TRAF6, a Key Regulator of Tgfβ-Induced Oncogenesis in Prostate Cancer

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS

New Series No. 1739 ISSN 0346-6612 ISBN 978-91-7601-315-1

TRAF6, a key regulator of TGFβ-induced oncogenesis in prostate cancer

Reshma Sundar

Department of Medical Biosciences, Pathology Umeå University, Umeå 2015

Responsible publisher under Swedish law: The Dean of the Medical Faculty This work is protected by the Swedish Copyright legislation (Act 1960:729) Copyrights © Reshma Sundar New Series No: 1739 ISSN: 0346-6612 ISBN: 978-91-7601-315-1 E-version available at: http://umu.diva-portal.org/ Cover picture: TGFβ type I receptor nuclear translocation Cover picture design: Harvinder Singh, ZD technologies Cover design: Print and Media Printed by: Print and Media, Umeå, Sweden, 2015

To my father, who gave me the greatest gift anyone could give—he believed in me.

Some people grumble that roses have thorns;

I am grateful that thorns have roses.

Abstract

Prostate cancer is the most common cancer in men, with the incidence rapidly increasing in Europe over the past two decades. Reliable biomarkers for prostate cancer are currently unavailable. Thus, there is an urgent need for improved biomarkers to diagnose prostate cancer at an early stage and to determine the best treatment options. Higher expression of transforming growth factor-β (TGFβ) has been reported in patients with aggressive cancer.

TGFβ is a multifunctional cytokine that acts as a tumor suppressor during early tumor development, and as a tumor promoter at later stages of cancer. TGFβ signals through the canonical Smad or non-Smad cascade via TGFβ type II and type I receptors. The TGFβ signaling cascade is regulated by various post-translational modifications of its key components. The present investigation aimed to identify a potential function of TRAF6 in TGFβ-induced responses in prostate cancer.

The first two articles of this thesis unveil the proteolytic cleavage of TGFβ type I receptor (TβRI), and the biological importance of the liberated TβRI intracellular domain (TβRI-ICD) in the nucleus. We found that tumor necrosis factor receptor- associated factor 6 (TRAF6) polyubiquitinates TβRI, which leads to cleavage of TβRI by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta (PKCζ)-dependent manner. Following ectodomain shedding, TβRI undergoes a second cleavage by presenilin 1 (PS1), which liberates TβRI-ICD. TβRI-ICD translocates to the nucleus, where it regulates its own expression as well as expression of the pro-invasive gene Snail1, thereby promoting invasion. We further found that TβRI-ICD associates with Notch intracellular domain (NICD) to drive expression of the pro-invasive gene Snail1, as well as Notch1 ligand Jag1.

The third article provides evidence that TRAF6 promotes Lys63-linked polyubiquitination of TβRI at Lys178 in a TGFβ-dependent manner. TβRI polyubiquitination was found to be a prerequisite for TβRI nuclear translocation, and thus for regulation of the genes involved in cell cycle, differentiation, and invasion of prostate cancer cells.

In the fourth article we investigated the role of the pro-invasive gene Snail1 in TGFβ-induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cells.

List of Papers

This thesis is based on the following papers:

1. Yabing Mu*, Reshma Sundar*, Noopur Thakur*, Maria Ekman*, Shyam Kumar Gudey, Mariya Yakymovych, Helena Dimitrou, Annika Hermansson, Maria Teresa Bengoechea-Alonso, Johan Ericsson, Carl-Henrik Heldin, Marene Landström. TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer. Nature Communications. 2011;2:330.

2. Shyam Kumar Gudey, Reshma Sundar, Yabing Mu, Anders Wallenius, Guanxiang Zang, Anders Bergh, Carl-Henrik Heldin, Marene Landström. TRAF6 Stimulates the Tumor-Promoting Effects of TGFβ Type I Receptor Through Polyubiquitination and Activation of Presenilin 1. Science Signalling. 2014 Jan 7;7(307).

3. Reshma Sundar, Shyam Kumar Gudey, Carl-Henrik Heldin, Marene Landström. TRAF6 promotes TGFβ-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFβ type I receptor. Cell Cycle. 2015; 14(4):554–65

4. Shyam Kumar Gudey, Reshma Sundar, Carl-Henrik Heldin, Marene Landström. Proinvasive Snail1 targets TGFβ Type I receptor to promote epithelial-to-mesenchymal transition in prostate cancer. Manuscript

* Indicates that these authors contributed equally to the work Reprints have been made with permission from the respective publishers.

1. Reshma Sundar, Marene Landström. Chapter: TRAF6 in book Encyclopedia of Signaling Molecules, Edition: 2013. XLVIII, Publisher: Springer, pp.1916-1921. 2. Anneli Jorsback, Maria Winkvist, Asa Hagner-McWhirter, Marene Landstrom, Reshma Sundar, Ulla Engstrom. Abstract LB-276: 2-D DIGE and siRNA to find new cancer targets. 2012. Cancer Research 3. Terese Karlsson, Reshma Sundar, Marene Landström, Emma Persson. Osteoblast-derived factors increase metastatic potential in human prostate cancer cells, an effect partially mediated by non-canonical, TRAF6-dependent TGFβ signaling. Manuscript

Contents

Introduction 1 1. Prostate cancer 2 2. TGFβ 3 3. TGFβ superfamily 4 4. TGFβ isoforms and receptors 5 1. Smad proteins 6 2. TGFβ ligand activation 8 3. TGFβ canonical Smad signaling 8 4. Non-Smad TGFβ signaling 9 5. Non-Smad signaling via TRAF6 10 1. TRAF family and TRAF6 10 2. Tumor necrosis factor-α-converting enzyme (TACE) 12 3. Presenilins (PSs) 14 4. PKCζ 15 5. Snail1 16 6. Post-translational modification 18 1. Ubiquitination 18 2. Sumoylation 21 3. Phosphorylation 22 7. Notch signaling 23 Present Investigation 26 Aims 26 Materials and methods 27 Cell culture Transient transfection Protein analysis Immunoprecipitation Nuclear cytoplasmic fractionation assay In vivo ubiquitination assay In vitro ubiquitination assay Western blotting Immunofluorescence Proximity ligation assay (PLA) Quantitative real-time RT-PCR Chromatin immunoprecipitation assay Site-directed mutagenesis Invasion assay FACS analysis Immunohistochemistry Animal studies Patient material Statistical analysis Results and discussion 34 Paper I 34 Paper II 35 Paper III 36 Paper IV 37 Conclusions 38 Future perspectives 40 Acknowledgements 43 References 47

Abbreviations

ADAM A disintegrin and metalloproteinase ALK Activin receptor-like kinase AMH Anti-mullerian hormone AP-1 Activator protein-1 APH Anterior pharynx-defective APP Amyloid precursor protein AREG Amphiregulin BMP Bone morphogenetic protein CBP CREB-binding protein CTF C-terminal fragment CYLD Cylindromatosis DNA Deoxyribonucleic acid DUB Deubiquitinating enzyme EMT Epithelial-mesenchymal transition ER Endoplasmic reticulum ERK Extracellular signal-regulated kinase GDF Growth and differentiation factor GS Glycine and serine GTP Guanosine-5′-triphosphate HAT Histone acetyltransferase HECT Homologous to the E6-AP Carboxyl Terminus HMGA High mobility group HIF Hypoxia-inducible factor ICAM Intercellular adhesion molecule ICD Intracellular domain IKK IkappaB kinase JNK c-Jun N-terminal kinase kDa Kilodalton LAP Latency-associated peptide LTBP Latent TGFβ binding protein LUBAC Linear ubiquitin assembly complex MAML Mastermind-like MAPK Mitogen-activated protein kinase MH Mad homology MMP Matrix metalloproteinase NES Nuclear Export signal NFκB Nuclear factor kappa B NTF N-terminal fragment PC-3U Prostate cancer-3-Uppsala PEN Presenilin enhancer PS Presenilin PTEN Phosphatase and tensin homolog RING Really Interesting New Gene RIP Regulated intramembrane proteolysis SARA Smad anchor for receptor activation SBE Smad binding element siRNA Small interfering RNA Smurf Smad ubiquitylation regulator factor SNAG Snail/Gfi-1 STRAP Serine threonine kinase receptor associated protein SUMO Small ubiquitin-like modifier TACE TNFα-converting enzyme TAK1 TGFβ-activated kinase 1 TGFβ Transforming growth factor beta TNFα Tumor necrosis factor alpha TRAF Tumor necrosis factor receptor-associated factor TβR TGFβ receptor TβRI TGFβ receptor type I TβRII TGFβ receptor type II UBP Ubiquitin-specific processing proteases VCAM Vascular cell adhesion molecule

Introduction

Cells communicate with surrounding cells by sending and receiving various signals. Chemical cellular signals include growth factors, neurotransmitters, hormones, and extracellular matrix components. Sensory cells in the skin respond to mechanical stimuli like touch. In such cells, signals are transmitted across the cell membrane via cell surface-bound receptor proteins that send signals from the outside to the inside of the cell. Subsequently, these signals are transmitted to the nucleus or other cellular components via activation of other proteins. Post-translational protein modification can regulate intracellular signaling by controlling protein function. At any one time, several different signal transduction cascades are operating in the cytoplasm, and signaling pathways crosstalk with each other at multiple levels, allowing cells to coordinate signals and regulate their growth, survival, differentiation, and migration. Molecular interactions between different networks produce a steady state of cellular integrity. Any interruption or over-activity of cell-to- cell communication may cause disease conditions. For example, changes in transforming growth factor-β (TGFβ) signaling pathways can alter cell communication, resulting in uncontrolled cell growth that leads to cancer. This thesis focuses on TGFβ and its downstream activation of non-Smad signaling pathways in cancer. The present study aims to advance our knowledge regarding the roles of the E3 ligase tumor necrosis factor receptor- associated factor 6 (TRAF6) and the zinc finger transcriptional repressor Snail1 in TGFβ-induced oncogenesis, with a particular focus on prostate cancer.

