Novel Regulators of the TGF-Β Signaling Pathway

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 57

Novel Regulators of the TGF-˟ Signaling Pathway

MARCIN KOWANETZ

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You will rest when you die. Clint Eastwood, as Frankie Dunn.

Winners are simply willing to do what losers won’t. ‘Million Dollar Baby’.

List of Publications

This thesis is based on the following publications, which will be referred to in the text by their Roman numerals:

I. Valcourt U.*, Kowanetz M.*, Niimi H., Heldin C.-H., Moustakas A. (2005) TGF-ȕ and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol. Biol. Cell, 16(4):1987-2002. * equally contributed authors

II. Kowanetz M., Valcourt U., Bergström R., Heldin C.-H., Moustakas A. (2004) Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor ȕ and bone morphogenetic protein. Mol. Cell. Biol., 24(10):4241-54.

III. Kowanetz M., Lönn P., Kowanetz K., Heldin C.-H., Moustakas A. (2005) The inducible kinase SNF1LK regulates TGF-ȕ receptor signaling and degradation. Manuscript.

Reprints were made with the permission of the publishers.

Related Publications

i. Pardali K., Kowanetz M., Heldin C.-H., Moustakas A. (2005) Smad pathway-specific transcriptional regulation of the cell cycle inhibitor p21(WAF1/Cip1). J. Cell Physiol., 204(1):260-72. ii. Itoh S., Thorikay M., Kowanetz M., Moustakas A., Itoh F., Heldin C.-H., ten Dijke P. (2003) Elucidation of Smad requirement in transforming growth factor-ȕ type I receptor-induced responses. J. Biol. Chem., 278(6):3751-61. iii. Kurisaki A., Kurisaki K., Kowanetz M., Sugino H., Yoneda Y., Heldin C.-H., Moustakas A. (2005) Nuclear export of Smad3 is mediated by exportin4 and the Ran GTPase. Manuscript.

Contents

Introduction...... 13 1 TGF-ȕ superfamily signaling...... 13 1.1 Signaling effectors - Smads ...... 15 1.2 To Smad or not to Smad? ...... 18 1.3 Ending the signal ...... 19 2 Physiological functions of TGF-ȕ signaling in epithelial cells...... 23 2.1 TGF-ȕ inhibits growth of epithelial cells...... 23 2.2 EMT and metastasis...... 25 2.3 What triggers the switch from tumor suppressor to tumor promoter?...... 28 3 BMP and disease...... 29 4 Gene regulation by TGF-ȕ family members ...... 30 5 Id proteins ...... 31 6 SNF1LK...... 34 Aims of the present study ...... 38 Present investigation ...... 39 1 Paper I ...... 39 2 Paper II...... 41 3 Paper III ...... 43 Future perspectives ...... 46 Acknowledgements…...... 51 References...... 53 Abbreviations

ALK Activin receptor-like kinase AMP Adenosine 5’-monophosphate AMPK AMP-activated protein kinase ATP Adenosine 5’-triphosphate bHLH Basic helix-loop-helix BMP Bone morphogenetic protein CA Constitutively active CDK Cyclin-dependent kinase DN Dominant negative ECM Extracellular matrix EGFR Epidermal growth factor receptor EMT Epithelial-mesenchymal transition ERK Extracellular-signal-regulated kinase GFP Green fluorescent protein HLH Helix-loop-helix Id Inhibitor of differentiation/Inhibitor of DNA binding LZ Leucine zipper MAPK Mitogen activated protein kinase MET Mesenchymal-epithelial transition MH Mad-homology pRb Retinoblastoma protein SARA Smad anchor for receptor activation siRNA Small interfering RNA Smurf Smad ubiquitination regulatory factor SNF1 Sucrose-non-fermented 1 SNF1LK SNF1-like kinase TGF-ȕ Transforming growth factor-ȕ UBA Ubiquitin associated domain Į-SMA Į-Smooth muscle actin Introduction

For a single cell to function normally in a multi-cellular organism, such as the human body, it is critical to receive proper messages from the surrounding environment. Growth factors are the messengers that guarantee proper growth, function and programmed death of the cell. Therefore, for a whole organism to function the complex network of signals coming from and going to millions of cells needs to be orchestrated. Destroying this delicate signaling balance might result in several diseases, with cancer being the most pronounced one. This thesis is focused on a very powerful, multifunctional growth factor, transforming growth factor-ȕ (TGF-ȕ). It is now widely accepted that TGF-ȕ regulates several biological processes including early embryonic development, cell growth, differentiation, motility, apoptosis, angiogenesis and many others. Aberrant responses to TGF-ȕ are frequently found in cancers of epithelial origin. However, TGF-ȕ cannot be clearly defined either as tumor suppressor or promoter. The aim of this introduction is to shed light on the mechanism of TGF-ȕ signaling in normal tissues as well as in disease.

1 TGF-ȕ superfamily signaling

The transforming growth factor-ȕ (TGF-ȕ) superfamily consists of related multifunctional cytokines, which include TGF-ȕs, activins, and bone morphogenetic proteins (BMPs) (Piek et al., 1999a). The analysis of complete genomes revealed that the members of the family are encoded by 42 open reading frames in humans, 9 in flies and 6 in worms (Lander et al., 2001). Members of this superfamily act by binding to two different types of transmembrane serine/threonine kinase receptors, called type I and type II receptors (Fig. 1) (Shi and Massagué, 2003; ten Dijke and Hill, 2004). Sig- naling is initiated when ligand binds to the type II receptor, which induces formation of a hetero-tetrameric receptor complex composed of the two homodimeric receptors. The current model indicates that in such complexes the type II receptor is constitutively auto-phosphorylated and it transactivates the type I receptor by phosphorylation on serine and threonine residues within a type I receptor-specific sequence termed ‘GS domain’, a highly

13 conserved region close to the kinase domain of the receptor. Thus, the type I receptors act downstream of the type II receptors, and determine the specificity of intracellular signals (Attisano and Wrana, 2002). Up to date, seven type I receptors, commonly called activin receptor-like kinases (ALK) 1 to 7, and five type II receptors have been identified. Specific combinations of type I and type II receptors lead either to differential ligand binding or to differential signaling in response to the same ligand (Fig. 2) and the tissue-specific expression pattern of ligands and receptors dictates the final sensitivity of cells to particular members of the TGF-ȕ superfamily.

Figure 1. Overview of TGF-ȕ signaling pathway. Arrows indicate signal flow. Orange arrows: ligand and receptor activation; gray arrows: Smad and receptor inactivation; green arrows: Smad activation and formation of transcriptional complex; blue arrows: Smad nucleocytoplasmic shuttling. Phosphate groups and ubiquitin are represented by green and red circles, respectively (reprinted from Cell, Vol 113, Yigong Shi and Joan Massagué, Mechanisms of TGF-ȕ Signaling from Cell Membrane to the Nucleus, Pages No 685-700, Copyright (2003), with permission from Elsevier).

The ligand binding to the heterotetrameric receptor is also regulated by ligand traps and accessory receptors, so called type III receptors (Fig. 2) (Shi and Massagué, 2003). The ligand binding traps are soluble proteins that bind the specific ligand and prevent it from binding to the membrane receptor. They include decorin and Į2-macroglobulin (both bind free TGF-ȕ), fol-

14 listatin (binds activins and BMPs) and three distinct protein families that bind to BMPs: Noggin, Chordin/SOG and DAN/Cerberus (Shi and Massa- gué, 2003). Accessory receptors (such as betaglycan, endoglin or Cripto) have an opposite role to ligand traps as they promote activation of signaling by increasing ligand concentration close to the signaling TGF/BMP receptor complexes (Shi and Massagué, 2003).

Figure 2. TGF-ȕ-superfamily ligands, receptors and ligand traps (reprinted from Cell, Vol 113, Yigong Shi and Joan Massagué, Mechanisms of TGF-ȕ Signaling from Cell Membrane to the Nucleus, Pages no 685-700, Copyright (2003), with permission from Elsevier).

1.1 Signaling effectors - Smads

The intracellular signaling pathway initiated after receptor activation is gov- erned by effectors called Smads and so far Smad proteins are the only known signaling molecules that are directly phosphorylated by the type I receptor kinase. Firstly identified in Drosophila as Mad (Sekelsky et al., 1995) and in C.elegans as Sma (Savage et al., 1996) gene products, the vertebrate homo- logues were identified shortly after and named Smad (Derynck et al., 1996). Smads are subdivided into three subclasses based on their function: receptor-regulated Smads (R-Smads), common-partner Smad (Co-Smad) and inhibitory Smads (I-Smads) (Moustakas et al., 2001). So far, eight Smads have been identified: five R-Smads (Smad1, 2, 3, 5 and 8), one Co-Smad (Smad4,

15 but some organisms has more than one Co-Smad, for example Xenopus has two Smad4s: Smad4Į and Smad4ȕ encoded by two different genes (Howell et al., 1999; Masuyama et al., 1999), and common carp Cyprinus carpio has four types of Smad4 (Wang et al., 2005)) and two I-Smads (Smad6 and 7). All R-Smads and Co-Smads share similar structural organization, which consists of an N-terminal MH1 (Mad homology 1) domain, a middle linker domain and a C-terminal MH2 domain, whereas I-Smads differ by lacking the MH1 domain (Fig. 3). Each domain plays distinct role in signaling regulation. The MH1 domain is responsible for Smad-DNA and Smad- transcription factor interactions, whereas the MH2 domain takes part in Smad-receptor and Smad-Smad interactions and interaction with other transcription factors, including co-activators, such as p300 (Moustakas et al., 2001). The linker region is involved in interaction with E3 ubiquitin ligases, Smurf1 and 2 (or Smad ubiquitination regulatory factor 1 and 2) (Zhu et al., 1999) through a ‘PY’ motif.

Figure 3. Structure and role of Smads domains (reprinted from Nature, Vol 425, Rik Derynck and Ying E Zhang, Pages no 577-584, Copyright (2003) Nature Pub- lishing Group, http://www.nature.com/).

After ligand binding and hetero-oligomerization and activation of the receptors, R-Smads bind to the type I receptor. The interaction occurs between the L45 loop of the receptor and the L3 loop within the MH2 domain of R- Smad (Chen et al., 1998). Subsequently, the type I receptor directly phosphorylates the bound R-Smad at its very C-terminal serine-rich sequence SXS (Kretzschmar et al., 1997; Souchelnytskyi et al., 1997). The specific R-

