cancer
Rossum, Agnes Gerarda Sophia Helena van
Citation
Rossum, A. G. S. H. van. (2006, March 29). Cortactin couples dynamic actin networks to
cell migration and breast cancer. Retrieved from https://hdl.handle.net/1887/4349
Version:
Corrected Publisher’s Version
to cell migration and breast cancer
Karen Thijssen
Lay-out: Marieke Vianen
Illustraties
Hoofdstuk 1: Liselotte van der Zwet
Drukwerk: Ponsen & Looijen B.V., Wageningen
to cell migration and breast cancer
Cortactin verbindt dynamische actine netwerken
aan cel migratie en borstkanker
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magnificus Dr. D.D. Breimer,
hoogleraar in de faculteit der Wiskunde en Natuurwetenschappen en die der Geneeskunde,
volgens besluit van het College voor Promoties, te verdedigen op woensdag 29 maart 2006
klokke 14.15 uur
door
Agnes Gerarda Sophia Helena van Rossum
Promotoren: Prof. Dr. W.H. Moolenaar Prof. Dr. Ph.M. Kluin
Universiteit Groningen
Co-promotor: Dr. E.M.D. Schuuring Universiteit Groningen
Referent: Dr. A. Sonnenberg
Het Nederlands Kanker Instituut, Amsterdam Overige leden: Prof. Dr. P. ten Dijke
Prof. Dr. C.J. Cornelisse
The research described in this thesis was performed at the Leids University Medical Center (LUMC), Department of Pathology, Leiden, The Netherlands and at the Netherlands Cancer Institute (NKI-AVL), Division of Cellular Biochemistry, Amsterdam, The Netherlands.
Financial support for this research was provided by The Dutch Cancer Society (Koningin Wilhelmina Fonds), grant NKB-RUL 98-1647.
moet je het geloven. Ma
Page
Abbreviations 8
Chapter 1 General introduction 11
Chapter 2 Alternative splicing of the actin-binding domain of 51 human cortactin affects cell migration
(J Biol Chem. 2003 Nov 14;278(46):45672-45679)
Chapter 3 Comparative genome analysis of cortactin and HS1: 73 the significance of the actin binding repeat domain
(BMC Genomics. 2005 Feb 14 ;6(1):15)
Chapter 4 Cortactin affects cell migration by regulating 105 intercellular adhesion and cell spreading
(Conditionally accepted for publication in Exp. Cell Res)
Chapter 5 Transgenic mice with mammary gland targeted 129 expression of human cortactin do not develop (pre-
malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice (Conditionally accepted for publication in BMC Cancer)
Chapter 6 Summary and general discussion 151
Nederlandse samenvatting 163
List of publications 171
Curriculum vitae 173
Abbreviations
11q13 chromosome 11 band q13
aa amino acid(s)
ABD actin-binding domain
ABP actin-binding protein
ATP adenosine triphosphate
Arp2/3 actin related protein 2 and 3
BCR B-cell receptor
bp base pair(s)
BSA bovine serum albumin DCIS ductal carcinoma in situ CDK cyclin-dependent kinase CLSM confocal laser scanning microscope
DAB 3,3 diaminobenzidene-tetra-hydrochloride DMEM Dulbecco´s modified Eagle´s medium DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
ECL enhanced chemiluminescence ECM extracellular matrix
EDTA ethylene-diaminetetraacetic acid EGF(R) epidermal growth factor (receptor)
EMS1 chromosome eleven, mammary and squamous, cortactin ER oestradiol/oestrogen receptor
ERK extracellular regulated kinase EST expressed sequence tag F-actin filamentous actin FAK focal adhesion kinase FCS fetal calf serum
FITC fluorescein isothiocyanate
FN fibronectin
G-actin monomeric actin GAP GTPase activating protein GDP guanine diphosphate
GEF guanine nucleotide exchange factors GFP green fluorescent protein
GST glutathione S-transferase GTP guanine triphosphate GTPase guanine triphosphatase h hours
HAN hyperplastic alveolar nodules H2O2 hydrogen peroxide
HEK human embryonic kidney
HS1 haematopoietic lineage cell-specific protein 1 IDC infiltrating ductal carcinoma
Ig immunoglobulin ILC infiltrating lobular carcinoma kB kilobase pairs
kD, kDa kilodaltons
LCIS lobular carcinoma in situ LPA lysophophatidic acid MAb monoclonal antibody MEK Erk kinase or MAPK kinase
min minutes
MIN mammary intraepithelial neoplasia MLCK myosin light chain kinase
MMP matrix metalloproteinase MMTV mouse mammary tumour virus mRNA messenger ribonucleic acid nd not determined NLS nuclear localization signal NTA N-terminal acidic PAb polyclonal antibody
PAGE polyacrylamide gel electrophoresis PAK1 p21 activated kinase
PBS phosphate-buffered saline PCR polymerase chain reaction
PDGF(R) platelet derived growth factor (receptor) PI3K phosphatidylinositol 3´kinase PIP2 phophatidylinositol-4,5-biphosphate
RIPA radio immuno precipitation assay (weet niet zeker) PKC protein kinase C
PMA phorbol 12-myristate 13-acetate PMSF phenylmethyl sulfonyl fluoride RNA ribonucleic acid
RNAi RNA interference PRAD1 cyclin D1, CCND1, cyl1 RSV Rous sarcoma virus
RT-PCR reverse transcriptase polymerase chain reaction s seconds
S (ser) serine residue
SCC squamous cell carcinoma SD standard deviation SDS sodium dodecylsulfate SEM standard error of the mean SH2 Src homology 2 SH3 Src homology 3 SV splice variant T (thr) threonine residue TBS tris-buffered saline TPA 12-O-tetradecanoylphorbol-13-myristate Tris tris(hydroxymethyl)aminomethane TRITC texas red isothiocyanate
UTR untranslated region
WASP Wiskott-Aldrich syndrome protein Y (tyr) tyrosine residue
Chapter 1
1.1 Introduction
1.2
The biology of metastasis
1.2.1 Cancer is a multistep process 1.2.2 Cell-substratum adhesion 1.2.3 Extracellular matrix degradation 1.2.4 Intercellular adhesion1.3 Breast
cancer
1.3.1 Classification of human breast cancer
1.3.2 Classification of mammary tumors in the mouse 1.3.3 Identification of genes involved in breast cancer
1.4
The role of cortactin in cancer progression
1.4.1 Amplification of the chromosome 11q13 region: the identification of the responsible gene(s)
1.4.2 Cyclin D1 1.4.3 EMS1/cortactin
1.5
The role of cortactin on actin cytoskeleton organization
and cell migration
1.5.1 Actin polymerization
1.5.2 Classification of actin binding proteins
1.5.3 The actin cytoskeleton emerges in three forms
1.5.4 Membrane protrusions and cellular processes driven by actin polymerization
1.5.5 Rho-GTPases regulate the organization of the actin cytoskeleton 1.5.6 GTPases and cortactin
1.5.7 Cortactin couples dynamic actin networks to cell migration and breast cancer
1.1 Introduction
Breast cancer is the most frequent type of cancer among women in Western countries. One out of nine Dutch women develops breast cancer and one third of the breast cancer patients eventually die of this disease, most often as a result of distant metastases. Local metastasis in the axillary lymph nodes of the breast, is the most powerful prognostic factor for early recurrence and decreased survival in breast cancer patients [1;2].
