Novel Roles of Erbb3 Receptor Tyrosine Kinase in Vesicular Trafficking

Home , ABL (gene), ERBB3, Integrin, Receptor tyrosine kinase, Tyrosine kinase, Tyrosine kinase 2

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1535

Novel roles of ErbB3 receptor tyrosine kinase in vesicular trafficking

ANA ROSA SAEZ-IBAÑEZ

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0562-2 UPPSALA urn:nbn:se:uu:diva-373844 2019 Dissertation presented at Uppsala University to be publicly examined in B8; BMC, Husargatan, 752 37, Uppsala, Friday, 8 March 2019 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Johanna Ivaska (Turku University, Finland).

Abstract Saez-Ibañez, A. R. 2019. Novel roles of ErbB3 receptor tyrosine kinase in vesicular trafficking. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1535. 100 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0562-2.

ErbB3 is a catalytically impaired receptor tyrosine kinase (RTK) from the EGFR family. Upon ligand binding, ErbB3 forms heterodimers with other members of the family and triggers phosphorylation cascades that promote crucial cellular functions as proliferation, differentiation and survival. ErbB3 is also an important player in cancer progression, where it mediates resistance to EGFR inhibitors and promotes metastasis. In this thesis we have identified, first, a role of ErbB3 in promoting vesicular recycling of different cargoes, as β1-Integrin or E-cadherin. Vesicular recycling is the process by which cargo proteins are internalized and delivered back to the plasma membrane. This process allows fine tuning of the cargoes function, adjusting their availability on the membrane to different conditions. In the case of β1-Integrin, an important adhesion receptor that mediates association of cells with the extracellular matrix, proper recycling is required for cellular adhesion and migration, among other functions. E-cadherin, on the other hand, is the major cell-to-cell junctional molecule in between epithelial cells, and therefore its availability at the plasma membrane is required for the formation and maintenance of epithelial tissues. In addition, we also present the finding that ErbB3 plays a role in exosome release. Exosomes are small extracellular vesicles originated from the fusion of multivesicular bodies with the plasma membrane. Exosomes are crucial for intercellular communication and are able to act as cargo delivery units both under physiological conditions and in the context of disease. Our work shows that ErbB3 inhibits exosome release and dictates their cargo composition, which may have important implications for their function. Finally, we have also investigated novel mechanisms for regulation of ErbB3 function. Therefore, this thesis also includes our work on non-receptor tyrosine kinase Ack1, a multifaceted kinase which has been extensively linked to tumour progression and cancer cell survival. We show for the first time that Ack1 acts as an important negative regulation of ErbB3 protein level. Altogether, this thesis contributes to our understanding of ErbB3 cellular functions and modes of regulation, unveiling new roles of this RTK in vesicular trafficking and exosome release, and proposing Ack1 as a novel modulator of its function.

Keywords: ErbB3, Integrin, GGA3, Rabaptin5, PKD, Ack1, endocytic recycling, exosome

Ana Rosa Saez-Ibañez, Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, SE-75123 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-513-0562-2 urn:nbn:se:uu:diva-373844 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-373844) To my parents. A mis padres.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I. Saez-Ibañez, A.R., Scheffler, J.M.*, Terabayashi, T.*, Idevall-Hagren, O., Ferby, I. (2019) Ligand-independent role of ErbB3 in endocytic recycling. Manuscript.

II. Saez-Ibañez, A.R., Terabayashi, T., Scheffler, J.M., Ferby, I. (2019) Lig- and-independent role of ErbB3 in recycling of E-cadherin and assembly of adherens junctions. Manuscript.

III. Saez-Ibañez, A.R., Scheffler, J.M., Ferby, I. (2019) ErbB3 regulates release of extracellular vesicles. Manuscript.

IV. Saez-Ibañez, A.R.*, Pilia, G.*, Linderoth, E., Ferby, I. (2019) Ack1 is a negative regulator of ErbB3 that acts both by promoting its degradation and suppressing its expression. Manuscript.

Other articles co-authored but not included in this thesis:

Gilabert-Juan, J., Belles, M., Saez-Ibañez, A.R., Carceller, H., Zamarbide- Fores, S., Moltó, M.D., Nacher, J. (2013) A "double hit" murine model for schizophrenia shows alterations in the structure and neurochemistry of the me- dial prefrontal cortex and the hippocampus. Neurobiol Dis. 59:126-40.

Gilabert-Juan, J., Saez-Ibañez, A.R., Lopez-Campos, G., Sebastiá-Ortega, N., González-Martínez, R., Costa, J., Haro, J.M., Callado, L.F., Meana, J.J., Nacher, J., Sanjuán, J., Moltó, M.D. (2015) Semaphorin and plexin gene expression is altered in the prefrontal cortex of schizophrenia patients with and without auditory hallucinations. Psychiatry Res. 229(3):850-7.

* Asterisks indicate equal contribution

Cover illustration: Immunofluorescence of a 2D culture of Comma-D mouse epithelial cells. Internalized E-cadherin is shown in pink; the cell nucleus is depicted in yellow; the long fibers represent actin filaments from the cell cytoskeleton. Picture taken with Ζeiss Axio Imager M2 microscope and pro- cessed using Zeiss ZEN 2011 and Adobe Lightroom softwares. Ana Rosa Saez.

Examining Committee

Faculty Examiner (Opponent) Professor Johanna Ivaska Department of Biochemistry and Turku Center for Biotechnology University of Turku, Turku, Finland.

Evaluating Committee Associate Professor Sebastian Barg Department of Medical Cell Biology Uppsala University, Uppsala, Sweden.

Assistant Professor Samir El-Andaloussi Department of Laboratory Medicine, Clinical Research Center Karolinska Institute, Stockholm, Sweden.

Associate Professor Lars Jakobsson Research Division of Vascular Biology Karolinska Institute, Stockholm, Sweden.

Professor Staffan Johansson Department of Medical Biochemistry and Microbiology Uppsala University, Uppsala, Sweden.

Professor Staffan Strömblad Department of Biosciences and Nutrition Karolinska Institute, Stockholm, Sweden.

Contents

1. Introduction ...... 15 2. The EGFR family of receptor tyrosine kinases ...... 17 2.1. Structure and mechanism of activation ...... 17 2.2. Signal attenuation upon ErbBs activation ...... 21 2.3. ErbB3, a not-so-dead receptor tyrosine kinase ...... 23 2.4. ErbBs in cancer and antitumour drug resistance ...... 26 2.4.1. ErbB3 role in cancer drug resistance ...... 28 3. A brief introduction to vesicular trafficking ...... 29 4. Adhesion molecules: function, trafficking and their interplay with ErbB receptors ...... 31 4.1. E-cadherin, the main adherens junction molecule in epithelial tissues...... 31 4.1.1. E-cadherin vesicular trafficking ...... 32 4.1.2. E-cadherin crosstalk with signalling pathways ...... 35 4.1.3. E-cadherin in cancer progression ...... 36 4.2. Integrins, the main cell-to-ECM receptors ...... 37 4.2.1. Integrin structure, ligands and modes of activation ...... 37 4.2.2. Vesicular trafficking and recycling of integrins ...... 40 5. Molecular regulators of vesicular trafficking ...... 46 5.1. The PKD family of serine/threonine kinases ...... 46 5.2. The Rabaptin-5 effector protein ...... 49 5.3. GGA family of Arf-dependent clathrin adaptors ...... 51 5.4. Ack1 non-receptor tyrosine kinase ...... 54 6. Extracellular vesicles and exosome biogenesis ...... 57 6.1. Types of extracellular vesicles ...... 57 6.2. Extracellular vesicles biogenesis ...... 60 6.3. Extracellular vesicles in cancer and metastasis ...... 62 7. Present investigations ...... 64 7.1. Paper I. Ligand independent role of ErbB3 in endocytic recycling ...... 64 7.2. Paper II. Ligand-independent role of ErbB3 in recycling of E- cadherin and assembly of adherens junctions ...... 64

7.3. Paper III. ErbB3 regulates release of extracellular vesicles ...... 65 7.4. Paper IV. Ack1 is a negative regulator of ErbB3 that acts both by promoting its degradation and suppressing its expression ...... 65 8. Future perspectives ...... 67 Acknowledgements/ Agradecimientos ...... 69 Bibliography ...... 74

Abbreviations

ACAP1 Arf6-GTPase activating protein ACK1 Activated Cdc42-associated kinase 1 AJ Adherens junctions AKT RAC-alpha serine/threonine kinase ALA Alanine ALIX ALG-2 interacting protein X AP-2 Adaptor protein 2 AREG Amphiregulin ARF ADP ribosylation factor ARH Autosomal recessive hypercholesterolemia protein ARRDC1 Arrestin domain-containing protein 1 ASN Asparagine ASP Aspartic acid ATP Adenosine triphosphate BACE1 β-site APP cleaving enzyme 1 BTC Betacellulin CAMK Calcium/calmodulin-dependent protein kinase CBL Casitas B-lineage lymphoma proto-oncogene CC Coiled-coil CDK Cyclin-dependent kinase CERT Ceramide transfer protein CHEVI Class C Homologs in Endosome-Vesicle Interaction complex CHMP Charged multivesicular body proteins CK2 Casein kinase 2 CLAP Clathrin-associated sorting protein CLIC3 Chloride Intracellular Channel 3 CORVET Class C core vacuole/endosome tethering complex CRIB Cdc42- and Rac-interactive binding domain CYS Cysteine DAB1 Disabled-1 DAG Diacylglycerol DEP-1 Density-Enhanced Phosphatase-1

ECM Extracellular matrix EEA1 Early Endosome Antigen 1 EGF Epidermal growth factor EGFR Epidermal growth factor receptor EGFRvIII Epidermal growth factor receptor variant III EGTA Egtazic acid EHD1 Eps15-homology domain-containing protein EMT Epithelial-to-mesenchymal transition EPGN Epigen ER Endoplasmic reticulum ERAD ER-associated degradation ErbB1-4 Erythroblastic leukemia viral oncogene homolog 1-4 EREG Epiregulin ERK Extracellular signal-regulated kinase ERRP EGFR-related peptide ESCRT Endosomal sorting complexes required for transport EV Extracellular vesicle FAK Focal adhesion kinase FGFR Fibroblast growth factor receptor GAE γ-adaptin ear domain GAT GGA and TOM domain GEF Guanine nucleotide exchange factor GGA Golgi-localized, γ-ear-containing, Arf-binding proteins GIPC1 GIPC PDZ domain containing family, member 1 GLU Glutamic acid GLUT4 Glucose transporter type 4 GRB2,7 Growth factor receptor-bound protein 2/7 GSK-3β Glycogen synthase kinase 3 beta GTP Guanosine triphosphate HB-EGF Heparin Binding EGF Like Growth Factor HER1-4 Human epidermal growth factor receptor 1-4 HGFR Hepatocyte growth factor receptor HIS Histidine HOPS Homotypic fusion and protein sorting complex HRS Hepatocyte growth factor-regulated tyrosine kinase substrate HSP70,73 Heat-shock protein 70,73 ICAM Intercellular Adhesion Molecule 1 ILV Intraluminal vesicle ISEV International Society of Extracellular Vesicles LAMP1 Lysosomal-associated membrane protein 1 LFA-1 Lymphocyte function-associated antigen 1 LRIG1 Leucine-rich repeats and immunoglobulin-like domain-1 MAPK Mitogen-activated protein kinase MDGI Mammary-derived growth inhibitor

MHCII Major histocompatibility complex II MHR Mig6 homology region MIG6 Mitogen-inducible gene 6 MLCK Myosin light chain kinase MMP Matrix metalloproteinase mTOR Mammalian target of rapamycin complex MVB Multivesicular body NCBI National Center for Biotechnology Information NRDP1 Neuregulin receptor degradation protein 1 NRG1-6 Neuregulin 1-6 NSCLC Non-small-cell lung cancer OSBP1 Oxysterol-binding protein 1 PDGF Platelet-derived growth factor PH Pleckstrin homology domain PI3K Phosphoinositide 3-kinase PI4KIIIβ Phosphatidylinositol 4-kinase III beta PKC Protein Kinase C PKD Protein Kinase D PLC Phospholipase C PLD Phospholipase D PPFIA1 PTPRF Interacting Protein Alpha 1 PSI Plexin-semaphorin-integrin domain PTB Phosphotyrosine-binding domain PTK6 Protein Tyrosine Kinase 6 PTPN Protein tyrosine phosphatase PTPRJ Protein Tyrosine Phosphatase, Receptor Type J Rab11-FIP2 Rab11-interacting protein 2. RALT Receptor associated late transducer RCP Rab-coupling protein RNF41 Ring Finger Protein 41 RTK Receptor tyrosine kinase S1P Coupled sphingosine 1-phosphate SAM Sterile alpha domain SEC Size exclusion chromatography SH2/3 Src homology domain 2/3 SHARPIN SHANK Associated RH Domain Interactor protein SHC SH2-containing collagen-related protein SHP1/2 Src Homology region 2 domain-containing Phosphatase-1/2 SKD1 Suppressor of K(+) transport growth defect protein 1 SNX Sortin nexin SRC Sarcoma kinase STAM1/2 Signal Transducing Adaptor Molecule 1/2S STAT Signal Transducer and Activator of transcription t/v-SNARE Soluble NSF Attachment Protein Receptor

TGF Transforming growth factor TGN Trans-Golgi network TJ Tight junctions TNK1/2 Tyrosine Kinase Non-Receptor 1/2 TRAIL TNF-related apoptosis-inducing ligand TRKA Tropomyosin receptor kinase A TSG101 Tumour susceptibility gene 101 VAMP3 Vesicle-associated membrane protein 3 VCAM Vascular cell adhesion protein VHS Vps27, Hrs and Stam domain VPS Vacuolar protein sorting

1. Introduction

Multicellular organisms require an intricate communication system able to ensure cell and tissue homeostasis. During evolution, complex organisms have developed an extensive array of proteins, lipids and nucleic acids that take part in a myriad of interconnected circuits that ensure the ability to integrate different extracellular signals and generate the proper cellular response. Under physiological conditions, cells receive mechanical and chemical inputs from their surroundings and respond to them accordingly, in a constant dialogue that allows proper functioning not only of each cell, but of the entire tissue and organism. Concurrently to this communication system, cells and tissues require control mechanisms that keep in check its own proper functioning: proteins, lipids and nucleic acids need to be in the right place, at the right time and in the right amount. Compartmentalization of the eukaryotic cell has allowed specific cellular functions to take place within distinct lipid-membrane sub-compartments. Hand in hand with the development of such a spatial division, comes the need for a transport system that connects the different intracellular compartments. The vesicular trafficking machinery allows the internalization (endocytosis) of a variety of molecules (cargoes), including nutrients and macromolecules from the extracellular space and receptors and adhesion molecules sitting on the plasma membrane. After internalization, and by means of a sophisticated network of pathways leading towards different fates, the different cargoes may be recycled, degraded, activated or inactivated, transported to a different cellular organelle or even released to the extracellular space as components of exosomes or other extracellular vesicles. Growth factor receptors are among the proteins that initiate cellular responses to environmental signals and their malfunctioning is a major cause of cancer initiation and progression. Their extensive study over the years has proven that, upon ligand binding, growth factor receptors initiate phosphorylation cascades that culminate in a number of cellular changes, for example at the transcriptional level. However, their involvement in the vesicle trafficking aspects of the cell machinery is much less studied. With this thesis work, I aim to contribute to the knowledge on how growth factor receptors collaborate with the vesicle transport machinery to regulate cellular functions. Towards this aim, we have described two novel functions of ErbB3 growth factor receptor: to promote recycling of important cargoes such as E-cadherin and β1-integrin and inhibition of exosome release, which

15 implicate it for the first time in the endocytic and exocytic transport systems. Furthermore, we provide evidence that spatiotemporal control of ErbB3 itself is distinct from other members of the EGFR family, and occurs through regulation by the non-receptor tyrosine kinase Ack1. In this introduction, my goal is to contextualize our findings so that the reader can assess critically their scope and implications.

16 2. The EGFR family of receptor tyrosine kinases

The EGFR (or ErbB) family of receptor tyrosine kinases comprises four different receptors: EGFR (also called ErbB1 or HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4). They bind different subsets of a dozen of ligands and trigger activation of a variety of downstream cascades. The discovery of this signalling system dates back to the 1950s, when Dr. Stanley Cohen and coworkers isolated the epithelial growth factor (EGF) from mouse submaxil- lary glands (Cohen, 1965; Levi-Montalcini and Cohen, 1960). This discovery was followed by years of studies on the diverse physiological effects of EGF stimulation, while the identification of its receptor, EGFR, did not come until the late 1970s (Cohen and Carpenter, 1975; Cohen et al., 1980). Human ErbB2 (Her2) and its rodent homolog (Neu) were discovered next (King et al., 1985; Schechter et al., 1985; Shih et al., 1981), and, finally, the cloning of ErbB3 (Kraus et al., 1989) and ErbB4 (Plowman et al., 1993) would complete the family as we know it today. Notably, the EGFR family consists of four members only in vertebrates, while C. elegans and D. melanogater contain only one EGFR orthologue (Let-23 and DER, respectively). The number of ligands they bind is also smaller: one in the case of C. elegans and four in the case of D. melanogaster (Bogdan and Klämbt, 2001), as compared with twelve in mammals. In this section, an overview of our current knowledge on the structure, activation and function of the different members of the EGFR family will be given. It will be followed by a deeper analysis into ErbB3, the focus of this thesis work, and its function and regulation, its known interactors and its importance in cancer progression and resistance to cancer therapy.