16 Smad protein recruited into the signal transduction pathway depends on the type I receptor. Thus, TGF-ȕ, activins and nodals activate Smad2 and Smad3, because they signal through the type I receptors ALK4, ALK5 and ALK7, whereas BMP-related ligands activate Smad1, Smad5 and Smad8, because they signal through the type I receptors ALK1, ALK2, ALK3 and ALK6 (Fig. 2). Activated R-Smads dissociate from the receptor and form complexes with the Co-Smad (Fig. 1). The R-Smad/Co-Smad complexes translocate into the cell nucleus through interaction with importin-ȕ1 (Xiao et al., 2000a; Xiao et al., 2000b; Kurisaki et al., 2001) or components of the nuclear pore, Nup214 and Nup153 (Xu et al., 2002; Xu et al., 2003). R- Smad/Co-Smad nuclear complexes are then able to bind DNA and to regulate the expression of genes involved in the control of cell proliferation, apoptosis, differentiation and development. The rapidly growing number of studies shows involvement of many transcriptional co-regulators that act together with Smad complexes to regulate expression of target genes (Moustakas et al., 2001). These include: 1) sequence-specific transcription factors such as Sp1 (Pardali et al., 2000b), FAST-1 (Chen et al., 1997), Mixer and Milk (Germain et al., 2000), AP-1 (Wong et al., 1999), AML/Runx (Pardali et al., 2000a; Zhang and Derynck, 2000), YY1 (Kurisaki et al., 2003), c-Myc (Feng et al., 2002), ATF3 (Kang et al., 2003a) and others; 2) co-activators: p300/CBP (Feng et al., 1998; Janknecht et al., 1998), SMIF (Bai et al., 2002) and ARC105 (Kato et al., 2002); 3) co- repressors: c-Ski/SnoN (Luo et al., 1999; Stroschein et al., 1999), TGIF (Wotton et al., 1999), SNIP1 (Kim et al., 2000b), SIP1 (Verschueren et al., 1999) and many others. In the resting state, R-Smads reside predominantly in the cytoplasm wait- ing for receptor activation, whereas Smad4 is known to freely shuttle between nucleus and cytoplasm (Pierreux et al., 2000; Watanabe et al., 2000). The cytoplasmic localization of Smad2 and Smad3 is controlled by SARA (Smad anchor for receptor activation), a protein of the early endosome that interacts with inactive R-Smads (Tsukazaki et al., 1998). Upon TGF-ȕ stimulation, SARA binds to the receptor, thus enabling recruitment of Smad2 and Smad3 to the type I receptor and subsequent phosphorylation of Smads (Fig. 1). The cytoplasmic form of promyelocytic leukaemia (cPML) tumor suppressor serves as a bridge between Smad2, Smad3 and SARA and it is required for the accumulation of SARA and TGF-ȕ receptors in the early endosome (Lin et al., 2004). Moreover, an E3-ubiquitin ligase Itch has been recently implicated in promoting TGF-ȕ signaling through ubiquitination of Smad2 and enhancing its activation by ALK-5 (Bai et al., 2004). In contrast, the interactions between BMP-activated Smads (Smad1, 5 and 8) and cytoplasmic molecules regulating their access to the receptors have not been deeply characterized. The cytoplasmic domain of CD44, the hyaluronan receptor, has been recently reported to interact with Smad1. This interaction promotes BMP-induced Smad1 phosphorylation and its nuclear transloca-

17 tion, suggesting that CD44 helps BMP signaling through increasing concentration of Smad1 in the proximity of the activated BMP-type receptors (Peterson et al., 2004). The current model suggests that R-Smads, bound to specific promoter elements, become dephosphorylated by a not yet identified phosphatase, they then dissociate from Smad4 and move back to the cytoplasm, where they are phosphorylated again by receptor complexes that remain continuously activated. They associate again with Smad4, localize back to the nucleus and bind again to gene specific transcription factors and DNA. That implies that activation or repression of gene targets is the result of repeated activation of the same Smad molecules (Pierreux et al., 2000; Inman et al., 2002b; Nicolas et al., 2004).

1.2 To Smad or not to Smad?

Smad proteins are central signal transducers in TGF/BMP pathway. How- ever, one should not ignore the additional signaling triggered by Smad- independent events such as TGF-ȕ-induced activation of MAPK pathway components (Engel et al., 1999; Yu et al., 2002; Itoh et al., 2003), a MAPK kinase kinase TAK1 (TGF-ȕ-activated kinase) (Yamaguchi et al., 1999), small GTPases RhoA, Rac, Cdc42 (Bhowmick et al., 2001a; Bakin et al., 2002; Edlund et al., 2002), and RhoB (Engel et al., 1998), and many others. The biological consequences of such activation remain not fully characterized and in many cases they seem to be important for events unrelated to transcription, such as cell cytoskeletal changes. Furthermore, the activity of Smads can be regulated not only by ALK receptors but also by other pathways. Ras acting via extracellular-signal- regulated kinase (ERK) phosphorylates Smad2 and Smad3 within the linker domain. Such phosphorylation of Smad3 negatively modulates the transcriptional activity of Smad proteins and attenuates the cytostatic response to TGF-ȕ (Kretzschmar et al., 1999). In contrast, Smad2 phosphorylation by ERK has been indicated to positively regulate its activity due to its greater stability and higher ratio of complex formation with Smad4 (Funaba et al., 2002). Smad2, Smad3 and Smad4 are phosphorylated by calmodulin- dependent kinases (CamK), which negatively regulates the TGF-ȕ signaling pathway. CamKII phosphorylation of Smad2 within MH1 and linker domain results in decreased Smad2 transcriptional activity (Wicks et al., 2000). In contrast, JNK (c-Jun N-terminal kinase) phosphorylation of Smad3 has a positive impact on the signaling and it facilitates Smad3 activation and its nuclear accumulation (Engel et al., 1999). Smad3 was also found to be phosphorylated within the MH1 domain by protein kinase C (PKC) (Yakymovych et al., 2001). The PKC-dependent phosphorylation of Smad3

18 abrogates direct DNA binding of Smad3 and affects its transcriptional activity (Yakymovych et al., 2001). The increasing number of reports identifying novel signaling components of TGF-ȕ pathway suggests that Smad activity is not only regulated by TGF- ȕ receptors. The interdependence of Smad and non-Smad signals is required for proper regulation of TGF-ȕ signaling activity and evoking its biological effects.

1.3 Ending the signal

1.3.1 Negative regulation of Smad signaling

Levels and time of residence of activated Smads in the nucleus dictate the strength of signal coming from the receptors. The already discussed model of R-Smad dephosphorylation regulates duration of the residence of Smads in the nucleus and serves the purpose of recycling R-Smads without perma- nently switching TGF-ȕ signaling off (Inman et al., 2002b). Permanent switch off of the signaling is closely related to the function of I-Smads, Smad6 and Smad7. I-Smads are potently induced by the signaling pathway and they participate in a negative feedback loop to control TGF-ȕ responses (Nakao et al., 1997; Afrakhte et al., 1998). Both Smad6 and Smad7, antagonize R-Smad activation by the receptors. I-Smads bind through their own MH2 domain to the receptor L45 loop thus antagonizing the binding of R-Smads to the receptor (Mochizuki et al., 2004). Moreover, I-Smads can complex with R-Smads, antagonizing R-Smad/Smad4 complex formation (Imamura et al., 1997; Nakao et al., 1997; Hata et al., 1998). Lastly, protein ubiquitination and proteasomal degradation are very powerful mechanisms regulating almost all biological processes (Glickman and Ciechanover, 2002; Ciechanover and Schwartz, 2004), including negative regulation of signaling pathways such as TGF-ȕ (Izzi and Attisano, 2004). Two E3-ubiquitin ligases, Smurf1 and 2, were shown to be directly involved in this process. Smurf proteins belong to a HECT domain family of E3- ubiquitin ligases and their structure consist of three distinguishable domains: 1) an N-terminal protein kinase C conserved 2 (C2) domain involved in interaction with phospholipids, 2) two (Smurf1) or three (Smurf2) WW domains involved in protein-protein interaction and 3) a C-terminal HECT domain that catalyzes transfer of ubiquitin molecules to its target substrates (Zhu et al., 1999; Kavsak et al., 2000). BMP-activated Smads, Smad1 and 5, are targets of Smurf1 (Zhu et al., 1999), whereas Smurf2 was shown to ubiquitinate Smad1/5 and Smad2 (Lin et al., 2000; Zhang et al., 2001). The interaction between Smurfs and Smads is mediated through the WW domain of Smurfs and a ‘PY’ motif within the linker region of R-Smads (Zhu et al., 1999). Increasing number of reports shows that Smurfs are not the only E3-

19 ubiquitin ligases regulating Smad signaling. Smad3 ubiquitination seems to be regulated in a Smurf-independent manner involving a different E3 ligase, the SCF/Roc1 complex (Fukuchi et al., 2001). Moreover, Smad2 has been shown to be targeted for degradation through ubiquitination by Tiul1 and NEDD4-2 (Seo et al., 2004; Kuratomi et al., 2005). Smad4 lacks a ‘PY’ motif and it therefore can not interact directly with Smurf proteins. However, its turnover can be regulated through a mechanism involving Smurf-mediated ubiquitination. Recent study has shown that Smad2, Smad6 or Smad7 can mediate this process through binding to Smad4 and bringing to its proximity a HECT E3-ubiquitin ligase, such as Smurf1 or Smurf2 (Moren et al., 2005). Smads act then as a bridge between Smad4 and Smurf, which triggers ubiquitination of Smad4 and its subsequent proteasomal degradation (Moren et al., 2005). Skp2, the F-box component of the SCF (Skp-Cul-F-box) ubiquitin E3 ligase complex, has been recently shown to interact with Smad4 and to control the turnover of Smad4 cancer mutants (Liang et al., 2004). In addition, other proteins, such as Jab1 and Ras have been shown to be involved in proteasomal degradation of Smad4 (Saha et al., 2001; Wan et al., 2002) but the exact mechanism and function of these alternative pathways remains to be solved. Smad7 is also regulated by ubiquitination. Arkadia, an E3-ubiquitin ligase, interacts with Smad7, which leads to Smad7 poly-ubiquitination, degradation and subsequent amplification of TGF-ȕ signal transduction (Koinuma et al., 2003). Smad7 can also be ubiquitinated by Smurfs, which probably occurs both in the nucleus and the cytoplasm. However, in the latter case subsequent Smad7 degradation does not lead to enhanced TGF-ȕ signaling, since it occurs during the Smurf-mediated receptor ubiquitination and degradation, which leads to a decrease receptor level at the plasma membrane (Kavsak et al., 2000; Ebisawa et al., 2001; Suzuki et al., 2002). This process is discussed in the next paragraph. Moreover, the level of Smad7 has been shown to be regulated through competition between acetylation and ubiquitination. The acetyltransferase p300 has been shown to target two lysine residues in the amino-terminus of Smad7. The acetylated lysi- nes can not be ubiquitinated anymore by Smurf1, and Smad7 becomes pro- tected from TGF-ȕ-induced degradation (Grönroos et al., 2002). The negative regulation of TGF-ȕ and BMP signaling pathways is still a poorly understood mechanism. The complexity of the pathway suggests that additional regulatory molecules such as phosphatases, kinases and components of the proteasomal degradation machinery might be involved. The questions, such as how Smurfs are activated and how proteasomal degradation of Smads is initiated, remain open for future investigation.

20 1.3.2 TGF-ȕ receptor endocytosis and degradation

As previously mentioned, the duration and strength of many signal transduction pathways are regulated by ubiquitin-mediated proteolysis. Accordingly, Smurfs and other ligases together with I-Smads have been implicated in regulation of turnover of the TGF-ȕ/BMP receptors. In unstimulated cells, both Smad7 and Smurf2 preferentially localize to the nucleus (Kavsak et al., 2000). After ligand stimulation, the levels of both proteins increase, leading to Smad7 and Smurf2 association through the PY and WW domains, respectively, and such complex is exported from the nucleus (Kavsak et al., 2000). In the cytoplasm, the Smad7-Smurf2 complex is recruited to the TGF-ȕ receptors, which become ubiquitinated by Smurf2 and subsequently degraded in not yet identified cellular compartments. Smurf2 can not bind directly to the receptor and the association is mediated via Smad7 that serves as a bridge between receptor and Smurf2 (Kavsak et al., 2000). A similar mechanism has been demonstrated for Smurf1, which in the same way as Smurf2 interacts with Smad7 and targets the TGF-ȕ type I receptor for ubiquitination (Ebisawa et al., 2001). The role of the C2 domain of Smurf1 in receptor turnover has been elucidated (Suzuki et al., 2002). The C2 domain, which interacts with phospholipids, helps the Smad7-Smurf1 complex to hook at the plasma membrane in the proximity of the receptors. Deletion of the C2 domain disables the binding of the complex to the plasma membrane and prevents the efficient ubiquitination and degradation of the TGF-ȕ receptors (Suzuki et al., 2002). Similarly to Smad7, Smad6 together with Smurf1 targets BMP type I receptors for ubiquitination and degradation (Murakami et al., 2003). Several other E3-ubiquitin ligases appear to regulate the degradation of TGF-ȕ receptors and these include WW-containing protein 1 (WWP1)/Tiul1 (Komuro et al., 2004; Seo et al., 2004). The unexplored question remains why cells require so many different E3-ubiquitin ligases that target the same substrate, i.e. the TGF-ȕ type I receptor. In many other signaling systems, the ubiquitin-mediated receptor endocytosis, followed by degradation in lysosome, is the ultimate endpoint of signaling (Dikic, 2003). Trafficking of the TGF-ȕ receptors has not been studied in sufficient detail yet. Interestingly, TGF-ȕ receptor endocytosis plays a central role both in enhancing the signaling as well as receptor degradation (Zwaagstra et al., 2001; Hayes et al., 2002; Penheiter et al., 2002; Di Gug- lielmo et al., 2003; Mitchell et al., 2004). Moreover, TGF-ȕ receptors, in contrast to other types of receptors, such as epidermal growth factor or insulin receptors, have a very intriguing characteristic of being internalized and recycled in a ligand-independent manner (Di Guglielmo et al., 2003; Mitchell et al., 2004; Le Roy and Wrana, 2005).

