutations and translocations of MYC, Bcl-2 and Bcl-6 in diffuse large B-cell lymphoma 1459. Bose, Muthiah (2018) Molecular and functional characterization of ABRAXAS and PALB2 genes in hereditary breast cancer predisposition 1460. Klemola, Tero (2018) Flexible hallux valgus : Results of a new surgical technique 1461. Pasanen, Anu (2018) Genetic susceptibility to childhood bronchiolitis

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UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA

DMEDICA Qi Xu Qi Xu University Lecturer Tuomo Glumoff ROLE OF WNT11 IN KIDNEY

Planning Director Pertti Tikkanen

Professor Jari Juga

University Lecturer Anu Soikkeli