2.1. Structure and mechanism of activation The ErbB receptors are type Ia transmembrane proteins consisting of three parts: a heavy glycosylated and disulfide-bonded ectodomain that provides a ligand-binding site and is subdivided into four subdomains (I, II, III and IV); a single transmembrane domain; and a large cytoplasmic region that encodes a tyrosine kinase and a tail with multiple phosphorylation sites (Linggi and

17 Carpenter, 2006). Upon ligand binding, the extracellular domain of these receptors suffers a rearrangement, exposing a hairpin loop in domain II that is required for the formation of homo- or heterodimers (Ogiso et al., 2002). Since the activating ligand interacts with both subdomain I and III of the extracellular domain, and these two lie at either end of domain II, it has been proposed that different ligands might promote different degrees of domain II curvature, which could play a role in determining heterodimerization specificity (Lemmon, 2009).

Figure 1. Structure and dimerization model of the EGFR family members. The resting form of EGFR, ErbB3 and ErbB4 keeps the dimerization arm (domain II of the extracellular part) unexposed, and needs to bind ligand in order to adopt a conformation compatible with dimerization. On the contrary, ErbB2 is constitutively exposing the dimerization arm, and therefore does not require ligand binding.

Ligand-induced dimerization has been previously postulated to mediate kinase activation. For most receptor tyrosine kinases, dimerization allows positioning the two cytoplasmic domains in such a manner that transphosphoryla- tion can occur. This means that each monomer cross-phosphorylates specific tyrosine residues of the other member of the dimer, generating docking sites that will be recognized by downstream proteins. However, a different mode of activation has been proposed in the case of the EGFR family by the group of John Kuriyan (Zhang et al., 2006). According to this model, ErbB receptors are activated allosterically: Upon dimerization, the two kinase domains form an asymmetric dimer, analogous to the complex formed by cyclins and cyclin- dependent kinases (CDKs). This way, one of the kinases (the activator) stabilizes the active conformation of the other (the receiver), which in turn is able to transphosphorylate the cytoplasmic tail of the first one. A segment of the juxtamembrane domain has also been proposed to play an important role in kinase activation of EGFR receptors. According to this study, the C-terminal portion of the juxtamembrane segment of the receiver kinase (which comprises residues 664 to 671 in the EGFR sequence) stablishes extensive contact

18 with the C-lobe of the allosteric activator kinase, resulting in dimer stabilisa- tion (Brewer et al., 2009). Kinase activation result in phosphorylation of a number of residues in the cytoplasmic tail of the receptors, which causes the recruitment of diverse downstream proteins that contain phosphotyrosine interaction domains, such as Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains (Wagner et al., 2013). This way, different signal transduction pathways will be activated such as MAPK/ERK, Ras, PI3K/Akt, Src, PLCγ, JAK and mTOR, as well as several transcriptional regulators, like the STATs, c-Fos and others (Yarden, 2001). This series of events will cause, ultimately, a number of cellular responses such as survival, proliferation, differentiation and motility (Yarden and Sliwkowski, 2001).

Figure 2. Intracellular domains in the ErbB receptors and the asymmetric dimer model. Upon ligand binding, conformational changes in the extracellular domain allow dimerization of the receptors. After dimerization, the C-lobe of the allosteric activator kinase interacts with the N-lobe of the receiver kinase through a conserved hydrophobic patch, stabilizing it in a conformation catalytically active, which then can phosphorylate tyrosine residues on the tail of the activator kinase.

Although the four members of the EGFR family are closely related (40-50% of amino acid identity in the extracellular domains and 60-80% in the intracellular domains), there are substantial differences among them. For example, they have different ligand binding affinities: EGFR recognizes seven EGF- like ligands (EGF, TGF , HB-EGF, AREG, BTC, EREG, and EPGN) , while ErbB3 binds ligands from the neuregulin family (NRG1, NRG2 and NRG6) and ErbB4 interacts with both neuregulins (NRG1-5) and some EGF-like ligands (HB-EGF, BTC and EREG) (Iwakura and Nawa, 2013; Schneider and Wolf, 2009). On the contrary, ErbB2 does not bind any known ligand, but differently from the other members of the family, it does not require ligand- binding for dimerization, since its ectodomain is stabilized in an open conformation that resembles the ligand-activated state (Cho et al., 2003). Another

19 key difference between the ErbBs is that, contrary to the other members of the family, ErbB3 presents an impaired kinase activity due to substitutions of critical residues in its catalytic domain, involving the absence of the catalytic base aspartate (Guy et al., 1994; Sierke et al., 1997). Therefore, the kinase activity of ErbB3 is in the order of one thousand times lower than that of EGFR (Hynes, 2016) and it has been catalogued as a pseudokinase (Manning et al., 2002). Although recent reports suggest the importance of its residual kinase activity (Shi et al., 2010; Steinkamp et al., 2014), ErbB3 was until recently mostly overlooked due to its lack of catalytic activity, and insights into its function have emerged mainly in the context of ErbB2 or EGFR overexpression (Holbro et al., 2003; Lee-Hoeflich et al., 2008; Rexer and Arteaga, 2013). These particular properties of ErbB2 (it does not respond to any ligand) and ErbB3 (it can respond to ligands but is catalytically dead) have a fundamental consequence: these receptors should only be able to activate downstream signalling cascades in the context of heterodimerization with other members of the family (Citri et al., 2003). Yet in the case of ErbB2, overexpression can trigger downstream signalling by ligand-independent homodimerization (Hynes and Stern, 1994). Of note, heterodimers containing ErbB2 or ErbB3 are not less functional than other combinations. ErbB2, for example, is considered the most potent co-receptor, given its high affinity for other ErbB members (Graus Porta et al., 1997) and its ability to induce potent signalling, even oncogenic, in absence of ligand (Karunagaran et al., 1996). ErbB3, on the other hand, can also yield fully functional asymmetric dimers. Since in this model one of the kinases, the allosteric activator, does not require catalytic activity for its role, ErbB3 may have specialized for this function (Jura et al., 2009).

Figure 3. Signaling pathways downstream of ErbB dimers formation. Upon ligand binding, ErbB receptors dimerize and, sequentially, phosphorylate each other, creating docking sites for downstream effectors. To simplify, many effectors and cascade ramifications are not depicted in this illustration.

2.2. Signal attenuation upon ErbBs activation Attenuation of activated EGFR family members has to occur in order to avoid overactivation of the above-mentioned pathways. The most extensively studied mechanism for ErbB signal attenuation is the clathrin-dependent endocytosis and subsequent sorting of the activated ligand-receptor complex via the multivesicular body (MVB) to lysosomal degradation, a mechanism that has been extensively studied in the case of EGFR, but which is less explored for the other ErbB receptors. Upon ligand exposure, internalization of EGFR is enhanced (Herbst et al., 1994). In this context, the adaptor protein Grb2, which binds EGFR through its SH2 domain, recruits the E3 ubiquitin ligase Cbl, resulting in mono- and polyubiquitinations of the receptor (Huang et al., 2006; Jiang et al., 2003; Johannessen et al., 2006). This step seems to be required for receptor endocytosis, since EGFR ubiquitination is recognized by ubiquitin- binding proteins of the clathrin coat, although ubiquitination-independent internalization of EGFR has also been reported (Huang et al., 2007). In addition, fast clathrin-independent internalization of EGFR has also been observed in presence of high ligand concentration which, proposedly, saturate the clathrin-

21 dependent pathway (Bakker et al., 2017; Sigismund et al., 2005, 2008). Upon internalization, the receptor is trafficked through the endosomal pathway to the MVBs, where EGFR interacts with components of the ESCRT machinery and is later degraded in the lysosome (Madshus and Stang, 2009). Alterna- tively, internalization of EGFR can result in its recycling, and the choice of fate (recycling or degradation) is strongly linked to the ubiquitination status of the receptor (Eden et al., 2012). In addition, it should be noticed that internalization of the receptor, per se, is not a mechanism of signalling attenuation, since EGFR is able to signal from endosomal compartments (Murphy et al., 2009; Wang et al., 2002) Dephosphorylation of tyrosine residues in the C-terminal tail of the activated receptor is also a mechanism of attenuation of ErbB activation (Tonks, 2006). A growing list of tyrosine-specific phosphatases have been implicated in this process, such as PTPN6 (Shp1), PTPN11 (Shp2), PTPN9 (Meg2) and PTPRJ (Dep-1) (Tarcic et al., 2009; Yao et al., 2017; Yuan et al., 2010). More- over, their action has been proven important both for dephosphorylation of receptors on the cell surface or internal membrane compartments (Monast et al., 2012). Other negative regulators of ErbB activity have been found. For instance, a number of splice variants coding for secreted proteins that correspond to the extracellular domain of the different ErbB receptors are able to bind the full receptors and block their dimerization. Examples of this phenomenon are Her- statin that binds ErbB2, P85-s-ErbB3 that binds ErbB3, and ERRP that binds EGFR (although the latter is not a splice variant but the product of a different gene) (Doherty et al., 1999; Lee et al., 2001; Yu et al., 2001). In addition, there are other important effectors acting as negative regulators of the ErbB members, among them Mig6 (also known as RALT), Lrig1 and Ack1. Mig6 is a cytoplasmic proteins whose tumour suppressor role has been well stablished (Ferby et al., 2006; Hopkins et al., 2012) and which is able to interact directly with the ErbBs (Anastasi et al., 2003), inhibiting their activation by engaging with the interface of interaction in the asymmetric dimer (Zhang et al., 2007b). On top of that, Mig6 drives EGFR degradation by promoting interaction of EGFR with the clathrin adaptor AP-2, and therefore enhancing its endocytosis (Frosi et al., 2010). Lrig1 is also a negative regulator of EGFR signalling. This transmembrane protein contains a CBL-binding domain and interacts with the different ErbBs through its extracellular portion (Gur et al., 2004). The proposed model is that, by interaction with the activated receptor, Lrig1 acceler- ates the recruitment of the E3 ligase Cbl to EGFR (Wang et al., 2013b), and therefore its ubiquitination and degradation. Ack1 is another protein linked to regulation of EGFR trafficking. Ack1 is a non-receptor tyrosine kinase which has been found to interact with EGFR promoting its endocytosis, although others have proposed the opposite outcome (Grøvdal et al., 2008; Howlin et al., 2008; Shen et al., 2007). In paper IV of this thesis work we demostrate the

22 effect of Ack1 as a negative regulator of ErbB3. A deeper analysis on the structure and function of this protein can be found in section 5.4.

2.3. ErbB3, a not-so-dead receptor tyrosine kinase ErbB3 is a 180 kDa glycoprotein the primary ligands of which are the members of the NRG family. It is encoded by the ERBB3 gene at 12q13.2 (NCBI ID: 2065), which is about 23 kb in size and consists of 28 exons. The human gene is transcribed to a 6.2 kb mRNA. However, there are also truncated tran- scripts, many of them still with unknown functions. ErbB3 expression is detected at different stages of development and several groups have noticed that its pattern of expression is distinct from those of other ErbB receptors, suggesting unique functions (Fox and Kornblum, 2005; Iwakura and Nawa, 2013; Klonisch et al., 2001). ErbB3 is heavily glycosylated, with 10 potential N- linked glycosylation sites. Interestingly, only one of those glycosylation sites is conserved among all the members of the EGFR family, raising the question whether the glycosylation pattern may contribute to the unique functions carried out by this receptor (Stein & Staros, 2000). Although, nearly all possible combinations of ErbB receptors exist, ErbB3 does not form stable, ligand-bound, homodimers, in contrast to other members of the family. This is thought to be due, in part, to certain amino acid changes in a loop near of domain II of the ECD, which acts as a dimerization arm (Dawson et al., 2005). Other likely cause are the changes on the α-helix transmembrane domain of ErbB3. Interaction between the α-helices from two receptors are important for stabilization of receptor dimers, and the GXXXG consensus sequence has been implicated in this, also in the case of other families of RTKs. Interestingly, while the other members of the EGFR family contain two of these sequences, ErbB3 contains only one, which could be a key reason why this receptor is less prone to homodimerize (Mendrola et al., 2002). However, ErbB3 does form self-oligomers, both the full length protein and the extracellular domain only, although for this kind of interaction a different surface of the protein is required, instead of the surface involved in the dimerization process (Kani et al., 2005; Landgraf and Eisenberg, 2000). Inter- estingly, oligomerization models show that both inactive ErbB3 and ErbB4 can be part of these clusters (heterooligomers) (Jura et al., 2009), and that stimulation with neuregulin destabilizes them, suggesting that they represent a resting form. As introduced before, the catalytic activity of ErbB3 is impaired, and therefore, ErbB3 belongs to the 10% of human kinases catalogued as pseudoki- nases. Its kinase region contains a fully conserved ATP-binding site, but other substitutions at positions 740 (Ala instead of Cys), 759 (His instead of Glu) and 834 (Asn instead of Asp) may explain the lack of functionality. Another

23 important feature is that in ErbB3, the C-terminal portion of the of the intracellular juxtamembrane segment is not conserved. Since this region, as explained before, is important for the interaction of the receiver kinase with the C-lobe of the allosteric activator, this is consistent with ErbB3 being unable to perform as a receiver in this asymmetric dimerization model (Jura et al., 2009). Despite of these changes, the rest of the sequence of the cytoplasmic domain of ErbB3 is largely conserved when compared with other members of the ErbB family, and its ability to bind downstream effectors seems unaltered. ErbB3 C-terminal tail contains 13 tyrosines, which provides an important signalling platform for SH2 or PTB domain containing proteins. Tyrosine-phosphorylated ErbB3 is a major docking site for PI3K, having the highest affinity for it among all the ErbB receptors due to the presence of 6 binding sites for the p85 subunit of PI3K (Hynes and Lane, 2005). Therefore, activation of ErbB3 normally results in strong activation of AKT signalling, with the most characteristic outcome being increased proliferation and cell survival. Other classical examples of proteins binding to phosphotyrosines in ErbB3 are Shc, Grb7, PTK6 and c-Src (extensively reviewed in Sithanandam & Anderson, 2008). Grb2 has been found to interact with ErbB3 in some studies (Schulze et al., 2005), but not in others (Jones et al., 2006).

Figure 4. Main phosphorylation sites on the cytoplasmic tail of ErbB3 and the different signaling proteins that are recruited to them. ErbB3 presents six binding sites for the p85 subunit of PI3K. Neither EGFR or ErbB2 have docking sites for p85, but they are able to couple with this route through other interactors. Adapted from Hynes and Lane, 2005. For an additional description of the phospho-interactome of ErbB3, Jones et al., 2006 is recommended.

24 Alternative modes of ErbB3 phosphorylation should be also considered. For example, it is well established that overexpressed Met/HGFR receptor is able to interact with and phosphorylate ErbB3, and this mechanism has been linked to gefitinib resistance in lung cancer (Arteaga, 2007; Engelman et al., 2007). Interestingly, it has been recently shown that ErbB3 acts as a substrate for MET only in the context of MET overexpression, and that this interplay, which is ligand-independent, occurs in endomembranes more than on the cell surface (Frazier et al., 2018). In addition, several groups have documented that the cycling-dependent kinase 5 (Cdk5) interacts with ErbB3 and phosphorylates ser/thr residues, which somehow affects ErbB3 activation, as was shown by the effect on PI3K activation (Fu et al., 2001; Li et al., 2003). ErbB3 is subjected to a strict control of the protein level through independent mechanisms to those controlling the levels of other ErbBs. The biosynthesis time for ErbB3 is about 30 minutes, and its half-life is 2-3 h. Both lysosomal and proteosomal-dependent degradation have been documented for ErbB3, and the involvement of one or the other type may be connected with the subcellular localization of ErbB3. Surface ErbB3, for example, is predom- inantly degraded via lysosomes. ErbB3 is internalized, both constitutively and in response to ligand stimulation, in a clathrin-dependent manner (Reif et al., 2016; Sak et al., 2012). Although AP-2 is a major clathrin adaptor for EGFR, it does not seem to interact with the cytoplasmic tail of ErbB3, a fact that was initially understood as inability of ErbB3 to undergo endocytosis (Baulida et al., 1996). However, other clathrin-associated sorting proteins (CLAPs) can work, instead of AP-2, in promoting cargo loading into clathrin-coated pits. In fact, it was recently shown that epsin-1 plays such a role during ErbB3 endocytosis (Szymanska et al., 2016). Upon internalization, and in the context of ligand stimulation, ErbB3 has been shown to traffic from early to late endosomes and to interact with the ESCRT-0 subunit Hrs, as a pre-step to lysosomal degradation (Fosdahl et al., 2017), while endocytosis in absence of lig- and stimulation could instead lead to slow recycling of ErbB3. We and other have realized that, contrary to what could be expected, ErbB3 in unstimulated cells localizes mostly to intracellular vesicular compartments (Fosdahl et al., 2017). On the other hand, proteosomal-dependent degradation of ErbB3 is regulated by the E3 ubiquitin ligase Nrdp1 (also known as Rnf41), which stimulates ubiquitination of ErbB3 in a ligand-independent manner (Diamonti et al., 2002; Qiu and Goldberg, 2002). More recently, it has been proposed that Nrdp1 acts as part of the ERAD machinery of protein degradation of newly synthesized ErbB3 in the endoplasmic reticulum. This way, Nrpd1 would recognize specifically the immature, non-glycosylated, form of ErbB3 before it exits the ER and play an important role in keeping the cellular levels of ErbB3 in check (Fry et al., 2011). Indeed, Nrdp1 protein is suppressed in about 57% of breast cancers relative to the matched normal tissue from patients (Yen et al., 2006), and it shows an inverse correlation with ErbB3 levels in the same

25 samples. Interestingly, ErbB3 is the only example to date of a RTK subjected to quantity control at the ER, a checkpoint normally associated to metabolic enzymes (Hegde and Ploegh, 2010). The presence of mechanisms that control ErbB3 steady-state levels points towards the existence of a role for ErbB3 that is independent of ligand stimulation.