21 Figure 4. TGF-ȕ receptors are internalized through clathrin- or caveolin- dependent pathway. Model based on: (Di Guglielmo et al., 2003).

Recent work has shown that TGF-ȕ receptors can be internalized through two different routes, a clathrin-dependent and lipid raft/caveolar-dependent pathway (Fig. 4) (Di Guglielmo et al., 2003). The clathrin pathway serves two purposes. First of all, the receptors entering this pathway are recycled back to the plasma membrane. Secondly, entering the clathrin-dependent route is required for the TGF-ȕ receptors to initiate proper intracellular signaling. The receptors enter the early endosome where SARA is enriched, which enables the receptors to phosphorylate Smad2 recruited by SARA and propagate the signal (Tsukazaki et al., 1998; Di Guglielmo et al., 2003). In contrast, entering the lipid raft/caveolar compartment leads to TGF-ȕ receptor degradation (Di Guglielmo et al., 2003). The receptor directed to lipid- rich raft regions of plasma membrane binds Smad7-Smurf2 complexes, which preferentially associate with the components of lipid rafts via the C2 domain of Smurf2. The receptor, Smad7 and Smurf2 are internalized via caveolin-1-positive vesicles and this interaction leads to receptor ubiquitination that is followed by its degradation (Di Guglielmo et al., 2003). How- ever, this model is still under dispute since another group (Mitchell et al., 2004) has shown that degradation of the TGF-ȕ receptors occurs also in a clathrin-dependent manner and not through the lipid raft pathway. Moreover, no co-localization of the receptors with caveolin-1 could be detected, suggesting that lipid-rafts might not be involved in the regulation of TGF-ȕ receptor trafficking (Mitchell et al., 2004). The latter stand in contrast to the finding that the type I receptor can interact with caveolin-1 (Razani et al.,

22 2001). Adding to the confusion within the field, it remains unclear what causes receptors to segregate between clathrin-dependent and -independent pathways. Moreover, it is unknown in which compartments TGF-ȕ receptors are degraded. Inhibiting proteasomes or lysosomes delays and partially blocks the degradation of ubiquitinated TGF-ȕ receptors, suggesting that both pathways are involved (Kavsak et al., 2000). Recent data suggests however that lysosomes might be involved in degradation of the TGF-ȕ ligand, which dissociates from the receptor during clathrin-dependent trafficking in sorting endosomes, whereas the receptor alone is recycled back to the plasma membrane (Di Guglielmo et al., 2003; Mitchell et al., 2004). That would suggest that proteasomal components might play a central role in degradation of activated TGF-ȕ receptors.

2 Physiological functions of TGF-ȕ signaling in epithelial cells

The functional role of TGF-ȕ superfamily members depends greatly on the tissue type exposed to the specific ligand. It is well known that members of this family regulate many fundamental cell responses, such as growth, migration, adhesion, differentiation and modification of extracellular matrix components (Piek et al., 1999a). However, the effect of TGF-ȕ on epithelium remains probably of the highest interest, since carcinomas (tumors of epithelial origin) are the most common type of human cancer. TGF-ȕ is a well- known player controlling both positively and negatively tumor progression of epithelial cells. Its function seems to be bimodal: a) epithelial cells exposed to TGF-ȕ become rapidly growth inhibited via regulation of factors such as p21 and c-myc, which leads to cell cycle arrest, b) as neoplasia progresses, TGF-ȕ can act as a tumor promoter by changing the tumor cell’s differentiation status through the epithelial-mesenchymal transition (EMT) process (Roberts and Wakefield, 2003).

2.1 TGF-ȕ inhibits growth of epithelial cells

Regulation of the cell cycle by TGF-ȕ is a quite well understood process that involves orchestrated regulation of many factors (Moustakas, 2002; Siegel and Massagué, 2003). The induction of the cell cycle inhibitors p15 and p21 (Hannon and Beach, 1994; Datto et al., 1995; Reynisdottir et al., 1995; Par- dali et al., 2000b) and repression of factors promoting proliferation, for example c-myc or Id proteins (Pietenpol et al., 1990; Ling et al., 2002; Kang et

23 al., 2003a)(Paper I), are hallmarks of TGF-ȕ-induced growth inhibition in epithelial cells. TGF-ȕ activates growth inhibitory gene responses at any point in the cell- cycle phases G1, S or G2. However, TGF-ȕ inhibits the proliferation by ar- resting cells primarily in the G1 phase of the cell cycle (Laiho et al., 1990) and that is why TGF-ȕ addition to dividing cells that have already passed the restriction point of the cell cycle will not affect completion of the ongoing cycle. Cyclin-dependent kinases (CDKs) drive progression through the G1 phase of the cell cycle and inhibiting their activity is a major mechanism TGF-ȕ employs to block cell cycle progression. TGF-ȕ transcriptionally induces expression of the CDK inhibitors, p15 and p21 (Hannon and Beach, 1994; Datto et al., 1995; Li et al., 1995; Reynisdottir et al., 1995) via Sp1 binding sites and Smads were shown to contribute to this process since they can physically and functionally interact with Sp1 (Feng et al., 2000; Pardali et al., 2000b). For the cell cycle to progress, CDKs must be activated via association with specific cyclins (reviewed in: (Murray, 2004)). CDK4/6 activation requires association with cyclin D, whereas cyclin E activates CDK2. Binding of p15 and p21 inhibitors to specific CDK-cyclin complexes leads to inactivation of such complexes (in the case of p21 that binds CDK4/6-cyclin D complex) or disruption of such complexes (as happens when p15 binds CDK2-cyclin E complex). A major outcome of these activi- ties is accumulation of hypo-phosphorylated retinoblastoma protein (pRb), a major substrate of CDK. Hypo-phosphorylated pRb remains bound to E2F transcription factors, which leads to repression of major gene targets with crucial roles in progressing the cell cycle towards the S-phase (Mulligan and Jacks, 1998; Classon and Dyson, 2001; Trimarchi and Lees, 2002). Another mechanism governing the TGF-ȕ-induced cytostatic process is to repress expression of growth-promoting transcription factors. The TGF-ȕ- activated Smad protein complexes together with E2F4/5 and transcriptional repressor p107 target the promoter of the c-myc proto-oncogene (Chen et al., 2002). c-Myc, a ubiquitous promoter of cell growth and proliferation, is a member of the basic helix-loop-helix leucine zipper (bHLH-LZ) family of transcription factors (reviewed in: (Pelengaris et al., 2002)). c-Myc, together with another bHLH-LZ transcription factor, Max, promotes cell cycle progression through transcriptional induction of such genes as cyclin D2 and CDK4, which triggers G1-S progression of eukaryotic cells (Bouchard et al., 1999; Hermeking et al., 2000). The complex of cyclin-D2-CDK4 sequesters another CDK inhibitor p27, subsequently leading to its proteasomal degradation, which prevents p27 binding to cyclin-E-CDK2 complex (Steiner et al., 1995; O'Hagan et al., 2000). c-Myc was also shown to act as a transcriptional repressor with p15 and p21 being its primary targets. Negative regulation is achieved by Myc-Max complexes interacting with Miz1, a transcription factor positively regulating p15 and p21 promoters. Miz1 bound to Myc-Max can not associate with co-activator p300 and both p15 and p21 can

24 not be transcriptionally activated (Staller et al., 2001; Herold et al., 2002). Therefore, TGF-ȕ represses the expression of c-myc in order to deprive the cell of growth promoting functions and to enhance expression of cell cycle inhibitors – p15 and p21. TGF-ȕ blocks cell cycle progression also through repression and inactivation of Cdc25A, a phosphatase that acts as CDK activator. TGF-ȕ-mediated repression of cdc25A gene is mediated via E2F transcription sites with E2F4- p130 complexes playing a dominant role (Iavarone and Massagué, 1999). Cdc25A can also be inactivated by an inhibitory phosphorylation, executed by the RhoA-p160ROCK network (Bhowmick et al., 2003). Finally, TGF-ȕ represses expression of Id genes in order to inhibit proliferation of epithelial cells (Kang et al., 2003a)(Paper II). Id2 was shown to interact via its helix-loop-helix domain with the pocket domain of pRb (Iavarone et al., 1994). Such interaction, together with phosphorylation of pRb by CDKs, leads to release of E2F transcription factors from their repressive complex with pRb and enables free E2F to regulate the expression of genes involved in progression through the S-phase of the cell cycle. The role of Id proteins in TGF-ȕ-driven growth inhibition will be discussed in paragraph 4 of this thesis.

2.2 EMT and metastasis

As it was mentioned above, epithelial cell tumors are the most common human malignancies, but despite deep understanding of many aspects of carcinogenesis, the mechanisms governing invasion and metastasis are still poorly understood (Hanahan and Weinberg, 2000). Epithelial-mesenchymal transition, or EMT, a process in which epithelial cells acquire mesenchymal characteristics, is believed to be a crucial step during tumor expansion. In normal conditions EMT is a very important process, occurring predominantly during embryo- and organogenesis (Thiery, 2002; Tosh and Slack, 2002) when endoderm and mesoderm are formed, but EMT is also important for other processes such as tubulogenesis and branching in mammary glands and tissue reorganization during wound healing. EMT is a complex process in which epithelial cells undergo dramatic changes, such as acquisition a fibroblastoid phenotype, loss of expression of epithelial-specific proteins and induction of various mesenchymal markers, and finally digestion and migration through extracellular matrix (ECM) (Grünert et al., 2003). During this process epithelial cells loose polarity and acquire mesenchymal characteristics through down-regulation of proteins responsible for adherence and tight junctions, such as E-cadherin and ZO-1, and up-regulation of mesenchymal markers such as cytoskeletal proteins vimentin and Į-smooth muscle actin (Į-SMA). During the changes acquired during EMT, epithelial cells become more motile and migrate through ECM (Fig. 5) (Grünert et al., 2003).

25 The loss of epithelial cell contact with its surrounding cells and matrix is thought to be a critical part of EMT. Down-regulation of E-cadherin expression is a hallmark of EMT and most of our knowledge on how EMT is regulated at the molecular level comes from studies of E-cadherin gene regulation. Zing-finger transcriptional repressors, such as Snail, Slug and SIP1 were shown to bind E-boxes within the E-cadherin promoter leading to its repression (Nieto et al., 1994; Batlle et al., 2000; Cano et al., 2000; Carver et al., 2001; Comijn et al., 2001). The E2A gene products, E12 and E47, which belong to the basic-helix-loop-helix (bHLH) family of transcription factors, were also shown to be strong suppressors of the E-cadherin promoter via binding to E-boxes within the promoter (Perez-Moreno et al., 2001). Re- cently, Twist, another member of the bHLH transcription factor family, was shown to repress E-cadherin also via E-boxes and to contribute to metastasis through promoting EMT in breast cancer cells (Yang et al., 2004). But how important are those factors for tumor progression in vivo? Consistent with the mechanism of E-cadherin down-regulation, Snail is highly expressed in the infiltrating ductal carcinomas and some dedifferentiated tumors (Blanco et al., 2002) and high levels of Twist could be correlated with invasive breast carcinoma (Yang et al., 2004). Additionally, a recent study correlated the expression of Snail, Slug and SIP1 with highly metastatic ovarian and breast carcinoma (Elloul et al., 2005).

