Sandra kleiner, Joshi Venugopal, Yoshikuni Nagamine


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First, I would like to thank my parents who
supported me through all my life and are
always there for me when I need them.
I also wish to acknowledge Dr. Yoshikuni
Nagamine, who supervised me, and gave me the opportunity to develop my scientific
thinking and skills in his lab. While he offered scientific freedom, he was always available for discussions. I greatly appreciate that.

Thanks also to Prof. Gerhard Christofori and Prof. Fred Meins, the two other members of my thesis committee, for the advice they gave me during the committee meeting and for the time they will still have to invest to read and evaluate this thesis.

My special thanks go to Joshi Venugopal,
from whom I could learn a lot in many aspects of life and who became a close friend of mine. His critical and logical thinking inspired me in several things. I really appreciate the time we spent together.

I also want to acknowledge Malgorzata
Kiesielow for our fruitful collaboration and for
teaching me siRNA transfections.
In addition, I wish to thank my former and
current lab members Faisal, Hoanh, Fumiko,
Kacka, Sandra and Stephane. I always
enjoined working and spending some free time
with all of you.
Further, I want to acknowledge the technical
staff at the FMI who were always friendly and
helpful and made the scientific life at the FMI
much easier and productive. Thanks go to all
of the FMI members (especially from the
Hynes laboratory) and to all of those, who
provided me with scientific material: François
Lehembre, Kurt Ballmer, Tony Pawson, Peter
E. Shaw and Jerrold Olefsky. My thanks also
go to Pat King and Sara Oakley for critical
reading of my manuscripts.
My heartiest gratitude goes to Boris
Bartholdy who always supported me
scientifically with all of his skills and privately
with all of his love.

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Sandra kleiner, Joshi Venugopal, Yoshikuni Nagamine

  1. 1. ISOFORM-SPECIFIC ROLES OF THE ADAPTOR PROTEIN SHCA IN CELL SIGNALING Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von SANDRA KLEINER aus Weißenborn, Deutschland Dissertationsleiter: Dr. Yoshikuni Nagamine Friedrich Miescher Institute for Biomedical Research BASEL, 2005
  2. 2. Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultätauf Antrag vonProf. Fred Meins, Dr. Yoshikuni Nagamine, Prof. Gerhard Christofori und Prof.Patrick MatthiasBasel, den 22.11.2005 Prof. Dr. Hans-Jakob Wirz (Dekan) 2
  3. 3. TABLE OF CONTENT TABLE OF CONTENTSUMMARY 51. INTRODUCTION 6 1.1 The Shc adaptor proteins 6 1.1.1. Genomic and structural organization of Shc 6 Genomic organization and regulation of Shc expression 6 Structural organization of Shc proteins 8 1.1.2 Signaling and function of ShcA 9 Role of Shc in mitogenic Ras/Erk signaling 9 Role of Shc in c-myc activation and cell survival 11 Role of Shc in cell adhesion, migration, and cytoskeletal organization 11 Role of Shc in tumorigenesis 12 In vivo function of Shc 13 Conventional Shc knockout 13 Conditional T-cell specific knockout and transgenic mice 13 Shc Role of p66 14 1.2 Signaling of the E-cadherin cell-cell adhesion protein 19 1.2.1 E-cadherin-dependent cell-cell adhesion 19 E-cadherin: a member of the classical cadherins 19 Function of catenins in the E-cadherin adhesion complex 20 Function of the E-cadherin-catenin complex 21 1.2.2 E-cadherin as a tumor suppressor 22 1.2.3 E-cadherin-mediated signaling 23 1.3 RNA interference: a new and powerful tool in molecular biology 28 1.4 Research objectives 312. RESULTS 32 2.1 Research communication 32 Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering siRNA INTRODUCTION 32 EXPERIMENTAL 33 RESULTS 34 DISCUSSION 36 REFERENCES 37 2.2 Using siRNAs to study Shc function 39 3
  4. 4. TABLE OF CONTENT 2.2.1 Isoform-specific knockdown of p46/52Shc 39 2.2.2 Growth inhibition upon Shc knockdown 39 2.3 Role of Shc in EGF-induced signaling in epithelial cells 43 2.3.1 Role of Shc in EGF-induced Erk activation 43 Shc 2.3.2 Effect of p66 on EGF-driven proliferation and cell survival 44 2.4 Research Publication (under review) 46 Induction of uPA gene expression by the blockage of E-cadherin via Src- and Shc-dependent Erk signaling INTRODUCTION 46 MATERIALS AND METHODS 47 RESULTS 48 DISCUSSION 53 REFERENCES 56 2.5 Supplementary data to 2.4 59 2.5.1 Role of FAK in Decma-induced Erk activation 59 2.5.2 Disruption of cell-cell adhesion using EGTA in LLC-PK1 cells 59 2.6 Role of p66Shc in regulating cell survival in epithelial cells 613. DISCUSSION 63 3.1 Isoform-specific knockdown and knockdown-in of Shc using siRNA 63 3.2 Role of Shc in mediating Erk activation 65 3.2.1 Shc is dispensable for EGF-induced Erk activation 65 3.2.2 Shc mediates Erk activation downstream of E-cadherin 67 3.3 The role of p66Shc in stress response 70 3.4 Isoform-specific role of p46Shc 71 3.5 Conclusion 714. MATERIAL AND METHODS 735. REFERENCES 746. ACKNOWLEDGEMENTS 847. ABBREVIATIONS 858. CURRICULUM VITAE 86 4
  5. 5. SUMMARY SUMMARY ShcA is a bona fide adaptor protein without We used this technique to investigate theany enzymatic activity. Upon activation of contribution of individual ShcA isoforms toreceptor tyrosine kinases, ShcA associates EGF-induced MAPK activation in epithelialwith the receptor and becomes tyrosine cells. Knockdown of all or single ShcAphosphorylated. Phosphorylated ShcA recruits isoforms had no effect on EGF-induced Erk Shcthe Grb2/SOS complex to the membrane, activation. Moreover, overexpression of p66 Shcwhere SOS stimulates the small GTPase Ras, in non p66 -expressing MCF7 cells did notresulting in the activation of the Ras/MAPK change EGF-induced proliferation or viability.pathway. The fact that Grb2 binds directly to These data suggest that EGF-induced MAPKmost of the receptor tyrosine kinases raises activation in epithelial cells is ensured by athe question of how important is the role of Shc redundant coupling of Grb2 to the mediating MAPK activation? Moreover, In a quest for growth factor-independentbeside growth factor-induced MAPK activation, pathways involving Shc-mediated Erkare there other pathways in which ShcA- activation, we investigated signalingmediated MAPK activation is relevant? downstream of the cell-cell adhesion molecule ShcA is expressed in three different E-cadherin. We identified a previously Shc Shc Shcisoforms: p46 , p52 , and p66 . These unknown signaling pathway which is inducedisoforms are all derived from a single gene and upon disruption of E-cadherin-dependent cell-differ only in their N-terminal part. Although all cell adhesion This pathway involves Src- andisoforms are phosphorylated by receptor Shc-dependent Erk activation, which resultstyrosine kinases, and subsequently bind to subsequently in the expression of the ShcGrb2, the p66 isoform does not seem to urokinase plasminogen activator. Applying the Shcmediate MAPK activation. The individual knockdown-in technique revealed that p46contribution of p46Shc and p52Shc in mediating and p52Shc, but not p66Shc, were able toMAPK activation is also not clear. The fact that mediate MAPK activation upon disruption ofall isoforms are ubiquitously expressed, with cell-cell adhesion. This pathway directly links Shcsome restrictions for p66 , complicates the disruption of cell-cell adhesion with theexperimental investigation of each isoform. expression of proteolytic enzymes, both ShcRecently, p66 has been implicated in the processes involved in metastasis and woundregulation of apoptosis in response to oxidative healing. Shcstress. To learn more about the role of p66 in Using siRNA, we established a system which mediating oxidative stress-induced apoptosis Shcallows isoform-specific knockdown of ShcA in epithelial cells, the effect of p66 on cell Shcproteins in tissue culture. Further development viability was investigated. Although p66 hasof this technique enabled us to express a been shown to enhance stress-inducedsingle isoform in the absence of endogenous apoptosis in fibroblasts, endothelial cells, andprotein. This so-called “knockdown-in” T-cells, no effect on p66Shc expression wastechnique is applicable for most proteins which observed in two different epithelial cells,are expressed in multiple isoforms, and allows suggesting that the apoptotic response inthe investigation of specific mutations against epithelial cells is mediated in a p66Shc-a clear background without overexpression. independent manner. 5