1 1. Prostate cancer

Prostate cancer is the most commonly diagnosed cancer in men, with a lifetime prevalence of one in six men. In 2012, over 1,111,000 new cases of prostate cancer were diagnosed worldwide, and estimated 307,000 men died of this disease (Esfahani, Ataei et al. 2015). Since 1977, the prostate cancer incidence has increased three-fold, and the median age of diagnosis is decreasing (Hussein, Satturwar et al. 2015). Prostate cancer is the most common cancer among men in Europe, with highest world age-standardized incidence rate reported in Norway and the lowest in Albania (Cancer Research UK website). Prostate cancer development is influenced by a variety of factors, including ethnicity, environment, lifestyle, genetic factors, and the male sex hormone testosterone (Felgueiras, Silva et al. 2014).

Improving the life expectancy for patients with prostate cancer will require better prognostic markers and treatment methods. Currently, the most commonly used prognostic tools are the prostate-specific antigen (PSA) test and histopathological grading, and the more commonly applied optional treatments are surgical removal of the prostate gland, radiation, chemotherapy, and hormonal therapy (androgen-deprivation therapy). However, many tumors relapse within three years and develop androgen- independent regrowth. It may be possible to prevent and treat recurrent disease to increase patient survival, by targeting growth factors, thus more research should be focused on identifying such biomarkers (Wikstrom, Damber et al. 2001; Diener, Need et al. 2010).

TGFβ acts as a tumor suppressor in normal prostate epithelium and premalignant lesions. However, TGFβ1 overexpression has been detected in prostate tumor, and high TGFβ1 levels are associated with prostate cancer

2 progression (Steiner, Zhou et al. 1994). Advanced prostate cancers show elevated TGFβ levels and decreased TGFβ receptor expression, which are associated with bad prognosis (Jones, Pu et al. 2009). Prostate cancers also reportedly show loss of heterozygosity on chromosome 18q, where the genes encoding intracellular transducers for TGFβ signals (Smad2 and Smad4) are localized (Padalecki, Weldon et al. 2003). The available data suggest that molecular targeting of TGFβ signaling may be a promising therapeutic approach for prostate cancer treatment (Jones, Pu et al. 2009).

2. TGFβ

Over three decades ago, TGFβ was first purified and characterized (Roberts, Lamb et al. 1980; Moses, Branum et al. 1981). The TGFβ family now includes 33 related isoforms that are potent cytokines involved in regulating an array of cellular processes, such as cell proliferation, differentiation, morphogenesis, tissue homeostasis, motility, adhesion, and death (Massague 1998; Moustakas and Heldin 2009). The human genome encodes over 42 TGFβ family members, while the Drosophila melanogaster and Caenorhabditis elegans genomes encode 7 and 4, respectively. Remarkably, TGFβ signaling is evolutionarily conserved in the ancestral Trichoplax adhaerens.

TGFβ exerts different and opposing effects depending on the cellular context and condition, and acts as a double-edged sword in carcinogenesis and tumor progression. Initially, it suppresses cellular growth in normal epithelial and premalignant cells. However, increased levels of locally secreted TGFβ can induce pro-tumorigenic effects by switching the cell fate to invasion and migration (Roberts and Wakefield 2003; Heldin, Vanlandewijck et al. 2012; Papageorgis 2015). Numerous human cancers (e.g., colorectal, pancreatic,

3 gastric, and prostate cancer) have shown mutations in genes encoding TGFβ pathway components, including TGFβ receptor I (TβRI), TGFβ receptor II (TβRII), Smad2, and Smad4 (de Caestecker, Piek et al. 2000; Kim, Park et al. 2005).

3. TGFβ superfamily

The TGFβ superfamily encompasses TGFβs, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), activins, nodal, and anti- mullerian hormone (AMH) (Padua and Massague 2009). TGFβ superfamily ligands signal through heterotetrameric complexes comprising two pairs of type I and type II serine/threonine kinase receptors localized at the cell surfaces. There are seven type I receptors (activin receptor-like kinase and ALK 1–7) and five type II receptors (TβRII, ActR-IIA, ActR-IIB, AMHRII, and BMPRII) (Moustakas and Heldin 2009). The TGFβ superfamily is divided into four subfamilies with different receptor binding specifications (Figure 1).

Ligand Type I Type II receptor receptor R-Smad Co-Smad TGFβ1, 2, 3 TβRI (ALK5) TβRII Smad2 Activin ALK4, 7 ActRII-A, B Smad3 BMP/GDF ALK1, 2, 3, 6 BMPRII Smad1 ActRII-A, B Smad5 Smad4 AMH ALK2, 3, 6 AMHRII Smad8

Figure 1. Schematic representation of the transforming growth factor-β (TGFβ) superfamily components

4 4. TGFβ isoforms and receptors

The TGFβ subfamily comprises TGFβ1, 2, and 3. These proteins signal through the 53-kDa TβRI (ALK5) and the 70-kDa TβRII, which are both transmembrane protein serine/threonine kinases. TβRI and TβRII each have a transmembrane domain, an intracellular kinase domain, and a relatively short extracellular domain of approximately 150 amino acids that is N-glycosylated and contains cysteine-rich regions. TβRI includes a highly conserved, 30- amino acid, serine/glycine-rich sequence called the GS domain, which is localized just upstream of the serine/threonine protein kinase domain (Figure 2). Although the TβRI kinase is referred to as a serine/threonine kinase, it can also phosphorylate tyrosine residues (Lawler, Feng et al. 1997). In both TβRII and TβRI, the kinase domain is followed by a short C-terminal extension, which is larger in TβRII compared to in TβRI.

Figure 2. Transforming growth factor-β receptor I (TβRI) structure

5 TGFβ1 binding creates a heterotetrameric complex of two TβRI and two TβRII. Compared to TβRII, TβRI has a very weak affinity for the ligand. Ligand binding to TβRII results in a 300-fold increase of the binding affinity of TβRII and TβRI (Yamashita, ten Dijke et al. 1994). In the ligand-induced receptor complex, TβRII transphosphorylates TβRI in the TTSGSGSGLP sequence of the GS domain and activates downstream signaling through Smad proteins (Okadome, Yamashita et al. 1994; Wrana, Attisano et al. 1994; Franzen, Heldin et al. 1995).

4.1. Smad proteins

Smad proteins are intracellular mediators of TGFβ signaling. The term Smad was derived from the names of two proteins: small body size (Sma) in Caenorhabditis elegans and mothers against decapentaplegic (Mad) in Drosophila melanogaster (Sekelsky, Newfeld et al. 1995; Savage, Das et al. 1996). Based on structure and function, Smads are classified into three subfamilies: the receptor-associated Smads (R-Smads) Smad1, 2, 3, 5, and 8; the inhibitory Smads (I-Smads) Smad6 and 7; and the common mediator Smad (Co-Smad) Smad4. Smad2 and Smad3 are direct substrates of TβRI. Smad7 is an antagonistic Smad that inhibits the TGFβ signaling cascade and is transcriptionally induced in response to TGFβ (Hayashi, Abdollah et al. 1997; Nakao, Afrakhte et al. 1997).

Smad proteins (Figure 3) contain a highly conserved N-terminal Mad homology (MH1) domain, a C-terminal Mad homology (MH2) domain, and an intervening linker domain of variable sequence and length. The R-Smads and Co-Smad have a conserved MH1 domain that contains a nuclear localization signal and a DNA binding domain, and that facilitates protein- protein interactions. I-Smads lack the MH1 domain. In the basal state, the

6 MH1 and MH2 domains inhibit each other. The MH2 domain is involved in protein–protein interactions through hetero- and homo-oligomerization. This region binds to TβRI and contains the receptor phosphorylation motif Ser-X- Ser (SXS) at the C-terminal end in R-Smads. The MH2 region is bound by several transcription factors, such as androgen receptor (AR), LEF1/TCF, FAST Fos, etc. (Abdollah, Macias-Silva et al. 1997; Kretzschmar, Liu et al. 1997; Souchelnytskyi, Tamaki et al. 1997; Moustakas, Souchelnytskyi et al. 2001). The variable linker region contains phosphorylation sites for mitogen- activated protein kinases (MAPKs) and other kinases. Phosphorylation in this region inhibits Smad nuclear translocation and promotes Smad degradation. The linker region also contains a PPXY (PY) motif that acts as a binding motif for the WW domain of the ubiquitin ligase Smurf1/2. The linker region of the Co-Smad contains a nuclear export signal (NES) but no PY motif.