Figure 5. Schematic representation of EMT.

Nowadays there is growing evidence that EMT is an important process during tumor progression and that it is a critical step during tumor metastasis (Petersen et al., 2003; Thiery, 2003). Although it is difficult to distinguish tumor cells that underwent this process from host normal stromal cells, the increasing number of reports shows that EMT occurs also in vivo while carcinoma progresses. A colon carcinoma study showed the presence of E- cadherin negative cancer cells at the tumor invasive front, that were invading the surrounding stroma (Brabletz et al., 2001), suggesting that EMT occurs only at certain sites of tumors, from where de-differentiated, highly motile tumor cells can detach and begin the metastatic process. Similarly, in breast cancer, EMT can be measured at the margins of cancer cell groups of up to 20% of tumors (Dandachi et al., 2001). EMT in vivo is frequently described as the number of tumor cells that express mesenchymal markers such as

26 vimentin and tenascin-C (Dandachi et al., 2001) or stromelysin-3 (Ahmad et al., 1998). It was also suggested that human breast cancer epithelial cells are able to use the EMT process to differentiate into mesenchymal-like cells in order to form a non-malignant stroma that interacts with tumor cells in order to facilitate tumor growth (Petersen et al., 2003). TGF-ȕ is a well established growth factor that triggers EMT (Thiery, 2002). In murine mammary epithelial cells NMuMG, TGF-ȕ promotes strong EMT through type I and type II receptor complex and activation of Smads (Miettinen et al., 1994; Piek et al., 1999b)(Paper I), suggesting that TGF-ȕ-activated Smad proteins play a crucial role in EMT-induction. Other reports in the same cell type showed, however, that Smads alone are not sufficient to trigger EMT and that RhoA, Erk and/or p38 MAPK pathways in cooperation with integrin signaling are required (Bhowmick et al., 2001a; Bhowmick et al., 2001b; Zavadil et al., 2001; Bakin et al., 2002; Yu et al., 2002). Human keratinocytes HaCaT and canine kidney MDCK cells are other examples of well characterized models to analyze molecular mechanisms of changing epithelial cell plasticity in response to growth factors, such as TGF-ȕ. Mouse mammary polarized epithelial cells EpH4 undergo EMT in response to synergistic signaling pathways of TGF-ȕ and Ras (Janda et al., 2002a; Grünert et al., 2003; Gotzmann et al., 2004). In this cell model, oncogenic Ras, acting through Raf-kinase/ERK and PI3-kinase/PKB/Akt- kinase pathways (Downward, 1998), protects cells from TGF-ȕ-induced cell cycle arrest. Interestingly enough, Ras alone or TGF-ȕ alone are not able to induce EMT, but together they are sufficient to render EpH4 cells tumorigenic and a hyperactive Raf/MAPK pathway is required for TGF-ȕ-induced tumor progression (Janda et al., 2002a; Janda et al., 2002b). The most com- pelling non-Smad mechanism of induction of EMT by TGF-ȕ has been recently proposed by Ozdamar et al. (Ozdamar et al., 2005). The surface pool of the TGF-ȕ receptors has been found to reside around the apical cell pe- riphery that co-localized with ZO-1 (Ozdamar et al., 2005). The authors identified Par6 as an interacting partner of the TGF-ȕ receptors at tight junctions and suggested that this complex is important for EMT. Par6 interacts with TGF-ȕ type I receptor and it is phosphorylated at Ser345 by TGF-ȕ type II receptor after TGF-ȕ ligand binding (Ozdamar et al., 2005). The phosphorylated Par6 recruits the E3 ubiquitin ligase Smurf1 to the complex in order to control ubiquitin-dependent degradation of RhoA, a member of the Rho GTPase family. Thus, through degradation of RhoA, Smurf1 regulates EMT in response to TGF-ȕ (Wang et al., 2003a; Ozdamar et al., 2005). Despite the large number of in vivo models to study TGF-ȕ-driven EMT, the exact molecular mechanism of this process is poorly understood. Snail and SIP1, the negative regulators of E-cadherin, are transcriptionally induced by TGF-ȕ (Comijn et al., 2001; Peinado et al., 2003). Moreover, SIP1 was firstly identified as a Smad-interacting protein (SIP) (Verschueren et al., 1999) that together with Smad proteins can regulate transcription of genes

27 involved in EMT. On the other hand, repression of Id2 expression by TGF-ȕ was shown to be critical for TGF-ȕ to trigger EMT in many epithelial cells (Paper II)(Kondo et al., 2004). In this case, epithelial cells are depleted of the inhibitory effect of Id2 on E12/E47 bHLH transcription factors.

2.3 What triggers the switch from tumor suppressor to tumor promoter?

The described dual role for TGF-ȕ in tumor progression has been proven to occur in several in vivo models of tumorigenesis (Tang et al., 2003; Rein- acher-Schick et al., 2004; Tian et al., 2004). So, how is it possible that one signaling pathway can have opposite physiological effects on the same type of cells and tissues? The molecular mechanism of TGF-ȕ acting as a hero and traitor is still poorly understood (Roberts and Wakefield, 2003). During the course of tumor progression, cancer cells try to escape from the growth inhibitory pathway of TGF-ȕ without compromising its pro-oncogenic functions. Tumor cells try to disable the suppressor responses and enhance the pro-metastatic ones through many mechanisms (Wakefield and Roberts, 2002). Ras pathway hyper-activation was already mentioned before. Acti- vated Ras/MAPK attenuates the growth-inhibitory effect of TGF-ȕ and gives pro-mitogenic signals, hence it helps to promote TGF-ȕ-triggered EMT (Oft et al., 1996; Lehmann et al., 2000; Janda et al., 2002a; Janda et al., 2002b). Often tumor cells decrease levels or mutate TGF-ȕ receptors in order to at- tenuate the growth inhibitory response (Kim et al., 2000c; Bachman and Park, 2005). However, such deregulation of receptors usually results in loss of all TGF-ȕ responses and such cells are able to create hyperproliferating but non-metastatic tumors, pointing at TGF-ȕ as a tumor suppressor of non- aggressive adenomas (Markowitz et al., 1995; Chen et al., 2001; Pinto et al., 2003). Interestingly enough, disruption of TGF-ȕ signaling in highly aggressive breast cancer cells suppresses their metastatic potential, suggesting a pro-oncogenic role for TGF-ȕ in highly tumorigenic cancer cells (Tang et al., 2003; Tian et al., 2004). Furthermore, some pathways can modulate Smad activity or change the balance between Smad and other signaling pathways. For example, Ras can enhance the proteasomal degradation of Smad4, which modulates the growth inhibitory response to TGF-ȕ (Saha et al., 2001). The already discussed transcription factor SIP1 can bind Smad proteins and repress E-cadherin, promoting EMT and metastasis (Comijn et al., 2001). Lastly, Smad4 is the most commonly mutated or deleted member of the pathway in many tumors. Smad4 was found to be deleted in 50% of pancreatic carcinomas (Hahn et al., 1996) and Smad4 mutations were identified in more than 30% of metastatic colon cancers (Miyaki et al., 1999), supporting the notion that Smad4 is a potent tumor suppressor. In some cases the loss of

28 Smad4 not only correlates with loss of TGF-ȕ-regulated anti-mitotic responses, but also promotes highly aggressive behavior of tumor cells. The loss of Smad4 expression in colorectal carcinoma has been correlated with loss of E-cadherin expression and enhanced EMT and exogenous reintroduction of Smad4 into tumor cells led to induction of E-cadherin expression through a not yet deciphered mechanism (Reinacher-Schick et al., 2004). EMT is crucial in cancer progression since it makes tumor cells to change adhesive properties, allowing their migration and enhancing their metastatic properties. TGF-ȕ has additionally strong impact on tumor development, which we still do not understand. Therefore, there is a deep need to understand the exact mechanism of how TGF-ȕ regulates tumor progression and metastasis in order to specifically target its signaling pathway, which will inhibit EMT-stimulating functions without affecting its cytostatic effects on cancer cells.

3 BMP and disease

BMP-related research has been mostly focused on the role of BMPs in osteoblast differentiation and bone formation and only few studies decipher the role of these growth factors in disease. Several mutations within the BMP type I/II receptors genes have been associated with primary pulmonary hypertension (PPH), a disease characterized by an increase in vascular resis- tance, which results in right ventricular failure (Eddahibi et al., 2002; Takeda et al., 2004). The exact mechanism of BMP involvement in this disease remains unknown, however BMP-2 has been shown to inhibit growth of vascular smooth muscle cells, so that mutations in BMP signaling could lead to uncontrolled proliferation of those cells, leading to defective vasculature (Nakaoka et al., 1997; Willette et al., 1999). BMP over-expression has been found in several bone tumors, such as osteosarcomas or malignant fibrous histiocytomas, and has been correlated with de-differentiated cancer cells, resulting in a poorer prognosis for patients (Yoshikawa et al., 2004). Poorly differentiated human prostate tumors show a loss of expression of a number of BMP receptors, suggesting that such loss is important for prostate cancer progression (Kim et al., 2000a). Similarly, the loss of BMP type II receptor (Kim et al., 2004), BMP-2, Smad4 and Smad8 (Horvath et al., 2004) was correlated with progression to a more aggressive type of prostate cancer. The exact mechanism of anti-tumorigenic role of BMPs remains unknown, but recent data suggest that BMP can inhibit growth and proliferation of prostate cancer cells through induction of cell cycle inhibitors, such as p21 (Miyazaki et al., 2004). The opposite results were found in malignant melanoma cells, in which high expression of BMP-4 and -7 was detected and both BMPs were found to be required for melanoma tumor progression (Rothhammer et

29 al., 2005). Yet, the precise role BMPs play during progression of various carcinomas remains open.