  6. 6. INTRODUCTION 1. INTRODUCTION This chapter provides insights into three 1.1.1. Genomic and structuraldifferent topics: (i) function of Shc proteins, (ii) organization of ShcE-cadherin-mediated cell-cell adhesion and (iii)RNA interference. Genomic organization and regulation of Shc expression1.1 The Shc adaptor proteins The human shc locus maps to the Shc proteins are prototype adaptor proteins chromosome 1q21 (Huebner et al., 1994). Itwhich represent molecules that possess no contains 13 exons, which give rise to threeapparent catalytic domains or activities. different gene products: three isoforms ofAdaptor proteins contain modular protein- about 46, 52, and 66 kDa. All isoforms areprotein and protein-lipid interaction domains, generated either through RNA splicing orsuch as src-homology domain 2 (SH2) and 3 alternative translational initiation (Migliaccio et(SH3), phosphotyrosine binding domain (PTB), al., 1997; Pelicci et al., 1992) (Fig. pleckstrin homology (PH) domains, and While the p46Shc/p52Shc transcript originatesare essential in propagating signals from a from the assembly of the non-coding exon 1receptor in a coordinated fashion (Zhang et al., with the 3 portion of exon 2 (exon 2a), and2002). with exons 3−13, the p66Shc transcript is The adaptor protein ShcA was initially formed by the assembly of exons 2-13. Aidentified as an SH2-containing proto- second mechanism that regulates transcriptiononcogene involved in growth factor signaling. of the three Shc isoforms is the alternativeSince than, it has been shown to be an integral usage of in-frame translational start codons.component implicated in the action of a wide The transcript encoding p66Shc has three in-variety of receptors, including receptor tyrosine frame ATGs that are responsible for the Shckinases (RTKs), G protein-coupled receptors translation of p66 , and, to a lesser extent, Shc Shc(GPCRs), immunoglobulin receptors, and p52 , and p46 . The p52Shc/p46Shc transcriptintegrins, as well as non-receptor tyrosine contains two in-frame ATGs that are Shckinases such as Src and FAK. To date, three responsible for the translation of p52 and Shcmammalian shc genes have been identified: p46 (Migliaccio et al., 1997). The mouse shcshcA, shcB (sck), and shcC (N-shc/rai) locus is similarly organized and maps to(Nakamura et al., 1996; OBryan et al., 1996; chromosome 3 (Kojima et al., 2001; MigliaccioPelicci et al., 1996). All three shc genes et al., 1997).encode proteins that are highly related in Less is known about the moleculardomain and structure. In the following section, I mechanisms that regulate the differentialwill provide an overview of the genomic expression of the various Shc isoforms. Itorganization and structural architecture of seems that different mechanisms control theShcA, hereafter referred to as Shc, along with expression of the two main Shc transcripts in Shc Shcits known functions in signal transduction. different cell types. p46 /p52 are found 6
  7. 7. INTRODUCTIONubiquitously in every cell type, whereas p66Shc engagement of CD4 and CD3 (Pacini et al.,expression varies and is restricted to certain 2004). In vivo, p66Shc expression has beentissues and cell lines, being absent in brain, in found to be induced in circulating peripheralmost hematopoietic cell lines, in peripheral blood mononuclear cells of diabetic patientsblood lymphocytes (PBL), and in a subset of (Pagnin et al., 2005).breast cancer cell lines (Jackson et al., 2000; Overall expression analysis has shown thatPelicci et al., 1992; Stevenson and Frackelton, Shc is expressed at its highest levels in the1998; Xie and Hung, 1996). Ventura et al. placenta, adipocytes, bronchial-epithelial cells,(Ventura et al., 2002) have recently identified colorectal adenocarcinoma, cardiac myocytes,epigenetic modifications, namely histone and smooth muscle cells of humans (humandeacetylation and cytosine methylation, as GNF SymAtlas).mechanisms underlying transcriptional The family members ShcB and ShcC are shcsilencing of p66 in specific cell types. derived from different genes, and theirHistone deacetylase inhibitors, or expression is restricted to the brain anddemethylating agents, were capable of neuronal tissue (Nakamura et al., 1996; Shcrestoring p66 expression in primary, OBryan et al., 1996; Ponti et al., 2005). Unlikeimmortalized, and transformed cells. shcA, only two isoforms are encoded by the shcAdditionally, the p66 -encoding locus could shcB and shcC reactivated in human PBL and mouse T-cells by treatment with a variety of apoptogenicstimuli, such as H2O2, the calcium ionophoreA23187, Fas ligation, and sequentialFigure of humanShc locus and exonassembly of Shctranscripts. A schematicrepresentation of the exonassembly in the shc shc shcp52 /p46 and p66encoding transcripts. Shcexons are indicated byboxes (black boxes are translated exons), the exon numbers are given above, and the splicing eventsare shown by the zig-zag line. The position of the three Shc ATGs is indicated below the exons (asdescribed in (Migliaccio et al., 1997)). 7
  8. 8. INTRODUCTION1.1.1.2 Structural organization of Shc (IRS-1/2), tensin, the epidermal growth factorproteins receptor (EGFR) pathway substrate (Eps8), and the integrin cytoplasmic domain- Shc proteins are characterized by their associated protein-1 (ICAP-1) (Schlessingerspecific modular organization, consisting of an and Lemmon, 2003).amino-terminal phosphotyrosine-binding (PTB) The PTB domain shows remarkabledomain, a central proline- and glycine-rich structural similarity to pleckstrin homology (PH)collagen homology domain (CH1), and a domains, despite a very divergent primarycarboxy-terminal Src homology 2 (SH2) sequence (Zhou et al., 1995c). In a similar waydomain (Fig. The unique feature to PH domains, the Shc-PTB domain has beenthereby is the arrangement of the PTB and the shown to bind acidic phospholipids such asSH2 domain in an N to C order (Luzi et al., PI(4,5)P2 and PI(4)P (Zhou et al., 1995c), and2000). Shc proteins are evolutionarily well also PI(3,4,5)P3 (Rameh et al., 1997). Theconserved and can be found in mammals, high affinity (KD=10-50 µM) of this bindingfishes, flies and worms. suggests that the interaction of Shc with the membrane could occur independently of an interaction with tyrosine-phosphorylated receptors. Consistent with this idea was the identification of residues within the Shc-PTB domain that are critical for phospholipid binding and membrane localization and are distinct from the residues necessary for posphpo-Figure Domain structure of Shc tyrosine binding (receptor binding). Over theproteins. All Shc isoforms share the same last few years many different proteins, such asmodular organization: N-terminal PTB domain, F-actin, SHIP (SH2-containing inositolcentral collagen homology domain (CH1), and polyphosphate 5 phosphatase), IRS-1 and ShcC-terminal SH2 domain. p66 contains an PP2A (protein phosphatase type 2A), haveadditional collagen homology domain (CH2). been found to bind to the Shc-PTB domain in aAll known phosphorylation sites are indicated. phosphotyrosine-dependent or -independent manner (Kasus-Jacobi et al., 1997; Lamkin et A second phosphotyrosine-binding (PTB) al., 1997; Thomas et al., 1995; Ugi et al.,domain, distinct from the SH2 domain, was 2002).discovered in Shc proteins (Blaikie et al., 1994; On the N-terminal edge of the PTB domainKavanaugh and Williams, 1994). The unique of p52Shc and p66Shc there is a serinefeature of the Shc-PTB domain is that its phosphorylation site (Fig. (El-Shemerlybinding to target sequences is determined by et al., 1997). Further studies haveresidues N-terminal to the phosphotyrosine, demonstrated that phosphorylation of this siteand is not influenced by residues C-terminal to is necessary for Shc binding to thethe phosphotyrosine (Blaikie et al., 1997; Trub phosphatase PTP-PEST and downregulationet al., 1995; Zhou et al., 1995a). Today, more of insulin-induced Erk activation, most likelythan 160 proteins containing a PTB domain areknown, including insulin receptor substrate 1/2 8