In a significant portion of breast cancers, the chromosome 11q13 region is amplified [3-6]. Amplification of 11q13 correlates in cancer patients with poor prognosis, since this amplification is associated with the presence of lymph node metastasis and increased mortality [3;7-10]. One of the genes located within this amplified region is the EMS1 gene, which encodes the protein cortactin. In tumors with 11q13 amplification, cortactin appears to be overexpressed. Biochemical assays revealed that cortactin is a filamentous actin (F-actin) binding protein that has emerged as a molecular scaffold which mediates the assembly and organization of actin cytoskeletal networks. Cells overexpressing cortactin show enhanced migration [11;12], invasion [11] and increased metastatic potential in vivo [13], while RNA interference induced down-regulation of cortactin in highly invasive cells [14], deletion mutants [12] or microinection of anti-cortactin antibodies [15] decreased the invasive potential.Thus, in human cancer, increased cortactin levels might promote cellular processes such as cell motility, invasion and metastasis.
The characterization, regulation and cellular functions of cortactin in actin dynamics have been extensively reviewed elsewhere [16-20]. This chapter mainly describes the cellular processes in which cortactin is involved, in particular F-actin cytoskeleton organization, cell adhesion, migration, and invasion.
1.2 The biology of metastasis
1.2.1 Cancer is a multistep process
structure by histone acetylation/methylation and methylation of cytosine-residues within CpG-islands at promoter regions. Epigenetic events play essential roles in cancer initiation and progression [22;23]. Cells with a proliferative or survival advantage are able to undergo clonal expansion due to the selective advantage that the genetic alterations provide. These genetic alterations can affect cellular processes including cell division, apoptosis and DNA repair. Altered gene function is required for all types of cancer, but specific cancers have characteristic patterns of mutations. Thus, a particular gene or signaling pathway may be restricted to a subset of cancers. In addition, the effects of a particular gene or gene product may only manifest at a particular stage of the disease.
Figure 1. Schematic representation of the metastatic process. Progression of cancer to an invasive
and metastatic phenotype requires additional events. In order to metastasize, tumor cells have to (I) detach from the primary tumor, (II) degrade the basal membrane, (III) migrate through the tissue to enter the circulation (lymphatic system, blood stream), (IV) resist streaming forces, (V) combat the immune surveillance and (VI) subsequently to leave the circulation and invade a target organ (VII) to grow out as a tumor, referred to as a metastasis. Figure adapted from ‘Molecular biology of the cell’ from Alberts et al. [231].
cancer cells migrate from their site of origin to form deposits at distant sites (Figure. 1). In order to metastasize, tumor cells have to (I) detach from the primary tumor, (II) degrade the basal membrane, (III) migrate through the tissue to enter the circulation (lymphatic system, blood stream), (IV) resist streaming forces, (V) combat the immune surveillance and (VI) subsequently to leave the circulation and invade a target organ (VII) and finally to grow out as a tumor, referred to as a metastasis. Tumor cells migrate in response to environmental signals and for cell migration rearrangements of the actin cytoskeleton are necessary. Migration is not possible without alterations in normal adhesion, which is mediated by cell adhesion molecules. To be able to metastasize, cancer cells should alter the expression levels/patterns of genes that affect cell-cell interaction, cell-substratum adhesion, cell motility and degradation of the extracellular matrix (ECM) [24;25]. For example, mutations in E-cadherin [26;27], increased activation of matrix metalloproteinases [28;29] or increased activity of Rac1 GTPase, [30], have been associated with the metastatic potential of various cancers. From the different processes involved in cancer progression, three are described below in more detail: cell-substratum adhesion, ECM degradation, and intercellular adhesion.
1.2.2 Cell-substratum adhesion
Adhesion of cells to the ECM is mediated by integrins, heterodimeric cell surface receptors composed of two single-pass transmembrane subunits, that are designated α and β. Until now 19 α and 8 β subunits have been identified [31]. From these subunits, 22 integrins are known with different binding specificities. The cytosolic domains of integrins bind to adapter proteins (e.g., α-actinin, vinculin, talin, filamin) that link integrins to the actin cytoskeleton. Upon binding to the ECM, integrins form clusters, which lead to the recruitment of actin filaments and signaling proteins to the cytoplasmic domain of integrins [32] resulting in formation of large protein complexes called focal adhesions. Migrating cells continuously make and break contacts with the matrix. In order to allow the dissemination of cancer cells, alterations in cell-substratum adhesions are required for spreading of cancer cells. This is illustrated by the observations that the expression of different integrins are either down-regulated or absent in breast cancer cells [33;34].
1.2.3 Extracellular matrix degradation
metastatic cancer cells that actively migrate and invade surrounding tissues (see paragraph 1.5.4). These structures are actin, actin-binding proteins and metalloprotease-rich formations with underlying ECM degradation areas.
1.2.4 Intercellular adhesion
Cell-cell adhesion is essential for tissue morphogenesis and includes tight junctions, gap junctions, adherens junctions and desmosomes [35]. Tight junctions (also called zonula occludens) seal cells together to limit the diffusion of ions and small molecules between adjacent cells, allowing sheets of epithelial cells to form permeability barriers, which are essential for many physiological functions at the organ level. Gap junctions control communication between cells by providing channels for small molecules up to 1 kD that can move from the cytoplasm of one cell into the cytoplasm of a neighboring cell. Adherens junctions and desmosomes consist of various cadherins to link the plasma membranes of adjacent cells. Intracellularly, desmosomes are linked to intermediate filaments, whereas adherens junctions are linked to the actin cytoskeleton via catenins [36]. The cadherins are a family of calcium dependent homophilic transmembrane glycoproteins involved in cell aggregation, segregation, and migration through dynamic cell-cell contact remodeling [37;38], accompanied by dynamic actin cytoskeletal rearrangements. They are responsible for the strong cell-cell adhesion that promotes epithelial polarity and keeps epithelial cells at their appropriate location. There are over 40 different known cadherins, of which E-, P-, and N-cadherins are the most widely expressed. In addition, N-cadherins appear to initiate and modify intracellular signaling pathways. Loss of the epithelial (E-) cadherin function in breast cancers has been associated with increased invasiveness and shortened relapse-free survival [39]. Activation of the c-Src tyrosine kinase (see paragraph 1.4.3) regulates tyrosine phosphorylation of E-cadherin and β-catenin causing disassembly of adherens junctions. Src-transformed epithelial cells have been shown to display decreased cell-cell adhesion. The cadherins themselves are poor substrates for tyrosine kinases, but the cadherin-interacting proteins β-catenin, plakoglobin, and p120catenin are highly phosphorylated on tyrosine in Src-transformed epithelial cells. The actin cytoskeleton is involved in intercellular adhesion strengthening and turnover of receptors and membrane-bound proteins. The Arp2/3 protein complex, involved in actin polymerization, is related to adherens junctions by its association with E-cadherin [40], and, as a consequence, it links rearrangements of the actin cytoskeleton directly to adherens junctions.
