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The 5' inositol phosphatase SHIP2 regulates EGF-elicited protrusion in MTLn3 cells

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The 5' inositol phosphatase SHIP2 regulates

EGF-elicited protrusion in MTLn3 cells

by

Georgiana L. Kuhlmann

Submitted to the Department of Biology

in partial fulfillment of the requirements for the degree of

Master of Science in Biology

at the

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

June :2010

@Massachusetts

Institute of Technology 2010. All rights reserved.

ARCHIVES

Author_

Department of Biology

May 21, 2010

Certified by

Frank B. Gertler

Professor of Biology

Thesis Supervisor

Accepted by

Tania A. Baker

Co-Chair, Department Graduate Committee

MASSACHUSETTS INSTTUTE OF TECHNOLOGY

JUN

0

2 2010

LIBRARIES

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The 5' inositol phosphatase SHIP2 regulates EGF- elicited

protrusion in MTLn3 cells

by

Georgiana L. Kuhlmann

Submitted to the Department of Biology on May 21, 2010, in partial fulfillment of the

requirements for the degree of Master of Science in Biology

Abstract

In metastatic cancer, cells must be able to migrate from their original environment, move through the blood or lymphatic system, and colonize a distant organ. Mena, a member of the Ena/VASP family of proteins, is upregulated in invasive populations of breast cancer cells. The Ena/VASP (enabled/vasodilator-stimulated phosphoprotein)

family of proteins regulate both the geometry and dynamics of actin filament networks. Mena specifically is alternatively spliced with an invasive isoform, MenaINV, upregu-lated in metastastic cells, while an epithelial isoform, Menalla, is downreguupregu-lated. SH2-domain containing 5-inositol phosphatase (SHIP2) interacts with Mena and is thought to play a role in breast cancer. SHIP2 is a 5-phosphatase that catalyzes the

dephospho-rylation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) to phosphatidylinositol

3,4-bisphosphate (PI(3,4)P2) as well as the dephosphorylation of phosphatidylinositol

4,5-bisphosphate (PI(4,5)P2). PI(3,4,5)P3 and P][(4,5)P 2 are two major phosphoinositides at

the plasma membrane and regulate a variety of cellular functions, including receptor sig-naling, membrane-cytoskeleton interactions and clathrin-mediated endocytosis. Here I have looked at the effects of knocking down SHIP2 in the MTLn3 cell line, a metastatic rat breast carcinoma line. I found that when SHIP2 is knocked down in cells, there is an increase in membrane protrusion upon stimulation with EGF, and that recruitment of Mena to the leading edge is enhanced, implying that this increase in protrusion may be due to a change in Mena localization.

Thesis Supervisor: Frank B. Gertler Title: Professor of Biology

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Acknowledgements

Thanks to Frank Gertler, for having me as a member of the lab, for advising me through this thesis, and for being supportive in all my decisions regarding my career. He has been an excellent advisor in this past year. Thanks as well to the Biology Department, for providing a fantastic graduate program. Thanks especially to Tania Baker for all her support and to Betsey Walsh for always having the answers to every single one of my questions. I have greatly enjoyed my time at MIT.

Thanks to all members of the Gertler lab for being patient and thoughtful teachers, and for helping me find my way around the lab. This thesis would not be in existence if it were not for lab members' support this year, in particular Shannon Alford, Elaine Pinheiro, Michele Balsamo and Jose Medrano.

Thanks to my supporters outside of MIT, including my parents, who have always encouraged me to do what makes me happy, despite their concerns about job security. This thesis was written up with the help of my cats, who tried their best to add their own interpretations of the data, despite not being able to spell very well.

Finally, I am most grateful to my husband, who has been extremely patient while I agonized over my thesis while becoming increasingly less helpful around the house. His support has made all my decisions possible. I could not ask for a better partner in crime.