  9. 9. INTRODUCTIONthrough dephosphorylation of Shc (Faisal et The third conserved region maps as aal., 2002). binding site for adaptins which links the The SH2 domain of Shc is located at the C- endocytic machinery of clathrin-coated pitsterminus and was thought to be the only with integral membrane proteins, suggesting adomain responsible for the recruitment of Shc potential role of Shc in endocytosis. Thisto activated growth factor receptors before the region is only weakly conserved in Drosophilaidentification of the Shc-PTB domain. It folds in (Lai et al., 1995). Shca very similar manner to other SH2 domains p66 contains an additional N-terminal CH-(Mikol et al., 1995; Zhou et al., 1995b). Unlike like domain (called CH2) (Migliaccio et al.,the Shc-PTB domain, the target binding of the 1997), which is also found in the longerShc-SH2 domain is determined by residues C- isoforms of ShcB and ShcC, but not in theterminal to the phosphotyrosine Drosophila Shc protein (Luzi et al., 2000). In(Ravichandran, 2001). contrast to the CH1 domain, the CH2 domain Between the PTB and the SH2 domain is the can be serine/threonine phosphorylated incollagen homology (CH) 1 domain. This region response to several stimuli such as oxidativeis characterized by a large number of glycine stress (Migliaccio et al., 1999), 12-O-and proline residues, but does not feature tetradecanoylphorbol-13 acetate (TPA) (El-typical collagen-like repeats. While the PTB Shemerly et al., 1997), and epidermal growthand the SH2 domains share high similarity, factor (EGF) (Okada et al., 1997). The78% and 68% respectively, the CH1 domain is phosphorylation of serine 36 (S36) has been Shcgenerally less well conserved between linked to the role of p66 in oxidative stressdifferent species. However, within the response (Migliaccio et al., 1999) and will bemammalian Shc family members, three regions discussed later. The physiological relevance ofsharing a higher degree in homology are the threonine phosphorylation site (T29) haspresent in this domain. Two of these not yet been defined.conserved regions comprise three criticaltyrosine phosphorylation sites, Y239, Y240, 1.1.2 Signaling and function of ShcAand Y317, and additional amino acidssurrounding the amino-terminal Role of Shc in mitogenic Ras/Erkphosphorylation site suggesting an important signalingrole in the recognition of effector proteins(OBryan et al., 1996). Y317 is conserved in In vivo and in vitro studies from variousmammalian Shc proteins, but not seen in those laboratories have clearly established a role forof lower organisms. Y239 and Y240 are also Shc in Ras/MAPK activation (Lai and Pawson,present in Drosophila Shc (Lai et al., 1995), but 2000; Pratt et al., 1999; Salcini et al., 1994).Shc in C. elegans does not contain any of the This is the only function of Shc of which thetyrosine residues (Luzi et al., 2000). Both molecular mechanism is understood. Activationphosphorylation sites conform to the of RTKs results in the recruitment of Shcconsensus Grb2-binding site and have been proteins and, subsequently, in Shcdemonstrated to bind Grb2 (Velazquez et al., phosphorylation. Phosphorylated, hence2000; Walk et al., 1998). activated, Shc binds to the Grb2/SOS complex. 9
  10. 10. INTRODUCTIONThe Shc/Grb2/SOS complex is then localized preferentially in complexes that also containto the membrane through the interaction of Shc Shc (Buday et al., 1995; Pronk et al., 1994;with the phosphorylated receptor via its PTB or Ravichandran et al., 1995). Still, manySH2 domain (Blaikie et al., 1994; Pelicci et al., receptors are able to directly recruit the1992; Ravichandran et al., 1993). At the Grb2/SOS complex, leading to Ras activationmembrane in vicinity to Ras, SOS stimulates without the involvement of Shc (Arvidsson etnucleotide exchange on Ras and, thereby, al., 1994; Batzer et al., 1994; Schlaepfer et al.,activation of Ras (Fig. (Ravichandran, 1998). In response to integrin ligation,2001). GPCR, integrins, and cytokine however, Shc is necessary and sufficient forreceptors without intrinsic tyrosine kinase activation of the MAP kinase pathway (Wary etactivity utilize other soluble and associated al., 1996). The ability of Shc to mediate Rastyrosine kinases to phosphorylate Shc activation is largely dependent on the three(Sayeski and Ali, 2003; Velazquez et al., 2000; tyrosine residues within its CH1 domain.Wary et al., 1996). In addition to translocating Phosphorylation-deficient mutants exertthe Grb/SOS complex to the membrane, Shc dominant-negative activity, whereby theseems to influence the extent of Ras importance of distinct Shc tyrosines differsactivation. The Shc/Grb2 interaction increases between the cell types and receptorsthe level of SOS bound to Grb2 in some (Ravichandran, 2001).systems, and SOS has been foundFigure Model for Shc-mediated Ras activation downstream of RTK. Shc binds to RTKsand recruits the Grb2/SOS complex which activates Ras. See text for details. 10
  11. 11. INTRODUCTION1.1.2.2 Role of Shc in c-myc activation and phosphorylation via the Shc/Grb2/Gab2/PI3Kcell survival pathway, and might therefore be involved in the regulation of IL-2-mediated cell survival The observation that Shc is involved in c- (Fig. (Gu et al., 2000).myc activation has led to two suggestions. The involvement of ShcB and ShcC inFirst, Shc might play a role in signaling other survival of neuronal cells has become morethan mediating Ras/MAPK activation and, evident. Whereas ShcA is only expressed insecond, the downstream signaling of proliferating neuroblasts and is downregulatedY239/Y240 and Y314 might have distinct in post-mitotic neurons, ShcB and ShcCproperties (Fig. In BaF cells, Gotoh et remain expressed (Cattaneo and Pelicci, 1998;al. (Gotoh et al., 1996) showed that Shc could Conti et al., 1997). Mice with no ShcB and/orinduce c-myc expression in response to IL-3 ShcC expression display a loss of certain typesstimulation which was dependent on of peptidergic and nociceptive neurons (SakaiY239/Y240, but not on Y137. The same et al., 2000). It appears, therefore, that ShcAsituation was demonstrated for EGF signaling plays a role in neuronal proliferation, but ShcBin NIH3T3 cells (Gotoh et al., 1997). and ShcC isoforms play a role in survival ofSubsequently, a role for Shc in c-myc gene post-mitotic neurons.activation has been shown in IL-2 signaling(Lord et al., 1998), in PDGF signaling (Blake etal., 2000), and in T-cell antigen receptor (TCR)signaling (Patrussi et al., 2005). However, itremains unclear how Shc mediates c-mycactivation and what target genes are in turnaffected by c-Myc. Induced c-myc expression downstream of IL-2/3 and TCR correlated with survival signals in Figure Distinct signaling capacitieshematopoetic cells (Gotoh et al., 1996; Lord et of the major tyrosine phosphorylation, 1998; Patrussi et al., 2005), suggesting an The three tyrosine phosphorylation sites andinvolvement of Shc in the regulation of a pro- the signaling linked to these tyrosines aresurvival pathway via c-myc. Lord et. al. (Lord et, 1998) observed Shc-dependent inductionof proliferation and expression of c-myc, bcl-2 Role of Shc in cell adhesion,and bcl-x in response to IL-2. Nevertheless, migration, and cytoskeletal organizationthe proliferative response and the expressionof bcl-family genes were not sufficient to The implication of Shc in processes such asmediate sustained cell survival and cell adhesion, migration, and cytoskeletalantiapoptotic effects associated with a organization originates from diverse reports incomplete IL-2 signal in murine T-cells. In a different contexts.different study, a Shc chimera fused to the IL-2 Embryonic fibroblasts derived from Shc-receptor β chain that lacks other cytoplasmic knockout mice have defects in spreading ontyrosines was able to evoke PKB/AKT fibronectin (Lai and Pawson, 2000). Similarly, 11