2.4. ErbBs in cancer and antitumour drug resistance Normal tissues develop into neoplastic tissues through a complex multi-step process whose origin is largely multifactorial. Several cancer hallmarks have been proposed in order to provide an organized framework for the study of this disease (Hanahan and Weinberg, 2000, 2011). Aberrant function of the EGFR family of RTKs links directly with two of those hallmarks: the sustained proliferative signalling and the self-sufficiency in growth signals. Therefore, although the four members of this family are necessary for the correct development and maintenance of a variety of tissues (heart, lung, skeletal muscle and nervous system, among others), their overexpression and aberrant activation have been implicated in the development of numerous cancer types, of which a few examples will be now commented. Overexpression of EGFR or its oncogenic splice variants is found in more than 50% of malignant gliomas, 30 to 90% of advanced colorectal tumours and 40 to 90% of non-small cell lung cancer (NSCLC) (Ekstrand et al., 1991; Gupta et al., 2009; Ljuslinder et al., 2011; Wong et al., 1992). In addition to those cases where overexpression or mutations in the receptor are the source of the aberrant signalling, also excess of ligand production (either by the cancer cells or the cells from the surrounding stroma) is the cause of hyperactive EGFR signalling in some cancers (Salomon et al., 1995). Furthermore, ErbB2 gene amplification is detected in about 30% of breast cancer patients, where the degree of overexpression can reach up to 100-fold in cancer cells and correlates with poor prognosis (Lofts and Gullick, 1992; Slamon et al., 1987), and is also prominent in ovarian, gastric and salivary cancers (Holbro and Hynes, 2004). Furthermore, mutations in its kinase domain have been detected in lung cancer (Stephens et al., 2004). ErbB3 overexpression has also been reported in solid tumours and, importantly, 63% of primary breast tumours show overexpression of ErbB3 relative to normal tissues (Yen et al., 2006). In addition, oncogenic mutations of ErbB3 have been identified in diverse types of tumours (Jaiswal et al., 2013) and ErbB3 expression has been linked to metastasis formation in melanoma, breast and cervical cancer (Du et al., 2018; Li et al., 2018; Tiwary et al., 2014; Xue et al., 2006). In the case of ErbB4, its involvement in cancer is less clear. Although activating mutations have been linked to NSCLC (Kurppa et al., 2016) and this receptor is overexpressed in a fraction of colon cancers (Williams et al., 2015), it should be noticed that, in

26 the mammary gland, ErbB4 is involved in inhibition of proliferation and induction of apoptosis (Das et al., 2010; Feng et al., 2007; Muraoka-Cook et al., 2008). From all the possible combinations, the ErbB2-ErbB3 pair is the one with strongest oncogenic potential, and even in absence of the corresponding ligand (and at normal levels of ErbB3 expression), ErbB2 overexpression is sufficient to spontaneously recruit autoinhibited ErbB3 into heterodimers. The formed dimers may assume the ligand-induced conformation, resulting in weak but prolonged activation (Citri et al., 2003). Those patients whose cancers show alterations in EGFR family function tend to present poorer prognosis. Therefore, there is a lot of interest into developing clinical therapies targeting members of this family, either based on small tyrosine-kinase inhibitors or antibodies which recognize their extracellular domains. The following table presents a list of the most important ErbB inhibitors to date:

Table 1. Main ErbB-targeted therapies approved for commercialization. Agent Type Target Mechanism of action Development status

Trastuzumab mAb ErbB2 Interacts with domain IV of Approved for ErbB2-pos- the receptor. Only effective itive breast cancer against overexpressed ErbB2 Pertuzumab mAb ErbB2 Binds dimerization arm Approved in some coun- (domain II) blocking di- tries for ErbB2-positive merization breast cancer

Cetuximab mAb EGFR Competitive inhibition of Approved for colorectal EGF and other ligands cancer and head and neck binding to EGFR squamous cell carcinoma Gefitinib EGFR Reversible tyrosine kinase Approved for non-small small inhibitor (ATP-site di- cell lung cancer after fail- molecule rected) ure of other treatments Erlotinib small EGFR Tyrosine kinase inhibitor Approved for non-small molecule (ATP-site directed) cell lung cancer after fail- ure of other treatments. First-line treatment, in combination with gem- citabine for pancreatic cancer Lapatinib small EGFR/ Reversible dual tyrosine ki- Approved for treatment molecule ErbB2 nase inhibitor (ATP-site di- of ErbB2-positive meta- rected) statis breast cancer in combination with cape- citabine

27 2.4.1. ErbB3 role in cancer drug resistance Despite of the initial optimism surrounding therapeutic benefits of ErbB2 and EGFR-specific drugs, the development of resistance to these drugs has become a major problem in which ErbB3 is a crucial component. It is now well documented that resistance to these inhibitors is accompanied by a compen- satory upregulation of ErbB3, resulting in increased signalling via the PI3K/Akt pathway (Sergina et al., 2007). Alternatively, increased production of neuregulins can also mediate this resistance, as is the case for colorectal cancer patients treated with cetuximab (Yonesaka et al., 2011). In addition, and as mentioned above, ErbB3 interaction with MET is pivotal for gefitinib resistance in NSCLC patients (Engelman et al., 2007). ErbB3-targeted therapies are also under development, although none of them are yet approved for cancer treatment (Lyu et al., 2018). Because of the impaired kinase activity of ErbB3, the usage of small kinase inhibitors is not an option, so efforts are put instead into developing blocking antibodies against this receptor. Examples worth mentioning are MEHD7945A, a dual EGFR/ErbB3 inhibitor which showed reduced tumour growth in both tumour xenografts and cell lines resistant to cetuximab and erlotinib (Huang et al., 2013b), and MM-111, a bispecific antibody that forms a tertiary complex with ErbB2 and ErbB3, resulting in growth inhibition of ErbB2-overexpressing cancer cell lines (McDonagh et al., 2012). ErbB3-exclusive antibodies have been also developed, such as MM-121, a monoclonal that, in combination with other drugs, blocks ligand-dependent ErbB3/ErbB2 dimerization, causing a dramatic downregulation of the PI3K/Akt signalling (Huang et al., 2013a; Jiang et al., 2014; Wang et al., 2013a), and also Patritumab (previously known as U3-1287/AMG-888) which is a monoclonal antibody that causes ErbB3 internalization and is now in phase III clinical trials for a variety of advanced solid tumours (Horinouchi, 2016).

28 3. A brief introduction to vesicular trafficking

The role of the ErbB3 receptor tyrosine kinase in controlling vesicular trafficking of different cargo proteins is one of the main themes of this thesis. Therefore, in this short section, I would like to give an overview on the different mechanisms and protein families involved in these trafficking pathways, as they will be referred to in the next sections. Although the literature defines the endosomal trafficking system in slightly different manners, we can distinguish four main compartments in it: the early endosome, which is the organelle that takes up the incoming endocytosed material; the perinuclear recycling compartment, specialized in the recycling of internalized cargo back to the cell surface; the late endosome or multivesicular body, an acidified organelle in which invagination of the outer membrane gives rise to intraluminal vesicles (ILVs); and the lysosome, a very acid compartment equipped with hydrolysing enzymes that carry out degradation of incoming material (Hu et al., 2015; Hutagalung and Novick, 2011; Stenmark, 2009; Wandinger-Ness and Zerial, 2014). All these different endosomal compartments are characterized by specific resident proteins and distinct lipid composition. The Rab GTPses are a family of low molecular weight GTP-binding proteins which are either cytosolic or membrane-associated, and which not only mark different organelles but also act as master regulators of their trafficking process (Grant and Donaldson, 2009; Mizuno-Yamasaki et al., 2012). GTPases cycle between the GDP and GTP-bound states, and the transition is facilitated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs turn on the GTPases by catalysing conversion of GDP into GTP, while GAPs termi- nate the activity by inducing the hydrolysis of GTP (Bos et al., 2007). GTP- bound Rabs can interact with effector proteins that mediate a variety of processes, from cargo selection and vesicle formation to fusion, without forget- ting vesicle translocation and tethering (Hutagalung and Novick, 2011). A graphic summary of the best characterized Rabs and their organelle localization can be seen in Figure 5.

Figure 5. Rab GTPases coordinate vesicular trafficking. The illustration depicts recycling routes after endocytosis and the endosomal maturation process, which involves acidification of the intraluminal space. Each compartment (or subdomains of the same compartment) are enriched in different Rab GTPases that regulate the fate of the endocytosed cargo.

The vesicular trafficking system also relies on a mechanism that ensures that vesicles only fuse with the right compartment. This mechanism is enforced by the SNARE family of proteins, which tethers the donor and receptor membranes before fusion takes place by pairing a specific v-SNARE (vesicular) with its corresponding t-SNARE (target membrane). Examples of SNARE proteins that will later be mentioned in this introduction are VAMP-3 or syntaxin-6, which mediate fusion events in between the early endosome, the trans-Golgi network (TGN) and the late endosome (Wang et al., 2017). The specific assembly between v- and t-SNAREs is mediated by different tethering factors, as the CORVET complex or the exocyst, which will also appear later in the text. Many other proteins are required during the trafficking process but are beyond the scope of this thesis: effectors that interact and recognize specific cargoes, coat proteins of different nature and adaptor proteins that mediate the interaction with actin filaments or microtubules, among others. Each of them contributes to this finely tuned system and, accordingly, malfunction of any of them may contribute to multitude of pathologies, including cancer.

30 4. Adhesion molecules: function, trafficking and their interplay with ErbB receptors

Cell-to-cell junctions and cell-to-extracellular matrix adhesions are essential structures for the formation and maintenance of tissues. Their function is not only to establish physical connection in between cells and anchorage to the substrate, but also to organize and regulate the cytoskeleton, mediate cell movement and modulate signalling pathways, therefore controlling tissue development, structure and cell physiology. In paper I and II of this book I will present our findings on the importance of ErbB3 for the proper vesicular trafficking of two important types of adhesion molecules, β1-integrin (paper I) and E-cadherin (paper II), to the plasma membrane of epithelial cells. In general, cadherins and integrins mediate two different types of cell adhesion: Cad- herins are important for cell-to-cell junctions, while integrins are involved in cell adhesion to the extracellular matrix. In this section, these proteins will be discussed with focus on their functions and our current knowledge about their vesicular trafficking.

4.1. E-cadherin, the main adherens junction molecule in epithelial tissues. There are three main types of cell-to-cell junctions: tight junctions (TJ), desmosomes and adherens junctions (AJ). The main function of tight junctions is to provide a physical barrier that restricts the permeability of epithelial layers, which is essential for polarization and for preventing water loss and free cir- culation of growth factors, etc., while desmosomes provide mechanical strength to epithelia. These two types of junctions are beyond the scope of this thesis, but a comprehensive review about them can be found in: (Balda and Matter, 2008; Delva et al., 2009; Johnson et al., 2014; Zihni et al., 2016). Adherens junctions (AJ) are a cell-to-cell adhesion characterized by the presence of Cadherin or Nectin proteins, which bridge the membranes of two adjacent cells, and a variety of cytoplasmic proteins linking the whole complex with condensed actin filaments (Miyaguchi, 2000). In epithelial tissues, E-cadherin is the primarily expressed cadherin. Similar to the other members of the family, E-cadherin extracellular domain is divided into five repetitive subdomains, each one containing a calcium-binding sequence. The interaction

31 with calcium ions controls the conformation of the extracellular region and it is required for the establishment of homophilic interactions with same-type cadherins present in the neighbouring cell. Therefore, calcium chelators, such as EGTA, are widely used to promote the dissociation of cells in monolayer culture (Meng and Takeichi, 2009). The E-cadherin cytoplasmic tail binds to proteins collectively called catenins. The juxtamembrane portion binds to p120-catenin, which belongs to the family of the Armadillo proteins, whereas the carboxy-terminal part binds β-catenin. β-catenin associates with -catenin and the classical view is that this protein mediates the interaction of the whole E-cadherin-β-catenin- -catenin complex with actin filaments (Rimm et al., 1995). This model has been called into question (Drees et al., 2005; Gates and Peifer, 2005; Yamada et al., 2005) since the quaternary complex cadherin-β- catenin- -catenin-actin filament has not been purified. However, it was more recently reported that mechanical forces are required for stabilization of this interaction (Buckley et al., 2014), which may explain why efforts into purify- ing this complex have been unsuccessful.

Figure 6. Schematic representation of E-cadherin-based adherens junctions (AJs) in epithelial cells. On the left, typical organization of an epithelial monolayer (desmosomes not included). To the right, details of the E-cadherin structure and homodimerization. Although other proteins are involved in the interaction with the cytoskeleton, only the core E-cadherin/catenin complex is illustrated.

4.1.1. E-cadherin vesicular trafficking Although mature tissues and confluent monolayers look like static structures, their adherens junctions are under constant turnover. The surface levels of E- cadherin and other proteins of the complex are controlled by various mechanisms. Targeting of newly synthesized E-cadherin from the Golgi to the plasma membrane occurs by mediation of the so called exocyst complex, containing the Sec6/8 proteins (Yeaman et al., 2004a). The exocyst is a protein complex, conserved among eukaryotes, that is required for sorting and the final fusion step in the secretory pathway, both for vesicles derived from the

32 Golgi or from recycling endosomes (Brüser and Bogdan, 2017). Knocking down of its members causes vesicles to accumulate underneath of the plasma membrane (Finger and Novick, 1998). The process of sorting E-cadherin from the trans-Golgi network (TGN) is of critical importance: since E-cadherin is a major player in the establishment of epithelial cell polarity, it itself must be transported to the membrane in a polarized manner, specifically to the basolateral surface of the cell. A dileucine sorting signal has been found in the cytoplasmic tail of E-cadherin and has been implicated in its targeting to basolateral membranes. However, this signal is not present in all the cadherin species, indicating the existence of alternative sorting mechanisms (Miranda et al., 2001). In addition, cotransport of β-catenin together with E-cadherin from the TGN seems to be important for the efficient trafficking of the latter (Chen et al., 1999). The post-Golgi carrier Golgin-97 is also required for the initial step of E-cadherin exit from the TGN (Lock et al., 2005). After exiting the Golgi, different trafficking pathways can be taken. In a Drosophila melanogaster model, two membrane trafficking pathways for delivery of newly synthesized DE-cadherin (the fruit fly orthologue to E-cadherin) have been described: lateral and apicolateral exocytosis, both mediated by Rab11 GTPase (Woichansky et al., 2016). Rab11 is a marker of the recycling endosomal compartment, meaning that newly synthesized E-cadherin, instead of going directly to the cell surface, goes first to this intermediate compartment (Lock and Stow, 2005). Apart from de novo synthesis, the rate of internalization and recycling of E-cadherin from the cell surface is to a high degree responsible for E-cadherin turnover. Early experiments showed that the half-life of surface E-cadherin is in the range of 5 to 10 h (McCrea and Gumbiner, 1991) while its biosynthesis exhibits slower kinetics (Cavey et al., 2008). Our own results demonstrate that, upon artificial disruption of AJs, newly synthesized E-cadherin does not account for the recovery of junctions during the first 6 h, although they start reforming almost immediately after disruption (paper II). Therefore, E-cadherin internalization and subsequent recycling is an important mechanism for AJs stabilization. E-cadherin may be internalized in response to several stimuli: when cell-cell junctions are artificially disrupted, when certain signalling cascades are activated, or in response to several transcriptional and posttran- scriptional regulators. Several possible routes of endocytosis have been described, both clathrin-dependent, clathrin-independent and caveolae-mediated (Brüser and Bogdan, 2017). Clathrin-dependent endocytosis is the most studied mechanism for E-cadherin internalization. In the case of E-cadherin, the dileucine sequence in the cytoplasmic tail acts as a binding motif for the clathrin adaptor AP-2 (Kelly and Owen, 2011). Interestingly, this motif overlaps with the sequence recognized by p120-catenin, consistent with mutually exclusive binding: in presence of p120-catenin, AP-2 cannot interact with E-cadherin and its clathrin- dependent internalization is prevented (Davis et al., 2003; Nanes et al., 2012).

33 In turn, in absence of p120-catenin, E-cadherin is constitutively internalized and subjected to lysosome-mediated degradation (Miyashita and Ozawa, 2007). Dynamin, a large GTPase which conducts the scission of invaginations on the cell surface, is also required for endocytosis of E-cadherin, and in its absence surface E-cadherin levels are increased (Kirchhausen et al., 2014). Common vehicles of E-cadherin internalization are Rab5- and EEA1-positive endosomes (Bryant and Stow, 2004), which play a role in both clathrin- dependent and independent endocytosis (Wandinger-Ness and Zerial, 2014). Interestingly, Rab5 overexpression and, in general, increased vesicular trafficking, have been recently shown to alter epithelial monolayer rigidity and to promote organized collective cell migration (Malinverno et al., 2017). Upon internalization, E-cadherin may be recycled or degraded. Recycling of E-cadherin can happen through different pathways: fast and direct recycling mediated by Rab4 (de Madrid et al., 2015), or slower, via the Rab11-positive recycling endosome (Loyer et al., 2015). In addition, RabIX has been identified in Drosophila as another important mediator of DE-cadherin recycling, although knowledge on this GTPase is still scarce (Woichansky et al., 2016). Others have found that Rab8 and its effector JRAB/MICAL-L2, also associated with Rab13 and the recycling of the TJ protein occludin, are required for the recon- stitution of E-cadherin-based junctions after chemical disruption by EGTA treatment (Nishimura and Sasaki, 2008; Yamamura et al., 2008). For completion of the recycling process, as in the case of surface targeting of the newly synthesized E-cadherin, the exocyst complex is also required. In fact, β- catenin and Rab11 are known to interact with several proteins from the exocyst complex, and blocking this interaction provokes E-cadherin to accumulate in the recycling endosomal compartment (Langevin et al., 2005). Internalized E-cadherin that is not recycled is instead sent for lysosomal degradation, although the exact process of fate determination is not completely understood. E-cadherin degradation is an important step in metastatic progression, and studies show that, upon growth-factor induced activation, Src tyrosine kinase phosphorylates E-cadherin and the E3-ubiquitin ligase Hakai marks it for degradation (Fujita et al., 2002). In this process, Src-induced activation of Rab5 and Rab7 seems crucial, as does the E-cadherin interaction with Hrs, an ESCRT-complex subunit that mediates its lysosomal degradation (Palacios et al., 2005). A similar observation was made upon extracellular calcium depletion, which caused activation of Cdc42 and consequent activation of EGFR, which in turn activated Src, leading to the same degradative fate (Shen et al., 2007). In addition, the phosphatidylinositol phosphate kinase PIP- KIγi5 has been shown to associate with E-cadherin and promote its lysosomal degradation. In this context, sortin nexins 5 (SNX5) displayed an inhibitory role on E-cadherin degradation, by associating with PIPKIγi5 and inhibiting its function (Schill et al., 2014). These proteins are involved in a similar manner in the transport of internalized EGFR receptor (Sun et al., 2013). Another sortin nexin implicated in protecting E-cadherin from degradation is SNX16,

34 whose interaction with E-cadherin has been shown to promote its recycling (Xu et al., 2017).