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Contents

1 Introduction 9

1.1 Metastatic Disease in Breast Cancer . . . . 9

1.2 The Motility Pathway . . . . 12

1.3 The Ena/VASP Family of Proteins: Actin Cytoskeletal Regulators . . . . 17

1.4 Mena in Breast Cancer . . . . 21

1.5 SHIP2, an SH2-Domain Containing 5'-Phosphoinositide Phosphatase 23 2 Methods 27 2.1 Cell Culture and Fluorescence-Activated Cell Sorting . . . . 27

2.2 Transient Transfections . . . . 27

2.3 Protrusion Assays . . . . 28

2.4 Immunofluorescence. . . . . 28

3 Results 31 3.1 SHIP2 Is Knocked Down in GFP-Positive MTLn3 Cells . . . . 31

3.2 SHIP2 Knockdown Increases Membrane Protrusion as Compared to Con-trol Cells... . . . . . . . . . . . ... 31

3.3 The Amount of Mena Localized to the Leading Edge Upon Stimulation with EGF Increased in SHIP2 Knockdown Cells . . . . 33

3.4 Loss of SHIP2 Expression Results in a Reduction in Focal Adhesions 37

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Chapter 1

Introduction

1.1

Metastatic Disease in Breast Cancer

Breast cancer diagnoses make up one-third of all cancer diagnoses in women, and account for 15% of deaths due to cancer in the United States, second only to lung cancer [50]. Globally, breast cancer is the most commonly diagnosed form of cancer in women, with over 1 million cases diagnosed annually [20], and is the leading cause of death [71], with incidence being most prevalent in industrialized countries, though it is increasing in other areas as well [70]. According to the American Cancer Society, an estimated 192,000 new cases of invasive breast cancer and 62,000 cases of in situ cancer will have been diagnosed in 2009. While the incidence of diagnosis has gone up worldwide, mortality has decreased over the past 30 years [4]. Much of this improvement can be attributed to the introduction of widespread mammograms in the early 1980s [70], which decreased mortality 23-30% in the years following the increased screening [96] due to the early detection of tumors. Since the 1990s, however, mortality rates for breast cancers have plateaued [114], at least in part due to the lack of effective markers that identify which patients will develop metastatic disease and which will not.

Metastatic disease is the major cause of death in breast cancer patients [10] and 90% of breast cancer deaths are due to metastases [113]. Of patients with breast cancer,

10-15% have aggressive disease, and will develop distant metastases within 3 years, though

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been made to identify a gene expression signature for metastatic disease using microarray analysis of patient samples [104,107]. These studies have identified a 70-gene expression signature that stratifies patients into a poor prognosis group, with a 50 percent chance of remaining metastasis free in ten years, and a good prognosis group, with an eighty-fiver percent chance of remaining metastasis free in ten years [104]. However, due to the expense and technical difficulty of microarrays, this approach has not gained traction in the clinic. Instead, histology and lymph node metastases are used as predictors of distant metastases, but these methods are not completely predictive.

Further complicating the issue is the lack of a detailed understanding of the process of tumor initiation and metastasis. Ten years ago, Hanahan and Weinberg published a seminal review highlighting six essential alterations in cell physiology that must occur for malignancies to develop: self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless reproductive potential, sustained angiogenesis, and tissue invasion and metastasis [36]. While this succinctly highlights the changes that must occur, the mechanisms underlying these changes have yet to be clearly elucidated. It is known that for metastases to form, cells from the primary tumor must acquire the ability to exit the tumor, intravasate into either the blood or lymph system, and then exit and colonize distant organs [36]. This process of acquiring increased motility and invasive characteristics is hypothesized to involve an epithelial-to-mesenchymal transition (EMT) [431, though this has not been proven in breast cancer. During EMT, an epithe-lial cell undergoes multiple biochemical changes that allow it to assume a mesenchymal phenotype. Hallmarks of a mesenchymal phenotype include increased migratory capabili-ties, decreased proliferation, increased invasiveness, resistance to apoptosis, and enhanced ability to produce extracellular matrix components [43]. This process happens both dur-ing normal development as well as in metastasis initiation. In metastasis, miesenchymal cells are capable of exiting the primary tumor and breaking through the basement mem-brane. These migrating tumor cells , in murine mammary carcinomas, are accompanied

by macrophages as they move toward blood vessels [117]. The tumor cell and macrophage

form a paracrine loop in which the tumor cell secretes colony stimulating factor-1

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Cancerous cell in situ

Breaking through the basement membrane

EGFR

Paracrine Loop

F-1

intravasation

Figure 1-1: The process of invasion and metastasis. A normal cell becomes cancerous. An invasive cell gains the capability of exiting the primary tumor and breaking through the basement membrane. Cancer cells participate in a paracrine loop with macrophages, with the tumor cell secreting CSF-1 and the macrophage secreting EGF. Tumor cells intravasate with the help of perivascular macrophages.

carcinoma cells [117]. It has also been shown that intravasation (when a tumor cell enters the blood stream) occurs in association with perivascular macrophages [118]. Figure 1-1 outlines these steps.