  12. 12. INTRODUCTIONthe regulation of cell adhesion and EGF- Role of Shc in tumorigenesisinduced migration on fibronectin required theinteraction of Shc and α5β1 integrin in MCF7 The ability of Shc to mediate mitogenicbreast cancer cells (Mauro et al., 1999; Nolan signaling raises the question of whether Shcet al., 1997). In addition, Shc has been shown can drive tumorigenesis. Although Shc proteinsto localize to focal adhesions and to interact do not contain any enzymatic activity, Shcwith the focal adhesion kinase (FAK) (Barberis overexpression of p46/52 was able toet al., 2000; Gu et al., 1999). Although Shc can transform mouse fibroblasts and to enablebe a substrate of FAK (Schlaepfer et al., 1998), them to form tumors in nude mice (Pelicci ettheir effects on cell migration seem to be al., 1992). In tumor cells with known tyrosinedistinct. While Shc stimulates random cell kinase gene alteration, Shc proteins weremotility through activation of the Erk signaling found to be constitutively phosphorylated andpathway, FAK regulates directional persistent complexed with Grb2 and activated tyrosinemigration via p130Cas (Gu et al., 1999). In kinases (EGFR, PDGFR, ErbB-2, Met, BCR-ErbB2-driven migration, Shc seems to be Abl, and Ret) (Pelicci et al., 1995b).required for lamellipodia formation Underscoring the role of Shc in oncogenic RTK(reorganization of the actin cytoskeleton) and signaling, dominant negative Shc has beenfor mediating the interaction between the shown to block proliferation of ErbB-2 positivereceptor and Memo, which is necessary for cell human breast cancer cell lines (Stevenson etmigration-required reorganization of the al., 1999).microtubule network (Marone et al., 2004). In More recently, an in vivo study has unveiledsupport of this report, inhibition of EGF- an unsuspected role for the Shc in RTK-induced cell migration upon downregulation of mediated vascular endothelial growth factorShc has also been observed in a different (VEGF) production and tumor angiogenesisstudy (Nolan et al., 1997). In response to HGF, (Saucier et al., 2004). Using RTK engineeredoverexpression of Shc enabled enhanced to recruit a defined signaling protein, it wasmigration and growth of melanoma cells shown that the direct recruitment of either Grb2(Pelicci et al., 1995a). Whether Shc stimulates or Shc to an RTK oncoprotein is sufficient toproliferation or migration seems, at least induce transformation and metastasis (Saucierpartially, to be determined by external stimuli. et al., 2002). The authors then extended thisIn the presence of growth factors, Shc study in order to compare and define the roleregulates DNA synthesis, but under growth of Shc and Grb2 in RTK oncoprotein-drivenfactor-limiting conditions, Shc stimulates cell tumorigenesis (Saucier et al., 2004).migration (Collins et al., 1999). To what extent Fibroblasts expressing Shc-binding RTKboth responses depend on Shc-induced MAPK oncoproteins induced tumors with short latencyactivation, or activation of and cross talk with (approximately 7 days), whereas cellsother signaling pathways, is not clear. expressing Grb2-binding RTK oncoproteinsHowever, in one case, a direct interaction induced tumors with delayed latencybetween Shc and F-actin has been observed in (approximately 24 days). The early onset ofPC12 cells in response to NGF (Thomas et al., tumor formation resulted in the ability of Shc-1995). binding RTK oncoproteins to produce (VEGF) 12
  13. 13. INTRODUCTIONin culture and an angiogenic response in vivo. growth factors. Shc-deficient mouse embryonicMoreover, the use of fibroblasts derived from fibroblasts (MEFs) also showed changes inShc-deficient mouse embryos demonstrated focal contact organization and actin stressthat Shc was essential for the induction of fibers when plated on fibronectin, underscoringVEGF by the Met/hepatocyte growth factor the role of Shc in cytoskeletal organization.RTK oncoprotein and by serum-derived growthfactors. Conditional T-cell specific knockout and transgenic mice1.1.2.5 In vivo function of Shc Efforts over the past 10 years have1. Conventional Shc knockout demonstrated that Shc plays a critical role in T- cell receptor (TCR) signaling. The earliest The conventional knockout mouse created evidence linking Shc to TCR-mediatedby Lai and Pawson (Lai and Pawson, 2000) signaling was the observation that Shcclearly established a role for Shc in vivo. becomes tyrosine phosphorylated rapidly afterAblation of exons 2 and 3, which encode the TCR/CD3 crosslinking (Ravichandran et al.,PTB domain, by gene targeting resulted in a 1993). Several studies followed showing thatloss of expression of all three Shc isoforms in expression of dominant negative mutants ofhomozygous mutants. The homozygous Shc inhibited TCR-mediated downstreammutant embryos died at day 11.5 with severe signaling (Milia et al., 1996; Pacini et al., 1998;defects in heart development and Pratt et al., 1999). To examine the relativeestablishment of mature blood vessels. The significance of Shc compared to several othercardiovascular system showed defects in adaptors in T-cells, two genetic approachesangiogenesis and cell-cell contacts. Consistent were taken in mice (Zhang et al., 2002). Thewith this, Shc was mainly expressed in the first approach involved the generation of acardiovascular system of wild-type embryos. transgenic mouse with thymocyte-specificThe Shc∆ex2/3 mutants also provided evidence expression of a dominant negative form of Shc,for Shc in MAPK signaling in vivo. There was a where all tyrosine residues were mutated toloss of MAPK activation within the phenylalanine (ShcFFF). The ShcFFF transgenic mice had a reduced thymus size,cardiovascular system of the Shc∆ex2/3 mutants, with significant reduction in thymocyteas revealed by whole mount immunostaining numbers. Further analysis revealed that T-with phospho-specific Erk antibodies, when ShcFFF cells were blocked at the double negativecompared to wild-type embryos. Studies with stage (DN) of their development, which wasShc∆ex2/3 embryonic fibroblasts have characterized by the absence of CD4 and CD8demonstrated that Shc is necessary for MAPK markers (reviewed in (Zhang et al., 2003)). Thesignaling induced by a low concentration of authors did not observe any increase in thegrowth factors, but at a high concentration of apoptotic fraction of the DN cells in ShcFFFgrowth factors (50 ng/ml EGF or 25 ng/ml transgenic mice compared to wild-type mice.PDGF) no detectable difference in MAPK More recent studies using pulse BrdU injectionactivation was observed. These data suggest have demonstrated a defect in proliferation ofthat Shc sensitizes cells to low amounts of 13