Figure 3. Structure of Smad family proteins

7 4.2. TGFβ ligand activation

TGFβ ligands are secreted as latent complexes comprising an N-terminal propeptide and a C-terminal mature TGFβ polypeptide. The propeptide is termed the latency-associated peptide (LAP) and it noncovalently binds to the TGFβ polypeptide to form the small latent TGFβ complex (SLC). The SLC then interacts with a large secretory glycoprotein called the latent TGFβ-binding protein (LTBP), forming a large latent TGFβ complex (LLC). LTBP is required for secretion and storage in the extracellular matrix (ECM). The latent TGFβ ligands are activated by several proteases and interact with integrin and other proteins. LAP can be cleaved by matrix metalloproteases-2 (MMP-2), releasing mature TGFβ (Koli, Saharinen et al. 2001; Annes, Munger et al. 2003; Keski-Oja, Koli et al. 2004).

4.3. TGFβ canonical Smad signaling

TGFβ first binds to the extracellular domain of the constitutively active TβRII. This enhances TβRI binding affinity, leading to the formation of a symmetric heterotetrameric complex comprising two TβRII and two TβRI molecules. The constitutively active TβRII is autophosphorylated at Ser231 and Ser409, triggering its kinase activity. The kinase-active TβRII phosphorylates serine and threonine residues in the GS domain, thus transactivating TβRI. In turn, activated TβRI phosphorylates downstream effectors (the R-Smads Smad2 and Smad3) at their conserved C-terminal serine residues (SXS). The L3 loops of the R-Smads interact with the L45 loop in the TβRI kinase domain. R-Smad phosphorylation is inhibited by the I-Smad Smad7, which competitively interacts with TβRI (Hayashi, Abdollah et al. 1997; Imamura, Takase et al. 1997; Hata, Lagna et al. 1998). The receptor-phosphorylated R-Smads associate with the Co-Smad Smad4,

8 forming a trimeric complex comprising phosphorylated homo- or heteromeric R-Smads and Smad4. Smad7 can also inhibit trimeric complex formation (Hata, Lagna et al. 1998).

The trimeric complex navigates to the nucleus and regulates transcription in a context-dependent manner. R-Smads and Co-Smads shuttle between the cytoplasm and the nucleus. Phosphorylation of MAPK sites in the R-Smad linker region inhibits Smad nuclear transport, leading to proteasomal degradation, and thus down-regulating TGFβ signaling (Massague 1998). In the nucleus, R-Smads regulate transcriptional responses by binding to certain gene promoters for DNA binding transcription factors (Pierreux, Nicolas et al. 2000; Xu, Kang et al. 2002). Smad3 and Smad4 predominantly bind to the CAGAC DNA motif known as canonical Smad-binding element (SBE), while Smad2 does not bind directly to DNA. The canonical TGFβ–Smad signaling pathway regulates genes controlling proliferation, differentiation, migration, and apoptosis through associations with transcriptional coactivators (CBP, HATs, and p300) and corepressors (Ski and SnoN) (Roberts 1999; Ross and Hill 2008; Massague 2012).

4.4. Non-Smad TGFβ signaling

TGFβ also transmits signals through other pathways, including those involving extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (MAPK) (Derynck and Zhang 2003; Moustakas and Heldin 2009; Landstrom 2010). TGFβ can activate phosphatidylinositol-3-kinase (PI3K) and the downstream Akt through RhoA, as well as the tyrosine kinase Src (Bakin, Tomlinson et al. 2000; Vinals and Pouyssegur 2001; Derynck and Zhang 2003).

9 TβRI can phosphorylate serine and tyrosine residues in the adaptor protein ShcA. This leads ShcA to recruits the adaptor protein Grb2 to form a complex with Ras guanine exchange factor and son of sevenless (SOS), thereby activating Ras and the downstream Erk1/2 MAP kinase (Lee, Pardoux et al. 2007; Moustakas and Heldin 2009). Moreover, TβRII can phosphorylate the polarity protein Par6, promoting its interaction with the E3 ubiquitin ligase Smurf1. In turn, Smurf1 regulates the degradation of Rho small GTPase, resulting in loss of intercellular tight junction (Ozdamar, Bose et al. 2005).

This thesis primarily focuses on non-Smad signaling pathways in which the ubiquitin ligase TRAF6 is a key protein. The TGFβ-induced TRAF6–TAK1 signaling pathway can induce both apoptosis and epithelial–mesenchymal transition (EMT) (Sorrentino, Thakur et al. 2008; Yamashita, Fatyol et al. 2008; Landstrom 2010; Mu, Gudey et al. 2012).

5. Non-Smad signaling via TRAF6

5.1. TRAF family and TRAF6 The tumor necrosis factor receptor-associated factor (TRAF) family is a class of adapter proteins that activate various signaling pathways, including those involving nuclear kappa-light-chain-enhancer of activated B cells (NF-κB), JNK p38, and mitogen-activated protein kinases (MAPKs). TRAFs associate with the tumor necrosis factor receptors (TNFR) superfamily as well as other receptors (Inoue, Ishida et al. 2000; Kobayashi, Walsh et al. 2004; Landstrom 2010; Mu, Gudey et al. 2012). The TRAFs are genetically conserved in Dictyostelium discoideum (Regnier, Tomasetto et al. 1995), Caenorhabditis elegans (Wajant, Muhlenbeck et al. 1998), Drosophila melanogaster (Liu, Su et al. 1999), and mammals (Arch, Gedrich et al. 1998).

10 Seven different types of TRAFs have been found in mammals (TRAF1–7). TRAF1 and TRAF2 were initially identified as adaptor proteins of TNFR2 (Cao, Xiong et al. 1996; Ishida, Mizushima et al. 1996). TRAF2, TRAF3, and TRAF6 are ubiquitously expressed, while TRAF1, TRAF4, and TRAF5 expressions are more restricted. TRAF1 is generally expressed in the spleen, lung, and testis and TRAF4 in the spleen, lung, and thymus. TRAF5 is highly expressed during embryogenesis, and in the hippocampus and the olfactory bulb of adults (Rothe, Wong et al. 1994; Mosialos, Birkenbach et al. 1995; Arch, Gedrich et al. 1998).

All TRAF family members contain a conserved TRAF domain comprising a highly conserved carboxyl-terminal region (C-domain) and a coiled-coil amino-terminal region (Figure 4). This TRAF domain mediates homo- and hetero-dimerization of TRAF family members. Except for TRAF1, all TRAFs also contain an amino-terminal really interesting new gene (RING) finger motif, which is rich in cysteines and histidines that coordinate Zn2+ ion binding (Borden, Lally et al. 1995; Arch, Gedrich et al. 1998). TRAF6 was independently identified as an IL-1 signal transducer using yeast two-hybrid systems with an EST expression library (Cao, Xiong et al. 1996; Ishida, Mizushima et al. 1996). TRAF6 is a cytosolic protein that includes a cysteine- containing C3HC3D-type RING finger, and five zinc finger repeats. TRAF6- null mice have an abnormal phenotype with defective bone formation. They die at early age due to severe osteoporosis with defects in tooth eruption and bone remodeling, resulting from impaired osteoclast formation (Lomaga, Yeh et al. 1999; Naito, Azuma et al. 1999). The role of TRAF6 in IL-17 signaling was confirmed using embryonic fibroblasts from TRAF6 knockout mice (Bradley and Pober 2001).

Figure 4. Tumor necrosis factor receptor-associated factor 6 (TRAF6) structure

In its function as an E3 ubiquitin ligase, TRAF6 interacts with the E2- conjugating enzyme Msm2 (comprised of Ubc13 and Uev1A domains) to synthesize Lys63-linked polyubiquitin chains on target proteins (Deng, Wang et al. 2000; Wang, Deng et al. 2001). Recent studies have reported that TRAF6 binds to the conserved consensus motif basic residue-X-P-X-E-X-X- aromatic or acidic residue in TβRI (Sorrentino, Thakur et al. 2008). This TβRI–TRAF6 interaction leads to TRAF6 autoubiquitination and subsequent Lys63-linked polyubiquitination of TAK1 and, in turn, the activated TAK1 activates the downstream p38 MAPK pathway (Sorrentino, Thakur et al. 2008; Yamashita, Fatyol et al. 2008; Mu, Gudey et al. 2012; Gudey, Wallenius et al. 2014; Sundar, Gudey et al. 2015).