1.3 Breast cancer
1.3.1 Classification of human breast cancer
The classification of human breast cancer has a time-honored, clinically validated tradition that is widely accepted and applied around the world [41]. The two most common types of human breast cancer were originally classified by their place of origin within the breast: in ductal carcinoma, cancer cells develop in one of the milk ducts, whereas in lobular carcinoma cancer, cells originate from a milk-producing lobule of the breast. However, all carcinomas are thought to arise from the terminal duct lobular unit, and terms ´ductal´ and ´lobular´ do not imply a site or cell type of origin [42]. The two types of cancer are further subtyped depending on whether they remain in situ or invade in the surrounding tissue. In ductal carcinoma in situ (DCIS) the abnormal cells remain in the lining of a milk duct in the breast while in lobular carcinoma in situ (LCIS) the abnormal cells are in a lobule of the breast, and in both cases they have not invaded through the basement membrane into the surrounding breast tissue. DCIS is the most common form of non-invasive breast cancer (80%) and is considered as a precursor of invasive carcinoma in some cases, or indicator of elevated risk for development of carcinoma [43]. In infiltrating ductal carcinoma (IDC) and infiltrating lobular carcinoma (ILC,) cancer cells have invaded beyond the basement membrane into surrounding stroma, fatty and connective tissues of the breast. After invasion, the cancer cells can spread (metastasize) further throughout the body, being transported by the bloodstream or lymphatic system. IDC accounts for as many as four out of five invasive breast cancers whereas ILC is less common (10%) [41]. According to the WHO classification [41], there are 17 other types of primary infiltrating breast carcinomas, but also other intraductal neoplasias, various types of primary sarcomas (tumors originating from mesenchymal cells of the breast) and mixed tumors that are thought to represent genuine biphasic tumors with an epithelial and mesenchymal component.
1.3.2 Classification of mammary tumors in the mouse
1.3.3 Identification of genes involved in breast cancer
Less than 10% of breast cancer cases are genetically inherited, indicating that the remaining breast cancers are non-inherited (sporadic) [41] In other words, the majority of women diagnosed with breast cancer do not have an alteration in breast cancer predisposition genes. In hereditary cancers, the first mutation is inherited and the second (successive) is somatic, whereas in sporadic cancer all mutations are somatic. Breast cancer results from stepwise geneticalterations in individual cells and epigenetic changes in the behavior of not only malignantcells but also host cells that interact with the tumor suchas immune, vascular and stromal cells [45-48]. The involvement of molecules and signaling pathways in the transformation of mammary epithelia is illustrated by the elevated expression/activity or loss of expression of a number of proteins in breast carcinomas relative to normal breast tissues. Aberrant expression of such molecules might function as markers to predict the prognosis of a patient and estimate the risk of tumor recurrences.
Table 1: Genes involved in breast cancer and their normal functions Upregulated Normal function
HER-2/neu/c-erb B2 Tyrosine kinase receptor: growth and differentiation [49] EGFR Tyrosine kinase receptor: growth and differentiation [60]
IGF-IR Tyrosine kinase receptor:growth and differentiation, anti-apoptotic [61]
HGFR/c-Met Tyrosine kinase receptor: growth and differentiation [62]
TGF-β II Cytokine: growth and differentiation [63]
SRC Non-receptor tyrosine kinase: signal transduction [64]
PI3K Phosphatidylinositol-3-kinase: cell growth, survival, motility [49]
AKT/PKB Serine/threonine kinase: cell growth, survival [49]
RAS G-protein: signal transduction [49]
GRB-2 Adapter protein: signal transduction [64]
Cyclin D1 Cell-cycle mediator [65]
Cyclin E Cell-cycle mediator [49]
VEGF Growth factor: angiogenesis [66]
BCL-2 Cell survival, anti-apoptotic [67]
Ki-67 Nuclear antigen: proliferation [68]
PCNA Nuclear antigen: proliferation [68]
c-MYC Transcription factor: DNA transcription [49]
c-FOS Transcription factor: DNA transcription [49]
c-JUN Transcription factor: DNA transcription [69]
ETS-1 Transcription factor: DNA transcription [70]
EIF-4E Initiation factor: initiator of protein translation of mRNAs [49]
FAK Non-receptor tyrosine kinase: cell-matrix adhesion [71]
N-cadherin Cell-cell adhesion [72]
Ep-Cam Cell-cell adhesion [73]
CD44 Cell-cell adhesion [74]
MMP2 Metalloproteinases: ECM degradation [75]
MMP9 Metalloproteinases: ECM degradation [75]
uPA Urokinase-type plasminogen activator: ECM degradation [76]
PAI-1 Plasminogen activator inhibitor: ECM degradation [76]
AMAP1 Arf GTPase Activating Protein [77]
S100A4/MTS-1 Angiogenesis [78]
Fascin Actin bundling protein: organization actin cytoskeleton [79]
β-thymosin Actin monomer binding protein: organization actin cytoskeleton [80]
p130CAS Adapter protein: cell-matrix adhesion [81]
EMS1/cortactin Actin cross-linking protein: organization actin cytoskeleton [82]
Downregulated Normal function
p53 Cell-cycle checkpoint activation, induces cell-cycle arrest, pro-apoptotic [49]
p27 Inhibits cyclin-dependent protein kinases, arrest cell cycle in G1 phase [49]
BRCA-1 Regulates DNA transcription, DNA repair [49]
BRCA-2 DNA repair [49]
CHK2 Cell cycle checkpoint kinase, activates p53 after DNA damage [49]
ATM Checkpoint kinase: activates CHK2, DNA repair [49]
PTEN Phosphatase: negative regulator of AKT kinase, pro-apoptotic [49]
Rb Retinoblastoma gene: repressor of cell cycle and protein translation [49]
E-cadherin Cell-cell adhesion [26]
α2β1 Integrin: cell-matrix adhesion [83]
Paxillin Cell-matrix adhesion [81]
Tropomyosin Actin side binding protein: organization actin cytoskeleton [84]
Gelsolin Actin severing protein: organization of actin cytoskeleton (downregulation due to promoter methylation, an example of epigenesis) [85]
1.4 The role of cortactin in cancer progression
1.4.1 Amplification of the chromosome 11q13 region:
the identification of the responsible gene(s)
DNA amplification is a common mechanism of oncogenic activation in human cancer: e.g. amplification of chromosome 17q11 (HER2/neu), 8q24 (c-MYC) and 11q13 (CCND1/EMS1) [6;87-89] are often found in breast cancer. Band q13 of chromosome 11 is a site of frequent genetic aberration in a number of human malignancies, particularly in breast cancers (15%) [3-6], squamous cell carcinomas of the head and neck region (HNSCC) (45%) [8;90;91], bladder cancer (20%) [92;93] and in various other human carcinomas (reviewed by [94] and [95]. Positive correlations exist between 11q13 amplification and the presence of lymph node metastasis, poor prognosis and estrogen receptor (ER) positivity in breast cancer [3;96;97]. The 11q13 locus is up to 7 Mb large and exists of at least four distinct cores of amplification [98] suggesting the activation of more than one gene. The locus contains a number of candidate oncogenes including MYEOV, CCND1, FGF3, FGF4, EMS1/cortactin and EMSY. Initially, two genes, CCND1 and EMS1, localized 0.8 Mb from each other, encoding cyclin D1 and cortactin, respectively, were the best candidate oncogenes within this amplicon [82;99], because increased expression of both cyclin D1 and cortactin correlated well with 11q13 amplification [8;82;100], in contrast to FGF3 and FGF4 that are not overexpressed upon DNA amplification [101].