  14. 14. INTRODUCTIONthe late DN stage cells mediated by the pre- transform mouse fibroblasts (Migliaccio et al.,TCR (Fig. The same phenotype was 1997), suggesting a function distinct from the Shcalso obtained using the second approach, other two isoforms. Indeed, p66 does notconditional Shc knockout mice, with a nearly increase EGF-induced MAPK activation,complete loss of Shc protein expression in although it is tyrosine-phosphorylated uponthymocytes. Thus, both Shc expression and its EGF stimulation, binds to activated EGFRs,tyrosine phosphorylation play an essential and and forms stable complexes with Grb2non-redundant role in thymic T-cell (Migliaccio et al., 1997) (Fig. and proliferation. Furthermore, it has been shown that p66Shc expression inhibits EGF-induced c-fos promoter activation (Fig. The molecular mechanism is not understood, taken Shc into account that p66 expression did not inhibit Erk activation. However, the inhibition was attributed to the CH2 domain, since it Shc retained the inhibitory effect of p66 on the c- fos promoter (Migliaccio et al., 1997). In contrast, an independent study has shown that Shc p66 can function in a dominant-interferingFigure Role of Shc in T-cell manner and inhibits Erk activation downstreamdevelopment. Inducible expression of ShcFFF of EGFR signaling (Fig. (Okada etas a transgene or inducible loss of Shc protein al., 1997). These authors demonstrated notexpression arrests thymic development at the only tyrosine but also serine/threoninedouble negative (DN) stage. The block is seen Shc phosphorylation of p66 in response to EGF,where signaling from the pre-TCR occurs. The which impairs its ability to associate with therole of Shc during selection at the double tyrosine-phosphorylated EGFR, but not withpositive (DP) stage has not yet been Grb2. Co-immunoprecipitation of Shc and Grb2determined. SP: single positive; CD4 and CD8 from cells overexpressing the p45/52Shcare T-cell markers (adapted from (Zhang et al., Shc isoforms, versus p66 , directly demonstrated2003)). a competition of binding for a limited pool of Grb2 proteins (Fig. Inhibition of the1.1.2.6 Role of p66Shc Shc Ras/MAPK pathway by p66 in an S36 phosphorylation-dependent manner has also The cDNA encoding the largest isoform, shc been found following TCR downstreamp66 , was cloned in 1997, 5 years after the signaling (Pacini et al., 2004). Furthermore,discovery of the two smaller isoforms p66Shc-deficient T-cells have been reported to(Migliaccio et al., 1997). As already mentioned, proliferate faster than their normal counterpartsit encompasses an additional CH2 domain on in response to limiting ligand concentration,its N-terminus containing a serine (S36) and supporting an antagonistic activity of p66Shc onthreonine (T29) phosphorylation site. Unlike Shc Shc mitogenic signaling (Pacini et al., 2004). Thep46/52 , overexpression of p66 does not 14
  15. 15. INTRODUCTION mechanism whereby p66Shc-bound Grb2 becomes uncoupled from Ras remains to be Shc determined. It is possible that p66 binds Grb2 or the Grb2/SOS complex in a conformation which does not allow SOS to act as a guanine exchange factor for Ras (Fig. However, the finding that p66Shc participates in a complex which also includes RasGAP during early morphogenetic events in Xenopus gastrulation (Dupont and Blancq, 1999) suggests a different mechanism for the negative control of Ras/MAPK activation by this protein (Fig. Whatever the Shc mechanism is, p66 does not mediate growth factor-induced MAPK activation, and its expression might provide a mechanism for fine-tuning the Ras/MAPK pathway. More recently, loss-of-function studies have Shc unveiled an unexpected role of p66 in ageing and in the apoptotic response to oxidative stress (Migliaccio et al., 1999). Shc p66 -deficient mice exhibit a lifespan about 30% longer than wild-type. Moreover, they survive longer after treatment with paraquat, a drug that increases the production of reactive oxygen species (ROS) and, therefore, oxidative stress. Increased resistance to oxidative stress or oxidative stress-inducing agents such as UV and H2O2 can be correlated with a reduction in the apoptotic responses to these stimuli in p66Shc-/- fibroblasts. A Shc protective effect of p66 ablation againstFigure Possible mechanism of apoptosis in thymocyte and peripheral T- Shcp66 function in Ras/MAPK signaling. See lymphocyte has also been reported recently Shctext for details (A) p66 binds Grb2 in a (Pacini et al., 2004). Conversely, p66 Shcconformation which does not allow activation of overexpression results in enhanced stress- Shc ShcRas. (B) p66 competes with p46/52 for induced apoptosis in fibroblasts, endothelial ShcGrb2 binding. (C) p66 binds to RasGAP and cells and T-cells (Pacini et al., 2004; Trinei etnegatively influences Ras activation. al., 2002). The proapoptotic activity of p66Shc is strictly dependent on phosphorylation of S36 in the CH2 domain. S36 phosphorylation is 15
  16. 16. INTRODUCTIONobserved in response to many stimuli, mitochondria, and subsequent caspase 3including H2O2, UV (Migliaccio et al., 1999), activation (Fig. Again, the capacity ShcFas ligation (Pacini et al., 2004), and taxol of p66 to mediate p53-dependent apoptosis(Yang and Horwitz, 2002), but also in response requires phosphorylation of S36. The releaseto EGF (Okada et al., 1997) and insulin (Kao et of cytochrome C in oxidative stress is theal., 1997). Depending on the cellular context endpoint of the p53-dependent transcriptionaland on the identity of the stimulus, either Erk, activation of redox related genes. The resultingJNK, or p38 MAPK is responsible for S36 rise of ROS levels affects the mitochondrialphosphorylation (Le et al., 2001; Okada et al., membrane potential, leading to membrane1997; Yang and Horwitz, 2002). Taken permeability transition and cytochrome C Shctogether, these results suggest that p66 acts release (Li et al., 1999; Polyak et al., 1997).as a sensor of intracellular concentration of Cyclosporin A, an inhibitor of the mitochondrialROS (Fig. permeability transition pore which blocks Further experiments aimed at understanding oxidative stress-induced apoptosis of wild-type Shcthe mechanisms underlying the role of p66 MEFs, is able to prevent re-expressed p66Shcin regulating oxidative stress-induced from restoring apoptotic responses to oxidants Shcapoptosis have revealed that p66 is a in p66Shc-/- MEFs, suggesting that p66Shc maydownstream effector of the tumor suppressor regulate mitochondrial permeability transition,p53 (Trinei et al., 2002). It is required for p53- andinduced release of cytochrome C from ShcFigure p66 senses ROS and mediates oxidative stress-induced apoptosis. ROS Shcactivate one of the MAPKs, which in turn phosphorylates p66 on S36. S36 phosphorylation isnecessary for cytochrome C release and subsequent apoptosis. p53 acts upstream of p66Shc and Shc Shcenhances p66 protein stability, leading to p66 accumulation. p53-induced apoptosis is dependent Shcon p66 expression. 16