4 Gene regulation by TGF-ȕ family members

Recent genomic-scale studies have shown that TGF-ȕ superfamily members are able to regulate many genes whose function is involved in cell cycle regulation, apoptosis, differentiation and development. The growing number of reports proves that, especially TGF-ȕ1, can regulate more than 5% of the total transcribed genes in a given cell. So far, microarray screens have been performed in many TGF-ȕ1-responsive cell lines. cDNA microarray analysis of TGF-ȕ1-treated normal human keratinocytes HaCaT revealed that this cytokine regulates more than 700 genes, many of which are involved in regulating epithelial plasticity (Zavadil et al., 2001). Combined cDNA microarray and promoter transactivation studies in human dermal fibroblasts suggested that TGF-ȕ1 is a potent inducer of extracellular matrix components such as collagens (Verrecchia et al., 2001). Also a direct transcriptomic analysis of tumors metastasing to bone was performed and a potential group of genes involved in TGF-ȕ1-driven metastasis was identified (Kang et al., 2003b). Finally, the groups of genes expressed during EMT, metastasis and scattering in connection to TGF-ȕ signaling in mesenchymal-like or mammary gland epithelial cells were identified (Jechlinger et al., 2003; Xie et al., 2003). However, most of these studies concentrate on identifying new clusters of genes regulated by TGF-ȕ1 and related factors, without explain- ing the mechanisms by which newly revealed transcriptional targets are involved in TGF-ȕ-regulated biological processes. The increasing number of reports suggests the importance of non-Smad signaling pathways in regulation of TGF-ȕ-dependent processes such as differentiation and inhibition of proliferation in epithelial cells (reviewed in: (Derynck and Zhang, 2003)). Questioning the importance of Smads for TGF-ȕ signal transduction and gene regulation underscores the need to an- swer whether Smads play a central role in TGF-ȕ-driven biological events. Large scale studies of gene expression performed so far using antisense downregulation of Smad2, 3 and 4 (Kretschmer et al., 2003), Smad2 and Smad3 knock-out fibroblasts (Yang et al., 2003), conditional Smad4 knockout in the breast (Li et al., 2003), or our own work using Smad4-deficient epithelial cells (Paper II) or murine mammary epithelial cells (Paper I), support that Smads play a central role in the TGF-ȕ signaling pathway that leads to cell cycle regulation and differentiation. Interestingly, recent study in a pancreatic cancer cell line indicated that TGF-ȕ-driven cell cycle inhibition via up-regulation of p21 might be Smad-independent (Ijichi et al., 2004),

30 pointing that in certain cell types TGF-ȕ can employ alternative pathways to perform its biological function. The BMP-driven responses have not been studied as extensively yet and the majority of research is focused on the role of BMPs in osteoblast differentiation and bone formation. The increasing number of microarray studies aim to identify clusters of genes specifically involved in those processes (de Jong et al., 2002; Clancy et al., 2003; Korchynskyi et al., 2003; Peng et al., 2003; de Jong et al., 2004). An open question remains about the involvement of Smad proteins per se and the role of newly characterized target genes during BMP-driven processes, such as bone formation but also during cancer progression. Smads seem not to be alone in executing their biological responses. A cross-talk between TGF-ȕ signaling components and other pathways is well documented. The already mentioned activation of p38/MAPK pathways by TGF-ȕ in order to regulate together with Smad proteins genes involved in EMT can serve as one example (Zavadil et al., 2001; Bakin et al., 2002). Also, Jagged1/Notch signaling together with the TGF-ȕ/Smad3 pathway act together in epithelial cells in order to execute EMT through regulation of expression of Hey1 transcriptional repressor (Zavadil et al., 2004).

5 Id proteins

Among the genes identified as transcriptional targets of the TGF-ȕ superfamily members, Id (inhibitor of DNA binding or inhibitor of differentiation) proteins gain a growing interest in the field. Id family members (Id1-4) were first identified as negative regulators of basic helix-loop-helix (bHLH) transcription factor proteins (Murre et al., 1989; Benezra et al., 1990). bHLH proteins belong to a large family of HLH transcription factors consisting of more than 240 members. The members of this family play crucial roles in many cell growth and differentiation processes (Benezra et al., 2001; Lasorella et al., 2001; Rivera and Murre, 2001; Yokota, 2001; Zebedee and Hara, 2001). bHLH proteins were divided into two subgroups: class A, that contains ubiquitously expressed transcription factors (also referred as E proteins: E2A gene products or E2-2 and HEB proteins), and class B comprising tissue restricted proteins (such as MyoD and Myogenin, NeuroD, Mash, neurogenin, HES, SCL and Lyl-1 and many others) (Norton et al., 1998). All bHLH proteins share the characteristic feature of forming homo- and heterodimers through their highly conserved HLH domain. Typically, heterodimeric complexes consist of one bHLH protein of class A and one of class B. Dimerized bHLHs through their DNA- binding domain interact with the characteristic DNA E-box sequence: CANNTG, N-box sequence: CACNAG or Ets sites: GGAA/T and they act as

31 transcriptional activators or repressors. bHLH negative regulators, Id proteins, lack the basic DNA-binding region but they still carry the HLH domain. As a consequence of high affinity Id-bHLH interaction, bHLH proteins dissociate from each other and from DNA and are no longer able to regulate transcription (Fig. 6). In addition to bHLH proteins, Ids target also other transcription factors and cell cycle regulators, namely Ets and Retino- blastoma (pRb) family members. Regulation of the levels of Id family members during cell cycle progression has been widely observed (Hara et al., 1994; Iyer et al., 1999) and interaction of Id2 with Rb family members (p107, p130 and pRb) might explain the positive effects of Id on cell cycle regulation (Iavarone et al., 1994; Lasorella et al., 1996; Lasorella et al., 2000). This interaction is dependent on the pocket domain of pRb and the HLH domain of Id2. Together with phosphorylation of pRb by cyclin- dependent kinase 4/6 (CDK4/6) or CDK2, this interaction will lead to release of pRb from its repressive complex with E2F transcription factors. Therefore, free E2F is able to regulate the genes involved in progression through the S-phase of the cell cycle. Furthermore, Id2 and Id3 are also phosphorylated by CDK2 in late G1 phase which might alter Id2 and Id3 activity and possibly allowing them to interact with other proteins (Deed et al., 1997; Hara et al., 1997). In addition, Id proteins undergo rapid turnover in the cell with their levels regulated by the ubiquitin-proteasome pathway (Bounpheng et al., 1999). The importance of Id proteins have been highlighted through generation Id knockout animals (Ruzinova and Benezra, 2003). Single Id1 and Id3 knockouts mice are viable and fertile and do not have any dramatic defects (Yan et al., 1997; Pan et al., 1999). However, Id1 Id3 double knockout mice are embryonic lethal, mostly due to defected brain angiogenesis and prema- ture neuronal differentiation (Lyden et al., 1999). Id4-/- animals show reduc- tion in brain size, implying a critical role for Id4 in regulating neural stem cell proliferation and differentiation (Yun et al., 2004). The most severe phenotype can be observed in Id2-null animals (Yokota et al., 1999). They are characterized by very low survival rate, reduced population of natural killer cells (Yokota et al., 1999), Langerhans and splenic dendritic cells (Hacker et al., 2003). In addition, they lack lymph nodes and Peyer’s patches (Yokota et al., 1999). Moreover, Id2-null female mice reveal a striking phenotype in the mammary glands. Lobulo-alveolar development during pregnancy of knockout animals is severely defected, which results in impaired maturation of lactating cells (Mori et al., 2000), suggesting that Id2 plays a central role in differentiation of epithelial cells in the mammary gland. The positive influence Ids have on cell proliferation makes Id genes good candidates to be oncogenes and deregulation of Ids has been detected in several types of tumors (Perk et al., 2005). Their expression level is affected by several oncogene products, such as Ras (Tournay and Benezra, 1996; Bain et al., 2001) and EWS-ETS (Nishimori et al., 2002; Fukuma et al., 2003) but so

32 far mutations within the Id genes have not been identified in tumors (Arnold et al., 2001; Casula et al., 2003), suggesting that Ids are not classical oncogenes. However, several tumors express elevated levels of Id proteins and in many cases increased Id levels correlate with tumor proliferating indices and often with invasiveness (Fong et al., 2004), suggesting an important role of Id proteins in tumor progression. However, the exact mechanism and involvement of Ids in carcinogenesis requires more rigorous testing.

Figure 6. Id proteins act as negative regulators of bHLH-mediated transcription. In the absence of Id, class A and B bHLH proteins form complexes that bind DNA at specific E-box sequences to regulate groups of genes involved in differentiation. Growth factor stimulation (for example BMP) can lead to increased level of Id proteins in the cell. Id binds class A bHLH and disrupts transcriptionaly active bHLH heterodimers. E-box-dependent gene targets can not be regulated any longer.

The interaction of Id proteins with class A bHLH factors, such as HEB, E2-2 and E2A gene products (E2-5, E12 and E47), defines Id as negative regulators of differentiation. The Id-class A bHLH complex has high affinity and leads to dissociation of heterodimeric bHLH complexes from the DNA. First such evidence came from studies in Drosophila, where the emc gene product (ortholog of mammalian Id) was found to antagonize Daughterless and Achaete-scute bHLH proteins and to inhibit sex determination and neurogenesis (Ellis et al., 1990; Garrell and Modolell, 1990). Subsequent studies have shown that Id can negatively regulate differentiation of many mammalian cells and, since BMP is a potent regulator of Id (Miyazono and Miya- zawa, 2002), a clear connection between both pathways can be drawn. Sev-

33 eral members of the BMP family (mostly BMP-2 and BMP-7) have been recognized among the most prominent growth factors increasing the expression of Ids in many cell types including osteoblasts (Ogata et al., 1993), embryonic stem cells (Hollnagel et al., 1999), breast cancer cells (Clement et al., 2000), vascular smooth muscle cells (Dorai and Sampath, 2001) and other cell types. BMPs can determine the cell fate of mesenchymal cells by inducing Id1 that through interaction with E2A protein might interfere with MyoD-driven myogenesis (Neuhold and Wold, 1993; Katagiri et al., 1994). In neuronal cells, BMP-2 induces expression of Id1 and Id3 that leads to inhibition of neurogenesis through counteracting the transcriptional activation by Mash1, neurogenin and NeuroD (Nakashima et al., 2001). Regula- tion of Id expression by BMP in endothelial cells (Valdimarsdottir et al., 2002) implicates the important role of BMP and Id proteins in activation of endothelial cells and angiogenesis. Consistent with this data, Smad1 and Smad5-null mice (Chang et al., 1999; Yang et al., 1999; Lechleider et al., 2001) and Id1/Id3 double knockout mice (Lyden et al., 1999) exhibit similar abnormalities in the vascular system, suggesting strong links between Smad and Id proteins in angiogenesis. Recently, it has been found that TGF-ȕ1 is also involved in the transcriptional regulation of Id proteins (Kee et al., 2001; Ling et al., 2002; Chambers et al., 2003; Hacker et al., 2003; Kang et al., 2003a; Sugai et al., 2003)(Paper II), but the biological consequence of such regulation remains poorly understood. Recent reports imply that TGF-ȕ-driven down-regulation of Id1, 2 and 3 is associated with TGF-ȕ-mediated inhibition of proliferation in epithelial cells (Kang et al., 2003a)(Paper II). However, it remains unclear if this effect is the result of the Id-mediated regulation of pRb activity or transcriptional regulation of cell cycle regulators such as p15, p16, p21 (Prabhu et al., 1997; Pagliuca et al., 2000) or c-myc (Lasorella et al., 2000). In addition, the ini- tially identified involvement of Id2 in regulation of the c-myc promoter (Lasorella et al., 2000) is under dispute (Vandesompele et al., 2003; Wang et al., 2003b). Finally, our own study (Paper II) strongly suggests a critical role of Id2 and Id3 as negative regulators of TGF-ȕ-dependent EMT. However, the number of known genes that are regulated by Id proteins remains low and further studies to understand the function of Id in TGF-ȕ/BMP signaling must be performed.

6 SNF1LK

Sucrose-non-fermented 1-like kinase (SNF1LK) has been originally identified during a search for novel protein kinases expressed during heart development in the mouse and named Msk (myocardial SNF1-like kinase) (Ruiz et al., 1994), due to its high sequence similarity to a yeast SNF1, a kinase

34 regulating gene expression in response to metabolic stress (Lo et al., 2001). Based on inspection of the human kinome, SNF1LK belongs to a AMPK- related family of Ser/Thr kinases comprising 13 structurally related proteins (Fig. 7). Despite their sequence similarity, members of this family appear to have differential functions within the cell.

Figure 7. Dendrogram of AMPK subfamily of protein kinases. Based on: (Manning et al., 2002).

The name of the family comes from a yeast homologue of SNF1 and a member of the family, AMP-activated protein kinase (AMPK). Similarly to SNF1, AMPK is activated by a low energy charge of the cell reflecting an increase in the ratio between AMP and ATP. Activated AMPK phosphorylates certain regulatory proteins in order to switch off ATP-consuming processes and switch on ATP-producing catabolic pathways and restore the normal ATP level in the cell (Hardie et al., 2003). Microtubule regulating kinases (MARKs), other members of the AMPK-related family, play key roles in controlling cell polarity. MAPKs through phosphorylation of microtubule associated proteins (MAPs), increase dynamics of microtubules, which form a network regulating cellular transport, cell shape and polarity (Drewes et al., 1998). The function of the remaining members of the family is poorly understood. NUAK1 (also known as ARK5) is thought to be activated by Akt and suppresses death caused by glucose starvation (Suzuki et al., 2003). NUAK2 (or SNARK) has been described as a component of the cellular stress response (Lefebvre and Rosen, 2005). Finally, all members of AMPK family of kinases have been shown to be activated by direct phosphorylation by the master kinase and tumor suppressor LKB1, suggesting a possible role in tumor suppression (Lizcano et al., 2004).