5.2. Tumor necrosis factor-α-converting enzyme (TACE)

Tumor necrosis factor-α-converting enzyme (TACE)—also known as A disintegrin and metalloprotease (ADAM) 17—was initially identified as a protease that cleaves the transmembrane molecule TNF-α from the plasma membrane (Blobel 1997; Black 2002). TACE is a ubiquitously expressed, multi-domain, type I transmembrane protein comprising a cysteine-rich pro-

12 domain, a zinc-dependent catalytic domain, a cysteine-rich disintegrin region, and a cytoplasmic tail (Figure 5).

Figure 5. Tumor necrosis factor-α-converting enzyme (TACE) structure

Most TACE-null mice die at birth, but a few survive with hair and skin defects and with eyelid fusion failure (Peschon, Slack et al. 1998; Schlondorff and Blobel 1999; Black 2002). Ectodomain shedding by TACE leads to the release of several transmembrane molecules from the cell surface, including TGFα, notch1, CSF1, AREG, VCAM1, sIL6R, ICAM1, ErbB2, and EGFR (Kenny and Bissell 2007; Talmadge, Donkor et al. 2007; Scheller, Chalaris et al. 2011; Gudey, Wallenius et al. 2014). On the other hand, under in vitro conditions, recombinant TACE leads to little or no cleavage of synthetic peptides of HER4, APP, TNFR-I, RANKl, Notch, and IL-6R (Mohan, Seaton et al. 2002).

TGFβ reportedly activates TACE via the ERK1/2 MAPK signaling pathway. Activated TACE cleaves TβRI, thus decreasing TGFβ-induced growth inhibition due to reduced TβRI levels at the cell membrane (Liu, Xu et al. 2009). Mu et al. (2011) also report that TACE cleaves TβRI at the

13 transmembrane region, liberating the TβRI intracellular domain, which enters the nucleus and regulates EMT-associated genes, thereby promoting cancer cell invasion.

5.3. Presenilins (PSs)

Presenilins (PSs) are polytransmembrane aspartyl proteases that were initially identified through linkage analysis of Alzheimer’s disease (Medina and Dotti 2003), and that are highly conserved in both invertebrates and vertebrates. Mammalian PSs include two ubiquitously expressed homologous proteins: presenilin 1 (PS1) and presenilin 2 (PS2). PS1-null mice die immediately after birth and exhibit retarded embryonic growth, while PS2 knockout mice are viable and fertile, showing only mild pulmonary fibrosis and hemorrhage with increasing age (Brunkan and Goate 2005). PS1 localizes in the endoplasmic reticulum (ER), Golgi apparatus, endosomes, lysosomes, phagosomes, plasma membrane, and mitochondria of cells (De Strooper, Beullens et al. 1997; Brunkan and Goate 2005; Vetrivel, Zhang et al. 2006).

The most widely accepted PS1 structure contains eight transmembrane domains, the N-terminus, the C-terminus, and a cytosolic hydrophilic loop located between transmembrane segments 6 and 7 (Figure 6). A newly synthesized PS1 holoprotein undergoes endoproteolytic cleavage by an unknown presenilinase, generating an N-terminal fragment (NTF) of 27–28 kDa and a C-terminal fragment (CTF) of 16–17 kDa (Esler, Kimberly et al. 2000; Li, Xu et al. 2000). The PS1 holoprotein, PS1-NTF, and PS1-CTF each undergo ubiquitination and proteasomal degradation (Li, Pauley et al. 2002).

Figure 6. Presenilin 1 (PS1) structure

PS1 associates with TRAF6, leading to TRAF6 autoubiquitination and further ubiquitination of the p75 neurotrophin receptor in an NGF-dependent manner (Powell, Twomey et al. 2009). PS1, nicastrin, anterior pharynx defective-1 (Aph-1), and presenilin enhancer 2 (pen-2) together form the active γ- secretase complex, and co-expression of these components leads to increased presenilin heterodimerization and γ-secretase activity. The γ-secretase complex cleaves various type I transmembrane receptors, including amyloid precursor protein (APP), Notch, and CD44 (Edbauer, Winkler et al. 2003; Wakabayashi and De Strooper 2008; Gudey, Wallenius et al. 2014). PS1 is also implicated in TβRI proteolytic cleavage (Gudey, Sundar et al. 2014).

5.4. PKCζ

Protein kinase C (PKC) represents a family of protein serine/threonine kinases that serve as transducers of various signaling networks. PKCs play important roles in cancer progression and metastasis (Koivunen, Aaltonen et al. 2006). PKC isozymes are grouped into three subfamilies based on their structure and

15 function: classic or conventional PKCs (α, βΙ, βΙΙ, and γ), novel PKCs (δ, ε, η, and θ), and atypical PKCs (ζ and λ/ι). PKCζ and PKCλ/ι lack a Ca2+-binding domain and autoinhibitory region. PKCδ contains an N-terminal PB domain, a pseudosubstrate (PS), a cysteine-rich zinc finger motif containing a C1 domain, and a C-terminal kinase domain.

Figure 7. Protein kinase C-ζ (PKCζ) structure

PKCζ is involved in the ERK1/2 MAPK signaling pathway and functions as an adaptor in the ERK5–MAPK pathway. Its adaptor function is important in regulating NF-κB and the innate immune response. PKCζ also promotes EGF- stimulated chemotaxis of human breast cancer cells (Berra, Diaz-Meco et al. 1995; Griner and Kazanietz 2007; Xiao and Liu 2013). We have also found that TGFβ can cause PKCζ activation (Mu, Sundar et al. 2011)

5.5. Snail1

Snail1 is a C2H2 zinc finger protein belonging to a transcription factor family that also includes Slug and Scratch proteins. Snail1 was first characterized in 1984 through a mutagenesis screen of Drosophila melanogaster. The 2-kb Snail1 gene is localized on chromosome 20q.13.2 and contains three exons. SNAI1P is a Snail1 retrogene. Snail1 is a 264-amino acid protein with a molecular mass of 29.1 kDa. Its structure includes four zinc finger domains, an NES domain, a serine-rich domain (SRD), a destruction box (DB), and a SNAG domain (Figure 8).

Figure 9. Snail1 structure

Transcriptional regulators—including Notch-ICD, NF-κB, Smad, HMGA2, Erg-1, Prap-1, Stat-3, MTA3, IKK-α, and HIF-1-α—affect Snail1 expression by directly binding to its promoter (Thuault, Tan et al. 2008; Kaufhold and Bonavida 2014). Snail1 also binds its own promoter to upregulate its own expression. Yin Yang 1 (YY1) induces Snail1, whereas p53 represses Snail1 expression through induction of miR-34a, miR-34b, and miR-34c (Kim, Kim et al. 2011; Kaufhold and Bonavida 2014). In prostate cancer, Snail1 expression is correlated with high Gleason score (Poblete, Fulla et al. 2014).

Snail1-null mutations are lethal in mice due to a lack of gastrulation. EMT is an important part of normal embryonal development, and also occurs in metastatic cancer. EMT requires Snail1 activation by extracellular molecules, including EGFR ligands, TGFβ, and Wnts (Heldin, Landstrom et al. 2009; Fuxe, Vincent et al. 2010; Sou, Delic et al. 2010; Mu, Sundar et al. 2011). Snail1 directly binds to the E-boxes (5′-CACCTG-3′ motifs) in the proximal promoter region of E-cadherin, repressing E-cadherin expression (Peinado, Olmeda et al. 2007). Snail1 also represses other epithelial markers (e.g., occludin, claudins, and mucin-1) and upregulates the expressions of mesenchymal markers (e.g., vimentin, fibronectin, MMP-2, and MMP-9).

17 Additionally, Snail1 represses the tumor suppressors RKIP and PTEN (Kaufhold and Bonavida 2014). Snail1 is overexpressed in many cancer types and it promotes tumor cell migration, invasion, and metastasis. In breast carcinoma cells, Snail1 overexpression induces TβRII expression (Dhasarathy, Phadke et al. 2011). In lung cancer cells, Snail1 is repressed by TTF1, thus reversing EMT (Saito, Watabe et al. 2009; Moustakas and Heldin 2012).

6. Post-translational modification

Post-translational modification involves the covalent addition of a functional group to a protein after its biosynthesis. TGFβ receptors undergo multiple types of post-translational modifications, including the well-characterized processes of phosphorylation, sumoylation, and ubiquitination (Kang, Liu et al. 2009).

6.1. Ubiquitination

Ubiquitination is the reversible labeling of a target protein with a small modifier called ubiquitin. Ubiquitin is a 76-amino acid protein with a molecular mass of 8.5 kDa. Ubiquitination influences different cellular processes, including cell cycle regulation, DNA repair, endocytosis, autophagy, protein degradation, and gene regulation (Emmerich, Schmukle et al. 2011). The ubiquitination process involves three different enzymes: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin-ligating enzyme (Figure 9).