  17. 17. INTRODUCTIONhence cytochrome C release, by modulating apoptotic signals, suggesting that S36the production of ROS (Orsini et al., 2004). phosphorylation might serve other, ShcIndeed, intracellular ROS levels are drastically nonmitochondrial, activities of p66 which are Shc-/-reduced in p66 cells and enhanced in also needed to exert its proapoptotic function. Shc Shcp66 overexpressing cells (Nemoto and A second mechanism by which p66 couldFinkel, 2002; Orsini et al., 2004). Furthermore, influence ROS levels was suggested by Shcp66 has been found to localize to Nemoto et al. (Nemoto and Finkel, 2002) (Fig. Shcmitochondria and to be associated with Hsp70. They linked p66 expression to the(Orsini et al., 2004). The best evidence was transcriptional activity of the forkhead familyderived from a recent report by Giorgio et al. transcription factor, FKHRL1. In quiescent(Giorgio et al., 2005), which clearly established cells, FKHRL1 localizes predominantly in the Shca role for p66 in the generation of ROS. nucleus where it positively regulates Shcp66 was found to function as a redox transcription of genes such as catalase,enzyme that generates mitochondrial ROS as implicated in ROS scavenging. Oxidativesignaling molecules for apoptosis (Fig. stress most probably promotes FKHRL3). It does so by utilizing reducing equivalents phosphorylation in a PKB-dependent manner,of the mitochondrial electron transfer chain and subsequent exclusion from the nucleusthrough the oxidation of cytochrome C. results in a reduction of its transcriptionalInterestingly, S36 phosphorylation was not activity. Phosphorylation and cytoplasmic Shcobserved in the mitochondrial pool of p66 : localization of FKHRL in response to H2O2 was Shcinstead a different region was necessary for abrogated in p66 -deficient MEFs. Shcthe redox activity of p66 . It seems, therefore, Accordingly, FKHRL-dependent transcription of Shcthat p66 exists in two different pools, a the catalase gene was augmented in these Shccytoplasmic one and a mitochondrial one. cells, suggesting a pivotal role of p66 in the ShcSignificant translocation of p66 from cytosolto mitochondria does not occur followingFigure Model of p66Shc redoxactivity during mitochondrialapoptosis. Proapoptotic signals inducerelease of p66Shc from a putative Shcinhibitory complex. Active p66 thenoxidizes reduced cytochrome C (red) andcatalyzes the reduction of O2 to H2O2.Permeability transition pore opening byH2O2 then leads to swelling andapoptosis. NADH-Cyt B5 reductase isindicated as an additional putative source of reduced cytochrome C (taken from (Giorgio et al., 2005)). 17
  18. 18. INTRODUCTIONredox-dependent inactivation of FKHRL1 and, perspective, inhibition of p66Shc may bethereby, in the control of ROS. envisioned as a novel way to prevent the deleterious effects of ROS-mediated diseases in general and of Ang II on the heart in particular.Figure p66Shc regulates FKHRL1 Shctranscriptional activity. p66 expressionenhances PKB phosphorylation via anunknown mechanism. This leads to a decreasein FKHRL1 transcriptional activity due tophosphorylation by PKB which causes itsretention in the cytoplasm. Finally, ROS-detoxifying enzymes such as catalase are lessexpressed. The ability to generate ROS and to regulateexpression of scavenger proteins makes Shcp66 an attractive target for therapies againstvascular diseases, which are strongly Shcmediated by ROS. Indeed, deletion of p66reduces systemic and tissue oxidative stress,vascular cell apoptosis and earlyatherogenesis in mice fed a high-fat diet(Napoli et al., 2003). p66Shc-deficient micewere also resistant to theproapoptotic/hypertrophic action of AngiotensinII (Ang II). Consistently, in vitro experimentshave shown that Ang II causes a higher rate ofapoptotic death in cardiomyocytes isolatedfrom p66Shc(+/+) hearts than in those isolated Shc(-/-)from p66 hearts (Graiani et al., 2005). In 18
  19. 19. INTRODUCTION junctions represents a specialized form of1.2 Signaling of the E- cadherin-based adhesive contacts which helpscadherin cell-cell adhesion cells to form a tight, polarized cell layer that can perform barrier and transport functionsprotein (Gumbiner, 2005). The cadherins constitute a major class ofadhesion molecules that support calcium-dependent, homophilic cell-cell adhesion in allsolid tissues of the body. They mediate cell-cellrecognition events, bring about morphologicaltransitions that underlie tissue formation, andmaintain tissue architecture in the adultorganism. The next paragraph will give a briefintroduction of E-cadherin-dependent cell-celladhesion with major emphasis on its tumorsuppressing function and its signalingcapacities. Figure Epithelial junctional1.2.1 E-cadherin-dependent cell-cell complex. Adhesion between vertebrate cells isadhesion generally mediated by three types of adhesion junction: adherens junction (zonula adherens), E-cadherin: a member of the tight junction (zonula occludens), andclassical cadherins desmosomes. Electron micrograph of an epithelial junctional complex containing zonula Cadherins represent a large superfamily adherens (ZA), zonula occludens (O), andwhich includes classical cadherins, desmosome (D). The ZA junction completelydesmosomal cadherins, atypical cadherins, encircles the apex of the epithelial cell, but onlyproto-cadherins and cadherin-related signaling a section through the junction is shown. Themolecules (Gumbiner, 2005). E-cadherin is a membranes of the two cells align tightly at theprototype family member and belongs to the junction, with an extracellular gap of 250Å. Theclassical cadherins. Classical cadherins were cytoplasmic surface of the junction appears asoriginally named for the tissue in which they a dense plaque, presumably made up ofare most prominently expressed. Later, it cytoskeletal proteins, which associates withbecame clear that most cadherins can be actin filament (taken from (Gumbiner, 2005)).expressed in many different tissues. E-cadherin (epithelial cadherin) is expressed Classical cadherins are single-passprimarily in epithelial cells and is associated transmembrane proteins. They contain fivewith the zonula adherens (which is also known cadherin domains on their extracellular partas adherens junctions) of the epithelial which confer specific adhesive binding, andjunctional complex (Fig. Adherens homophilic protein-protein interactions 19
  20. 20. INTRODUCTION cytoplasmic proteins, the catenins, is a second characteristic which distinguishes classical cadherins from other members of the cadherin superfamily (Fig. (Takeichi, 1995). α-catenin interacts, through β-catenin, with the distal part of the cadherin cytoplasmic domain. γ-catenin (also known as plakoglobin) can bind to the same site as β-catenin in a mutually exclusive way, whereas another catenin, p120- catenin, interacts with a more proximal region of the cytoplasmic domain. Function of catenins in the E- cadherin adhesion complex The main function of catenins is the conversion of the specific homophilic bindingFigure The classical cadherin- capacity of the E-cadherin extracellular domaincatenin complex. Cadherin is a parallel, or into a stable cell-cell adhesion. Although the E-cis, homodimer. The extracellular region of cadherin extracellular domain alone possessesclassical cadherins consists of five cadherin- homophilic binding properties, stable celltype repeats (extracellular cadherin domains) adhesion requires the cadherin cytoplasmaticthat are bound together by Ca2+ ions (yellow tail and associated proteins (Yap et al., 1997).circles) to form stiff, rod-like proteins. The core α-catenin can mediate physical linksuniversal-catenin complex consists of p120- between cadherin and the actin cytoskeleton,catenin, bound to the juxtamembrane region, either by directly binding actin filaments orand β-catenin, bound to the distal region, indirectly through other actin-binding proteinswhich in turn binds α-catenin. In a less well such as vinculin and α-actinin (Fig. way, α-catenin binds to actin and Besides linking cadherins to the actinactin-binding proteins, such as vinculin, α- cytoskeleton, catenins are believed to playactinin, or formin-1 (taken from (Gumbiner, additional roles. β-catenin is a well known2005)). signaling molecule in the Wnt pathway (see below), and catenins can interact with otherbetween two cadherin molecules on two cells. signaling molecules, such as GTPasesThe exact structure of the homophilic bond is (Goodwin et al., 2003), PI3K (Woodfield et al.,still a matter of debate (Gumbiner, 2005), but 2001), and formin-1 (known to nucleate actinan intriguing possibility is that some of the polymerisation) (Kobielak et al., 2004), toexisting models represent different influence the state of the actin cytoskeletonconformational states that are important for the (see below) (Fig. of adhesion. The presence of a The core function of p120-catenin is toconserved cytoplasmic tail that associates with regulate cadherin turnover (Reynolds and 20