1.4.2 Cyclin D1
Cyclin D1 [105] was first described as a candidate oncogene in parathyroid adenomas (as PRAD1 [106]and plays a critical role in the timing of the initiation of DNA synthesis in the normal cell cycle of mammalian cells [107]. The main function of cyclins that operate in the G1 phase including cyclin D1, is to phosphorylate and
thereby inactivate members of the pocket-protein family (retinoblastoma Rb, p107, p130). The cyclins exert their actions through association with, and activation of cyclin dependent protein kinases (CDKs). Activated (= unphosphorylated) Rb binds transcription factor E2F (six members, E2F1-6), thereby preventing DNA synthesis. The cyclin D1/CDK4-activated complex phosphorylates and inactivates Rb causing the release of E2F, which subsequently results in transcription of genes implicated in DNA replication. This sequence of events allows the cell to enter the S-phase. Overexpression of cyclin D1 curtails the duration of the G1 but does not prevent the
onset of quiescence that normally occurs in the absence of growth factors [108]. Cyclin D1 is expressed in most tissues but its levels are higher in breast epithelial cells. In agreement with this, aberrant expression of cyclin D1 was documented in several human malignancies, but most frequently in human breast cancers [109-111]. Cyclin D1 gene amplification is reported in up to 20% of human breast cancers, whereas its gene product, the cyclin D1 protein has been shown to be overexpressed in over 50% of the breast carcinomas [6;112];Kandel, 2001 532 /id}. Overexpression of cyclin D1 is frequently observed in estrogen receptor (ER) positive breast cancers and cyclin D1 has been shown to stimulate ER-mediated transcriptional activation through direct binding to the ER [113]. Several studies showed cyclin D1 overexpression in invasive ductal carcinoma (IDC) [114] and in invasive lobular carcinoms (ILC) [115] but also in ductal carcinoma in situ (DCIS), but not in ductal hyperplasia [116]. These studies suggest that cyclin D1 expression may play an important role in the early stages of carcinogenesis. Thus, CCND1 is an interesting candidate gene for cancer initiation of tumors with an 11q13 amplification. This hypothesis is supported by experiments with MMTV-cyclin D1 transgenic mice, which are prone to mammary hyperplasia and adenocarcinomas [117]. However, mammary tumors developed stochastically at late age suggesting the need of additional genetic hits.
1.4.3 EMS1/cortactin
found in cytoskeletal proteins, whilst the SH2 domain is required for its interaction with tyrosine phosphorylated proteins [124]. The best-known Src substrates are key components in integrin-mediated signal transduction found at focal adhesions and bound to actin or integrin, such as cortactin, vinculin, talin, paxillin, FAK, tensin, ezrin, and p130cas. In addition, intercellular junctional proteins, such as β- and γ-catenin, ZO-1, occludin, p120catenin and connexin 43 are also identified as major sites of tyrosine phosphorylation by Src kinase [123;125;126].