E1 activates ubiquitin through a two-step ATP-dependent process. First, the E1 activating enzyme binds both ATP and ubiquitin, forming an ubiquitin-

18 adenylate intermediate. In the next step, ubiquitin is transferred to the sulfhydryl group of the cysteine residue of E1 through a thio-ester linkage. The E2 conjugating enzyme then transfers the activated ubiquitin from E1 to a cysteine of E2 via a trans-thio-esterification reaction. The E3 ubiquitin- ligating enzyme acts as a substrate recognition module, catalyzing the transfer of ubiquitin from E2 to the target protein. This enzymatic process creates an isopeptide or peptide bond between the C-terminal glycine of ubiquitin and a lysine of the target protein (Pickart 2001; Groettrup, Pelzer et al. 2008; Schulman and Harper 2009; Dikic and Robertson 2012). Ubiquitin can also bind the target protein through the N-terminus or methionine, cysteine, serine, and threonine residues (Hochstrasser 2009). Moreover, ubiquitin molecules can covalently bind to each other, forming polyubiquitin chains. The human genome encodes two E1 activating enzymes, 37 E2 conjugating enzymes, and over 600 E3 ligases (Komander 2009).

Figure 8. The ubiquitination process

19 Different types of ubiquitination control a variety of cellular processes by regulating protein localization, protein–protein interaction, protein activation, and proteasomal or lysosomal degradation of proteins. Marking a protein with a single ubiquitin on a single lysine residue of a target protein is termed monoubiquitination, while the addition of multiple ubiquitin moieties on multiple lysine residues is called multi-monoubiquitination. These types of ubiquitination trigger receptor endocytosis and DNA repair (Komander 2009). Polyubiquitination is the formation of a ubiquitin chain on a single lysine residue of the target protein. Ubiquitin has seven lysine residues at positions 6, 11, 27, 29, 33, 48, and 63, each of which can mediate a ubiquitin chain. Lys48-linked polyubiquitin chains target proteins for proteasomal degradation. On the other hand, Lys63-linked polyubiquitin chains target proteins for activation of signal transduction, endocytic trafficking, or translation (Dikic, Wakatsuki et al. 2009; Komander 2009; Kravtsova- Ivantsiv and Ciechanover 2012). Linear ubiquitination or M1 linkage is a recently discovered process in which a ubiquitin chain is linked by a methionine residue created by the linear ubiquitin assembly complex (LUBAC) (Rieser, Cordier et al. 2013).

In canonical TGFβ signaling, basal Smad protein basal levels are regulated by ubiquitination and proteasomal degradation. Smurfs are E3 ligases of the homologous to the E6-AP carboxyl terminus (HECT) family, which contain an N-terminal C2 domain, WW domains, and a C-terminal HECT domain. Smurf1 regulates Smad1 and Smad2 abundance. SCF/Roc1 E3 ligase targets phosphorylated Smad3 for proteasomal degradation in the cytoplasm (Fukuchi, Imamura et al. 2001; Moustakas, Souchelnytskyi et al. 2001). Smad4 transcriptional activity is enhanced by monoubiquitination at lysine 507 in the MH2 domain (Moren, Hellman et al. 2003). Smurfs also target the TGFβ receptor complex and the transcriptional co-repressor SnoN. Smurf1

20 and Smurf2 form a complex with nuclear Smad7, which is exported to the cytoplasm where it targets the TβR for proteasomal and lysosomal degradation (Moustakas, Souchelnytskyi et al. 2001). STRAP is a WD domain protein that promotes Smurf-mediated TβRI ubiquitination (Liu, Elia et al. 2000). The E3 ligase TIF1γ disrupts the nuclear–Smad complex through Smad4 ubiquitination (Hesling, Fattet et al. 2011; Moustakas and Heldin 2012). Moreover, the E3 ligase Arkadia stimulates TGFβ signaling by targeting the repressors Smad7, c-Ski, and SnoN. Fbxw also positively regulates TGFβ signaling by promoting the degradation of phosphorylated TGFβ-induced factor 1 (TGIF1) (De Boeck and ten Dijke 2012).

In non-canonical TGFβ signaling pathways, the E3 ligase TRAF6 associates with TβRI at a conserved consensus motif, activating downstream TAK1– p38/JNK MAPK pathways (Sorrentino, Thakur et al. 2008; Yamashita, Fatyol et al. 2008). In this context, we have identified Lys178 as an acceptor lysine for TGFβ- and TRAF6-induced polyubiquitination of TβRI (Sundar, Gudey et al. 2015).

Deubiquitinating enzymes (DUBs) remove ubiquitin or ubiquitin chains from the substrate. UCH37 is a DUB that deubiquitinates TβRI by binding Smad7, and thereby promoting TGFβ-induced migration (Cutts, Soond et al. 2011). Another DUB, cylindromatosis (CYLD), inhibits TAK1-p38 activation by deubiquitinating Smad7 in T cells (Zhao, Thornton et al. 2011).

6.2. Sumoylation

Small ubiquitin-related modifier (SUMO) is a reversible post-translational protein modifier that is covalently attached to acceptor lysines of target proteins via sumoylation. SUMO proteins are approximately 10 kDa in size

21 and share 18% amino acid sequence identity with ubiquitin. The human genome encodes four SUMO proteins: SUMO1, SUMO2, SUMO3, and SUMO4. Sumoylation involves formation of an isopeptide bond between the C-terminal glycine of SUMO and a lysine residue of the target protein. Unlike ubiquitin, SUMO proteins have a consensus acceptor site: ΨKxE, where Ψ is a hydrophobic amino acid, K is lysine, x is any amino acid, and E is glutamic acid (Hannoun, Greenhough et al. 2010).

Sumoylation can alter target protein localization, stability, and activity (Bayer, Arndt et al. 1998; Mossessova and Lima 2000; Geiss-Friedlander and Melchior 2007). The majority of SUMO targets are nuclear proteins, but sumoylation can also reportedly regulate cell surface receptors and channels at the plasma membrane. TβRI is sumoylated by Ubc9 on a lysine residue that is not a classical SUMO consensus acceptor site. TβRI sumoylation increases TβRI kinase activation and enhances Smad2 and Smad3 activation (Kang, Liu et al. 2009). Moreover, TGFβ reportedly down-regulates the sumo-specific E3-ligase PIAS1, in turn, destabilizing SnoN by reducing sumoylation (Netherton and Bonni 2010; Moustakas and Heldin 2012).

6.3. Phosphorylation

Phosphorylation is a post-translational modification involved in many cellular processes. The covalent binding of a phosphate group to a target protein can alter protein function by changing the protein conformation or by promoting protein–protein interactions. TGFβ binding to TβRII induces phosphorylation of serine and threonine residues in the TβRI GS domain, causing a TβRI conformational change. TβRI phosphorylation activates its kinase activity, resulting in phosphorylation of downstream R-Smads and activating the Smad signaling cascade. Phosphorylation of TβRI also prevents binding of the

22 TGFβ signaling-inhibitor FKBP12 (Huse, Chen et al. 1999; Moustakas, Souchelnytskyi et al. 2001; Xu, Liu et al. 2012). TβRI and TβRII are dual- specificity kinases that can also phosphorylate tyrosines. Such phosphorylation activates the Erk1/2–MAPK signaling pathway through association between TβRI and the adaptor protein Shc (Lee, Pardoux et al. 2007). Dephosphorylation also regulates the TGFβ cascade, in which Smad7 interacts with GADD34 (a regulatory subunit of protein phosphatase 1), resulting in TβRI dephosphorylation (Xu, Liu et al. 2012).

7. Notch signaling

In 1919, the laboratory of Thomas Hunt Morgan characterized a notched wing phenotype in Drosophila melanogaster with loss of wing margin tissue from the distal tip of the wing (Mohr 1919), which was determined to be caused by a mutation in the newly discovered gene encoding the Notch protein (Yamamoto, Schulze et al. 2014). The Notch signaling pathway has since been determined to be important for cell–cell communication and cell-fate determination during metazoan development, embryogenesis, cell lineage progression, and differentiation (Andersson, Sandberg et al. 2011).

Notch is a single-pass transmembrane protein comprising two non-covalently bound polypeptides. The mammalian genome encodes four Notch molecules (Notch1–4). Canonical Notch signaling is initiated by the binding of one of the five cell-bound Notch ligands: Jagged1, Jagged2, delta-like ligand (DLL) 1, DLL-3, or DLL-4. Ligand-bound Notch undergoes a sequential proteolytic cleavage called regulated intramembrane proteolysis (RIP) (Andersson, Sandberg et al. 2011; Ayaz and Osborne 2014). RIP reportedly regulates a wide variety of cellular processes, including protein activation, intercellular

23 signaling, intracellular signaling, and protein degradation (McCarthy, Twomey et al. 2009; De Strooper and Annaert 2010).