  21. 21. INTRODUCTIONRoczniak-Ferguson, 2004). Loss of p120- an inactive, or less adhesive, conformationcatenin leads to significantly reduced levels of (Fig. in epithelial cells (Davis et al.,2003). Thus, p120-catenin directly influences Function of the E-cadherin-cateninadhesive strength by controlling the amount of complexE-cadherin available at the cell surface for The E-cadherin-catenin complex is essentialadhesion. for the formation of epithelia in the embryo, Furthermore, the adhesive strength of and maintenance of epithelial structure in thecadherins is changed by posttranslational adult. It carries out different functions, includingmodifications of p120-catenin and β-catenin. cell-cell adhesion, cytoskeletal anchoring, andAlthough poorly understood, tyrosine signaling. The expression of different types ofphosphorylation of catenins is believed to cadherins mediates selective cell recognitionregulate the conformation or organization of events that are responsible for the sorting ofcadherins. It is thought that phosphorylation of different groups of cells in developing tissues,catenins could lead to a disruption of and the formation of selective connectionsdimerization and reduced clustering of the between neurons in the developing nervouscadherin molecules at the surface, resulting in A B CFigure Function of catenin proteins in the E-cadherin-catenin complex. There are threeways in which catenins contribute to the cadherin function. (A) α-catenin provides a direct physical linkto the actin cytoskeleton through interaction with E-cadherin-bound β-catenin and actin or actin-binding proteins such as vinculin and α-actinin. (B) Catenins bind to or influence signaling molecules(GTPases, formin-1, PI3K) known to control the actin cytoskeleton. (C) Phosphorylation of cateninsmight control the adhesive strength of the cadherin-catenin complex. Depicted is a hypotheticalexample where phosphorylation of catenins could lead to a disruption of dimerization and reducedclustering of cadherin molecules at the cell surface, resulting in an inactive or less adhesiveconformation. Ca2+ ions are indicated by yellow circles. EC: extracllular cadherin domain (taken from(Gumbiner, 2005)). 21
  22. 22. INTRODUCTIONsystem (Gumbiner, 2005). In cell culture, a 1994; Hirohashi, 1998). This observation hasmixed population of cells expressing different prompted an examination of the functional rolecadherins become sorted by adhering only to of E-cadherin in tumor progression. Behrens etthose cells expressing the same cadherin (Yap al. (Behrens et al., 1989) showed that epithelialet al., 1997). During development, segregation cells acquire invasive properties whenof cells into distinct tissues is accompanied by intercellular adhesion is specifically inhibited bychanges in the complement of cadherins the addition of E-cadherin function-blockingexpressed by the cells. The specificity of antibodies; the separated cells then invadehomophilic binding is therefore a fundamental collagen gels and embryonic heart tissue.mechanism by which cadherins influence the Subsequently, several groups haveorganization of various cell types into tissue demonstrated that re-establishing the(Yap et al., 1997). However, different functional cadherin complex by forcedcadherins can be promiscuous with regards to expression of E-cadherin results in a reversiontheir adhesive binding properties, with of an invasive, mesenchymal phenotype to aevidence for heterophilic adhesion between benign, epithelial phenotype of cultured tumordifferent classical cadherins. The level of cells (Birchmeier and Behrens, 1994; Navarrocadherin expression, and presumably therefore et al., 1991; Vleminckx et al., 1991). Based onthe overall strength of adhesion, has also been these data, it has been proposed that the lossfound to strongly influence cell-sorting of E-cadherin-mediated cell-cell adhesion is abehavior, independently of the type of cadherin prerequisite for tumor cell invasion andexpressed (Gumbiner, 2005). metastasis formation. The in vivo proof that The importance of E-cadherin-mediated cell loss of E-cadherin is not a consequence of de-adhesion is also highlighted by the fact that its differentiation, but rather the cause of tumordisturbance is causally involved in cancer progression, was made by Christofori anddevelopment. colleagues (Perl et al., 1998). Intercrossing RipTag2 mice, which provide a model of1.2.2 E-cadherin as a tumor pancreatic carcinogenesis, with transgenicsuppressor mice that maintain E-cadherin expression in β- cell-derived tumor cells resulted in the arrest of tumor development at the adenoma stage, The majority of human cancers (ca. 80-90%) whereas expression of a dominant-negativeoriginate from epithelial cells. In most, if not all, form of E-cadherin induced early invasion andof these epithelial-derived cancers, E-cadherin- metastasis. Very recently, a second study hasmediated cell-cell adhesion is lost, concomitant demonstrated causal evidence for thewith the transition from benign, non-invasive involvement of E-cadherin in tumortumor to malignant, invasive tumor. Although progression. A group from the NetherlandsE-cadherin expression is maintained in most introduced a conditional loss-of-functiondifferentiated tumors, including carcinomas of mutation in the E-cadherin gene into mice thatthe skin, head and neck, breast, lung, liver, carry p53 mutations. Although tissue-specificcolon, and prostate, there seems to be an inactivation of E-cadherin alone did not resultinverse correlation between E-cadherin levels in tumor formation, the combined inactivationand cancer grade (Birchmeier and Behrens, 22
  23. 23. INTRODUCTIONof E-cadherin and p53 led to the accelerated transcription factors, such as Snail and Slug,development of mammary gland and skin has been observed downstream of RTKtumors. Moreover, loss of E-cadherin induced signaling (Thiery, 2002). Snail, Slug, SIP1, anda phenotypic change from non-invasive to E12/47, as well as Twist, are factors whichhighly invasive mammary gland tumors, and a repress transcription from the E-cadherinconversion from ductal to lobular carcinomas promoter via the E-boxes (Cavallaro and(Birchmeier, 2005). These results show that Christofori, 2004; Yang et al., 2004).the loss of E-cadherin-mediated cell-cell β-catenin is also actively involved in EMTinvasion is one rate-limiting step in the (Fig. 1.2.2) and its role as a signaling moleculeprogression from adenoma to carcinoma and will be discussed later.subsequent formation of tumor metastases. In addition to EMT, which is a rather Downregulation of E-cadherin is often part of organized process leading to downregulationa process called epithelial-to-mesenchymal of E-cadherin expression, various othertransition (EMT), which is characterized by the mechanisms are involved in the disruption ofloss-of-expression of epithelial genes and the cell-cell adhesion during tumor progression. Again-of-expression of mesenchymal genes variety of genetic mechanisms, such as(Thiery, 2002). EMT is a crucial event during deletion or mutational inactivation of the gene,tumor metastasis but also occurs in normal or gene mutations which result in theembryonic development, for