Figure 2. Schematic representation of the structural domains of cortactin. Cortactin contains four
main distinguishable domains (I-IV). (I) The N-terminal acidic (NTA) domain, that directly can activate the Arp2/3 complex, which consist of two actin-related proteins (Arp2 and Arp3) tightly bound to five other proteins: ARPC1, ARPC2, ARPC3, ARPC4 and ARPC5. Cortactin and N-WASP can bind simultaneously to Arp2/3 complex; cortactin binds to Arp3 while N-WASP can bind at the same time to Arp2 and ARPC1. (II) The 6½-fold 37 amino acids F-actin binding repeat domain (numbered 1-6), which is required for Arp2/3 activation and for the localization of cortactin at the actin cytoskeleton. Splicing of
cortactin occurs at the 6th or 5th and 6th repeat (see Chapter 2). (III) The central region consisting of an
α-helix and a proline-rich region. The proline-rich region contains functional threonine (T), serine (S) and tyrosine (Y) phosphorylation sites and is thought to act as a molecular hinge that determines the conformation of cortactin. (IV) The SH3 domain which interacts with several proteins involved in actin polymerization, cell–cell adhesion and membrane dynamics (see Table 2, page 36). Different signal transduction cascades regulate cortactin function in response to stimuli, such as growth factors. This regulation appears to involve phosphorylation of specific threonine, serine and tyrosine residues in the
proline-rich domain by Pak/Erk or Src kinases, respectively. Increased PIP2 levels induced in response
Cortactin is an F-actin binding protein involved in regulation of the actin cytoskeleton. The protein cortactin contains four main, distinguishable domains: (I) the N-terminal acidic (NTA) domain containing a DDW-Arp2/3 binding motif followed by (II) a 6.5-fold 37-amino acid F-actin binding repeat domain, (III) a central region, and (IV) an SH3 domain at the C-terminal (Figure 2). The DDW-Arp2/3 binding site and the actin-binding domain together regulate F-actin polymerization and dynamics by activating the Arp2/3 complex [127]. Both are necessary for translocation of cortactin to sites of actin polymerization [128]. The central part of the protein, between the F-actin repeat domain and the SH3 domain, contains an α-helix sequence and a proline-rich region that can bind to SH3-domains of other proteins. Three serine/threonine phosphorylation sites [129] and three Src tyrosine phosphorylation sites [130;131] are located in the central region. Tyrosine phosphorylation of cortactin by Src occurs at tyrosine residues 421, 466, and 482, according to a fixed sequence with initial phosphorylation at tyrosine 421 followed by Y466 [131]. Tyrosine-phosphorylated cortactin (I) occurs in response to growth factor treatment, integrin-mediated cell adhesion, bacterial invasion and cell shrinkage (reviewed in [19]), (II) exhibits reduced F-actin cross-linking activity [130], (III) is required to enhance cell migration [12] and metastasis (Li, cancer res, 2001), (IV) may help to stabilize kinase-cortactin association (e.g. Src and Fer kinase [132;133] and/or (V) provide docking sites for other SH2 domain-containing proteins (reviewed by [20]). Exactly how tyrosine phosphorylation of cortactin would influence its ultimate cellular functions still remains largely unknown. However, the importance is underlined by the strong positive correlation between a high level of tyrosine phosphorylation and enhanced cell migration and metastasis [12;13;134;135]. Cortactin can be serine/threonine phosphorylated by Erk or Pak kinases and occurs at residues T401, S405 and S418 (Figure 2). Serine/threonine phosphorylation of cortactin promotes its binding and activation of N-WASP and subsequently leads to increased Arp2/3 mediated actin polymerization [136]. Once incorporated into a stabilized F-actin meshwork, cortactin might become less accessible for kinases. The actin-binding domain is essential for the efficient serine/threonine phosphorylation induced by Fer kinase-mediated F-actin depolymerization [137]. No direct evidence is available yet to confirm whether F-actin-bound cortactin can be phosphorylated in situ and that this leads to actin destabilization and disassembly. The cortactin-SH3 domain mediates the interaction with various proteins (Figure 2, listed in Table 2, page 36, and reviewed in [19]). Since cortactin operates mainly in cytoskeletal rearrangements, it may link other proteins via its SH3 domain to sites of actin polymerization or may itself be directed to the site of actin polymerization by those other proteins (see section below)
show enhanced migration [11;12](Chapter 2), invasion [11] and increased metastatic potential in vivo [13]. Other mechanisms related to cortactin function that affect cell migration are alternative splicing (Chapter 2), tyrosine phosphorylation (as discussed above) and competition for binding with other cortactin-binding proteins such as AMAP1 [77] (as discussed in paragraf 1.5.6) or BPGAP [139]. Thus, there seems to be a strong correlation between changes in the level and/or function of cortactin and cancer progression. Cortactin is involved in the reorganization of the actin cytoskeleton in several cellular processes. In this way, cortactin might promote cancer cell migration and invasion by influencing the integrity of the actin cytoskeleton. Since both cyclin D1 and cortactin are overexpressed in tumors with 11q13 amplification, their contribution to the genesis and progression of breast cancer may be additive or even synergistic.
1.5 The role of cortactin on actin cytoskeleton
organization and cell migration
1.5.1 Actin polymerization
1.5.2 Classification of actin binding proteins
The actin cytoskeleton contributes to many processes in cells, including cell shape change, motility, polarization, contraction, cytokinesis, signal transduction, endocytosis, and intracellular vesicle trafficking. Each of these processes involves different actin-binding proteins (ABP) and different arrangements of actin filaments [141;142]. The ABPs allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals that are essential in abovementioned cellular processes, both normal and pathological. The activities of ABPs can be modulated by their protein expression levels itself, calcium levels, phosphoinositide levels, pH, salt concentrations or post-translational modifications such as phosphorylation. Their expression pattern can be cell type specific in accordance with special functions of cells. The ABPs can be classified into one (or more) group(s) of proteins depending on their activity towards actin [143] (Figure 3):
(I) Actin monomer-binding proteins inhibit or accelerate nucleotide exchange. Their major function is sequestration of G-actin from the monomer pool and the subsequent prevention of polymerization. The sequestering protein profilin accelerates exchange of ADP for ATP and thereby attracting more monomers to the (+) end resulting in an increased assembly of actin filamentous actin, while β-thymosin binds preferentially to ATP bound G-actin and as such inhibits spontaneous nucleation of actin and polymerization of F-actin.
(II) Capping proteins bind at the ends of actin filaments and depending on whether they bind at the (+) or (-) end, they may either prevent further actin polymerisation or promote disassembly of actin filaments. Uncapping of capping proteins from barbed ends is enforced by certain inositol phospholipids (=phosphoinositides), resulting in promotion of actin assembly. Similarly, tropomodulins are pointed end capping proteins that stabilize actin filaments.
(III) Severing proteins, such as gelsolin, bind along existing filaments and cut them into short fragments to create free barbed ends to support polymerization. Many of the proteins classified as severing proteins are also barbed end-capping proteins. The barbed ends need to be prevented from being capped (by phosphoinositides) to support further elongation.
(IV) Actin motor proteins, such as myosins, bind one another to form antiparallel complexes with actin-binding ´heads´ on either side. As such, myosin II bundles F-actin in antiparallel arrays and generates contractile structures under tension such as the actomyosin stress fibers.
(V) Side-binding proteins, such as tropomyosin, bind to the sides of actin filamentsto stabilize the actin filaments and (together with troponins) regulate the interaction of the filaments with myosin in response to calcium.
Figure 3. Classification of actin-binding proteins. Monomeric (G-actin) can spontaneously
(VIIa) Bundling and (VIIb) cross-linking proteins, such as α-actinin and filamin respectively, arrange actin filaments in higher order structures. Functionally, bundles and networks have identical roles in a cell: both provide a framework that supports the plasma membrane and, therefore, determines the shape of the cell. Structurally, bundles differ from networks mainly in the organization of actin filaments. In bundles, the actin filaments are closely packed in parallel arrays (VIIa), whereas in a network the actin filaments crisscross (VIIb), often at oblique angles, and are loosely packed. Cells contain two types of actin networks. One type of actin network, associated with the plasma membrane, is planar or two-dimensional, like a net or a web; the other type, present within the cytosol is three-dimensional, giving the cytosol gel-like properties. Actin crosslinking increases the viscosity and stiffness of the actin filament network, stabilizing the actin-rich cortex, and it is necessary for cell motility to form a rigid mesh structure to generate force in the forward direction and advance the leading edge of the cell. Without cross-linking and bundling, effective actin-based motility is not possible. The function of many of these proteins appears to be controlled by extracellular signals, such as chemotactic signals, that lead to reorganization of cytoskeletal structure. In all bundles and networks, the filaments are held together by actin cross-linking proteins. Most bundling and cross-linking proteins are dimeric (e.g. α-actinin) or have two actin-binding domains to connect two filaments (e.g. fimbrin). The length and flexibility of a cross-linking protein or the distance between actin-binding domains critically determine whether bundles or networks are formed. Short cross-linking proteins hold actin filaments close together, forcing the filaments into the parallel alignment that is characteristic for bundles. When the parallel bundles are held apart far enough, it allows interaction with other proteins such as myosin. In contrast, long, flexible cross-linking proteins are able to adapt to any arrangement of actin filaments and tether orthogonally oriented actin filaments in networks.