4.1.2. E-cadherin crosstalk with signalling pathways Signalling by tyrosine kinases can also regulate the function, trafficking and levels of E-cadherin expression. Tyrosine phosphorylation of the E-cadherin- catenin complex results in the endocytosis of E-cadherin and subsequent disruption of the cell-cell junctions. Cytoplasmic kinases, such as the Src family, have emerged as important regulators of E-cadherin dynamics. Src activation causes disruption of AJs in at least two manners: on one hand, Src, as already mentioned, phosphorylates E-cadherin in the juxtamembrane domain, creating a binding site for Hakai, a c-Cbl-like ubiquitin ligase, which results in proteosomal degradation of E-cadherin (Fujita et al., 2002; Hartsock and Nelson, 2012); on the other hand, Src activation causes translocation of β-catenin to the cell nucleus, where it activates a transcriptional program associated with cellular migration and the process of epithelial-to-mesenchymal transition (Benham-Pyle et al., 2015; Fernández-Sánchez et al., 2015). It has been proposed that β-catenin translocation to the nucleus is the result of tyrosine phosphorylation of β-catenin by Src, which lowers the affinity of β-catenin for E- cadherin (Roura et al., 1999). Newer evidence, however, suggests that this phosphorylation is not crucial, but the activation by Src of the focal adhesion kinase (FAK) is, which results in relaxation of E-cadherin surface tension through actomyosin remodelling (Gayrard et al., 2018). In addition to its role in destabilizing AJs, it has also been shown that Src activation can promote E- cadherin-based contacts and favour E-cadherin recycling, and that the strength of the signal may be decisive for the occurrence of one or the other outcome (Förster and Luschnig, 2012; McLachlan et al., 2007). In addition, physical interaction with the E-cadherin-catenin complex has been reported for RTKs, such as hepatocyte growth-factor receptor (c-Met), fibroblast growth-factor receptor (FGFR) and EGFR, which seems to involve co-endocytosis of the cadherin and the RTK upon stimulation of the receptor (Cavallaro and Christofori, 2004). Early studies showed that, upon ligand stimulation, EGFR phosphorylates β-catenin and gamma-catenin, which induces disassembly of the cadherin-catenin complex and lead to internalization (Hoschuetzky et al., 1994). Additionally, E-cadherin interaction with EGFR at the cell surface has been proposed to decrease EGFR mobility and ligand-affinity (Andl and Rustgi, 2005; Qian et al., 2004), and its control over EGFR signalling has proven crucial for the establishment of cell polarization in the epidermis of new born mice (Rübsam et al., 2017). Despite several functional links between the function of EGFR and E-cadherin, the crosstalk of this adhesion molecule with other members of the ErbB family has not been extensively studied, apart from reports showing that overexpression of ErbB2 causes downregulation of E-cadherin gene

35 (Cheng et al., 2013; Palacios et al., 1995). Interestingly, interaction between E-cadherin and ErbB3 has only been observed in the context of overexpression and in stimulated cells (Witta et al., 2009). Our work is therefore the first proof of interaction between ErbB3 and E-cadherin at endogenous levels and in the context of ligand deprivation (paper II).

4.1.3. E-cadherin in cancer progression Most human cancers originate from epithelial tissue. This fact brings to light the capital importance of all the cellular mechanisms that play a role in main- taining the epithelial features, among them the cell-cell junction components. Noteworthy, during tumour progression and metastasis, E-cadherin expression is frequently lost and cells exhibit defects in the formation of cell-cell contacts (Cano et al., 2000). The resulting reduced adhesion in between cells facilitates dissemination of cancer cells from the primary tumour. Accord- ingly, already in the 1990s it was proposed that the loss of E-cadherin-mediated cell-cell adhesion is a prerequisite for metastasis formation (Birchmeier and Behrens, 1994) and in fact, E-cadherin downregulation is one of the hallmarks of the EMT process (Valastyan and Weinberg, 2011; Yang and Weinberg, 2008). In agreement, it has been shown that re-establishing fully functional cadherin complex (by forced expression of E-cadherin, for instance) results in reversion from an invasive, mesenchymal phenotype to a benign, epithelial phenotype of cultured tumour cells (Perl et al., 1998). However, the tumour suppressive role of E-cadherin has encountered, over the years, many exceptions that invite us to reconsider such a role. On one hand, EMT is not a pre-requisite for metastasis formation (Fischer et al., 2015; Zheng et al., 2015), and distal metastases generally preserve E-cadherin expression (Shamir et al., 2014). Additionally, it has been observed that clusters of cells disseminating from the primary tumour can form distal metastases (Aceto et al., 2014; Cheung et al., 2016). Consequently, in this scenario, E- cadherin does not act as a tumour suppressor, but as a factor that facilitates cancer spreading, giving rise to a new type of malignant invasion: the collective invasion, which is an important feature of metastatic breast cancer (Cheung et al., 2013). The notion that E-cadherin expression can also have a pro-metastatic function is relevant in the context of our work, where we discover that absence of ErbB3, known for its pro-tumorigenic and pro-metastatic role, can result into impaired E-cadherin cell-to-cell junctions.

36 4.2. Integrins, the main cell-to-ECM receptors Integrins are cell surface receptors that mediate and regulate the interaction between cells and their surrounding extracellular martix. They belong to an evolutionary preserved family of type I transmembrane receptors composed of and β subunits, that always act together in the form of non-covalently bound heterodimers. In vertebrates, there are 18 -integrin and 8 β-integrin subunits, and the different combinations give rise to 24 possible heterodimers (Barczyk et al., 2010; Hynes, 2002). Integrins mediate both adhesion and migration, and therefore their correct functioning is crucial for epithelial homeostasis and avoidance of invasion and metastasis. Integrins are regulated both at the level of their protein amount at the cell surface and through their activation status. In this section, the most important features of integrin structure, activation and vesicular trafficking will be discussed, with special attention to their importance to cancer. In epithelial cells, β1-integrin is the predominant subunit that forms heterodimers with different -integrins. Since our work in paper I focuses on recycling of β1-integrin, trafficking of this integrin will be discussed in more depth than for other subunits.

4.2.1. Integrin structure, ligands and modes of activation The structures of both and β subunits share an extracellular head, a leg section, a single transmembrane domain and a short cytoplasmic tail, but there are important differences both in the head and in the leg. The subunit presents four/five extracellular domains: a 7-bladed β-propeller domain, which constitutes the head, and a ‘thigh’ domain followed by two ‘calf’ domains, which constitute the leg. Some -Integrins (9 out of 18) include an insertion of 200 residues in the β-propeller domain, termed the I domain (Larson et al., 1989) and which, when present, acts as the major ligand-binding site (Xiong et al., 2001, 2004). In addition, the lower side of the head contains calcium binding domains which influence the ability to bind ligand (Humphries et al., 2003). The thigh and calf domains are immunoglobulin- like structures made of β-sandwich folds, which present interdomain segments that confer flexibility to the entire integrin structure (specifically, the linker between the head and the thigh, and the interdomain between the thigh and the calf I, called ‘genu’) (Campbell and Humphries, 2011). The β subunit consist of the same domains, although they present a different structure: the head segment is composed of a βI domain (which is homologous to the I insertion in some of the -integrins), a hybrid segment (with a β-sandwich structure) and a plexin-semaphorin-integrin (PSI) domain (Xiao et al., 2004). The leg domain that follows consists of four EGF modules which connect with a β-ankle domain that is the final extracellular piece before the transmembrane domain (Anderson et al., 2013; Zhu et al., 2008). The EGF domains are quite plastic,

37 conferring the β-subunit higher flexibility than the -integrins (Campbell and Humphries, 2011).

Figure 7. General structure of the

Integrins are synthesized in the endoplasmic reticulum and already there as- sembled into heterodimers, a step necessary for their subsequent transportation towards the Golgi apparatus (Ho and Springer, 1983). In the Golgi, integrin heterodimers undergo extensive glycosylation before going to the cell surface, which affects their ligand binding properties and their roles in migration and adhesion (Bellis, 2004; Cai et al., 2017; Kariya et al., 2017). Different integrin heterodimers show affinity for different ligands, and for each ligand there is more than one heterodimer that can recognize it. Integrin ligands are either extracellular matrix components (collagen, fibronectin, pro- teoglycans, laminin, etc.) or non-ECM components, as well as other proteins on the cell surface (ICAMs, VCAMs and E-cadherin, in haematopoietic cells), plasma proteins or cytokines, among others (Humphries et al., 2006; Moreno- Layseca et al., 2019). For example, β1-integrin, which is the subtype studied in paper I, is able to bind, depending on the -subunit that it associates with, up to 10 different ligands.

Figure 8. β1-integrin heterodimers and the components of the extracellular matrix substrates that they bind, according to Moreno-Layseca et al., 2019.

Despite of variations in size, subunits are larger than β, although β-subunits, through their cytoplasmic tails, mediate most of the intracellular protein interactions of each heterodimer. It must be noted that neither type of subunit has direct actin-binding properties or catalytic activity. Therefore, integrin heterodimers act as hubs for adaptor proteins, which mainly bind three specific sites on the β-subunit tail: a membrane proximal HDRK motif, recognized by FAK, paxilin and Fyn (Reddy et al., 2008); a membrane proximal NPxY motif, recognize by talin through its phosphotyrosine-biding domain (PTB) (Wegener and Campbell, 2008); and a membrane distal NPxY motif, recognized by kindlin also through its PTB domain (Moser et al., 2008). The different adaptor proteins that can bind integrins act in different manners; some have catalytic activity, like FAK, that upon activation initiates downstream cascades; others have a structural function, like talin, linking integrins to the actin cytoskeleton; finally, the third group act as scaffolds, like the kindlin proteins, which mediate the interaction with a second adaptor protein (Legate and Fässler, 2009). Importantly, there are other adaptor proteins that inhibit integrin activation, like SHARPIN or MDGI, that are two -subunit binding proteins that, when associated with integrins, block interaction with adaptors like talin (Bouvard et al., 2013). Integrins allow bidirectional signalling, which means that they can undergo activation by two different and independent means: inside-out and outside-in activation. Inside-out signalling implies that adaptor proteins trigger a conformational change in the integrin heterodimer when they bind the cytoplasmic tail, increasing the affinity of the integrin head domains for their ligand at the

39 ECM (Takagi et al., 2001). For example, binding of talin to the β-tail causes separation of the and β tails, and this triggers the conformational change that will result in integrin activation. Outside-in signalling, on the contrary, occurs when binding of ECM proteins to the integrin heterodimer generates the conformational change that allows integrin clustering and recruitment of adaptor proteins to the cytoplasmic tail. Phosphorylation cascades are also important in this type of activation. In fact, Src tyrosine kinase and its inhibitor Csk are constitutively bound to the β-integrin tail, but upon ligand binding, the Csk is displaced allowing Src activation (Arias-Salgado et al., 2005). Integrins can also act by transactivating other receptor proteins. For example, both Vβ3 and β1-integrins can associate with the EGFR receptor and induce its phosphorylation at distinct sites, in cooperation with a macromolec- ular complex including Src tyrosine kinase and p130Cas adaptor protein (Ivaska and Heino, 2011; Moro et al., 1998, 2002). Moreover, β1-integrin- dependent phosphorylation of EGFR has been shown at cell-cell contacts, where the RTK and the integrin dimer establish close contact (Yu et al., 2000). Ligand-independent transactivation of MET by β1-integrin has also been reported in ovarian cancer cells. In this context, fibronectin binding to the integrin heterodimer triggered -integrin association and activation of Met, followed by downstream activation of Src and FAK and increased invasive potential (Mitra et al., 2011).

4.2.2. Vesicular trafficking and recycling of integrins Integrins on the cell surface can be internalized by both clathrin-dependent and independent mechanisms and the same heterodimers may use multiple routes for internalization. Among the different adaptor proteins that can bind the NPXY-like motifs in the cytoplasmic tail of β-integrins, there are some, like Dab2, ARH and Numb, which act as clathrin-adaptors, therefore mediating clathrin-dependent endocytosis (Onodera et al., 2013). Dab2 has been found to mediate internalization of β1, 1, 2 and 3-integrins in HeLa cancer cells, and Dab2-depleted cells showed reduced migration and reduced targeting of β1-integrin to the migratory leading edge (Teckchandani et al., 2009). Involvement of Dab2 and ARH in 5β1-integrin endocytosis upon microtubule disassembly has also been proven in fibroblasts (Ezratty et al., 2009). Notably, although the work from Teckchandani et al. indicates that Dab2 is specifically involved in internalization of disperse inactive β1-integrin, the work from Ezratty et al. shows that Dab2 is also involved in the clathrin-dependent internalization of β1-integrin ocurring at focal adhesions, in agreement with what others have also found (Chao and Kunz, 2009). Whether the activation status of β1-integrins is relevant for its internalization has been addressed by using antibodies that recognize either the active or inactive integrin form, concluding that internalization rate of active β1-integrin is higher than

40 that of inactive one, and that active and inactive forms undergo distinct trafficking routes (Arjonen et al., 2012). Internalization via clathrin-independent mechanisms has been observed in different contexts. For example, caveolae-dependent internalization of 5β1 integrin occurs constitutively and in absence of fibronectin (Shi and Sottile, 2008), while, in ovarian cancer cells, activated EGFR can trigger internalization of 2-integrin in a caveolae/lipid raft dependent manner (Ning et al., 2007). Alternatively, rapid internalization of integrins by micropinocytosis has been proposed as an additional mechanism to keep up with the formation and release of focal contacts during cellular migration (Gu et al., 2011). Internalization of integrins is also mediated by proteins that interact with the membrane proximal segment of the -cytoplasmic tail, as is the case for the Rab21 GTPase. Rab21, when locked in the GDP-bound form, provoked accumulation of active β1-integrin at focal adhesions, and this affected negatively the adhesive and migratory capabilities of breast and prostate cancer cells (Pellinen et al., 2006). Furthermore, Rab21-dependent trafficking of β1- integrin to the cleavage furrow of dividing cells is crucial for completion of cytokinesis, and malfunctioning of this route is linked to occurrence of multi- nucleated cells in different types of cancer (Pellinen et al., 2008). In addition, some -subunits can also interact with PDZ-domain containing protein adaptors, like GIPC1 (El Mourabit et al., 2002), which through interaction with myosin VI can mediate both clathrin-dependent and independent internalization (Naccache et al., 2006). Upon internalization, integrins can either be recycled or degraded, and this decision is pivotal for the regulation of integrin dynamics. A number of routes have already been elucidated for the recycling of integrins. Just after internalization, integrins and other cargoes enter the Rab5-positive early endosome compartment (Zerial and McBride, 2001). From here, integrins can undergo two major recycling routes: Rab4-dependent short-loop/fast recycling or Rab11-dependent long-loop/slow recycling, although there are other routes and proteins implicated that will also be discussed below. Importantly, these pathways are not exclusive for integrin trafficking, and other protein cargoes also use them, such as the already discussed E-cadherin, or transferrin receptor, glucose transporter GLUT4 and MET, among others (Brüser and Bogdan, 2017; Jaldin-Fincati et al., 2017; Parachoniak et al., 2011; van der Sluijs et al., 1992). The Rab4-mediated route allows rapid recycling of internalized cargo directly from the early endosome compartment and without passing through the perinuclear recycling compartment (Caswell and Norman, 2006). It has been shown in fibroblasts that this route allows fast recycling of vβ3 (in 5 to 10 minutes), and that this is enhanced upon stimulation with platelet-derived growth factor (PDGF). Importantly, although a limited amount of 5β1 is also recycled through this route, this heterodimer does not show any response to PDGF stimulation (Roberts et al., 2001). Furthermore, protein kinase D