35 The first study described the expression and potential role of SNF1LK (or Msk) in heart development (Ruiz et al., 1994). Later, SNF1LK has been identified to be induced in the adrenal gland of rat by high-salt diet and in PC12 cells by depolarization and at this time it was named SIK (for salt- inducible kinase) or KID-2 (for kinase induced by depolarization-2) (Wang et al., 1999; Feldman et al., 2000). The tissue distribution study has showed that SNF1LK is expressed not only in the brain but also in other tissues (Feldman et al., 2000; Horike et al., 2003). Subsequent studies have identified two isoforms of SNF1LK, termed SIK2 (or SNF1LK2) and SIK3 (Katoh et al., 2004). SIK2 is expressed in adipose tissue, whereas SIK3 has a ubiquitous expression pattern. Structurally SNF1LK consists of an N-terminal serine/threonine kinase domain, occupying about 1/3 of the protein that is followed by an ubiquitin- associated domain (UBA) (Fig. 8). Comparison of the C-terminal stretch of SNF1LK with all known domains found in public databases (InterPro from EMBL-EBI) has not yielded in any positive score (Kowanetz M., unpub- lished observation), suggesting that the C-terminal part of SNF1LK might contain a novel domain. The substrate of the kinase domain has not been identified yet, although in vitro kinase essays using commercially available substrates have shown that a canonical phosphorylation motif of SNF1LK might be (Hy)((B)X/X(B))XX(S/T)XXX(Hy), where S/T is the phosphorylation site on Ser or Thr, (Hy) and (B) are hydrophobic and basic residues, respectively (Horike et al., 2003). The UBA domain is a small, about 40 amino-acid long domain, shown to function in ubiquitination processes (Buchberger, 2002). It is believed that it mediates interaction with ubiquitin and ubiquitinated proteins, however its exact function remains poorly understood.

Figure 8. The domain structure of SNF1LK. KINASE, Ser/Thr kinase domain; UBA, ubiquitin associated domain.

The function of SNF1LK has not been studied extensively and so far its role has been described only in the processes related to steroidogenesis and adipogenesis (Okamoto et al., 2004). The limited number of studies has shown that SNF1LK can repress ACTH-induced expression of steroidogenic genes, such as CYP11A and StAR in mouse adrenocortical tumor-derived Y1 cells (Lin et al., 2001; Doi et al., 2002). These reports have shown that SNF1LK negatively regulates the transcriptional activity of cyclic AMP response element binding protein (CREB), by acting on the basic leucine zipper domain of CREB and most probably preventing the proper activation of the CREB-containing transcriptional complex (Lin et al., 2001; Doi et al.,

36 2002; Takemori et al., 2002). However, the exact mechanism of how SNF1LK would interfere with CREB and CREB-activated transcription has not yet been described. Interestingly enough, recent reports have suggested that SNF1LK might be involved in other pathways than regulation of CREB activity. Kin-29 (or Sma-11) that is believed to be a SNF1LK homolog of C. elegans, has been found to be involved in the Sma pathway in nematodes (Lanjuin and Sen- gupta, 2002; Maduzia et al., 2005). KIN-29, similarly to other members of TGF-ȕ signaling pathway in C. elegans, regulates the body size and KIN-29 mutants result in small animals, however KIN-29 does not affect the tail size of nematodes. Moreover, KIN-29 is able to suppress the long mutant phenotype created by overexpression of dbl-1, a C. elegans TGF-ȕ-like ligand, suggesting that KIN-29 genetically interacts with the Sma signaling pathway (Maduzia et al., 2005). In the second report, the involvement of human kinases in clathrin- and caveolae/raft-mediated endocytosis has been ex- plored (Pelkmans et al., 2005). Using high-throughput RNA interference and using viral entry screens to monitor different types of vesicular trafficking in HeLa cells, Pelkmans et al identified a large number of kinases that are po- tentially involved in endocytic transport (Pelkmans et al., 2005). HeLa cells with decreased level of SNF1LK showed changes in distribution and mor- phology of caveolin-1–GFP-positive structures and their accumulation in a condensed perinuclear spot. This striking phenotype of down-regulation of SNF1LK by means of RNA interference technology suggests that SNF1LK plays a critical role in endocytic processes. The role of SNF1LK isoforms is even more ill understood. SIK2 (or SNF1LK2) has been shown to be involved in insulin signaling through phosphorylation of insulin-receptor-substrate-2 (IRS-2) (Horike et al., 2003). Moreover, a recent study has shown that SIK2, similarly to SNF1LK, is involved in regulation of CREB activity through interaction and phosphorylation of CREB coactivator called transducer of regulated CREB activity (TORC) (Screaton et al., 2004).

37 Aims of the present study

To understand the specific role of R-Smads and Co-Smad in TGF- ȕ/BMP-regulated proliferation of epithelial cells or epithelial- mesenchymal transition.

To identify and characterize novel gene targets, whose regulation is dependent on the presence of Smad4 and to link their function to TGF- ȕ/BMP-mediated biological effects.

To identify and characterize the function of novel TGF-ȕ-regulated gene targets in regulation of the signaling pathway per se.

38 Present investigation

1 Paper I

TGF-ȕ and the Smad Signaling Pathway Support Transcriptomic Repro- gramming during Epithelial-Mesenchymal Cell Transition.

The central role of Smads in executing EMT is still under dispute (Derynck and Zhang, 2003). In this study we investigated the effects of various TGF-ȕ family members on the EMT process of several epithelial cell lines. We investigated the potency of several members of the TGF-ȕ superfamily to induce growth inhibition and EMT in epithelial cells of different origin. We have found that only TGF-ȕ1, -ȕ2 and -ȕ3 were able to induce EMT in mammary and lung epithelial cells, whereas this effect was less pronounced in normal keratinocytes. Activin-A did not stimulate any mor- phological changes in mammary cells and keratinocytes, however it induced scattering in normal lung epithelial cells. Finally, BMP-7 did not induce EMT or scattering of any cell type tested. Similar to the EMT results, only TGF-ȕs could induce a potent cell cycle arrest of epithelial cells, whereas BMP was unable to evoke a potent cytostatic effect. We moved on to test whether the differential response of epithelial cells to different ligands is due to the expression pattern of the specific receptors. In mouse mammary epithelial cells NMuMG we could detect expression of several receptors for all tested ligands (TGF-ȕs, BMP-7 and activin-A). NMuMG do not express ALK-1 (endothelial TGF-ȕ type I receptor), ALK-6 (BMP receptor type IB) and ALK-7 (activin receptor type IC or nodal receptor). However, we have detected expression of four type II receptors for all ligands tested, ALK-2 (activin receptor type IA), ALK-3 (BMP receptor type IA), lower levels of ALK-4 (activin receptor type IB) and ALK-5 (TGF-ȕ receptor type I). Therefore the inability of activin-A and BMP-7 to induce EMT in NMuMG cells cannot be explained by the lack of expression of receptors for these ligands, suggesting that induction of EMT is an individual characteristic of TGF-ȕ1, -ȕ2 and -ȕ3. To confirm that activin and BMP are unable to promote EMT, we analyzed the effect of adenoviral over-expression of all constitutively active (CA) type I receptors: ALK-1(CA) to -7(CA). In agreement with the above, BMP branch receptors (ALK-1, -2, -3 and -6) did not induce EMT in NMuMG cells. However, TGF-ȕ branch receptors (ALK-4 for ac-

39 tivin, ALK-5 for TGF-ȕ and ALK-7 for nodal) induced a potent EMT. To confirm that also endogenous ALK-4 can contribute to EMT, we over- expressed in NMuMG cells dominant-negative versions of all type I receptors that carry a lysine to arginine mutation in the kinase active site. Over- expression of such mutant of ALK-4, ALK-5 and ALK-7 could block TGF- ȕ1-induced EMT, suggesting that in mammary epithelial cells all TGF-ȕ branch receptors are able to promote EMT; however ALK-7 is not expressed at all in NMuMG cells. We confirmed this finding using a small-molecular- weight inhibitor (SB431542) which selectively inactivates the ALK-4, ALK- 5 and ALK-7 receptor kinases (Inman et al., 2002a). When added to the cells, this inhibitor could block TGF-ȕ1-induced EMT in NMuMG cells. In order to investigate the involvement of the Smad-dependent pathway into TGF-ȕ1-mediated EMT, we over-expressed dominant-negative forms of Smad2, Smad3 or Smad4. The presence of these proteins could efficiently inhibit the EMT process in NMuMG cells despite the stimulation with TGF- ȕ1. Similar results were obtained when one of the inhibitory Smads, Smad7, was introduced to the cells. In contrast, Smad6, which mainly blocks the BMP signaling pathway, was not able to block EMT mediated by TGF-ȕ ligand. Moreover, over-expression of wild-type forms of Smad2, Smad3 and Smad4 but not Smad1 or Smad5 led to potent EMT. Thus endogenous TGF- ȕ-type Smad activity is required for the EMT process initiated by TGF-ȕ1 stimulation. In order to identify novel markers of EMT and novel genes involved in EMT, and to understand the mechanism by which TGF-ȕ1 and its receptors regulate cellular differentiation and lead to EMT in mammary epithelial cells, we performed a large scale transcriptomic analysis. Measuring of expression level of genes after 2, 8 and 36 hours of TGF-ȕ1 treatment led to formulating a short list of TGF-ȕ-responsive genes, whose functional properties and expression pattern strongly link them to the EMT process. Since BMP-7 or BMP type I receptors fail to induce EMT, we postulated that genes with the same pattern of regulation by both TGF-ȕ1 and BMP-7 might not be crucial for the EMT process. Based on differential regulation of selected genes by TGF-ȕ1 and BMP-7, we narrowed the list to genes responding only to TGF-ȕ1 but not to BMP-7 and we proposed that this group of genes is involved in TGF-ȕ1-driven EMT in NMuMG cells. We also confirmed that regulation of these genes is dependent on Smad signaling by over-expressing a mutant form of the ALK-5 receptor that is unable to interact with R-Smads (ALK-5(TD)mL45). In the presence of this mutant the selected genes could no longer be regulated. Finally, in order to make our statement, that selected genes are important for EMT, we have analyzed the expression of our candidate genes in two other cell lines: human normal mammary epithelial cells (HMEC) that undergo very potent EMT when treated with TGF-ȕ1 and human keratinocytes HaCaT that exhibit weak scattering and cytoskeletal changes in response to

40 TGF-ȕ1. Comparison of the expression pattern of the selected target genes in NMuMG, HMEC and HaCaT cells allowed us to propose that Muc1, Fbln2, Hmga2, Lmcd1, Ssb1, Zyx, and Msn are newly identified targets of the TGF- ȕ pathway that might play a functional role in the process of EMT. In this work we provided a comprehensive analysis of most components of the TGF-ȕ superfamily signaling pathways using the NMuMG cell model system and we identified a group of novel gene targets of this pathway that might be involved in TGF-ȕ-mediated transdifferentiation leading to metastasis. We showed that mammary epithelial cells can undergo EMT in the presence of TGF-ȕ1 stimulation or introduction of constitutively-active ALK-4, ALK-5 or ALK-7 receptors. Analysis of endogenous receptor expression revealed that only ALK-5 is expressed in NMuMG cells at the level that is required for normal function of the signaling pathway. Moreover, through over-expression of dominant-negative Smad2, Smad3 or Smad4 or fully active Smad7, we demonstrated that TGF-ȕ-driven transdifferentiation of epithelial cells is a Smad-dependent process. In addition, we identified a small group of novel target genes, and we proposed that their regulation is important in order for TGF-ȕ1 to achieve its pro-tumorigenic program.