(VIII) Nucleation promoting factors. Nucleation process: two actin molecules bind weakly, but addition of a third stabilizes the complex. This trimer then adds additional molecules and forms a "nucleation site". This is the slow, or lag phase of the polymerization process. Nucleation promoting factors help to speed this process. On the one hand, formins are proteins that nucleate formation of unbranched actin filaments, such as those in stress fibers. Formins are located adjacent to the plasma membrane. The actin filaments that they nucleate have their barbed ends, to which actin monomers add, anchored to the formin.
Figure 4. Model for the assembly and disassembly of the dendritic actin filament network at the leading edge of a cell. At the leading edge of a migrating cell, a large pool of profilin-bound actin-GTP
monomers are ready to assemble into filaments, but requires the Arp2/3 complex and additional cellular components like N-WASP to catalyze nucleation (1). The activated Arp2/3 complex, while bound to an existing actin filament (2), nucleates the rapid growth of new filaments (3) that push the plasma membrane forward (4). N-WASP predominantly binds to the free form of the Arp2/3 complex while cortactin has much higher affinity for the Arp2/3 complex once it is incorporated into actin filaments. Once the branch point is established, N-WASP is released from the complex and is replaced by cortactin, which stabilizes the branching site by simulaneously binding to the actin filament. Filaments grow until they are capped with actin capping protein (5). Polymerized actin hydrolyses its bound ATP (6), and ADP-F-actin is severed and depolymerizes (7). ADP-F-actin is a substrate for the actin depolymerizing factor ADF/cofilin. Free ADP-G-actin binds profilin, which in turn catalyzes nucleotide exchange to regenerate ATP-GTP. As a consequence, profilin ´primes´ the actin for another round of assembly and simultaneously inhibits spontaneous nucleation of ´free´ actin filaments, thus ensuring
(WASP and N-WASP) and Scar/WAVE (WAVE1, WAVE2, WAVE3) family (proteins that bind monomeric actin) or by proteins that bind F-actin (cortactin or Actin binding protein 1 (Abp1)). When activated, the Arp2/3 complex binds to the side of an existing actin filament and nucleates assembly of a new actin filament. The resulting branch structure is Y-shaped. WASP, WAVE and cortactin proteins have a DDW acidic domain that binds and activates Arp2/3, plus domains that recognize and bind to various signaling factors that might recruit other proteins necessary for proper/specific actin-polymerization to sites of active actin polymerization. Cortactin binds to WASP via its SH3 domain and activates N-WASP-mediated actin polymerization. Thus, WASP/WAVE/cortactin proteins may determine where in a cell actin polymerization will occur. Recently, a model was proposed [147] in which the association of N-WASP leads to activation of Arp2/3 complex facilitating its binding to an existing actin filament and initiating new actin assembly. Subsequently, activated Arp2/3 then becomes less accessible to WASP but is more prone to associate with cortactin. Cortactin then replaces N-WASP, stabilizes branched actin filaments, and inhibits disassembly of the actin network. Thus, aside from promoting actin polymerization directly or indirectly via N-WASP, cortactin stabilizes actin branches and alters the structure, rigidity or persistence of the filamental network. In addition, serine/threonine phosphorylation of cortactin promotes its binding and activation of N-WASP leading to increased Arp2/3 mediated actin polymerization [136]. The WASP/WAVE/cortactin proteins itself are regulated by binding to proteins of the Rho family (Cdc42, Rac1, see paragraph 1.5.5) and/or to PIP2 (phosphatidylinositol-4,5-bisphosphate). Arp2/3
complex mediated actin polymerization is necessary in processes such as lamellipodia formation at the leading edge of motile cells [128;148], cell spreading [149], intercellular adhesion [40;150], endocytosis [148;151] and bacterial comet tail formation after bacterial fagoctyosis [152].
(IX) Adapter proteins. Cells use multidomain proteins, such as CD2AP, Crk and Nck, as adapters between actin and other (signaling) proteins.
1.5.3 The actin cytoskeleton emerges in three forms
Upon extracellular signals, including growth factors, cells change their cell shape by reorganizing the actin filaments in structures including actin bundles, actin networks, and stress fibers [155]. The cytoskeletal alterations include the production of cell surface protrusions (filopodia, lamellipodia, and membrane ruffles) as well as the formation of focal adhesions and stress fibers.
Filopodia (also called microspikes) are typically found at the edge of moving
or spreading cells. Filopodia are long thin transient structures that extend from the cell surface and that are present only during the time required to establish stable contacts with the underlying substratum. Bundles of parallel actin filaments, oriented with their plus ends toward the filopodial tip are cross-linked within filopodia by the small actin-binding protein fascin. The closely spaced actin filaments provide stiffness to the filopodia.
Lamellipodia and membrane ruffles. Lamellipodia are dynamic structures,
constantly changing shape. Lamellipodia are thin but broad projections at the edge of a mobile cell that contain extensively branched arrays of actin filaments, oriented with their barbed ends toward the plasma membrane. At the leading edge of a lamellipodium, actin severing and capping proteins keep the actin filaments short, whereas Arp2/3 keeps initiating new branches that grow to propel the edge of the cell forward. An actin gel-like network of short, branching actin filaments is more effective in pushing the leading edge forward than unbranched filaments, given the flexibility of actin filaments. Lamellipodia and filopodia are required for the spreading and motility of cells. For movement, the cells use precursor contacts in ruffling lamellipodia or beneath filopodia. A membrane ruffle is a lamellipodium in the process of folding back onto the cell body from which it extended. When ruffles touch the surface of their own cell, they fuse with it; when they touch the surface of another cell, they retract.
Stress fibres. Stress fibers are a specific cytoskeletal organization of actin
monomers. Large bundles of actin filaments are attach to the plasma membrane via adapter proteins in focal adhesions where integrins mediate cell attachment to ECM. In stress fibers, F-actin and myosin II are in bipolar arrangement and this feature contributes to the tension and the ability of the fibers to contract. Contraction of the actin stress fibers allows the cell to exert tension on the substratum, an important part of controlling morphogenesis. Formation of stress fibers and focal adhesion complexes are a key regulatory event in cell growth and cell movement such as migration and invasion.