41 (PKD1), which binds to the C-terminal tail of β-integrin, was found to be required for Rab4-dependent recycling (Woods et al., 2004). Later work from the same group showed that this is due to phosphorylation of the Rab5/Rab4 effector Rabaptin-5 by PKD1, which ultimately controls vβ3 recycling and invasive migration of cancer cells (Christoforides et al., 2012). PKD and Ra- baptin-5 are two molecules directly implicated as targets of ErbB3 in our studies (paper I and II), and will therefore be discussed more extensively later. The long-loop route allows integrin recycling via the perinuclear recycling compartment, which is enriched with Rab11 and Arf6 GTPases (Powelka et al., 2004). Rab11 interacts, through its adaptor Rab11-FIP2, with myosin Vb, and this allows transportation along the actin cytoskeleton of vesicles carrying a variety of cargoes (Hales et al., 2002). Microtubules are also implicated in Rab11-mediated vesicle transport (Casanova et al., 1999; Yoon et al., 2005), and this could be controlled by Protein kinase B/GSK-3β (Morfini et al., 2002). Integrin β1 is known to follow the Rab11 pathway in many different contexts: upon EGF stimulation in cancer cells (Powelka et al., 2004), in unstimulated fibroblasts (Roberts et al., 2004), or in embryonic neural crest cells subjected to high ligand concentration (Strachan and Condic, 2004), among many others. In addition to Rab4 and Rab11-mediated recycling, other recycling routes have been elucidated in the last few years. Rab25, through direct interaction, has been shown to mediate recycling of β1-integrin to the pseudopodial tips of an ovarian cancer cell line, promoting invasive migration in 3D culture (Caswell et al., 2007). Although Rab25, also known as Rab11c, belongs to the Rab11 family, its expression pattern (exclusively epithelial) and its unique ability to interact directly with β1 justifies considering it a separate case (Goldenring et al., 1993). Moreover, Rab25 has been linked with trafficking of active β1-integrin to the lysosome, from where it recycles back to the cell surface, in cooperation with the CLIC3 chloride channel (Dozynkiewicz et al., 2012). Since Rab25 expression, but not Rab11, was found to induce expression of CLIC3, Rab25 further reinforces recycling of β1 also from the lysosome. The Rab13 GTPase has also been implicated in the recycling of β1-integrin during cell migration and invasion, together with the Golgi- localized gamma-ear containing Arf-binding protein GGA2 (Sahgal et al., 2018, preprint). Additionally, Rab13 is involved in the trafficking from the TGN to recycling endosomes (Nokes et al., 2008) and, although it has been linked to the trafficking of a lymphocyte-specific integrin type, LFA-1 (Nishikimi et al., 2014), the knowledge on its involvement on the trafficking of other receptors of this family is only beginning to grow. Recently, retrograde transport of β1-integrin to the trans-Golgi network has been implicated in the recycling of integrins. The retrograde trafficking machinery is controlled by several proteins including Rab6, Syntaxin-16 and the retromer sorting complex. The retromer is a multiprotein complex formed by

42 VPS26, VPS29 and VPS35 and different sortin nexins (SNX) and which, depending on the specific SNX, mediates return of cargo proteins to the plasma membrane or to the TGN (Lucas and Hierro, 2017). Although the retromer- dependent recycling is mostly considered to concern cargoes other than integrins, like lysosomal enzymes or Wnt glycoproteins (Franch-Marro et al., 2008; Zhang et al., 2012), retromer-dependent retrograde transport of inactive, non- ligand bound β1-integrin was recently found to play a major role in recycling of this subunit. In this study, using a vectorial proteomics approach, different integrins were found to traffic from the cell surface to the TGN and recycle back, among them β1, locking this route results in reduced cell adhesion and reduced persistent migration in different epithelial cell lines (Shafaq-Zadah et al., 2016). In addition, others had found before that the complex formation of t-SNARE Syntaxin-6, a type of SNARE involved in the retromer function, and the v-SNARE VAMP3 is involved in the retrograde transport and recycling of β1 and β1 in HeLa cells (Riggs et al., 2012). Another protein machinery, the CORVET, has been linked to endosomal trafficking of integrins. The CORVET is a hexameric complex important for fusion of different endosomal compartments. It is composed of a core shared with the HOPS complex (tethering complex at the late endosome) and two additional subunits, Vps8 and Vps3 (van der Kant et al., 2015; Perini et al., 2014). Vps8 and Vps3 have been found to localize in Rab4-positive vesicles and to mediate the recycling of β1-integrin, but not of the transferrin receptor, through the Rab11-positive recycling endosome, where they transiently interact with CHEVI, another vesicle-interaction complex that localizes to the perinuclear recycling compartment (Jonker et al., 2018). Alternatively, recycling of integrins can happen by interaction with the retriever machinery, a heterotrimeric complex structurally similar to the retromer but which consists of DSCR3 (paralogue to VPS26), C16orF2 (structurally related to VPS35) and VPS29 (also present in the retromer) and associates with different sorting nexins (like SNX17) than those associated with the retromer. The retriever complex is able to mediate the retrieval of a different set of endocytosed cargoes to the cell surface (Rabouille, 2017). One of them is the β1-integrin heterodimer, whose cell surface presence is reduced upon SNX17, CCDC93, CCDC22, C16orf62 or DSCR3 depletion, all of them components of the retriever machinery (McNally et al., 2017). SNX17 was earlier found to be required to rescue β1-integrin from lysosomal degradation and redirect it towards the recycling pathways (Steinberg et al., 2012). In this study, SNX17 was proposed to act by promoting Rab4-dependent recycling of β1, but if the retriever promotes recycling specifically through this route or if it collaborates with different Rabs depending on the cellular and tissue context, is still unaddressed. In general, any given integrin heterodimer seems to be able to take several different trafficking itineraries. However, despite the

43 fast expansion of the vesicular trafficking field, little is known about why in different contexts integrins take one route or another. The activation status of integrins has emerged as an important determinant of trafficking fate. The previously cited work from Arjonen et al. did not only show differences in the internalization rate of active and inactive integrins, but also demonstrated that their recycling is not equivalent: while inactive β1-integrin undergoes fast Rab4-dependent recycling, the active conformation, still bound to its ligand, traffics towards the Rab7-positive late endosome and is recycled less efficiently and in a Rab4-independent manner (Arjonen et al., 2012). Furthermore, in fibroblasts it has been shown that ubiquitination and sorting of active ligand-bound β1-integrin to multivesicular bodies and subsequent lysosomal degradation, is a pre-requisite for migration (Lobert et al., 2010). On the contrary, another group has reported that β1-integrin at focal adhesions, when internalized, associates with and is activated by focal adhesion kinase (FAK) in the early endosomes, from where it goes to the Rab11 compartment and is recycled to the leading edge of migrating cells (Nader et al., 2016). This disparity of results may be explained by different types of integrin activation acting in each case and by the presence or absence of ligand associated with the internalized receptor: while inside-out activated integrin, maybe still in clusters, may need to be recycled in an efficient manner to build up new focal adhesions, the recycling of outside-in activated integrin, still lig- and bound, presents no advantage for the formation of new contacts, and may instead be degraded. In any case, other examples across the literature describe differential trafficking of active and inactive integrins (Mana et al., 2016; Sahgal et al., 2018, preprint; Shafaq-Zadah et al., 2016) showing that the activation status is an important regulatory element of the stability and half-life of integrins. It seems clear that in the absence of recycling, endocytosed integrins are instead targeted to late endosomes and lysosomes for degradation, although it should be noted that transport to the late endosome does not always imply subsequent degradation (Rainero and Norman, 2013). Depletion of a number of proteins important for recycling of certain integrins causes misrouting of integrins towards the lysosomal compartment. An example is the previously mentioned SNX17, which protects β1 from degradation by binding to the distal NPXY motif and the adjacent TT788/789 residues in β1-integrin, the same region recognized by kindlins at the cell surface (Böttcher et al., 2012; Steinberg et al., 2012). Similarly, depletion of the t-SNARE syntaxin-6 causes ubiquitination and misrouting towards the lysosome of the β1-integrin heterodimer in endothelial cells. Although the exact mechanism by which syntaxin-6 is connected to integrin recycling was not elucidated, this protein was found to colocalize in the early endosomal compartment with β1 and to mediate cell spreading and migration on a fibronectin substrate (Tiwari et al., 2011). In endothelial cells, another factor, the adaptor protein PPFIA1, has been connected to polarized recycling of β1-integrin (Cheung et al., 2016).

44 Silencing of the C-terminal Eps15-homology (EH) domain-containing protein EHD1 disrupts recycling of β1-integrin in human cancer cells and fibroblasts, although in this specific case, lysosomal degradation was not observed, but only integrin accumulation in the perinuclear region (Jović et al., 2007). In addition, the Arf6-GTPase activating protein (GAP) ACAP1, previously known to mediate trafficking of the transferrin receptor (Dai et al., 2004) also colocalizes with endocytosed β1-integrin and is required for its recycling, in a mechanism dependent on growth factor stimulation and that involves ACAP1 phosphorylation by Akt (Li et al., 2005). Another molecule implicated in regulation of integrin trafficking is APPL1, an adaptor protein that reduces Rab5- dependent internalization of β1-integrin. However, while other adaptors that promote recycling of β1-integrin also promotes cell migration, APPL1 instead inhibits cell migration on fibronectin (Diggins et al., 2018). This dis- crepancy highlights the tight regulation of surface integrins levels required to achieve proper adhesion and migration dynamics. Notably, another study found a different effect of APPL1 on migration (Tan et al., 2016), thus further investigations are required to fully understand the role of this adaptor protein. Importantly, also a mutant version of the tumour suppressor p53 has been shown to accelerate recycling of β1-integrin and EGFR, via recruitment of the Rab11 effector RCP (Rab-coupling protein) to β1-integrin, which caused increased invasiveness of lung cancer cells (Muller et al., 2009). The ability of EGFR and β1-integrin to recycle together has also been observed in the context of β3 blockage and EGF stimulation, and its occurrence is linked to increased fibronectin-dependent migration (Caswell et al., 2008). Two members of the family of the GGA Golgi-localized gamma-ear containing Arf-binding proteins, GGA3 and GGA2, are also involved in integrin recycling. The case of GGA2 dictating the recycling of β1-integrin in cooperation to Rab13 was already commented earlier on this section (Sahgal et al., 2018, preprint). In addition, GGA3, through binding to Arf6, was found to promote Rab4-dependent recycling of β1-integrin in cancer cells. Upon GGA3 knockdown, both β1-integrin and SNX17 relocate to LAMP1-positive late endosomes and subsequently β1-integrin is degraded in the lysosome (Ratcliffe et al., 2016). Our work on ErbB3 regulation of β1-integrin recycling (paper I) also connects with GGA3 function in a similar fashion. Accordingly, this protein will be introduced in more detail in section 5.3.

45 5. Molecular regulators of vesicular trafficking

5.1. The PKD family of serine/threonine kinases The protein kinase D family (consisting of three proteins in mammals: PKD1, PKD2 and PKD3) is a group of Ser/Thr kinases acting downstream of PLC (phospholipase C) and PKC, and which responds to several stimuli (oxidative stress, hormones, growth factors and neurotransmitters, among others). PKD proteins have two N-terminal C1 domains (C1a and C1b) that bind to DAG, an auto-inhibitory pleckstrin homology (PH) domain and a C-terminal kinase domain (Cobbaut and Van Lint, 2018; Rykx et al., 2003). The three members of this family share high similarity, up to 85% amino-acid identity in the case of PKD1 and PKD2 (Azoitei et al., 2018), mainly in the kinase domain and the C1 region. They also present some divergence in the C-terminal tail (Papazyan et al., 2006), the N-terminal sequence and between C1b and the PH domain (LaValle et al., 2010). Despite overlapping in many functions, these sequence disparities between the three PKD proteins may account for some of the reported functional differences (Hausser et al., 2005; Huck et al., 2012; Matthews et al., 2010).

Figure 9. Domains structure of the three PKD isoforms. Key phosphorylation sites are indicated.

Originally, PKD proteins were classified as a subgroup belonging to the PKC family and, for this reason, PKD1 is also known as PKCµ and PKD3 is also named PKCν. Inclusion into the PKC family was due to homology of their C1

46 domain with the DAG-binding domain in PKC proteins, but eventually these proteins were reclassified as a subgroup of the calcium/calmodulin-dependent protein kinase (CAMK) family, in accordance with the sequence homology of their kinase domain (Manning et al., 2002). Activation of PKD proteins can happen in at least three different ways: one is the classical PLC-PKC-dependent mechanisms. When PLC is activated by external stimuli on associated receptors, it carries out the cleavage of membrane lipids, generating DAG and IP3. Membrane-associated DAG recruits both PKC and PKD through their C1 domains. Then, it has been proposed that PKC phosphorylates PKD at Ser744 and Ser748 in the activation loop, after which PKD is able to autophosphorylate at Ser916 and also Ser748, support- ing a much more sustained activation (Waldron and Rozengurt, 2003). How- ever, mutants disrupting Ser744/748 phosphorylation are still able to display Ser916 autophosphorylation, although they are catalytically inactive towards downstream substrates (Rybin et al., 2009), indicating that phosphorylation of this residue is not always a good reporter of PKD activity. In the same manner, it should be noticed that phosphorylation at Ser748 is both carried out by PKC and by PKD itself. Therefore, this is not a perfect residue to measure PKC- mediated activation of PKD. Another type of PKD activation is mediated by the Gβγ subunit of the heterotrimeric G protein. Gβγ activates PKD by direct interaction with the autoinhibitory PH domain (Jamora et al., 1999), and this is known to affect structure and vesicularization of the Golgi apparatus. The third PKD activation route is through caspase-3-cleavage of the autoinhibitory domain, which can occur during genotoxic agent-induced cell death (Endo et al., 2000). Upon activation, different isoforms of PKD regulate crucial aspects of cell physiology and cancer (for a general review on different functions: Fu and Rubin, 2011; or: Rykx et al., 2003). PKD proteins have been extensively linked to EMT and tumour progression (Azoitei et al., 2018; Durand et al., 2016; Roy et al., 2017), although in different ways depending on the isoform and the exact context. PKD1 was shown to inhibit cell invasion by negative regulation of several metalloproteinases and its expression was found to be epigenetically suppressed in invasive breast tumours from patients (Eiseler et al., 2009) and also in gastric cancer (Kim et al., 2008). In addition, PKD1 may be an important repressor of EMT by inhibitory phosphorylation of Snail, a transcriptional inhibitor of E-cadherin (Du et al., 2010; Zheng et al., 2014) and, as mentioned before, by phosphorylating β-catenin, which impacts negatively its nuclear translocation (Du et al., 2009). In contrast, PKD3 is overexpressed in triple-negative breast cancers (Huck et al., 2014) and prostate cancer (Chen et al., 2008). PKD2, although it does not show cancer-associated elevated expression in all studied cases, seems to contribute to the proliferation of cancer cells and mediate multi-drug resistance (Chen et al., 2011; Hao et al., 2013). PKD2 is the predominant isoform in colorectal cancer, where treatment with PKD inhibitors reduces cell proliferation (Wei et al., 2014),

47 and it also promotes glioblastoma cells growth, being often overexpressed in gliomas of higher grading (Azoitei et al., 2011). Furthermore, inhibition of PKD3 and PKD2 causes downregulation of typical EMT markers (such as MMP-9, Snail and N-cadherin), which links both proteins to the promotion of EMT, in contrast to PKD1 (Durand et al., 2016). Also important in the context of our work is the role of PKD proteins in vesicular transport. PKD1 and PKD2, but not PKD3, play important roles in the regulation of vesicular fission of basolateral membrane-targeted carriers from the TGN, according to the study of mutant versions of these isoforms (Bossard et al., 2007; Liljedahl et al., 2001; Yeaman et al., 2004b). Specifi- cally, overexpression of a kinase-dead form of PKD2 (PKD2-KD) causes accumulation of E-cadherin and β1-integrin together with mutant PKD2 in the TGN, while PKD1-KD only indirectly reduces E-cadherin targeting to the membrane by impairing the exit from the Golgi of components of the exocyst machinery (Yeaman et al., 2004b). PKD1 and PKD2 phosphorylation of PI4KIIIβ, a crucial kinase for the structure and dynamics of the TGN, has been proposed as the molecular mechanism for PKDs involvement in the regulation of transport dynamics from the TGN (Hausser et al., 2005), and phosphorylation by PKD1 and PKD2 of the ceramide transfer protein (CERT) (Fugmann et al., 2007) and oxysterol-binding protein 1 (OSBP1) (Nhek et al., 2010) provide further insights into this process. Phosphorylated PI4KIIIβ produces PI(4)P, which acts as a platform for recruiting other lipid-modifying enzymes, as CERT and OSBP1, that contribute to Golgi fragmentation. Phosphorylation on CERT decreases its affinity for the target lipid PI(4)P, while phosphorylation on OSBP1 impairs its Golgi localization. Altogether, this network of interactions may ensure proper lipid homeostasis in the Golgi, with PKD activity being required for vesicular transport. In line with this, PKD overactivation causes Golgi fragmentation and impaired cargo delivery (Nhek et al., 2010). Additionally, PKD proteins affect Golgi architecture by directly mediating the vesicle budding process. Through PKD binding to DAG and Arf1-GTPase on the outer leaflet of the trans-Golgi membrane, PKD recruits phospholipase D (PLD), generating the membrane curvatures required for the budding initiation (Azoitei et al., 2018). PKD proteins have also been involved in other trafficking processes, such as the Rab4-dependent endocytic recycling of β3 integrin, mediated by PKD1 in response to PDGF stimulation (Woods et al., 2004) through PKD1 phosphorylation of Rabaptin-5 (Christoforides et al., 2012). PKD1 is also involved in Rab4-dependent recycling of β3 integrin in endothelial cells, upon stimulation with vascular endothelial growth factor (Blasio et al., 2010). In addition, our own findings link PKD proteins to E-cadherin recycling and reestablishment of adherens junctions after calcium deprivation (paper II), indicating that PKD, in cooperation with ErbB3 receptor, may elicit recycling of also other cargoes. Alternatively, PKD proteins may affect E-cadherin stability at the plasma membrane by interacting with its binding partners, such as

48 β-catenin. PKD1 phosphorylation of β-catenin at Thr112 and Thr120 has been shown to promote β-catenin membrane localization (Du et al., 2009), while a Thr120 point mutation of β-catenin abolishes its binding to -catenin (Aberle et al., 1996). This connection invites speculation that PKD1 phosphorylation at that residue stabilizes the junctional complex, although more evidence is required to understand the specific molecular mechanism.