2 Paper II

Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor ȕ and bone morphogenetic protein.

In order to investigate the importance of Smad4 in TGF-ȕ signaling and gene regulation, we have performed a high-throughput gene expression analysis in epithelial cells. As cell model we decided to use human mammary epithelial cells MDA-MB-468 that lack endogenous Smad4. Adenoviral expression of an unrelated protein, GFP, or Smad4 led us to identify 173 genes, the regulation of which was dependent or independent on the presence of Smad4. The majority of them (161 genes) were found to be Smad4-dependent and regulation of only 12 genes did not require the presence of Smad4. Within the Smad4-dependent group of regulated genes, 41 transcripts were exclusively regulated by TGF-ȕ1 and 88 by BMP-7, whereas only 25 genes were common targets of both growth factors. We went on with our study with the aim to identify a group of genes, the regulation of which is important for TGF-ȕ- and BMP-regulated biological processes in epithelial cells. Among the common gene targets of TGF-ȕ1 and BMP-7 were Id2 and Id3, which have not been previously studied extensively in epithelial cells responding to these growth factors. We demonstrated that Id2 and Id3 expression in response to TGF-ȕ1 was bimodal, with

41 an early mRNA induction phase followed by a late phase of down- regulation. The BMP-7 response was continuously sustained and positive. Previous reports (Iavarone et al., 1994) suggested that members of the Id family are involved in regulation of the cell cycle through binding to pRb. Therefore, we investigated if regulation of both Id2 and Id3 is important for the cytostatic effect of TGF-ȕ in epithelial cells. Interestingly, adenoviral over-expression of Id2 and/or Id3 could block TGF-ȕ1-mediated growth inhibition in human keratinocytes HaCaT and mouse mammary epithelial cells NMuMG. Moreover, small interfering RNA (siRNA)-driven down- regulation of endogenous Id2 or Id3 sensitized the cells to BMP-7. What drew our attention was that Id proteins are well known inhibitors of differentiation. However, Id involvement in EMT has not been studied so far. Interestingly enough, ectopic over-expression of Id2 and to a lesser ex- tend Id3 could block TGF-ȕ1-driven EMT. Hence, down-regulation of endogenous Id2 and Id3 proteins using siRNA enabled BMP-7 to regulate EMT markers such as Į-SMA, ZO-1 or E-cadherin. Simultaneous stimulation of mouse lens epithelial Į-TN4 cells with TGF- ȕ1 and BMP-7 increased the Id2 and Id3 protein levels, suggesting that BMP-7 can antagonize the TGF-ȕ response in epithelial cells. Stimulation with both factors led to complete block by BMP-7 of TGF-ȕ1-driven EMT of Į-TN4 cells. Moreover, the antagonistic effect of BMP-7 over TGF-ȕ1 was abolished in the presence of specific siRNA against both Id2 and Id3, suggesting that BMP-7 can act as TGF-ȕ1 inhibitor through differential regulation of Id proteins.

Figure 9. Id proteins are critical regulators of growth and EMT responses to TGF-ȕ in epithelial cells.

To sum up, we have identified a number of TGF-ȕ1/BMP-7-regulated novel genes. Expression of these genes largely depends on the presence of

42 Smad4, suggesting that Smad4 plays a central role in executing TGF-ȕ- dependent responses in epithelial cells. The comparison of genes regulated either by TGF-ȕ1 or BMP-7 revealed a very short common transcriptional program, that included genes mostly implicated in cell cycle regulation (p21, c-myc, Id1-3). So far, it remains unclear if the growth factor-specific biological effects are dependent on common or distinct gene targets regulated either by TGF-ȕ or BMP. Importantly, we showed that differential regulation of Id2 and Id3 by TGF-ȕ1 and BMP-7 can explain distinct physiological responses of these two pathways in epithelial cells. Knockdown of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7, so BMP could act as potent growth inhibitor and inducer of EMT. In contrast, overexpression of Id proteins blocked TGF-ȕ1-mediated transdifferentiation and suppression of proliferation. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-ȕ pathways (Fig. 9).

Based on studies in papers I and II we now propose a novel operational tool for screening for TGF-ȕ-regulated factors critical for EMT. They must respond to TGF-ȕ but not to BMP. Alternatively, TGF-ȕ and BMP should lead to an opposite regulation of such factors as we showed for the Id proteins.

3 Paper III

The inducible kinase SNF1LK regulates TGF-ȕ receptor signaling and degradation.

Our previous work (Paper I and II) has strongly suggested that Smads are pivotal molecules transducing signals from the receptor to the nucleus, in order to execute a specific gene expression pattern. In addition, we showed that both Smads and specific TGF-ȕ receptors are critical for triggering EMT. Therefore, the proper regulation of the TGF-ȕ signaling flow is important in order to achieve the desired biological activity. The strength and duration of the signaling pathway is often regulated by receptor-mediated endocytosis. Endocytosis and trafficking of the TGF-ȕ receptors is a critical step, which, together with shuttling of activated Smads between cytoplasm and nucleus, regulates the proper flow of TGF-ȕ signal transduction. In paper III we describe the identification of a novel negative regulator of the TGF-ȕ signaling pathway, SNF1LK. We have identified SNF1LK as an immediate-early gene induced by the TGF-ȕ/Smad pathway in the course of the microarray screen in human mammary epithelial cells MDA-MB-468 (Paper II). The induction of

43 SNF1LK requires the presence of Smad4. Knocking-down the endogenous SNF1LK using siRNA enhanced the overall potency of endogenous TGF-ȕ signaling, as measured by expression of several known TGF-ȕ-regulated gene targets. Conversely, over-expression of exogenous SNF1LK led to a relative decrease of the TGF-ȕ-inducible 3TP-lux promoter-reporter and partially inhibited the activation of Smad2 and Smad3 as measured by their phosphorylation status. Since the levels of Smads were not affected, we tested the effect of over- expression of SNF1LK on the levels of TGF-ȕ type I receptor ALK-5. The over-expression of SNF1LK affected the turnover of the receptor and induced the receptor degradation. Furthermore, we have demonstrated that only the actively signaling, but not the inactive receptor was targeted for SNF1LK-mediated degradation. The turnover rate of molecules not related to TGF-ȕ signaling, such as CIN85, EGFR and GFP, was not affected by SNF1LK. Using specific inhibitors we showed that SNF1LK-induced degradation is mediated by both proteasomes and lysosomes.

Figure 10. The current model of the role of SNF1LK in the TGF-ȕ receptor degradation process.

In the course of biochemical studies we have shown that SNF1LK interacts with Smad7. Moreover, Smad7 brings SNF1LK into the proximity of both ALK-5 and Smurf2, in order to mediate a potent down-regulation of the receptor. Using a series of SNF1LK mutants we could show that both phosphorylation of Smad7 by SNF1LK and interaction of SNF1LK with ubiquit-

44 inated ALK-5 receptor via SNF1LK’s UBA domain mediate an efficient removal of the activated TGF-ȕ receptor. In addition, SNF1LK co-operates with Smurf2 in degradation of ALK-5. Smurf2 first ubiquitinates the receptor, then SNF1LK binds ubiquitinated ALK-5 and targets it for degradation. Moreover, we could show that SNF1LK actively interacts with proteasomes, suggesting that the degradation of an activated pool of TGF-ȕ receptors occurs via proteasomal complexes. In conclusion, we have identified SNF1LK as a novel gene target of the TGF-ȕ/Smad pathway. We propose that SNF1LK plays a critical part in degradation of activated TGF-ȕ receptors. First of all, SNF1LK associates and phosphorylates Smad7. Additionally, through interaction with Smad7, SNF1LK is able to reside close to the activated receptor. Moreover, Smurf2 ubiquitinates ALK-5 and once the receptor is ubiquitinated, SNF1LK associates with its poly-ubiquitin chains and targets the receptor for proteasomal degradation. Interestingly, we were able to show for the first time that degradation of activated TGF-ȕ receptors is mediated by proteasomes. Thus, in paper III we propose a novel function for SNF1LK, in which SNF1LK joins Smad7 and Smurf2 in the negative feedback loop of TGF-ȕ signaling (Fig. 10).

45 Future perspectives

Members of the TGF-ȕ superfamily of growth factors regulate a number of key processes influencing the fate of several cell types. The first part of my work focuses on the involvement of specific members of the family in regulation of differentiation of epithelial cells, EMT. The growing evidence that EMT is a critical step during tumor progression through initiation of metastasis emphasizes the need for deep understanding of its mechanistic aspects. The future design of anti-cancer drugs effectively targeting pro- tumorigenic aspects of TGF-ȕ signaling without the loss of TGF-ȕ-mediated cytostatic effect on epithelial cancer cells requires the detailed knowledge on how TGF-ȕ operates and evokes EMT. Several reports have suggested that TGF-ȕ triggers EMT also through other pathways than Smad activation (Derynck and Zhang, 2003). However, our work (Paper I) has suggested that Smads are critical and central players in TGF-ȕ-induced EMT in several epithelial cell lines. Despite the same cell model used, our results are in contrast with those of Bhowmick et al. (Bhowmick et al., 2001a), in which inhibition of Smads did not affect TGF-ȕ-induced EMT. However, in this report the authors used clones of NMuMG cells stably expressing wild-type Smad7 or dominant negative Smad3, and it is highly possible that such cells could adapt to the continuous presence of high levels of exogenous Smad7 and mutant Smad3 and differently respond to TGF-ȕ. Instead, we took advantage of adenoviral transient expression of Smad7 or mutant Smad2, Smad3 or Smad4 and our data clearly pointed to Smads as pivotal signal transducers in TGF-ȕ-triggered EMT. In agreement with this hypothesis, we were not able to detect a significant number of genes regulated in the absence of Smad4 (Paper II). Despite these discrepancies, based on our strong evidence for the involvement of Smads in the EMT process, and similarly to others (Bhowmick et al., 2001a; Bhowmick et al., 2001b; Zavadil et al., 2001; Bakin et al., 2002; Yu et al., 2002), we suggest that modulation of EMT is a complex process that requires orchestrated regulation by Smads and other signaling pathways. High throughput transcriptomic analysis performed in epithelial cells undergoing EMT in response to TGF-ȕ has resulted in identification of several hundred regulated genes. Expression of some of the identified genes requires a complex co-operation between Smads and other signaling molecules. It is also highly possible that Smads are required to initiate EMT, whereas non-Smad effectors are involved in sustaining the differentiation process. Therefore, a challenge for the closest future is to define the