In its initial proteolysis, known as S1 cleavage at trans-Golgi, Notch is cleaved by furin-like convertases, forming a bipartite receptor that translocates to the cell membrane. Notch RIP then starts with the binding of a ligand that is presented to Notch by a neighboring cell, triggering ectodomain shedding by ADAM17/TACE, termed S2 cleavage. This forms the Notch extracellular truncation fragment (NEXT), also known as notch extracellular domain (NECD). Ectodomain shedding depends on the distance of the substrate from the plasma membrane and on conformational changes of the cleavage site (Hayashida, Bartlett et al. 2010). Subsequently, S3 cleavage of NEXT/NECD is mediated by γ-secretase, which releases the Notch intracellular domain (NICD). PS1—the catalytic core of the γ-secretase complex—cleaves NEXT/NECD in the transmembrane region (Brou, Logeat et al. 2000; Hayashida, Bartlett et al. 2010; Andersson, Sandberg et al. 2011; Bi and Kuang 2015). The NICD then translocates to the nucleus, where it interacts with the transcriptional repressor RBPJκ/CSL, converting it into a transcriptional activator of downstream target genes, including hairy/enhancer of split 1 (HES1) and HES-related with YRPW motif families (HEY1) (Nam, Weng et al. 2003; Ayaz and Osborne 2014; Teodorczyk and Schmidt 2014). The growing list of Notch targets includes HES7, HEY2, PPAR, c-Myc, Sox2, Pax6, cyclinD1, NFκB, and p21/waf1. Nuclear NICD later undergoes proteasomal degradation by the E3 ligase F-box and WD repeat domain- containing 7 (FBW7). HES5 reportedly represses FBW7 transcription and creates a positive feedback loop in Notch signaling (O'Neil, Grim et al. 2007; Sancho, Blake et al. 2013; Teodorczyk and Schmidt 2014).

24 The Notch signaling pathway is implicated in the progression of breast, lung, prostate, and cervical cancers. The TGFβ and Notch signaling pathways are not completely independent of each other, as TβRI-ICD and NICD form a complex in TGFβ-stimulated prostate cancer cells (Gudey, Sundar et al. 2014). Moreover, TGFβ induces Jagged-1 expression, and Jagged-1 knockdown reduces TGFβ-induced p21 expression (Gudey, Sundar et al. 2014; Lindsey and Langhans 2014). TGFβ also induces HES1 and HEY1 expressions (Blokzijl, Dahlqvist et al. 2003; Zavadil, Cermak et al. 2004).

25 Present Investigation

Aims The aim of this thesis was to investigate the functional role of TGFβ-induced and TRAF6-mediated signal transduction in cancer, with the main focus on prostate cancer. The specific aims of each paper were as follows:

Paper I: To investigate the functional role of TRAF6 on the TβRI, and its biological significance.

Paper II: To elucidate the molecular mechanism underlying the transmembrane cleavage of TβRI by PS1, and its role in cancer.

Paper III: To identify the ubiquitin acceptor lysine in TβRI, as well as the biological significance of TRAF6-induced TβRI ubiquitination.

Paper IV: To demonstrate the functional role of the zinc finger protein Snail1 in TGFβ-induced tumor progression.

26 Materials and methods

Cell culture

All cells were cultured at 37°C in 5% CO2 in a humidified incubator.

Human prostate cells (including LnCaP cells and PC3U cells originated from PC3 cells) and human lung carcinoma cells (A549) were cultured in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin and streptomycin (PEST). Cells were starved for 16–18 hours in RPMI with 1% FBS, 1% L- glutamine, and 1% PEST.

Human breast cancer cells (MDA-MB-231), human embryonic kidney (HEK) cells containing SV40-T antigen (293T), TRAF6 knockout (TRAF6−/−) mouse embryonic fibroblasts (MEFs), PS1 knockout (PS1−/−) MEFs, and wild-type MEFs were grown in Dulbecco’s modified essential medium (DMEM) supplemented with 10% FBS, 1% L-glutamine, and 1% PEST. MDA-MB-231 and 293T cells were starved for 16–18 hours in DMEM containing 1% FBS, 1% L-glutamine, and 1% PEST. MEFs were starved for 16–18 hours in DMEM containing 0.5% FBS, 1% L-glutamine, and 1% PEST.

Mouse prostate epithelial cells (TRAMPC2) derived from C57BL/6 mice were cultured in DMEM with 5% FBS, 5% Nu-serum IV, 1% PEST, and 4 mM L-glutamine; adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose; and supplemented with 0.005 mg/ml bovine insulin and 10 nM dehydroisoandrosterone. Cells were starved for 16–18 hours in DMEM containing 1% FBS, 1% Nu-serum IV, 1% PEST, and 4 mM L-glutamine; adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose; and

27 supplemented with 0.005 mg/mL bovine insulin and 10 nM dehydroisoandrosterone. After starvation, cells were stimulated with TGFβ1 (10 ng/mL) for the indicated time periods.

Transient transfection PC3U and HEK 293T cells were transfected with plasmids using Fugene6 transfection reagent following the manufacturer’s instructions. PC3U cells were transfected with a non-targeting control RNA or a specific short interfering RNA (siRNA) using oligofectamine transfection reagent following the manufacturer’s instructions.

Protein analysis After TGFβ1 stimulation, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). For protein extraction, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer. Protein concentration was measured using a BCA kit and samples were subjected to SDS-PAGE on NuPAGE gels, followed by western blotting.

Immunoprecipitation Cell lysates were incubated overnight at 4°C with the indicated antibody. Next, Protein G Sepharose beads were added. The Protein G Sepharose beads with protein complexes attached were washed in RIPA buffer using centrifugation. After addition of SDS sample buffer with reducing agent, the samples were heated at 95°C for 10 min.

Nuclear cytoplasmic fractionation assay TGFβ1-stimulated cells were harvested, washed with ice-cold PBS, and centrifuged. To the pellet, we added Buffer I containing 20 mM Tris HCl (pH

7), 10 mM KCl, 2 mM MgCl2, 0.5% NP40, 1 mM aprotinin, and 1 mM

28 Pefabloc. The cells were then sheared with a needle and syringe, and then the samples were centrifuged and the supernatant was collected as the cytoplasmic fraction. The pellet was washed with Buffer II (Buffer I + 0.5 M NaCl) and centrifuged again. The supernatants were collected as the nuclear fraction. The cytoplasmic and nuclear fractions were subjected to protein measurement and western blotting.

In vivo ubiquitination assay PBS containing 1% SDS was added to the harvested cells, which were then washed with ice-cold PBS. Samples were heated at 95°C for 10 minutes, followed by the addition of PBS containing 0.5% NP40 and protease inhibitors. After the samples were centrifuged, the thick slimy layer was discarded and the remaining parts of the samples were incubated overnight with a specific antibody. Next, Protein G Sepharose beads were added, the samples were centrifuged, and the Protein G Sepharose beads were washed in PBS containing 0.5% NP40. We next added SDS sample buffer containing 1 mM dithiothreitol (DTT) reducing agent to the samples, which then were heated at 95°C. Samples were subjected to SDS-PAGE followed by western blotting.

In vitro ubiquitination assay An in vitro ubiquitination assay was performed with E1, E2 (Ubc13-Uev1A), and GST-TβRI or GST-K178R-TβRI proteins with or without GST-TRAF6.

The reaction mixture also contained 10 mM MgCl2, 50 mM NaCl, 20 mM Tris (pH 7.4), 10 mM ATP, 10 mM DTT, 2.5 µM ubiquitin, and 100 µM MG132. The reaction was stopped with addition of SDS sample buffer. Then the samples were subjected to SDS-PAGE, followed by western blotting.

29 Western blotting Samples were subjected to SDS-gel electrophoresis using NuPAGE Novex 4 12% or 10% gels, 12% Bis-Tris gels, or 3–5% or 7% Tris-Acetate gels. After electrophoresis, proteins were transferred to a nitrocellulose membrane (iBlot transfer stack) using an iBlot gel transfer device. The membranes were probed using specific primary and secondary antibodies, and developed using chemiluminescence and autoradiography.

Immunofluorescence Harvested cells were rinsed in PBS, and then fixed in 4% paraformaldehyde for 10 minutes, followed by permeabilization in 2% Triton X-100. PBS with 5% BSA was used for blocking, and then the cells were incubated with specific primary and secondary antibodies. Subsequently, the slides were mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI).

Proximity ligation assay (PLA) Following TGFβ1 stimulation, cells were harvested. The cells were then fixed in 4% paraformaldehyde, permeabilized in 2% Triton X-100, blocked in PBS with 5% BSA, and incubated with specific primary antibodies. The proximity ligation assay (PLA) was performed following the manufacturer’s instructions. Confocal microscopy was used to visualize the slides and pictures were taken. Signals were quantified using Blob-Finder software.

Quantitative real-time RT-PCR After TGFβ1 stimulation, cells were harvested and RNA was isolated using the Qiagen RNA isolation kit. Next, cDNA was synthesized using the Thermoscript RT-PCR cDNA synthesis kit. Real-time RT-PCR was

30 performed using the Power SYBR Green PCR Master Mix and appropriate forward and reverse primers.