5.2. The Rabaptin-5 effector protein Rabaptin-5 is a cytosolic protein of about 100 kDa that acts as effector of Rab5 and Rab4 GTPases (Stenmark et al., 1995). Also known as Rbpt5 or RABEP1, this homodimerizing cytosolic protein consists of four coiled-coil domains, two at the N-terminal side (CC1-1 and CC1-2) and two at the C-terminal side of its sequence (CC2-1 and CC2-2) (Kälin et al., 2016). Rabaptin-5 was initially identified as a Rab5-GTP effector protein, with which it mainly interacts through a binding domain in its C-terminus (Vitale et al., 1998), but also via a second interacting domain partially overlapping with the CC1-2 region (Korobko et al., 2006). In addition, Rabaptin-5 can recruit Rab5 guanine exchange factor Rabex5 (Horiuchi et al., 1997), to which it binds through the third coiled-coil segment (CC2-1). Rabaptin-5 was later found to interact as well with Rab4-GTP, via both the CC1-1 and the CC1-2 domains (Deneka et al., 2003; Korobko et al., 2005; Vitale et al., 1998). Rabaptin-5 also contains GAT and GAE binding domains, both present in the GGA proteins that will be discussed in the next section.

Figure 10. Structure of the Rabaptin5 dimer, showing the coiled-coil (CC) domains and the regions of interaction with several interaction partners. Adapted from Kälin et al., 2016.

Because of its ability to interact with both Rab5, Rabex5 and Rab4, Rabaptin- 5 has been proposed to mediate, on one hand, maintenance of endosomal Rab identity, since it recruits both Rab5 and its GEF, therefore sustaining a positive-feedback on Rab5 activation. On the other hand, Rabaptin-5 can coordinate two consecutive compartments in the endocytic itinerary, since it can bind

49 simultaneously to Rab5 and Rab4 (Kälin et al., 2015; Mizuno-Yamasaki et al., 2012; Stenmark, 2009). Although it is clear that Rabex5 and Rabaptin-5 cooperate in the activation of Rab5, the molecular details of the process have remained elusive. A mechanistic model has been proposed in which membrane-bound Rab5-GTP recruits Rabex-5 and Rabaptin-5. Interaction with Rabaptin-5 causes Rabex-5 to undergo a conformational change that exposes the substrate binding site, so that conversion of Rab5-GDP to Rab5-GTP takes place. In turn, generation of more Rab5-GTP causes recruitment of more Ra- baptin-5 and Rabex-5 to the membrane, constituting a perfect example of a positive feedback loop (Delprato and Lambright, 2007; Lippé et al., 2001; Zhu et al., 2007, 2010). In addition, it has been proposed that Rabex-5, through its coiled-coil domain, can associate weakly with the membrane on its own, without the presence of Rab5, and directly recruit Rabaptin-5, thereby initiating the feedback loop (Zhang et al., 2014), which could explain why silencing of Rab5 does not abolish Rabaptin-5 membrane localization (Kälin et al., 2015). Moreover, Rabex-5 has an ubiquitin-binding domain which is known to be required for Rabex-5 recruitment from the cytosol to the membranes (Mattera and Bonifacino, 2008), so it is possible that the initial recruitment of Rabex-5 is due to interaction with ubiquitinated cargo. However, the model still fails to explain why silencing of Rab4 blocks Rabaptin-5 recruitment to the membrane, as has been recently shown (Kälin et al., 2015). Therefore, Rab4 ap- pears more important for recruitment of Rabaptin-5 than Rab5 function, and it is therefore also required for full Rab5 activation. Rabaptin-5 function has been connected to both β3 and β1-integrin recycling in several studies (Christoforides et al., 2012; Do et al., 2017; Rios et al., 2008), in agreement with our own investigations, where we find that ErbB3 and Rabaptin-5 interact directly and that, in cooperation with GGA3, they control β1-integrin recycling (paper I). Intriguingly, the study from Christoforides et al. (2012), unveils that Rabaptin-5 is subjected to regulation by the PKD1 serine/threonine kinase. Upon PDGF stimulation, phosphorylation by PKD1 of Rabaptin-5 on Ser407 has been linked to Rab4-dependent recycling of vβ3 (Christoforides et al., 2012), although this phosphorylation seems to indirectly have a negative effect on 5β1 and EGFR recycling. Interestingly, Ser407 is located within the binding region that binds GAE domains, contained in both AP-1 clathrin adaptor and GGA proteins (Mattera et al., 2003), indicating that this modification could affect the interaction with GGA proteins. However, if this phosphorylation plays a role in our study system has not yet been addressed.

50 5.3. GGA family of Arf-dependent clathrin adaptors The GGA proteins (GGA1, GGA2 and GGA3 in mammals) are a family of monomeric clathrin adaptors. Their name stands for Golgi-localized γ-adaptin Ear-containing Arf-binding proteins, based on their domains structure, their ability to bind Arf GTPases and the fact that they were originally described in the TGN (Bonifacino, 2004). However, currently they are known to be in other locations as well and colocalize to a great extent with peripheral vesicles (Hirst et al., 2007; Kakhlon et al., 2006; Parachoniak et al., 2011; Sahgal et al., 2018). The GGA proteins contain three domains, named VHS (from Vps27, Hrs and Stam), GAT (from GGA and TOM) and GAE (from γ-adaptin ear), separated by two linker sequences (a short one between VHS and GAT, and a longer unstructured hinge segment between the GAT and GAE domains). The VHS domain recognizes sorting signals, specifically a DXXLL motif, in the cytoplasmic tail of some cargoes, such as sortilin and the mannose-6- phosphate receptors (MPRs) (Misra et al., 2002; Nielsen et al., 2001; Puertollano et al., 2001). Importantly, the hinge region in both GGA1 and GGA3 also contains this DXXLL sequence, indicating that GGA proteins are capable of autoinhibition (Doray et al., 2002). The GAT domain is able to interact with the GTP-bound form of Arf GTPases. Although originally it was thought that GGAs could only interact with class I Arf GTPases (Arf1, 2 and 3), it was later shown that they are also able to interact with Arf GTPases from class II (Arf4 and 5) and class III (Arf6), both in vitro and in vivo (Takatsu et al., 2002). The GAT domain is also responsible for the interaction with Ra- baptin-5, which again was thought to exist only in GGA1 and GGA2 (Zhai et al., 2003), but was soon after demonstrated to occur also in GGA3 (Mattera et al., 2003; Prag et al., 2005). The GGA interaction with Arfs and Rabaptin-5 is not mutually exclusive, since Arfs bind the N-terminal hook of the GAT segment, while Rabaptin-5 associates to the C-terminal triple helix bundle (Collins et al., 2003; Zhu et al., 2004). Moreover, the GAT domain of GGA1 and GGA3, but not of GGA2, can also bind ubiquitin (Shiba et al., 2004) via an hydrophobic patch around isoleucine 44, which does not overlap or inter- fere with the binding region to Rabaptin-5 (Prag et al., 2005). The hinge segment is a region of 80-286 residues without real secondary structure, and which shows a lot of variability between the different GGAs (Bonifacino, 2004), maybe explaining some of their non-overlapping functions. This region contains clathrin-box-like sequences responsible for the direct interaction with the terminal domain of clathrin heavy chain (Mullins and Bonifacino, 2001). It also includes the internal DXXLL sequence referred to before, and it has been reported that phosphorylation by casein kinase 2 (CK2) of a serine residue proximal to this sequence is required for the autoinhibitory interaction with the VHS domain (Ghosh and Kornfeld, 2003). The hinge region can also interact, via an WSFN sequence, with the γ-adaptin subunit in the AP-1 mul- timeric clathrin adaptor, and it has been proposed that interaction of GGAs

51 and Rabaptin-5 with AP-1 are mutually exclusive, since both recognise the same or overlapping regions in the γ-adaptin ear (Bai et al., 2004). Finally, the GAE domain is very much related to the γ1 and γ2-adaptin ear domains in AP- 1, and therefore it has many interactors in common with them. The GAE domain is involved in interaction with different accessory proteins, such as Ra- baptin-5 (which therefore interacts with GGAs via two different domains) (Mattera et al., 2003), the β-site APP cleaving enzyme 1 (BACE1) (Einem et al., 2015), the clathrin interacting protein CLINT (Kalthoff et al., 2002) γ- synergin or p56 (Lui et al., 2003). In addition, the GAE domain also estab- lishes contact with clathrin, in cooperation with the hinge segment (Zhang et al., 2007a). Although the three members of the GGA family share high similarity and functional redundancy, also non-overlapping functions have been demonstrated. GGA3, but not GGA1 or GGA2, has been shown to interact with TSG101, a component of the ESCRT complex for sortin ubiquitinated cargo into the multivesicular bodies, and to mediate lysosomal targeting of endocytosed EGFR (Puertollano and Bonifacino, 2004). In contrast, GGA3 has been shown, in collaboration with Arf6 GTPase, to protect MET from lysosomal degradation, inducing its recycling upon growth factor stimulation. In this context, GGA3 coimmunoprecipitated with MET, while interaction with GGA1 or GGA2 could not be found (Parachoniak et al., 2011). In addition, a recent investigation has shown interaction between GGA3 and the nerve growth factor receptor TrkA (but not with GGA1 and only weakly with GGA2) and linked it to its endocytic recycling, again in cooperation with Arf6. Interestingly, interaction between GGA3 and TrkA was found to be direct and through a DXXLL motif inside of the kinase domain of TrkA, which is not a common scenario, since this sorting domain is more usually located in the C- terminal tail (Li et al., 2015). Important for the topic of this thesis, GGA3 has been implicated in the recycling of β1-integrin, also in cooperation with Arf6 (Ratcliffe et al., 2016). Importantly, in both MET and β1-integrin cases, GGA3-driven recycling occurs via Rab4-dependent route, which has also been identified by others as an itinerary with strong GGA3 presence (D’Souza et al., 2014). On the other hand, also GGA1 and GGA2 present unique functions. GGA1 has been implicated in myogenesis via its role in stabilization of insulin receptor plasma membrane levels (Isobe et al., 2018). In addition, TGN localization of the p56 clathrin adaptor accessory protein was found to depend exclusively on GGA1 expression, but not on GGA2 or GGA3 (Uemura et al., 2018a). GGA2 is important for active β1-integrin recycling, while its depletion does not affect stability of inactive β1-integrin (Sahgal et al., 2018). This is in agreement with previous work showing that only GGA3 depletion, but not GGA1 or GGA2 depletion, reduced β1-integrin total protein levels (Ratcliffe et al., 2016). It was also recently proven that GGA2 stabilizes EGFR protein level, while this increases upon silencing of GGA1 or GGA3 (Uemura et al.,

52 2018b), in agreement with the previously mentioned role of GGA3 in driving lysosomal degradation of EGFR (Puertollano and Bonifacino, 2004). Interest- ingly, EGFR and GGA2 were shown to interact directly, and this interaction was mapped inside the VHS-GAT domains of GGA2 and the juxtamembrane region of EGFR (Uemura et al., 2018b). All these discoveries are very interesting in the light of our own results. In paper I, we have found that ErbB3 interacts with GGA3 and Rabaptin-5, promoting recycling of β1-integrin, and that in absence of ErbB3, the protein stability of GGA3, Rabaptin-5 and β1-integrin is compromised. Although we have not yet checked if the interaction between ErbB3 and GGA3 is direct, given the link found between GGA2 and EGFR, we could speculate that ErbB3 also interacts with GGA3 via its juxtamembrane domain. In addition, a DXXLL motif is also found in ErbB3 and, as in the case of TrkA, it is placed in the kinase domain of the receptor, and not at the very end of its cytoplasmic tail. Finally, the possibility that ubiquitination of the receptor is also involved in the ability of GGA3 to recognize it has not yet been ruled out and should also be studied.

Figure 11. Suggested model for Rabaptin-5, GGA3, β1-integrin and ErbB3 complex. Interactions between GGA3, Arf6 and Rabaptin-5 are illustrated according to Boni- facino et al., 2004.

53 5.4. Ack1 non-receptor tyrosine kinase Activated Cdc42-associated tyrosine kinase 1 (Ack1) is a non-receptor tyrosine kinase from the Ack family, which consists of two members in humans, Tnk1 and Ack1 (also known as Tnk2), and other homologous proteins in mouse, fruit fly and yeast (Prieto-Echagüe and Miller, 2011). Ack1 was originally identified for its capacity to physically interact with GTP-bound Cdc42 Rho GTPase and inhibit its activity (Manser et al., 1993) but many other functions in the regulation of cell physiology have been attributed to it. Ack1 is a 140 kDa ubiquitously expressed protein, although the highest levels can be found in brain, thymus, spleen and liver (Zhao et al., 2018). It is structured in nine domains: An N-terminal sterile alpha domain (SAM), the kinase region, an SH3 domain, a CRIB domain, two clathrin-binding regions, a Mig6-homology region (MHR) and finally, a ubiquitin-binding domain at the C-terminal side of the protein (Mahajan and Mahajan, 2015). The kinase domain of Ack1 is constitutively stabilized in an active form, but the SH3 domain, via interaction with the MHR domain is responsible for keeping it in a closed, auto-inhibited, conformation (Lin et al., 2012). It has also been proposed that symmetric homodimerization, mediated by two SAM domains, is required for the activation of Ack1 (Gajiwala et al., 2013).

Figure 12. Illustration on the top shows domain structure of Ack1 non-receptor tyrosine kinase. Below, Ack1 on its auto-inhibited conformation, mediated by a proline- rich region in the MHR domain, a well-stablished consensus motif for interaction with SH3 domains.

The presence of such a variety of protein interaction domains suggests that Ack1 has a central role coordinating multiple signalling effectors. As a non-

54 receptor kinase, Ack1 is activated downstream of a variety of membrane receptors, such as integrins, EGFR, PDGFR, bradykinin or Trk, among others. Ack1 function is connected to several cellular processes: regulation of gene transcription, via phosphorylation of STAT1 and STAT3 (Mahendrarajah et al., 2017) or activation of KDM3A demethylase (Mahajan et al., 2014), among others; pro-survival signalling, via phosphorylation of Akt (Mahajan et al., 2010) or ligand-independent activation of the androgen receptor (AR) (Mahajan et al., 2007); cytoskeletal remodelling, through interaction with Cdc42 and phosphorylation of WASP (Yokoyama et al., 2005); and vesicular trafficking, given its ability to interact with clathrin and clathrin adaptor proteins (Masdeu et al., 2017; Teo et al., 2001). Ack1 is also able to form a complex with β1-integrin and is involved in β1-integrin-mediated signalling cascades, in the context of cell spreading, adhesion and migration (Yang et al., 1999). Ack1 has been extensively connected to cancer development, and a tumour-promotor function has been proposed for it. Ack1 is overactivated in triple negative breast cancer cell lines and its depletion reduces invasion and proliferation in xenograft mouse models (Wu et al., 2016). It is also mutated or overexpressed in a variety of malignancies (such as lung, prostate or pancreatic cancer, among others) and its expression correlates with the metastatic potential of the tumour (van der Horst et al., 2005). Ack1 is implicated in cancer progression in many ways. As commented before, Ack1 phosphorylates Akt (Mahajan et al., 2010) and both activates AR (Mahajan et al., 2007) and induces its expression in prostate cancer (Mahajan et al., 2017). Ack1 also promotes prostate tumorigenesis via phosphorylation of the tumour suppressor WWox, which triggers its ubiquitination and degradation (Mahajan et al., 2005). Moreover, Ack1 regulation of p130cas is critical for growth and invasion in melanoma cells (Eisenmann et al., 1999). In addition, Ack1 induces survival of cancer stem-like cells (Mahajan et al., 2018) and interacts with demethylases that regulate the expression of mammary tumour oncogenes (Mahajan et al., 2014). On the contrary, Ack1 also presents pro-apoptotic functions, as it has been connected with the recruitment of TRAIL (TNF-related apoptosis-inducing ligand) receptors to plasma membrane lipid rafts for the induction of cell death (Linderoth et al., 2013). One of the most characterized functions of Ack1 is its role in EGFR trafficking, although investigations carried out by different groups point in different directions. On one hand, Ack1 has been shown to interact, via its Mig6- homology domain, with active EGFR upon EGF stimulation, and to drive its degradation (Shen et al., 2007). Given the ability of Ack1 to interact with the clathrin heavy chain, it has been proposed that Ack1 mediates EGFR clathrin- dependent internalization (Teo et al., 2001), and that the ubiquitin-binding domain at the N-terminus of Ack1 also contributes to interaction with EGFR (Shen et al., 2007). Interestingly, it was later reported that Ack1 localizes to autophagosomal structures upon EGF stimulation, and that it could mediate

55 degradation of EGFR via this compartment instead of the lysosome (Jones et al., 2014). However, others have found that upon depletion of Ack1, the expression on the plasma membrane of EGFR is reduced, which correlates with impaired cellular migration (Howlin et al., 2008). Although, all together, these results are difficult to interpret, they suggest that both excess and impairment of Ack1 may have a detrimental effect on EGFR desensitization. In agreement with this, others have discovered that both Ack1 overexpression and siRNA- mediated depletion reduce EGFR internalization rate (Grøvdal et al., 2008).