46 points of crosstalk between Smads and other effectors in processes of EMT and metastasis. It remains poorly understood whether epithelial cell growth inhibition is linked to EMT. Through high throughput transcriptomic analysis we revealed (Paper II) that members of Id family are important transcriptional targets of TGF-ȕ signal transduction. We were the first to demonstrate the existence of TGF-ȕ-regulated transcription factors that are capable of acting on the crossroads between proliferation and differentiation of epithelial cells. We showed that down-regulation of Id2 and Id3 by TGF-ȕ1 is required for cell growth inhibition and EMT and excess of endogenous Id2 and Id3 inhibit these processes. Interestingly, we could link the BMP-induced expression of both Id2 and Id3 with a poor biological effect of BMP on epithelial cells. We provided the first evidence suggesting that BMP does not have the same capabilities as TGF-ȕ because of the differential expression pattern of Id2 and Id3 triggered by these two growth factors. Hence, we enabled BMP to act as TGF-ȕ and induce growth arrest and EMT when we knocked-down the expression of endogenous Id2 and Id3 in epithelial cells. The important link between TGF-ȕ and Id2 is also coming from transgenic animals. Mice over-expressing TGF-ȕ1 or TGF-ȕ type I receptor in mammary gland have exactly the same phenotype as Id2-/- female mice. In both animals the final differentiation of mammary gland is defective, which leads to lack of lactation (Kordon et al., 1995; Mori et al., 2000). The potency of BMP to suppress TGF-ȕ-induced EMT has been indicated before (Zeisberg et al., 2003), however its mechanism has remained unknown. Importantly, in Paper II we suggest a novel mechanism, in which BMP antagonizes TGF-ȕ via opposite regulation of the same genes, i.e. Id2 and Id3. Hence, epithelial cells exposed to both BMP-7 and TGF-ȕ1, were not able to undergo EMT and we demonstrated for the first time that this antagonistic effect of BMP involves both Id2 and Id3. Thus, we propose that the proper regulation of Ids by TGF-ȕ family members defines the physiological outcome of responding epithelial cells. The exact mechanism by which Id proteins interfere with the Smad pathway remains unknown. Therefore there is an immediate need to identify genes whose regulation is affected by the presence or absence of Id family members in the context of TGF-ȕ and BMP signaling using cDNA microarrays. Over-expression of Id2/3 in NMuMG mouse mammary epithelial cells blocks EMT and the cytostatic effects of TGF-ȕ1 in this cell line. In contrast, knocking down of the endogenous Id2/3 coupled to BMP-7 treatment should evoke EMT and block cell cycle. Therefore, microarray analysis of differently treated cells might reveal novel gene targets, whose expression is al- tered by the presence or absence of specific Id proteins. Newly identified genes should belong mainly to differentiation and cell cycle regulators and their regulation will be meaningful for better understanding of Id involvement in TGF-ȕ superfamily-driven biological responses. Identification of Id

47 partners in epithelial cells might explain the negative regulatory mechanism of these factors. bHLH factors achieve their full transcriptional activity through heterodimerization and fully active dimers consist of one molecule of ubiquitously expressed class A bHLH and one molecule of tissue-specific class B bHLH. Id interacts predominantly with class A bHLH transcription factors and it is highly probable that in epithelial cells they achieve their effects through interaction with ubiquitously expressed factors such as E2A gene products (Kondo et al., 2004). It is also possible that Id can interact with some novel transcription factors, for which a connection to TGF-ȕ superfamily signaling has not been yet shown. However, one of the most challenging and interesting points is to identify epithelial-specific bHLH class B transcription factors. Surprisingly, such factors have not been identified yet.

Figure 11. The proposed role of Id2 for tumor progression. EMT, epithelial- mesenchymal transition; MET, mesenchymal-epithelial transition.

Several recent studies have shown that Id2 plays an important role during tumor growth (Itahana et al., 2003; Russell et al., 2004; Stighall et al., 2005) and several members of the Id family have been found to be over-expressed in many tumors (Ruzinova and Benezra, 2003; Perk et al., 2005). Id2 has been shown to act as a tumor suppressor in the intestinal epithelium (Russell et al., 2004). The loss of Id2 in epithelial tumors keeps the cancer cells in poorly-differentiated state that results in severe neoplastic lesions. Interest- ingly, tumor cells with no expression of Id2 were also hyper-proliferating, suggesting that the influence of Id2 on cell cycle is still not fully understood (Russell et al., 2004). In breast cancer cells, the high expression level of Id2 was correlated with reduced invasiveness of tumor cells and better survival of the patients (Stighall et al., 2005). Similarly, an earlier study in breast

48 tumors revealed that Id2 expression negatively correlates with cancer aggressiveness and metastasis. Hence, reintroduction of Id2 in aggressive breast cancer cells reduced their proliferative and invasive phenotype (Itahana et al., 2003). These interesting data confirm our finding that the down-regulation of Id2 by TGF-ȕ is required to initiate EMT and promote metastasis (Fig. 11). TGF-ȕ down-regulates Id2 expression in order to evoke EMT of epithelial cancer cells. Tumor cells that underwent EMT are now more mobile, invade the surrounding matrix and migrate in the direction of blood vessels. After intravasation they are taken by the blood stream to the target organ where they extravasate, form micrometastasis, undergo mesenchymal-to-epithelial transition (MET) and form rapidly growing macrome- tastasis that correlates with the reported increased expression level of Id2 in several types of tumors (Perk et al., 2005). So, is Id2 a tumor suppressor or a tumor promoter? This question remains still open and the future challenge is to monitor the delicate changes of Id2 expression patterns during tumor progression in animals. The deep understanding of how Id2 and bHLH transcription factors are regulated at each step of metastasis might lead to de- signing of effective anti-cancer drugs targeting the Id2-bHLH pathway. The biological effects of growth factors depend not only on the regulation of gene expression but also on the strength and length of the activated signal transduction pathway. In paper III we described identification of a novel negative regulator of the TGF-ȕ pathway, SNF1LK. We have demonstrated that SNF1LK is an important partner of Smad7 and Smurf2 in targeting activated ALK-5 receptor for ubiquitin-dependent degradation. We could show that SNF1LK regulates the turnover of the receptor through interaction with and phosphorylation of Smad7. However, at the time, we still do not understand what is the consequence of such phosphorylation. Our preliminary data indicated that Smad7 becomes phosphorylated by SNF1LK within its MH2 domain at serine 352. It is therefore possible that such modification of Ser352 might disrupt the interaction between positively charged lysine residues of the MH2 domain of Smad7 and the L45 loop of ALK-5 (Mochizuki et al., 2004). It remains unknown if the whole ALK-5-Smad7-Smurf2 complex becomes degraded together. Despite the complex formation with Smad7, Smurf2 does not alter its turnover (Kavsak et al., 2000). Instead, Arkadia has been suggested to ubiquitinate and promote degradation of Smad7 (Koinuma et al., 2003). Therefore, in our model we suggest that Smad7 becomes phosphorylated by SNF1LK in order to dissociate from the receptor and to guarantee the efficient receptor degradation. Moreover, free Smad7 now becomes available for Arkadia-mediated ubiquitination and degradation. We still do not know if SNF1LK can alter the interaction between Smad7 and Smurf2, but it is highly possible that Smurf2-mediated ubiquitination of ALK-5 is followed by SNF1LK-mediated phoshorylation of Smad7, which would promote dissociation of all components of the complex and degradation of only ubiquitinated receptor. However, this model

49 requires more rigorous testing. In addition, we demonstrated that SNF1LK is able to interact with the ubiquitinated receptor via its UBA domain. UBA domains are believed to have an interesting feature of interacting with poly- ubiquitin chains and ubiquitinated proteins (Buchberger, 2002), but the number of proteins with UBA domains and the understanding of the exact role of UBA-ubiquitin interactions are rather limited. We could show that the UBA domain of SNF1LK is important for the receptor turnover, because a SNF1LK mutant lacking its UBA domain could no longer interact with ubiquitinated receptor and it was less efficient in targeting ALK-5 for degradation. Deciphering the role of UBA-ubiquitin interaction in the contest of TGF-ȕ receptor trafficking is a challenging task for future investigation. Currently, it remains unknown if the activated TGF-ȕ receptors become degraded in proteasomes or lysosomes. However, we demonstrated that SNF1LK interacts with proteasomes. This interesting finding suggests that degradation of activated receptors occurs in proteasomes and SNF1LK might serve as a bridge between ubiquitinated receptors and proteasomes, helping the 19S subunit of proteasomes to recognize the cargo destined for degradation. The functionality and mechanism of SNF1LK and proteasome interaction will be addressed in future experiments. Also, the long C-terminal domain of SNF1LK has no obvious sequence motifs and understanding of its function in regulating SNF1LK activity is very important. Recently, SNF1LK has been identified as one of the several kinases involved in endocytosis (Pelkmans et al., 2005). At the time, its function in this process remains unexplored and future investigations will address the importance of SNF1LK for caveolin-dependent trafficking of receptors. Further elucidation of the function of SNF1LK and its implications in regulation of TGF-E signal transduction will lead to a deeper mechanistic understanding of TGF-ȕ signaling regulation, particularly how signal is ter- minated. It could also allow the design of novel anti-cancer drugs that specifically regulate the enzymatic activity of this kinase.

50 Acknowledgements…

...or the cream of the thesis.

So here we are, the only part of the thesis that will be read by 100% of people who will get it. So, focus well and try to find your name down here…

This work was carried out at the Ludwig Institute for Cancer Research in Uppsala in Sweden. I would like to thank many people who in one or another and not always intellectual way contributed to this work during several years of my stay in Sweden:

Firstly, I would like to acknowledge Aristidis Moustakas, for being my supervisor. It has been a privilege to work with you. Thank you for your endless enthusiasm and believing in science. You taught me how to do top quality science and always encouraged me to think independently. Thank you for believing in me, supporting my crazy ideas and promote my personal development. I think you are the perfect boss, who understands that success in science depends on the whole team. You had an enormous influence on me as a scientist as well as a person. THANK YOU!

Carl-Henrik Heldin, director of Uppsala branch of Ludwig, for support, encouragement and being a ‘pure scientist’.

Ivan Dikic, my co-supervisor during the first year of my PhD studies, for helping me to get to Ludwig, for teaching me what the top science is and showing what ‘to work hard’ means.

The TGF-ȕ Signaling Group was my second home in Uppsala. I would like to thank all current and former members of the group for the unforgettable atmosphere in the lab. First of all I want to thank people who contributed directly to my work and are co-authors on the papers, especially Ulrich, Peter and Rosita. I think we made a ‘dream team’ and learnt a lot from each other. I hope we will be working together once again in the future! Defi- nitely, the closest friend throughout this time in Ludwig was Ulrich. We had the unforgettable time working and having fun together. Thanks for the late night ‘on the stage’ lab parties and cell-lab singing contests! Katerina,

51 thanks for being such a cheerful and strong person, for all the laughs and tons of Greek halva, and for standing me and my mess in our small PhD office. Anita, for the tough discipline in the lab and the new meaning of the word ‘clean’. Rest of our amazing group: Sylvie, Hideki, Akira and Keiko.

Christer and Ihor for brilliant help on Paper III.

Becke, Lasse and Eva H. for making impossible things happen. CPU Uffe for volleyball competitions we have never won.

People who contributed to my work through several suggestions and re- agents: Johan E., Maite, Eva G., and Serhiy. Vasyl for being always very helpful.

Brilliant and ‘crazy’ scientists I have met throughout these years, especially Arne Östman, Johan ‘JoE’ Ericsson, and Christian Sundberg.

All other members of Ludwig in Uppsala: group leaders, post-docs, PhD students and technicians, cleaning personnel and other non-scientists, for making Ludwig a pleasant and an extremely functional place to work.

My close international friends (not necessary from Ludwig), for the great time we spent together and for keeping me away from the lab. Valeria and Magnus ‘Magus’, thank you for countless Italian-Swedish dinners and serious and funny discussions. French speaking part of the group: Ulrich, Phil- ippe and Annick, Jean-Baptiste, Samy and Trias for all non-serious mo- ments that kept me alive in Uppsala. Rest of the team: Anders ‘Kanin’, for sweating meetings in the gym, Ann-Sophie, Lola, Maria S. and Per. Pat- rick and Sarah, for fresh points of view on many subjects, Irish ‘touch’ and fantastic time we had together. Aga, Piotr, Peter, Nimesh and Paolo for keeping me smiling on the Ludwig corridors. Aris and Katerina for endless help when getting started in Uppsala metropolis and getting together in Upp- sala as well as in Kraków.

My family: Mama Gosia, Tata Leszek, Mama Krysia, Tata Andrzej and Michaá, for support in all my good or bad decisions and choices, tons of useful advices and always believing in me.

KaĞka, the most important person in my life. None of this would happen without you! Thank you for all your love, motivation to do all these things and being always right about everything. You bring sense to every day in my life. I love you…

Marcin

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69 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 57

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A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title "Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine".)

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