Chromatin immunoprecipitation assay The chromatin immunoprecipitation (ChIP) assay was performed using the Abcam ChIP kit following the manufacturer’s protocol. DNA and proteins were cross-linked in 4% formaldehyde, and the cells were sheared. Then the chromatin was precipitated using specific antibodies and was reverse cross- linked. After purification, DNA was amplified using quantitative RT-PCR with the Power SYBR PCR Master Mix and specific forward and reverse primers.

Site-directed mutagenesis Following the manufacturer’s instructions, we used the QuikChange Site- Directed Mutagenesis kit to create point mutants of different constitutively active TβRI constructs. Mutagenesis was confirmed by sequencing the plasmids.

Invasion assay Invasion assays were performed using CytoSelect Cell Invasion assay kits following the manufacturer’s instructions. Serum-free RPMI-1640 was used to rehydrate the upper basement membrane. Cells were seeded in the upper layer of the chamber in medium with or without TGFβ1. The lower chamber was filled with RPMI 1640 with 10% FBS. Non-invasive cells were removed, while invasive cells were stained with crystal violet staining solution and then photographed. Colorimetric quantification was performed by measuring the optical density at 560 nm using a spectrophotometer.

31 FACS analysis TGFβ1-stimulated cells were harvested, fixed, permeabilized, and blocked. The cells were then stained with primary antibody and incubated with secondary antibody. Next the cells were incubated with propidium iodide solution to label RNA, and with Hoechst dye to label DNA. Finally, the cells were sorted using FACS LSR II, and the data were analyzed using BD bioscience software.

Immunohistochemistry Tissue sections and a tissue microarray (TMA) containing multiple samples were deparaffinized, pre-treated, and blocked. The sections were incubated with primary antibody, and then HRP polymer. After counter-staining with hematoxylin, the tissue sections were analyzed using a bright-field microscope.

Animal studies This study was approved by the local animal review board in Umeå, Sweden (Approval ID: A110-12; Date: 21 August, 2012). TRAMPC2 cells were subcutaneously injected into C57BL/6 mice. Upon reaching palpable tumor volume, the mice were intraperitoneally injected with DMSO (control) or DBZ (a γ-secretase inhibitor) for 10 days. Then the mice were sacrificed and tumors were collected. Tissues were frozen or processed for RNA and protein analysis.

Patient material We used human tumor tissues from prostate, kidney, and urinary bladder cancers. These studies were approved by the Uppsala Ethical Review Board.

32 Statistical analysis Statistical analyses were performed using SPSS statistics. The Mann-Whitney U test was used to calculate p values in Papers II and III. One-way ANOVA and Student’s two-tailed t-test were used to determine statistical significance in Papers I, III and IV.

33 Results and discussion Paper I

TRAF6 ubiquitinates the TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer

An increasing number of transmembrane receptors reportedly undergo ectodomain shedding (Hayashida, Bartlett et al. 2010). TβRI ectodomain cleavage is mediated by TACE in an Erk1/2 MAP-kinase-dependent manner, resulting in reduced TGFβ signaling (Liu, Xu et al. 2009). We found that TGFβ activation caused Lys63-linked polyubiquitination of TβRI by TRAF6, as well as PKCζ activation, which promotes TβRI cleavage by TACE. Nuclear TβRI-ICD accumulation in human prostate cancer cells was confirmed using ectopically expressed HA-TβRI or GFP-TβRI. TGFβ stimulation activates TACE, which then cleaves TβRI between glycine 120 and leucine 121, releasing a 34-kDa TβRI-ICD.

We found that TβRI-ICD translocated to the nucleus, where it associated with the transcriptional co-activator p300 in nuclear promyelocytic leukemia (PML) bodies, promoting invasion in various types of cancer cells. Moreover, ChIP assays revealed TGFβ-induced binding of TβRI-ICD to the Snail1 promoter. Nuclear TβRI-ICD accumulation was observed only in malignant prostate cells, not in primary human epithelial cells. In conclusion, our data suggested that TRAF6-dependent non-canonical TGFβ signaling plays a specific role in tumor invasion.

34 Paper II

TRAF6 stimulates the tumor-promoting effects of the TGFβ type I receptor through polyubiquitination and activation of presenilin1

Regulated intramembrane proteolysis is a series of regulated events targeting transmembrane proteins to release their intracellular domains (ICDs). The γ- secretase complex, of which PS1 is the catalytic core, is involved in the transmembrane cleavage of various receptors, including Notch (McCarthy, Twomey et al. 2009). We investigated whether γ-secretase is involved in TRAF6-dependent TβRI-ICD formation by PS1 (Mu, Sundar et al. 2011; Mu, Gudey et al. 2012).

Our results suggested that TGFβ enhances PS1 expression and activation. Moreover, TRAF6 polyubiquitinates PS1 and recruits it to TβRI to cleave TβRI at the transmembrane region. Thus, TβRI is first cleaved by TACE in the ectodomain, and then by PS1 at the transmembrane region. After TGFβ stimulation, TβRI-ICD associated with NICD, driving the expressions of genes including TβRI, Jag1, and Snail1. In vitro treatment of cells with a γ- secretase inhibitor prevented TGFβ-induced invasion in various cancer cells. Moreover, using xenograft prostate cancer mice as an in vivo model, we found that γ-secretase inhibitor treatment prevented TβRI cleavage and reduced tumor growth.

35 Paper III

TRAF6 promotes TGFβ-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFβ type I receptor.

Ubiquitination is a post-translational protein modification that can alter protein function as well as control cellular processes, such as apoptosis, cell- cycle regulation, and gene regulation. We recently reported the association between TRAF6 and TβRI. Furthermore, TGFβ induces Lys63-dependent TβRI ubiquitination and prompts TβRI-ICD formation by promoting proteolytic cleavage by TACE and PS1 (Sorrentino, Thakur et al. 2008; Mu, Sundar et al. 2011; Gudey, Sundar et al. 2014).

In this study, we identified Lys178 of TβRI as the acceptor lysine for TRAF6- induced TβRI ubiquitination following TGFβ stimulation. Site-directed mutagenesis of Lys178 to arginine inhibited TβRI ubiquitination in vivo and in vitro. Moreover, a TβRI point mutant with Lys178 changed to arginine failed to form the ICD and to translocate to the nucleus. Our findings suggested that Lys63-linked TβRI polyubiquitination promoted TβRI-ICD formation and induced the expressions of EMT markers, such as Vimentin, Twist, N-cadherin, and the cell cycle regulator cyclin D1. Moreover, TβRI polyubiquitination promoted invasion in prostate cancer cells.

36 Paper IV

Proinvasive Snail1 targets TGFβ Type I receptor to promote epithelial to mesenchymal transition in prostate cancer

Epithelial-to-mesenchymal transition (EMT) leads epithelial cells to lose their polarity and cell–cell adhesion, and causes cancer cells to gain migratory and invasive properties (Hanahan and Weinberg 2000; Hanahan and Weinberg 2011). TGFβ plays an important role in promoting tumor growth by triggering EMT. The zinc finger protein Snail1 also plays a key role in the EMT process (Padua and Massague 2009; Heldin, Vanlandewijck et al. 2012).

In this study, we found that the transcriptional regulator Snail1 regulated TβRI expression at both the mRNA and protein levels. We also found that Snail1 regulated HES1 expression. Our results indicated that TβRI down- regulation reduced HES1 expression, suggesting that Snail1 also promotes EMT through TβRI in a TGFβ-dependent manner.

37 Conclusions

The following conclusions describe the molecular mechanism of TRAF6- induced TGFβ signal transduction in prostate cancer (Figure 9): • TGFβ causes TRAF6 activation. • TGFβ-induced TRAF6 activation causes Lys63-polyubiquitination of TβRI. • TGFβ induces activation of PKCζ, which in turn activates TACE. • TRAF6 polyubiquitinates and activates PS1. • TβRI is cleaved at the ectodomain region by TACE and at the transmembrane region by PS1, forming TβRI-ICD. • TβRI-ICD translocates to the nucleus, where it binds p300 and interacts with NICD. • TβRI-ICD promotes the expressions of Snail1, Jag1, Notch1, MMP2, p300, and TβRI. • TRAF6 ubiquitinates TβRI at lysine 178. • Lys63-linked polyubiquitination of TβRI promotes cell cycle regulation in human prostate cancer cells. • TβRI ubiquitination and TβRI-ICD nuclear translocation promotes invasion in prostate cancer cells, lung carcinoma, and breast carcinoma cells. • TβRI-ICD nuclear translocation occurs in various human cancers, but not in normal prostate epithelial cells. • In vivo experiments in a prostate cancer xenograft mouse model showed that γ-secretase inhibitor treatment reduced tumor growth and inhibited TβRI-ICD formation. • Snail1 regulates the expressions of TβRI and HES1.