56 6. Extracellular vesicles and exosome biogenesis

The first report on extracellular membrane-limited vesicles dates back to the 60s, with the identification by electron microscopy of small organelles released by platelets and involved in the coagulation of blood (Wolf, 1967). They were named ‘platelet dust’ and constitute the beginning of the nowadays exploding field of extracellular vesicles (EVs) research. A couple of decades later, it was shown in reticulocytes that endomembranes carrying transferrin receptor are released to the extracellular space after fusion of MVB with the plasma membrane, and the term “exosome” was coined to refer to these structures (Harding et al., 1983; Johnstone et al., 1987; Pan et al., 1985). Nowadays exosomes and other extracellular vesicles are known to be produced by virtu- ally all cell types, eukaryotic and prokaryotic, and to be involved in a wide array of processes, both in physiological and pathophysiological scenarios. Extracellular vesicles play important functions in cell-to-cell communication, immune response, tumorigenesis and metastasis, among many others. In this section, we will introduce some general knowledge in the field of extracellular vesicles, their biogenesis and their implication in cancer, in order to provide a frame for discussion of our findings that ErbB3 inhibits exosome release.

6.1. Types of extracellular vesicles Despite the overgrown terminology to refer to extracellular vesicles, these structures can be classified in three main categories, if considering their biogenesis process (EL Andaloussi et al., 2013; van Niel et al., 2018): apoptotic bodies, microvesicles and exosomes. Apoptotic bodies are large membranous blebs of 1 to 5 µm that occur only in dying cells. Microvesicles are particles of 100nm to 1µm, that bud directly from the plasma membrane, while exosomes usually have a size of 30 to 150nm, and are originated from extracellular release of intraluminal vesicles (ILV) upon MVB fusion with the plasma membrane (Bobrie and Théry, 2013). Alternative classification systems con- sider the tissue of origin (prostasomes, oncosomes, neurospheres…), or the size, therefore distinguishing small (30-100 nm), medium (100 nm-1 µm) and large (1-5 µm) EVs, although the exact size limit that separates one subgroup from another varies depending on the source of information (Németh et al.,

57 2017; van Niel et al., 2018; Zhang et al., 2018), and it is based, in any case, on approximated values. In addition, vesicle morphology can also be used for categorization (Lässer et al., 2018).

Figure 13. To the left, subcellular origin of microvesicles and exosomes; to the right, an overview of some of the components that can be found in exosomes.

Extracellular vesicles are not only present in all human fluids and conditioned media from cultured cells, but also in dairy products, yeast-derived beverages or different environmental samples (Biller et al., 2014; Rodrigues et al., 2016; Somiya et al., 2018). Several isolation methods are used to purify extracellular vesicles (Gardiner et al., 2016; Witwer et al., 2013). Differential ultracentrif- ugation is the most commonly employed technique, although other ap- proaches as ultrafiltration, size exclusion chromatography (SEC), separation in density gradient or immunoaffinity isolation have recently become more popular (Gardiner et al., 2016). Usage of each of these methods involves the possibility of bias toward a certain type of vesicle or the co-isolation of con- taminants with similar physical properties. Therefore, advantages and disad- vantages of each method should be evaluated beforehand, as well as their impact on experimental results (Coumans Frank A.W. et al., 2017; Simonsen Jens B., 2017; Van Deun et al., 2014). In addition, it is important to note that to date, the most used method to refer to extracellular vesicles is the one based on their subcellular origin (exosomes versus microvesicles). However, to our knowledge, there is no isola-

58 tion procedures that can fully distinguish between exosomes and microvesicles, as both types present a certain degree of overlap in size, density and markers. For example, although it is reported that during differential centrifu- gation large microvesicles are pelleted at 10,000 x g, while exosomes are enriched in the 100,000 x g pellet (Théry et al., 2006), the latter still contains a heterogeneous population of exosomes, small microvesicles and other con- taminants. Therefore, in absence of conclusive evidence on the subcellular origin of the particle, the term ‘extracellular vesicle’ is more appropriate than ‘exosome’ or ‘microvesicle’ (Bobrie and Théry, 2013; Gould and Raposo, 2013). Both exosomes and microvesicles transport a variety of proteins, lipids and nucleic acids that contribute to their functions. These components reflect the nature of the cell of source but, in many cases, are more enriched in EVs, proving the existence of a system for selective cargo loading (Ferguson and Nguyen, 2016; Record et al., 2014; Valadi et al., 2007). For example, proteins implicated in the process of MVB formation are more abundant in exosomes and serve as markers for their detection (van Niel et al., 2006); lipids that allow for higher membrane curvature are also enriched in extracellular vesicles, if compared with other membranous structures in the cell (Skotland et al., 2017); finally, EVs contain distinct subspecies of short RNAs and mRNAs and specific EV-sorting sequences have been identified (Batagov et al., 2011; Bolukbasi et al., 2012; Janas et al., 2015; Villarroya-Beltri et al., 2013). In addition, other parameters affect the composition of EVs, like the cell maturation status, culture confluence, various types of environmental stress or different culture conditions. A number of proteins are used as molecular markers of extracellular vesicles, including tetraspanins (CD63, CD9, CD81), heat shock proteins (Hsp70, Hsp73), the major histocompatibility complex II (MHCII) or members of the endosomal cargo sorting machinery (Alix, TSG101). Tetraspanins are a superfamily of proteins with four hydrophobic transmembrane domains and broadly expressed in human tissues (Andreu and Yáñez-Mó, 2014). Tetra- spanins act as scaffold units to anchor many different types of proteins to specific membrane regions and are also the most used markers for exosomes. However, it is now clear that microvesicles are also enriched in some tetraspanins such as CD63 or CD9 (Kowal et al., 2016). In the same manner, integrins were originally proposed as microvesicle markers (Heijnen et al., 1999) but a recent report showed them to be enriched in a subset of extracellular vesicles of 90 to 120 nm diameter, which can well have a MVB origin (Zhang et al., 2018). In conclusion, and although EV studies should always be supported by analysis of molecular markers, none of these markers, sepa- rately, can undoubtedly point to the subcellular origin of the particles. In fact, the International Society of Extracellular Vesicles (ISEV) avoids, in its latest guideline, to suggest any specific protein markers for the different subtypes of EVs (Théry et al., 2018).

59 6.2. Extracellular vesicles biogenesis Exosomes and microvesicles are produced by independent processes that, nev- ertheless, share some common elements. For the biogenesis of exosomes, formation of MVB is a fundamental step, and the function of the ESCRT machinery for endosomal cargo sorting has proven crucial for it. The ESCRT (endosomal sorting complex required for transport) is a complex of about 20 proteins which form five modules (ESCRT-0, -I, II, III and the Vps4 complex), together with a number of associated proteins (Henne et al., 2011; Olmos and Carlton, 2016). The different modules are recruited to the MVB sequentially. ESCRT-0 (composed of Hrs and STAM1/2 in mammals) is recruited to phosphatidylinositol 3-phosphate on the endosomal membrane and to ubiquitinated transmembrane cargo, and together with ESCRT-I (Tsg101, Vps28, VPS37 and hMvb12) participates in cargo clustering and initial in- wards budding of the late endosome membrane (Bache et al., 2003). Cargo clustering occurs below a flat clathrin coat, which has been proposed to act as a protective unit for diffusion of cargoes or their scape to an alternative pathway (Raiborg et al., 2006). The clathrin coat is later removed by the un-coating ATPase HSC70, granting access to the ESCRT modules II and III (Chang et al., 2002). ESCRT-II (EAP45, EAP30, EAP20) also participates in the budding process, while ESCRT-III (CHMP6, CHMP4, CHMP3 and CHMP2), together with the recruited Vps4 complex (SKD1, CHMP5 and LIP5), takes part in the membrane scission process, which gives rise to the ILVs (Palmulli and van Niel, 2018). In addition, the ESCRT-III module recruits deubiquitinating enzymes which remove ubiquitin from cargo proteins, but this is not a fundamental step in the process, since ubiquitinated cargoes are also found in exosomes (Buschow et al., 2005). Although most of the ESCRT subunits are on the MVBs limiting membrane, some, like TSG101, can also localize to the inside of ILVs. TSG101 is therefore present in exosomes, as previously indicated, but it is also present in those ILVs that are fated for lysosomal degradation. In fact, lysosomal degradation of TSG101, together with cargo receptors, has been proposed as a negative feedback mechanism for controlling the pace of endosomal maturation and receptor signal attenuation (Malerød et al., 2011). Exosomes can also form in a ESCRT-independent manner (Stuffers et al., 2009) via several mechanisms. One of them involves type II sphingomyelin- ase for the production of ceramide, which could generate spontaneous curvature at the late endosome membrane or trigger the activation of G-protein coupled sphingosine 1-phosphate (S1P) receptor, which is required for the maturation of MVB with exosomal fate (Goñi and Alonso, 2009; Kajimoto et al., 2013; Trajkovic et al., 2008). Clustering of tetraspanins such as CD9, CD81 and CD82 can also mediate ESCRT-independent MVB formation, given their ability to associate cholesterol and their 3D structure (cone-shaped and wider at the intraluminal side), which facilitates the initiation of inward budding

60 (Zimmerman et al., 2016). Lipid raft domains, rich in cholesterol and glyco- sphingolipids, can also contribute to the formation of ILVs (de Gassart et al., 2003). In addition, Arf6 GTPase has also been connected to exosome biogenesis through a process that requires interaction between syntenin and the ESCRT auxiliary subunit Alix (Ghossoub et al., 2014). The mechanisms that decide whether MVB are fated for lysosomal degradation or exosomal release are not completely understood, although it is likely that both outcomes are balanced. Accordingly, inhibition of lysosomal degradation has been reported to trigger increased exosome production (Eitan et al., 2016). It has been proposed that the implication of either ESCRT-dependent or independent mechanisms for MVB formation could favour different fates, in addition to post-translational modification on the protein cargo (Palmulli and van Niel, 2018). For example, ubiquitinated MHC-II in MVB is sent for lysosomal degradation, while non-ubiquitinated MHC-II is instead released in exosomes (Buschow et al., 2009). However, as previously mentioned, ubiquitinated cargoes are also found in exosomes (Buschow et al., 2005). Thus, the extent to which this is a decisive factor remains to be clarified. Ultimately, targeting of MVBs to the plasma membrane is required for exosome secretion. Depletion of different Rab GTPases, such as Rab11, Rab35, Rab9a, Rab5a, Rab2b, Rab27a and Rab27b affects release of exosomes (Beckett et al., 2013; Hsu et al., 2010; Ostrowski et al., 2010; Savina et al., 2002). However, it is plausible that some of these Rabs are not directly required for the membrane tethering and fusion step, but that their depletion affects upstream steps in the endosomal maturation process. In this respect, direct role in the docking of MVB to the plasma membrane has been demonstrated for Rab27a and Rab27b, as well as for Rab35 (Hsu et al., 2010; Ostrowski et al., 2010). In addition, both the microtubule and actin cytoskeleton play a role in MVBs positioning and delivery to sites of secretion. Rab7, by mediation of its effector protein RILP, has been shown to interact with dynein motor protein and facilitate the movement of MVBs towards the microtubule organizing centre, where the lysosome is (Wang et al., 2011). On the opposite, expression of the actin cytoskeleton regulatory protein cortactin favours Rab27a-mediated fusion of MVB with the plasma membrane (Sinha et al., 2016). This would suggest that involvement of different cytoskeleton- related effectors has also an impact on the MVBs fate. Formation of microvesicles is a less understood process, in which both cytoskeleton-associated proteins, phospholipids rearrangements, Arf6 GTPase and some ESCRT subunits play a role. On one hand, it is known that translocation of some lipids from the inner to the outer leaflet of the plasma membrane (‘flipping’) allows budding (Stachowiak et al., 2013) and that even protein accumulation on a region of the plasma membrane can trigger its curvature (Stachowiak et al., 2012). Upon initiation of budding, Arf6 GTPase is involved in phospholipase D-mediated ERK phosphorylation, which triggers activation of myosin light chain kinase (MLCK) at the neck of the emerging

61 microvesicle. This, together with actin-myosin-mediated cellular contraction allows completion of the microvesicle fission (D’Souza-Schorey and Clancy, 2012; Muralidharan-Chari et al., 2009). In addition, interaction of the ESCRT- I subunit TSG101 and arrestin domain-containing protein 1 (ARRDC1), together with the Vsp4 complex, can also mediate the budding and fission of microvesicles (Nabhan et al., 2012). Interestingly, TSG101 was also found to be required for budding of HIV from infected cells, a process that seems to share some similarities with that of microvesicle formation (Martin-Serrano and Neil, 2011). Biogenesis of both types of extracellular vesicles also depends to a great extent on the presence of cargo. For example, overexpression of some of the most common exosomal cargoes, such as MHCII, triggers formation of MVB and increased release of exosomes (Ostrowski et al., 2010). Additionally, it has been suggested that cargoes which normally recycle to the plasma membrane could end up in exosomes if their recycling is impaired, like the transferrin receptor in reticulocytes (van Niel et al., 2018; Vidal et al., 1997). Both these concepts are interesting in the context of our work in paper I and paper III. In paper III we discover that depletion of ErbB3 causes increased release of exosomes which carry β1-integrin on their surface. In paper I, we made the finding that depletion of ErbB3 impairs the recycling of β1-integrin, rerouting it for degradation. However, by inhibition of the lysosomal pathway we could only partially rescue the levels of β1-Integrin, suggesting that a fraction of integrin molecules could instead be diverted to exosomes for release to the extracellular space. Therefore, β1-integrin could be another example of a cargo whose impaired recycling triggers its release in exosomes, and it makes us wonder if other examples of faulty integrin recycling could also have, in addition to the reported increased degradation, a component of augmented rerouting to exosomes.

6.3. Extracellular vesicles in cancer and metastasis Extracellular vesicles play a fundamental role in tumour progression and metastasis, mediating both autocrine and paracrine communication (Tkach and Théry, 2016). EVs can be taken up by recipient cells, causing a number of functional changes via their cargo proteins and RNAs. Cancer cell-derived exosomes can increase directional cell movement and invasiveness of other tumour cells (Sung et al., 2015), and their release promotes chemotactic movement of cancer cells (Sung and Weaver, 2017). In addition, it has been shown that breast cancer cells with increasing metastatic potential secrete EVs which also have an increasing ability to promote cell migration (Harris et al., 2015). Tumour-secreted EVs can transfer oncogenic receptors, like the EGFR variant III (EGFRvIII), which is transported between glioma cells, inducing malignant transformation (Al-Nedawi et al., 2008). Also canonical EGFR can be

62 loaded into exosomes from gastric cancer cells and delivered to liver stromal cells, where it causes activation of MET by enhancing expression of its ligand HGF (Zhang et al., 2017). Moreover, it was recently shown that myoferlin, a protein involved in endocytosis and vesicle fusion, is present in exosomes from breast and pancreatic cancer cells, where it plays an important role by inducing migration and proliferation of recipient cells (Blomme et al., 2016). EVs can also provoke changes in the tumour microenvironment that facilitate the spreading of the malignancy and development of metastasis. Exo- somes and large microvesicles (oncosomes) have been found to carry metalloproteinases that participate in the degradation of the extracellular matrix (Clancy et al., 2015; You et al., 2015; Yue et al., 2014). Moreover, EVs can promote angiogenesis in the tumour, for example by inducing Akt signalling (Deregibus et al., 2007) or via regulation of angiogenesis-related genes in endothelial cells (Nazarenko et al., 2010). In addition, several lines of evidence prove the involvement of EVs in the formation of pre-metastatic niches, dis- tant from the original tumour. For example, melanoma-derived exosomes induce vascular leakiness at the lung pre-metastatic site and, via transfer of Met, provoke changes in bone marrow progenitors which support tumour vasculo- genesis (Peinado et al., 2012). Additionally, exosomes derived from pancreatic cancer cells are taken up by Kupffer cells in the liver, inducing secretion of TGFβ and fibronectin release into the liver microenvironment, which recruits bone marrow-derived macrophages and contributes to metastatic niche formation (Costa-Silva et al., 2015). Extracellular vesicles are also deeply involved in metastatic organotropism, the phenomenon by which different types of tumours tend to metastasise to specific target organs (Smith and Kang, 2017). It has been shown that tumour- derived EVs taken up by cells at the organ of destination prepare this for the formation of metastasis, and interestingly, integrins decorating the surface of these EVs are critical for the process (Hoshino et al., 2015). While α6β4- and α6β1-integrins dictate metastasis to the lung, the presence of exosomal αvβ5- integrin is associated with metastasis to the liver. Arrival of these exosomes to its corresponding target organ triggers a number of changes, like expression of specific ECM proteins that attract metastatic cells from the primary tumour (Hoshino et al., 2015). This example highlights the important presence of integrins in exosomes and other extracellular vesicles and suggests that altera- tion of the physiological levels of exosomal integrins, as we observe upon depletion of ErbB3 in paper III of this thesis work, may have important consequences in vivo. However, a deeper exploration of this phenotype needs to be executed in order to understand its biological consequences.

63 7. Present investigations

7.1. Paper I. Ligand independent role of ErbB3 in endocytic recycling ErbB3 is a catalytically-impaired receptor tyrosine kinase often overexpressed in different types of cancer and linked to metastatic progression. In this manuscript, we have uncovered a new role of ErbB3 which, in absence of ligand stimulation, drives Rab4-dependent endocytic recycling of β1-Integrin and transferrin receptor. Depletion of ErbB3 causes internalized β1-Integrin to go instead for lysosomal degradation and, as a consequence, targeting of β1-In- tegrin to the leading edge of migrating cells is defective, which results in reduced cellular movement. In addition, in this work we unveil the mechanistic details of ErbB3 involvement. ErbB3 interacts with and stabilizes the clathrin adaptor GGA3 and the Rab4/5 effector Rabaptin5, both of them with stablished functions in integrins and transferrin recycling. In conclusion, we show that ErbB3 is an integral part of the endosomal trafficking machinery, and that it constitutes the first example, to our knowledge, of a RTKs playing an in- strumental role in vesicular trafficking.