example during expression of a non-functional protein, causegastrulation (Fig. 1.2.2). Activation of RTK loss of E-cadherin expression or function,[fibroblast growth factor receptor (FGFR), especially in diffuse gastric cancer (BirchmeierEGFR family, transforming growth factor-β and Behrens, 1994; Bracke et al., 1996;(TGF-β) receptor, insulin-like growth factor Strathdee, 2002). Silencing of the E-cadherinreceptor (IGFR), hepatocyte growth factor gene by hypermethylation of promoter regionsreceptor (HGFR)] signaling is able to induce occurs frequently in carcinoma cell lines, inEMT via stimulation of PI3K, Src, Ras and thyroid carcinomas, and in several otherRac. Signaling downstream of EGFR, c-Met cancer types (Di Croce and Pelicci, 2003;and FGFR, as well as Src, results in tyrosine Hirohashi, 1998). More recently, proteolyticphosphorylation of E-cadherin, β-catenin and degradation of E-cadherin by matrix-metallop120-catenin, leading to a disassembly of the proteases (MMPs) has been described as acadherin-catenin complex, disruption of mechanism by which cell-cell adhesion can becadherin-mediated adhesion and cell disrupted. Cleavage of E-cadherin results inscattering. Tyrosine phosphorylation-mediated not only the disruption of cell-cell adhesion, butubiquitination and subsequent proteasomal also the production of a soluble 80-kDa E-degradation of E-cadherin or increased cadherin fragment that itself disrupts cell-cellendocytosis of E-cadherin seem to be adhesion in a dominant-interfering manner,mechanisms underlying this observed thereby promoting tumor progression (Noe etdisassembly (Fujita et al., 2002; Kamei et al., al., 2001; Wheelock et al., 1987).1999). Moreover, induction of expression of 23
  24. 24. INTRODUCTIONFigure 1.2.2: Epithelial-mesenchymal transition (EMT). Epithelial cells lose the expression ofepithelial-specific genes, such as E-cadherin, and acquire the expression of mesenchymal genes(vimentin, collagens, integrins). EMT causes cells to lose apical-basal polarity (shown on the left) andgain a fibroblast-like morphology, high motility and invasive properties (shown on the right). (A)Transcription factors (such as Snail and Slug) have been identified that control the expression of E-cadherin by binding directly to E-boxes in the gene promoter. Other factors, such as growth factorsand their receptors, the tyrosine kinase src, and cytoplasmic G-proteins (such as rac) can alsopromote EMT indirectly. (B) β-catenin was found to exert a dual role as an essential cytoplasmic-interaction partner of cadherins, which is essential for cell-cell adhesion, and as a nuclear partner ofthe T-cell factor (TCF)/lymphocyte-enhancer factor (LEF) family of transcription factors that regulategenes of the canonical Wnt signaling pathway. The switch of β-catenin from its action in cell adhesionto transcriptional control in the nucleus is controlled by binding to BCL9-2, which is the homologue of ahuman B-cell oncogene product, and is promoted by tyrosine phosphorylation of β-catenin (taken from(Birchmeier, 2005)). As already mentioned above, appropriate in a subset of E-cadherin-deficient tumors.cell-cell adhesion requires the cadherin-catenin However, direct evidence is lacking and itcomplex as a whole. Therefore, changes in the remains to be determined whether this wouldexpression of catenins, for example mutations represent a general process in tumorin α-catenin or expression of truncated α/β- progression.catenin, impair E-cadherin-mediated cell Proper E-cadherin function can also beadhesion and are often associated with overruled or replaced by the expression ofmalignant transformation (Hajra and Fearon, mesenchymal cadherins, such as N-cadherin,2002; Hirohashi and Kanai, 2003). Recently it which has been shown to promote cell motilityhas been shown that knockdown of p120- and migration. It becomes more and morecatenin results in the destruction of the entire evident that this “cadherin switch” is involvedcadherin complex (Reynolds and Roczniak- during the transition from a benign to anFerguson, 2004). Together with evidence of invasive tumor phenotype (Christofori, 2003).frequent p120-catenin loss in cancer, these Taken together, loss of E-cadherin-mediatedobservations suggest that p120-catenin cell-adhesion strongly contributes to tumordownregulation itself may be an initiating event progression, but it is unlikely that loss of E- 24
  25. 25. INTRODUCTIONcadherin by itself can account for the become confluent, but as cytosolic p120-metastatic phenotype, because loss of catenin becomes sequestered by the E-adhesiveness does not necessarily cause cells cadherin adhesion complex it cannot accountto become motile and/or invasive; additional for this decrease in Rho activity. Therefore,events are required. other mechanisms downstream of E-cadherin- mediated adhesion decrease Rho activity.1.2.3 E-cadherin-mediated signaling Noren et al. (Noren et al., 2003) reported that E-cadherin engagement in cell-cell adhesion An increasing body of evidence suggests suppresses Rho activity by inducingthat cadherins act at the cellular level as phosphorylation and activation ofadhesion-activated cell signaling receptors p190RhoGAP, probably through Src-family(Cavallaro and Christofori, 2004; Wheelock kinases. In other systems, E-cadherin wasand Johnson, 2003). Although signals that are found to communicate with Rho GTPases viaelicited by the formation of E-cadherin- PI3K signaling (Fig. 1.2.3). PI3K is andependent cell-cell adhesion have been upstream kinase of Rac and has previouslyextensively studied, signals that are induced by been found to interact with E-cadherin (Pece etthe loss of E-cadherin function, for example al., 1999; Woodfield et al., 2001). Yap andduring cancer progression, are only just being colleagues (Kovacs et al., 2002) showed thatelucidated. PI3K co-localized with E-cadherin at the Several studies have reported that leading edge of cadherin-based lamellipodia,establishment of E-cadherin-mediated contact and was necessary for full and sustainedinfluences the activity of Rho-family GTPases; activation of Rac. In contrast, another groupwith Rac and CDC42 being activated and Rho reported that Rac activation induced by E-being inactivated. The mechanisms underlying cadherin ligation was independent of PI3Kthis activation or inactivation vary depending activity, but dependent on EGFR signaling (seeon the model system used. One connection below) (Betson et al., 2002). Whatever thebetween cadherins and Rho GTPases is mechanisms are, E-cadherin-mediatedthrough p120-catenin. It has been shown that contacts influence the activity of Rho-familyp120-catenin activates Rac1 and CDC42, GTPases, which are believed to regulateperhaps by activating Vav2, which is a guanine dynamic organization of the actin cytoskeletonexchange factor for these GTPases (Fig. 1.2.3) and the activity of the cadherin/catenin(Grosheva et al., 2001; Noren et al., 2001). apparatus to modulate stabilization of theReynolds and colleagues showed that adhesive contact (Yap et al., 1997).cytosolic p120-catenin inhibits RhoA activity by Several studies have suggested functionalacting as guanine nucleotide dissociation interdependence of cadherins and RTK withinhibitor (Anastasiadis et al., 2000; Noren et respect to their signaling capacities. It hasal., 2000). It is worth noting that only cytosolic been demonstrated that initiation of de novo E-p120-catenin is able to modulate GTPase cadherin-mediated adhesive contacts canactivity; this function is abolished when p120- induce ligand-independent activation of thecatenin participates in the E-cadherin adhesion EGFR and subsequent activation of Erkcomplex. Rho activity decreases as cells (Munshi et al., 2002; Pece and Gutkind, 2000). 25