1.5.4 Membrane protrusions and cellular processes driven by
actin polymerization
The plasma membrane of many motile cells undergoes highly regulated protrusions and invaginations that support the formation of podosomes, invadopodia, circular dorsal ruffles and endocytic vesicles [156]. The name of the protrusion is dictated by its shape, which in turn depends on the organization of the actin cytoskeleton. Cortactin is localized to and involved in all these protrusions (listed below).
Podosomes are dot-shaped adhesion structures consisting of large circular
arrays with an actin core. Podosomes are highly dynamic structures that assemble and disassemble at least 10-fold faster than focal adhesions and have a role in cell adhesion, ECM degradation and motility. They are observed in osteoclasts and macrophages, but also in v-Src RSV-transformed cells [157] and human squamous cell carcinoma cells [138]. In podosomes, cortactin co-localizes with Arp2/3 complex in a ring of actin network around the core of actin bundles. In podosomes-like structures of smooth muscle cells, cortactin is enriched at specialized microdomains at the focal adhesion/stress fiber interface [158;159]}, but not in the focal adhesions or along stress fibers itself. Podosome-like structures in normal epithelial cells are distinct from focal contacts and also contain Arp2/3, cortactin, Src and, N-Wasp [160].
Invadopodia are filopodial-like membrane protrusions of the ventral plasma
membrane extending into sites of active ECM degradation. Invadopodia are prominent in cells that actively migrate and invade surrounding tissues. These structures are actin-rich and metalloproteases-rich formations with underlying ECM degradation areas. Cortactin localizes at the basis of invadopodia in a complex with paxillin and protein kinase C (PKC) µ [15;77]. Microinjection of anti-cortactin antibodies in an invadopodia-forming breast cancer cell line blocked the formation of invadopodia [15].
Circular dorsal ruffles/waves. Many of the same components as in
podosomes and invadopodia are involved in the formation of circular dorsal ruffles/waves, but the latter are transient formed on the dorsal surface of cells after receptor stimulation. The surrounding actin cytoskeleton is reorganized by first disassembling the actin stress fibers, which precedes the formation of lamellipodial protrusions [161].
Dendritic spines are small ´door knob´ shaped protrusions from the surfaces of
Endocytosis and vesicle movement. Cortactin binds directly to the large
GTPase dynamin-2 and thereby links the actin cytoskeleton to clathrin-dependent [167-169] as well as to clathrin-independent endocytosis [170]. Once the vesicles are pinched off, the endosomal vesicles are propelled by cytoplasmic cortactin-, F-actin- and Arp2/3-containing tails [148]. Analogous to this process, some intracellular pathogens, including the bacteria Listeria and Shigella and the Vaccinia virus, use the host cellular machinery to assemble networks of actin filaments that propel them through the cytoplasm [152]. The actin tails behind the endosomal vesicles or bacteria consist of actin, cortactin, Arp2/3, and N-Wasp. Eukaryotic cells use endocytosis to internalize plasma membranes, surface receptors and their ligands, viruses and various extracellular soluble molecules. Disturbances in the control of the endocytic machinery can contribute to carcinogenis: e.g. cortactin overexpression inhibits ligand-induced down-regulation of the EGF receptor [171]. In addition, dynamin-2 is, together with cortactin, also involved in post-Golgi transport [172] as well as in lamellipodia, podosomes, circular dorsal ruffles, and invadopodia formation [173].
Cell contractility is indispensable for cells to change their shape, to spread
and migrate. Local periodic contractions of lamellipodia appear to be dependent on myosin light-chain kinase (MLCK) [174] and enable the cell to rapidly expand to a maximum area before polarization. MLCK phosphorylates the myosin light chain (MLC) at the cell periphery resulting in stimulation of actin-myosin II assembly and, consequently, cell contraction. Cortactin has been shown to interact directly with MLCK and they co-localize in lamellipodia [175]. The interaction does not affect MLCK activity, but inhibits MLCK-F-actin interaction, whilst MLCK itself inhibits the cortactin-induced Arp2/3 polymerization in vitro. The MLCK inhibitor ML-7 inhibits cell spreading, indicating that spreading requires MLCK activity [176]. Cross talk exists between actin polymerization and actomyosin contraction [177], indicating that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton or vica versa. Cortactin might be the linker between the cytoskeleton and cell contraction by regulating localization of MLCK and/or MLCK activity and as such may affect cell spreading or cell migration.
1.5.5 Rho-GTPases regulate the organization of the actin
cytoskeleton
of external and/or internal stimuli they translocate to the plasma membrane. RhoGTPases function as conformational switches that cycle between a GDP-bound inactive and GTP-GDP-bound active form. Activated GTPases function by interacting with their specific downstream targets. GTP-hydrolysis and liberation of phosphate inactivate the GTPases. RhoGTPases can be activated by guanine nucleotide exchange factors (GEFs) or inactivated by GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) [179;180]. The GTP-GDP switch of GTPases forms an adequate control mechanism to rapidly activate and inactivate signaling pathways necessary to coordinate complex morphological changes [181].
The active GTP-bound forms of Cdc42, Rac1 and RhoA regulate a signal transduction pathway in cells that links extracellular signals to the formation of filopodia, lamellipodia and stress fibers, respectively [182-184]. The Cdc42, Rac1 and Rho proteins mutually affect each other showing that these proteins are functionally linked. For example, activated Rac1 inhibits Rho function resulting in decreased stress fiber formation. The GTPases Rac1 and Cdc42 regulate many actin binding proteins. Rac1 activates phosphatidylinositol (PI)-4 and PI-5 kinases that produce phosphatidylinositol-4,5-bisphosphate (PIP)2. PIP2 subsequently binds
to F-actin barbed-end capping proteins, thereby causing the uncapping of actin filaments and inducing a rapid actin polymerization at this end. PIP2 is widely
implicated in cytoskeleton regulation, although, the mechanisms by which PIP2
affects cytoskeletal changes have not been unraveled. GTP-Cdc42 and PIP2
activate N-WASP resulting in recruitment of the Arp2/3 complex to the leading edge thereby promoting formation of filopodia [185]. GTP-Rac1 can induce lamellipodia formation by activating its downstream targets IRSp53 or PAK1. As a result, Scar/Arp2/3 or cortactin/Arp2/3 respectively, are recruited to the cortical actin networks [186;187].