7.2. Paper II. Ligand-independent role of ErbB3 in recycling of E-cadherin and assembly of adherens junctions This work is a continuation of our investigation into the newly found role of ErbB3 in driving endocytic recycling. While in paper I we showed the importance of ErbB3 for recycling of β1-Integrin and transferrin receptor upon endocytosis, here we prove that recycling of another cargo, E-cadherin, is also subjected to regulation by ErbB3. ErbB3 depletion causes impaired recycling of E-cadherin in absence of ligand stimulation or cooperation with other members of the EGFR family. As a consequence, formation and assembly of adherens junctions (AJs) is defective in ErbB3-depleted cells, an observation that has not been previously reported. Moreover, we identify PKD serine/threonine kinase as an important ErbB3 effector in this scenario. We found that PKD and E-cadherin interact with ErbB3 upon artificial disruption of AJs, and

64 depletion of one of the PKD members, PKD2, also results into impaired formation of E-cadherin-based junctions, suggesting the PKD could be an important effector of ErbB3. These results broaden our knowledge on the role of ErbB3 in endocytic recycling and suggest that also other cargoes could be affected.

7.3. Paper III. ErbB3 regulates release of extracellular vesicles Exosomes are membrane vesicles released to the extracellular environment upon fusion of MVB with the plasma membrane. Exosomes and other extracellular vesicles perform important functions in cell-to-cell communication and cancer, although our knowledge on the mechanisms that control their formation and release is still limited. In this manuscript, we study the involvement of the ErbB3 receptor tyrosine kinase in the formation of exosomes in a mammary gland epithelial cell line. Our findings suggest that ErbB3 inhibits release of exosomes. In ErbB3-depleted cells, not only more exosomes are produced but they also present selective loading of β1-Integrin into a subpop- ulation of them. In addition, we have also elucidated that GGA3, which cooperates with ErbB3 in driving recycling of endocytosed β1-Integrin, also inhibits release of exosomes. Altogether, our results suggest that loss of ErbB3 and GGA3 provokes β1-integrin re-routing from recycling endosomes towards exosomes. These findings could very important in the context of cancer and metastasis, where exosome-borne β1-integrin has been connected with formation of the pre-metastatic niche and attraction of cancer cells from the primary organ towards secondary metastatic sites.

7.4. Paper IV. Ack1 is a negative regulator of ErbB3 that acts both by promoting its degradation and suppressing its expression The EGFR family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3 and ErbB4) initiate a myriad of cellular responses related to survival, cell proliferation and differentiation, and their dysregulation is observed in multitude of cancers. In this manuscript, we demonstrate that the non-receptor tyrosine kinase Activated Cdc42- associated kinase 1 (Ack1) controls protein levels of ErbB3 and antagonises its downstream signalling, but not that of EGFR or ErbB2. In Ack1-depleted cells, ErbB3 protein accumulates, showing sustained phosphorylation upon stimulation with neuregulin, and hyperactivation of downstream targets as Akt kinase. Moreover, in this work we elucidate that

65 Ack1 exerts a dual effect on ErbB3, both inducing its degradation and suppressing its transcription. These findings demonstrate that Ack1 is a specific negative regulator of ErbB3, uncoupling its control from that of other members of the family, as EGFR or Erbb2. These results are conceptually novel as they for the first time suggest that different EGFR family members are subjected to unique mechanisms of spatiotemporal control.

66 8. Future perspectives

This thesis work uncovers new roles of the ErbB3 receptor tyrosine kinase in modulating vesicular trafficking of β1-integrin and E-cadherin and in regulating the process of exosome release. It also unveils Ack1 as a specific negative regulator of ErbB3, and in general, contributes to the understanding of the processes that maintain epithelial homeostasis. As expected, our discoveries raise further scientific questions and can be taken as the starting point for future projects. In paper I, we have discovered that ErbB3 cooperates with Rabaptin-5 and GGA3 in driving endocytic recycling of β1-integrin in epithelial cells from mammary gland. However, the mode of interaction between ErbB3 and Ra- baptin-5/GGA3 is not fully clear and it should be mapped to specific regions in these proteins. If GGA3 and ErbB3 interact directly or not should also be elucidated. Eventually, all this information could help to understand the exact mechanism by which ErbB3 protects Rabaptin-5 and GGA3 from degradation. Moreover, ErbB3 acts in absence of growth stimulation and, therefore, it is possible that its presence on the plasma membrane is not a requirement for this function. Does ErbB3 meet with β1-integrin on the plasma membrane or do they meet in an intracellular compartment? Also, is direct interaction between ErbB3 and the cargo required or not for recycling of the latter? Addi- tionally, it would be intriguing to explore if this mechanism is also important in other cell types and what is its overall effect on cellular migration in vivo. ErbB3 expression is implicated in metastasis, could this be due to its effect on integrin-mediated migration? At the same time, abnormal integrin-mediated migration is associated to other pathologies, as immunodeficiency disorders. Could ErbB3 be required also in this context? In paper II we have discovered that ErbB3 is also required for E-cadherin recycling and we have proposed the PKD serine/threonine kinases as necessary effectors during this process. However, we still need to understand if all PKD proteins can cooperate with ErbB3 or if this is an isoform-specific function. Moreover, ErbB3 interacts with E-cadherin and PKD upon disruption of junctions, but we have not yet elucidated if the three proteins form a complex together. Additionally, others have shown that Rabaptin-5 phosphorylation by PKD is important for integrin recycling, which makes us wonder if Rabaptin- 5 could also be involved in the process of E-cadherin recycling. Finally, while recycling of integrins have been mostly connected to cellular migration, recycling of E-cadherin is required for the establishment of epithelial sheets.

67 Therefore, it would be interesting to investigate if ErbB3 function plays a role in collective cell migration during cancer progression, both in vivo and in vitro models. In paper III, we have uncovered a new role of ErbB3 as negative regulator of exosome release. In this ongoing work, we would like to elucidate the effects on other cells of the increased release of exosomes upon ErbB3 depletion. Moreover, we have found out that, in the context of ErbB3 silencing, β1- integrin is diverted to exosomes. Therefore, we would like to explore if this affects the exosome properties in terms of adhesion to recipient cells or in their ability to travel to organs rich in ECM in in vivo models. Moreover, we would like to bring light into other effects that ErbB3 depletion causes in the general composition of extracellular vesicles. Are other cargoes diverted to exosomes upon ErbB3 depletion? And do they have functional significance for the various roles of exosomes in intercellular communication? In paper IV, we have addressed the importance of Ack1 in the spatiotemporal regulation of ErbB3, both under steady state conditions and upon stimulation with neuregulin. However, the exact mechanism by which Ack1 controls ErbB3 expression, independently of that of EGFR or ErbB2, still remains to be elucidated. Ack1 seems to regulate ErbB3 both at the transcriptional level and promoting the degradation of ErbB3 protein, but if these are two independent mechanisms or both take part in a negative feedback loop needs to be further investigated. In addition, although Ack1 is reported to interact directly with EGFR through its Mig6-homology domain, ErbB3 is not recognized by Mig6 which may indicate that both proteins do not interact directly or do so through an alternative surface. Moreover, the importance of this regulatory checkpoint in vivo has not been investigated. Since both ErbB3 and Ack1 have been proposed as tumour-promoter proteins, what would be the effect of Ack1 depletion on ErbB3 status and susceptibility to cancer in different animal models?

68 Acknowledgements/ Agradecimientos

Doing a PhD has been the ultimate adventure for me. It has taken me to a new country, pushed me to learn, to teach, to fail and try again, to overcome some fears and, in short, to become better in many ways. This book would not be complete without thanking all the people who, in one way or another, have helped me to do all of this. First of all, I would like to express my gratitude to my main supervisor, Ingvar. Thank you for believing in the very-scared-master-student that I was the first time we met. I am very grateful that you have given me the space to grow as a scientist and that you are never afraid of looking into new research directions. Thanks for always keeping your door open and being welcoming to questions and brainstorming sessions. Thanks as well to my co-supervisor Calle, for all your advice and your dedication. You are truly an inspiration and every day you set an example with your professionality and your kindness. I feel very fortunate to have learned from you. Next, I would like to thank my fantastic colleagues at the Signalling Dy- namics Group: Giulia, I would need a second book to write down everything I have to thank you for, both as a colleague and a friend. You are not only a bright and hard-working researcher, but moreover, you are always willing to help those around you. Thank you being there with your support, your jokes and your contagious strength every time I was feeling down. I really feel lucky to have you. Aive, thank you for your guidance in the beginning of my PhD. You were always nice and patient with me, and we still miss you very much around here! Maria J., you were also one of the first persons that I got to work with when I arrived at Ludwig, thank you for those first lessons on western blot and all the nice chats we had. Noura, working with you has never been boring, thanks for being such an awesome woman, you bring good vibes wher- ever you go. Mari, thank you for all the tips on mouse work, our excursions to Yukikos and for being such a friendly colleague. I am sure you will have a very successful career back in Japan! Julia, you have helped me improve a lot, showed me new techniques and dragged me to yoga at 7am. Thank you for all of it, even the last part! I am also very thankful to Jasminka and Takeshi, from whom I learnt a lot, and to my students Marie and Tabassom, for being so fun and hardworking and, in general, the best students one could ask for.

69 I did not only have great lab colleagues, but also great corridor mates. I would like to start with those that make it all work: Aino, Anita, Maryia, Lasse and also in the past, Lotti and Ulla. Call me dramatic, but without you this place would have burned to ashes a long time ago! Thank you for being so nice and helpful and for never stopping improving our corridor, you all are really great professionals. Thanks as well to all the PhD students and post- docs that I have met during these years: Laia, thank you for so much. You are the busiest person I know and still you find the time to give me advice for the future and cheer me up. In many ways, you have been a great mentor to me and I never stop learning from your strength. Costas, you are a great researcher and an even better person, thank you for all the laughs in the cell lab, the Christmas songs on the radio are more endurable with you around. Panos, I wish you a lot of luck in France. Also, you are the first person I’ve ever seen going to the gym in jeans, please don’t do that! Maria T., you are a great colleague and friend and even now, when you are as busy and stressed as ever, you are still the most supportive and sweet person. Niki, it is always fun to share the electrophoresis room time with you, thanks for always being extra nice! Chrysa, it was great to meet someone that shares my interest in EVs, I wish you a lot of good results. Deepesh, thank you for all the help with the microscope and the fun times during the dish duty week. Chun, you are a very hardworking and inspiring person, I wish you a lot of luck during the rest of your PhD! Also, Melanie, Kehuan and Caroline, although we did not get to know each other very much, I wish you all the best during the rest of your PhD adventure. Oleks, thanks for reminding me I spend too much time in the cold room, I will end up hearing your voice when I open the fridge at home! Ria, I love your fun attitude, I wish you a lot of luck with your future EGFR enter- prises! Anahita, thank you for being always so nice and for all your support during our teaching this year. Kallia, you are so sweet and fun, we need more people like you in the world. Thank you also Natalia, for all the advice over the years, Ihor, for being a nice colleague and giving me a hand with the ul- tracentrifuge and Anders, for always being helpful and kind to everyone. I also want to thank Jessica, Yanyu, Yutaro, Siamak, Tianyi and Per for contributing to the nice atmosphere. Many others have been part of this corridor in the past and I would like to mention them, especially Glenda, Haisha, Sin- nisky and Inna. Thank you for all the laughs and every time you gave me advice. Claudia, who was also my flatmate, I miss your energy in the corridor and your no-carbs propaganda at home. I’m sure you keep spreading your message in Switzerland. Merima, I am happy that you stayed in Uppsala after Ludwig and that we could keep having fun and discover our common taste in books. I wish you a lot of success on your next adventure in London! Moreover, I have been very lucky to share corridor with a lot of wise senior scientists. Thanks, Aris, for all your guidance during teaching and for your efforts in making our corridor a great place to work. Thank you Staffan for always having time for my doubts on integrins and for kindly offering help

70 whenever I needed it. Thanks as well to Evi and Johan for your feedback during my seminars and your great sense of humour. Anna-Karin, thank you for your participation and insightful comments during my half-time evaluation. Jin-Ping, Lena and Dorothe and your respective research groups, thank you also for contributing to the nice environment in B11:3. I cannot pass on the opportunity to thank the administrative staff at IMBIM, who work efficiently to solve all our problems and, on top of that, always bring the best mood to retreats and parties. Specially, I would like to thank Alexis for his professionality and endless patience in helping me navigate all the defence bureaucracy. I also want to take the opportunity to thank all of those with whom I have collaborated over the years, and who have made my projects sharper and better. Thanks Olof I., for all the lessons on TIRF and tips on the postdoc life. Mikael S., thank you for your comments and suggestions for my projects. Jens, you really rock at live cell microscopy and I hope I can keep learning from you in the future. Also, thanks to Karin and Monika at the TEM service for teaching me so much on electron microscopy and having fun with me searching for exosomes on the grid. I also owe a lot to Taija and all her group, specially Simon, Magda, Inés, Nina and Yan. You all are the most welcoming crew and I have learnt loads from you. In this last year of PhD, I also found the time to get involved in the PhD student association of IMBIM. I want to thank all my colleagues there, specially Aida, Viktor, and Elin for all the hard work and everything we have accomplished together. Thanks also to Eva G. for being the first to talk me into it, as you also did with the photo-club and ACES. You always make my life more interesting! Without a bit of non-academic life my academic self would not have sur- vived all these years. Therefore, I need to thank all the friends that brought joy to my life in Uppsala. I want to start with two friendships that go a long way back in time. Andrea, it was already great to have you in my life back in Valencia and it only became better when you too moved to Sweden. Thanks for being that person that I could call in the middle of the night if I would need to and for sharing a bit of your everyday with me, even when it is only to be bored together. Daniel, I guess I would have never ended up in Uppsala if it were not for you! You helped me to adapt to the Nordic life, stoically accepted my moments of drama and became one of my most reliable friends. Thank you for years of my favourite combo: random jokes and meaningful chats. Mahsa, you are one of the most special friends that I met on this adventure. You have been there to travel the world with me, but also to give me company during the most boring night in a hospital room. I hope the end of this PhD brings the time and opportunities for many more adventures together! Car- men, thank you for our dinners and time together, and for getting to love me even if we are so different. You always make me feel strong, supported and capable of everything. All those things are also what I see in you and I have

71 no doubt you will achieve everything you aim for. Alejo, you are a great and caring friend, thank you for always cheering me up… I’m looking forward to our next concert! Gianni and Sophia, you guys are very special to me and I always feel good when I am around you, thank you for making me laugh, making me think and counting with me for fun plans. Joran, I always enjoy your company, but I don’t forgive that you got me trapped on top of a climbing wall like a scared cat. Guilhe, I tend to complain but I actually adore your ability for random conversations, don’t change! Ale, thank you for your sense of humour and your always great book recommendations. Eva F. and Anders, you two are incredibly fun, never stop being so authentic! Christian, I enjoyed discussing extracellular vesicles with you, thanks for sharing with me the lo- cation of all the great NTA machines at BMC! Fred, you have been the best flatmate! We had so much fun in the villa and, lucky for me, we stayed friends after it. Despite your weird taste in music, I enjoy spending time with you a lot, either taking pictures or laughing uncontrollably at Eurovision. Johan, you have also been a great flatmate, thanks for your lessons on how to cele- brate a kräftskiva properly and all our abstract conversations while cooking dinner. Davide, thank you for coming to the rescue every time I don’t understand something in physics or about turkeys. Ilektra, I always enjoy your company, I hope we would see each other more often! Ana M., your laughter is always contagious, thanks for being so sweet to me. Special mention to the rest of the Spanish crew: Jose, Guillermo, Laura M., Fernando, Ana Z., Jesus, Maria y Alberto, for always turning any gathering into a great party. Also, other friends in the distance have helped me staying sane all these years. Thank you to my favourite duo, Juan and Ville, because I adore you and our yearly trips are my favourite way to recharge my batteries. Alex, thanks for being the sweetest and taking care of me every time I go to Madrid; Michael and Jana, we have lived far from each other since we first met and still managed to create a great friendship; Amalia, I adore your audio mes- sages of twenty minutes and I really feel there is nothing I could not share with you, thanks for always being there; Nuria, you take care of your friends and always put a smile on my face; Lucia, one of my favourite hobbies is to solve world problems while talking with you over two beers. That is, without a doubt, one of the things I miss the most from Spain. And last, but not least, my childhood friend Laura and my high school friends Carolina and Pilar. Over the years you have defined the person I am and, no matter how little we see each other, you have a special place in my heart. Finally, I would like to thank my better half, Emilio, for being the most supportive partner I could have asked for. Although the last months have been incredibly stressful, you always managed to make them a bit easier and a bit happier, so that I go to sleep every day feeling like the most fortunate person for having you in my life. You also gave me the flu five days before submis- sion, but I can’t ask for perfection!

72 Gracias también a mi familia: a mis sobrinas, por las que siempre quiero intentar ser mejor persona, a mis tíos, tías, hermanos y cuñadas, por cuidar de mi hasta el día de hoy y sobre todo a mis padres, que son dos personas increíbles. Gracias, papá, por enseñarme, desde el ejemplo, el valor del es- fuerzo y la honestidad, dos pilares fundamentales en investigación científica. Gracias, mamá, por contagiarme tu creatividad y energía, también indispen- sables en ciencia, y por creer en mi tantísimo. Desde que soy pequeña habéis luchado por convertirme en una mujer independiente y me habéis brindado todas las oportunidades para conseguirlo. Por eso, esta tesis no es solo el fruto de mi sacrificio, también es el fruto del vuestro y siempre os estaré agradecida por ello.

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