Figure 9: Illustrated summary of Papers I, II, and III

39 Future perspectives

Prostate cancer is the most prevalent non-cutaneous cancer among men. Its multistage oncogenesis includes sustained proliferative signaling, evasion of apoptosis, growth suppressor resistance, uncontrolled replicative potential, sustained angiogenesis, and activation of tissue invasion and metastasis. These cancer hallmarks are caused by genetic or epigenetic modifications in tumor cells. Moreover, the tumor microenvironment contributes to prostate cancer tumorigenesis. Heterotypic cellular signaling also plays a predominant role in the oncogenic process (Hanahan and Weinberg 2000; Hanahan and Weinberg 2011; Scheller, Chalaris et al. 2011; Felgueiras, Silva et al. 2014).

TGFβ acts as both a tumor promoter and a tumor suppressor. The present thesis elucidates a molecular mechanism whereby TGFβ promotes prostate cancer oncogenesis. We identified a novel TGFβ-induced non-Smad signaling pathway in cancerous cells in which TRAF6 causes Lys63-linked TβRI polyubiquitination, promoting TβRI proteolytic cleavage by TACE and PS1, which releases TβRI-ICD. This liberated TβRI-ICD translocates to the nucleus in cancer cells, where it induces expressions of its own gene and the EMT markers Snail1 and MMP2, thus promoting invasion in cancer cells.

Moreover, we report that TβRI-ICD associates with NICD in a TGFβ- dependent manner. Site-directed mutagenesis of TβRI at the TACE and/or PS1 cleavage site inhibited its nuclear translocation. Experiments in a prostate cancer xenograft mouse model further revealed that treatment with a γ- secretase inhibitor (DBZ) reduced tumor growth and TβRI-ICD formation. We also identified Lys178 as the acceptor lysine in TβRI for TGFβ-induced Lys63-linked polyubiquitination by TRAF6. Mutagenesis of lysine 178 in

40 TβRI to arginine resulted in failure to form TβRI-ICD and also prevented invasion in prostate cancer cells (Mu, Sundar et al. 2011; Gudey, Sundar et al. 2014; Sundar, Gudey et al. 2015).

The non-Smad TGFβ signaling pathway that leads to TβRI-ICD formation may be a positive feed-back mechanism in cancer cells, in parallel with the classical Smad signaling cascade. Our findings suggest that this occurs in a TβR kinase-independent manner, which also promotes autoregulation of TβRI expression. This may enable cancer cells to be hyper-responsive to specific TGFβ signals. TβRI-ICD is not a classical transcription factor, and it remains unclear whether it directly binds to DNA or if it acts as a co-transcription factor. It will be important to identify any possible direct transcriptional targets of TβRI-ICD, and to determine whether TβRI-ICD regulates genes involved in regulating cell proliferation. It will also be important to determine where TβRI-ICD binds to the Snail1 and TβRI promoters.

Although we have detected TβRI-ICD nuclear translocation in cancer cells, it is remains unknown how TβRI-ICD translocates to the nucleus? Full-length TβRI reportedly translocates to the nucleus with the help of importin β1 (Chandra, Zang et al. 2012). Future investigations should examine whether TβRI-ICD uses a similar mode of translocation to the nucleus.

Histopathological examination of cancer specimens is a specific and conventional diagnostic method for grading prostate cancer (Felgueiras, Silva et al. 2014). It would be interesting to investigate a possible correlation between TβRI-ICD expression and Gleason score in prostate cancer tissue.

A major challenge in prostate cancer diagnosis is the difficulty of predicting the disease outcome prior to metastasis, and the existence of only limited

41 clinical symptoms. Elucidation of better predictive biomarkers will help to provide better prognostic information and the best treatment options for individuals.

Finally, it will also be interesting to analyze the possible effects of targeting TRAF6 to prevent TβRI ubiquitination and cleavage. Future studies should also examine tumor tissues for possible TβRI mutations—for example, on the amino acid residues G120, VI129, and K178, which we found to be important for activation of non-canonical signals.

42 Acknowledgements

This thesis is not only a culmination of my work at the keyboard—it represents a milestone of five years of my work at the Department of Medical Biosciences, Umeå University. During this time, my studies have been financially supported by grants from Umeå University, The Swedish Cancer Society, ALF-VLL, The Kempe Foundation, The Knut and Alice Wallenberg Foundation, and The Cancer Research Foundation in Northern Sweden: Lion’s Cancer Research Foundation.

I have not travelled alone on this journey to obtain my Ph.D. Numerous others have supported and encouraged me along the way—including my family, colleagues, and friends. I am grateful to everyone who made this thesis possible, and I would especially like to thank the following people.

First and foremost, I give my heartfelt thanks to my supervisor, Prof. Marene Landström. You have been motivating, encouraging, and enlightening. Your patience, flexibility, care, and faith in me have enabled my completion of this dissertation journey. You have been a tremendous mentor to me, and your scientific advice has been priceless. I am forever grateful to you for helping to make my Ph.D. studies enjoyable.

I would also like to give sincere thanks to my co-supervisor, Prof. Carl- Henrik Heldin. You have supported me since the day I began as a student at the Ludwig Institute of Cancer Research, Uppsala. You are a true inspiration to all aspiring scientists. Thank you particularly for your encouragement during our scientific discussions.

43 My special thanks to all of the people at the Ludwig Institute for Cancer Research, the Rudbeck lab, and the Department of Medical Biosciences. Thank you, Noopur, for your initial guidance in the lab when I first arrived as a student. Ola, thank you for teaching me mutagenesis and for taking such good care of me.

I cannot complete this thesis without expressing my gratitude to Annika Hermansson. Thank you for your great support. You are one of the nicest people I have met in Uppsala.

I have also been very happy to get to know and collaborate with many members of the Landström group at Ludwig and Umeå. Thanks to Yabing, Maria Ekman, Mariya, Marianna, Verena, Ihor, Linda, Guangxiang, Stina, Anders, Pramod, Anahita, Alexei, Chunyan Li, and Lena.

Song Jie, Gizem, Maria, and Kartik, I have greatly enjoyed sharing an office with you. Thank you for the many nice conversations.

I would also like to acknowledge Prof. Aris Moustakas and his group members for our interactions during journal club and scientific presentations at Ludwig.

Special thanks to Prof. Anders Bergh, Pernilla Wikström, Anders Widmark, Emma Persson, and all others who participated in prostate cancer meetings.

I am grateful to Prof. Karin Nylander and Prof. Gunilla Olivercrona for their encouragement.

44 I also thank the members of Bengt Westermark, and Karin Forsberg Nilsson’s group at Rudbeck. Thank you, Umash, for helping with GFP plasmid construction. Demet and Soumi, I give special thanks to you both for the friendly chats and assistance.

Thanks to Maria Brattsand, Maria Nilson, Susanne Gidlund, Pernilla Andersson, Hinnerk, Erik, Sofia, Xingru, Camilla, Zhara, Ela, Lee Ann, and Isil for all of the nice conversations.

I also recognize the support and help from Karin Boden, Carina Ahlgren, Clara, and Clas Wikström. Terry Persson and Åsa Lundsten, I have been privileged to get to know you. Thank you for all your great help.

Anika Jahns, there are no words that can sufficiently express my gratitude and appreciation to you. You have been there whenever I have needed any help. It was my greatest pleasure to meet you. I would also like to thank your mom, dad, and grandmother.

Thanks to our special friends Ravi, Vidya, and the little cutie Kanasu for the wonderful time that we spent together. I give my heartfelt acknowledgement for the friendly help that you have given at all times.

Thank to the Umeå malayalees, Sajna, Sinosh, Remya, Sujith, Nisha and Philge, for the great times we’ve had together. Maria and Edvin, I give my heartfelt thanks for your caring and help.

I further wish to acknowledge a friendship that began at Uppsala. Navya, you’re a special person. Thanks also to Nisha, Rajesh, Nimesh, and

45 Sandeep. Smitha, I also appreciate the time that you spent helping me with cloning.

I would also like to extend warm thanks to Sharvani, Munender, and Aadyom, Aunbhav, Dinesh, Kamla, Vijay, Uma, and Bala.

I also recognize the support and help from Shyam’s childhood buddies and their family: Jyothishwar, Harika, Karthik, Diana, Harish, Ratna-Sree, Uday, Saroja, and Kushru.

Thanks to all of my teachers from school to university. Especially, I must say thank you to Manoj Narayanan, Santhosh Kumar, and Gopa Kumar for your unconditional support. I am not sure that I would be writing this thesis without your encouragement.

Finally, I thank my family: my Mother, Sister, Brother, Brother-in-law, Kashy, Bhagya, Uncle, Aunty, Bhaskar, and Sandeep. You have always been beside me offering your support during both the happy and hard moments.

I have no adequate words to express my thanks for the support, care, and love I have recieved from my Father. You gave me the strength to reach for the stars and to chase my dream, but today I must look for you in the stars.

A journey is easier when you travel together. I owe everything to you, Shyam. During my time with you, I have been happier, stronger, and more content. Through your love, support, patience, and unwavering belief in me, I have been able to complete this Ph.D. journey. To our sweet little angel, Ameyaa, our love, thank you for being an adaptable daughter.

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http://www.cancerresearchuk.org/cancerinfo/cancerstats/types/prostate/incidence/#age

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