1.5.6 GTPases and cortactin
Activation of the GTPase Rac1 is required for translocation of cortactin to the cell periphery and this translocation is required for tyrosine phosphorylation of cortactin [131]. This signaling is likely to be regulated via the serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42, that binds and phosphorylates cortactin [186] (Figure 2). Cortactin contains one PIP2 binding
motive in the 4th repeat of its actin-binding repeat domain and both PIP2 and F-actin
compete for the same site [188]. This suggests that Rac1 induced PIP2-production
can cause dissociation of cortactin from actin filaments and might affect the cross-linking activity of cortactin [188].
with BPGAP1, a RhoGAP, resulting in facilitated translocation of cortactin to the cell periphery and increased cell migration [139]. Finally, cortactin-SH3 binds to AMAP1, a GAP of ADP- ribosylation factor (Arf) GTPase, which is expressed at high levels in invasive breast cancer cells [77]. Inhibition of AMAP1 decreased breast cancer cell invasion. AMAP1, Arf, paxillin and cortactin localize to invadopodia and AMAP1 act to bridge cortactin with the integrin protein paxillin. High levels of AMAP1 expression and its association with paxillin and cortactin are crucial for the invasive phenotypes.
Thus, cortactin expression levels might affect the activity of GTPases via GEFs or GAPs and subsequently the equilibrium between signaling proteins, which may finally influence cytoskeletal organization, cell migration, invasion and metastasis.
1.5.7 Cortactin couples dynamic actin networks to cell migration
and breast cancer
Cell migration on solid substrates (two-dimensional migration) is an integrated process [191]. Cells form a ´leading edge´ (lamellipodia) by preferentially protruding membrane in a single direction, usually in response to a directional or chemotactic signal. This is mediated by the localized polymerization of filopodia and Arp2/3 mediated actin polymerization in the cortex creating a branched array of actin filaments, in a directional manner. As the leading edge comes into contact with the extracellular matrix, small adhesions, called focal complexes, are formed that attach the membrane to the matrix. These focal complexes either turnover or mature into larger focal contacts (or focal adhesions) [192]. An internal complex of structural and signaling molecules forms on the cytoplasmic side of these adhesions, serving to link the extracellular matrix to the intracellular cytoskeleton [193]. Following the formation of focal adhesions, the cell is able to exert force on them by contraction of the actin cytoskeleton through activation of myosin II [194]. It is this force that propels the cell forward. The final step of cell migration occurs when adhesions at the rear of the cell are disassembled and the cell retracts the trailing edge. The Arp2/3 complex binds directly to the focal adhesion protein vinculin [149]. This association is transient and only observed in newly formed focal complexes at the cell periphery, but not in mature focal adhesions. Cortactin is highly tyrosine phosphorylated in response to integrin activation [195;196], suggesting a role for cortactin in cell-matrix adhesion: yet, there is no evidence that cortactin localizes to focal adhesions themselves.
disease development whereby microbial pathogens use the host actin cytoskeleton including cortactin function to enter the host cell [197-199]. The unique domain architecture of the cortactin functionally connects it to a variety of proteins (Table 2) and cellular functions. Its N-terminal part primarily links cortactin to cytoskeletal reorganization events. The C-terminal SH3 domain is able to distinguish between
Figure 5. Cortactin functions in several cellular processes. Cortactin functions in several cellular
different partners, which is important for functionally (un)coupling cellular processes at the required time and place in vivo.Although cortactin is ubiquitously expressed in all cells but the hematopoietic cells, this is not the case for all the interacting proteins. Thus, the effect of cortactin on cell migration and other cell properties might be dependent on the expression levels of these proteins in different cell types, different subcellular compartments and under different physiological circumstances. On the other hand, cortactin and several binding partners such as F-actin, N-WASP and Arp2/3 localize to many of the same sites, suggesting that actin polymerization may be a common mechanism in all these processes. From these findings, we hypothesize that the most important function of cortactin is to bridge a diversity of other proteins into the de novo Arp2/3 mediated actin polymerization and to stabilize the newly formed branched network. Cortactin can either recruit proteins or be recruited by proteins to the site of actin polymerization. Cortactin affects rearrangements of the actin cytoskeleton and links dynamic actin networks to several cellular processes involved in cell migration. As such, the deregulation of cortactin (levels) might be responsible for changed properties related to diseases such as invasion in cancer.
Getting closer to the identification and understanding of all the aspects of the multi-faceted role of cortactin as a molecular scaffold will eventually lead to a better understanding of the role of cortactin in the development of (breast) cancer. Although cortactin has been studies quite extensively in the last few years, it is still not know in which stage of cancer progression (as illustrated in Figure 1) cortactin overexpression is most crucial. Whether it is in during cell proliferation, during which cancer cells make invadopodia and break through the basement membrane, or at the stage in which cells detach from the primary tumor and migrate to the blood vessel, or even at later stages. In other words, does cortactin overexpression, as observed in human breast cancer, deregulate processes in the early hyperplastic lesions, in DCIS/LCIS, IDC/ILC or in metastasis formation and if so, how?
Table 2: Interaction of cortactin with proteins and lipids. Listed are proteins that physically interact
with cortactin or are in a complex with cortactin (indirect). Interacting proteins were grouped occording their function.
Residue Size (kD) via domain Reference(s)
Size (kD) via domain Reference(s)
Actin-related: Arp3 (Arp2/3-complex) 50 DDW [127;128] Dynamin2 100 SH3 [161;204] F-actin 43 ABD [153] MIM 120 SH3 [205] N-WASP 53 SH3 [147;206] MLCK 135/210 SH3 [175;207] WIP 55 SH3 [208] Adhesion: δ-catenin 160 N-term [209] E-cadherin (indirect?) 120 [210] N-cadherin (indirect?) 130 [211] N-syndecan (indirect?) 50/55/120 [212;213] ZO-1 200 SH3 [214] Receptor: EphA (indirect?) [215] Met (indirect?) 145 [216] TEM7 SH3 [217] TEM7related SH3 [217] Tir (indirect?) [218]
GTPases and GTPase-related:
BPGAP1 SH3 [139] Dynamin2 100 SH3 [161;204] Fgd1 -82 SH3 [189;190] RICH SH3 [219] AMAP1 SH3 [77] Adapter: Crk 40 SH3 [133;220] Grb2 24 PRD [216]] CD2AP 80 SH3 [221] Nck 47 PRD [133] WIP 55 SH3 [208] Endocytosis: Dynamin2 100 SH3 [161;204] HIP1R (indirect?) 120 [222] Apoptosis: HAX-1 35 N-term [223;224] Lipids: PIP2 ABD [188] Other:
Kv1.2 (potassium channel) (indirect?) 70 [225]
Brain:
CBP90 90 SH3 [226]
CortBP1/SHANK2 180 SH3 [227;228]
CortBP2 SH3 [229]
1.6 Outline of this thesis
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