In an attempt to better understand what accounts for the ability of cells to exit the tumor and invade into the blood stream, the Condeelis lab at Albert Einstein College of Medicine, developed an in vivo invasion assay [116] in order to collect the subpopulation of cells in a tumor with increased mobility and chemotactic ability. In this assay, a needle filled with matrigel and EGF is inserted into a primary breast tumor growing in rat, allowed to remain for 6 hours, and then removed. Cells that have actively migrated into the needle are collected, and can then be further analyzed by RT-PCR. Using this technique, Wang et al. [111, 112] were able to derive an "invasion signature," a gene

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expression data set based on mRNA collected from cells with invasive potential. This was done using both an orthotopic model of breast cancer, using MTLn3 cells, a metastatic rat breast carcinoma line, [111] as well polyoma middle T (py-MT) oncogene derived tumors [112]. The patterns of gene expression were similar across the two types, with 3 groups of genes being up-regulated: genes involved in the repression of proliferation, anti-apoptotic genes, and genes involved in the motility pathway [111]. Significant changes were observed in the Arp2/3 complex, capping protein, the cofilin pathway and Mena

[112].

1.2

The Motility Pathway

The process of invasion requires cell motility, as is demonstrated by the invasion signature derived by Wang, et al. [111,112]. Cell motility occurs in a cycle of four events: protrusion of the leading edge, adhesion, retraction of the rear of the cell and de-adhesion [52,80]. Membrane protrusion is considered to be a key event in motility [52] and many of the up-regulated genes in the motility pathway are involved in the process of membrane protru-sion, including Cdc42, parts of the Arp2/3 complex, Mena and its paralog EVL [111,112]. Membrane protrusions were first classified in a series of papers by Abercrombie et al. in

1970, in which the lamellipodium, a broad, fiat protrusion, and the filopodium, a long,

needle-line protrusion were observed and named [2,3]. The protrusive force is generated through directed polymerization of the actin cytoskeleton pushing on the cytosolic face of the cell membrane. Mogilner and Oster have proposed an elastic Brownian ratchet model to quantitatively describe the force generated by actin polymerizing just beneath the surface of a protruding membrane [62].

Actin filaments (F-actin or filamentous actin) are made of two parallel protofilaments that wrap around each other in a right-handed helix. These filaments are polarized, with the pointed, or minus, end being the site of slower polymerization and the barbed (or plus) end being the site if rapid polymerization. It is this barbed end that is directed towards the membrane [100]. Free actin subunits (G-actin, or globular actin) are bound to ATP, which is hydrolyzed to ADP soon after a monomer has been incorporated into

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a growing filament. Actin monomers are also bound to other proteins that make spon-taneous polymerization unfavorable. The most abundant of these is thymosin, which, when bound, locks actin into a state in which it cannot bind either the barbed or pointed end of a filament or exchange ATP for ADP, rendering it polymerization incompetent [5]. Recruitment of monomers for polymerization depends on another monomer-binding pro-tein, profilin, which blocks polymerization at the pointed end, but allows polymerization at the barbed end. Profilin also catalyzes the exchange of ADP for ATP. Profilin-actin complexes therefore represent the major source of actin monomers available for polymer-ization. When the monomer has been incorporated into a growing filament, it loses its affinity for profilin and the profilin dissociates. In the absence of external stimuli, cells normally keep actin filaments in a capped state through the action of capping protein, so that monomers are not continuously added on to the barbed end. This capping activity must be inhibited for long actin filaments to form [18,80].

The molecular mechanisms underlying actin dynamics are quite complex, and have been actively studied for many years, though a complete understanding is remains elu-sive. What is known is that extracellular signals received by cell-surface receptors are passed on to small GTPases, including the Rho, Rac and Cdc42. These signals are integrated by Wiscott-Aldrich syndrome protein (WASP) family of proteins, including WASPs and the SCAR/WAVE (WASP-family verprolin-homologous protein) complex. WASPs and SCAR/WAVE integrate signals from adaptor molecules such as Grb and

Nck, as well as phosphatidyl inositol 4,5-bisphosphate (PI(4,5)P2). Activated WASPs

and SCAR/WAVE then binds to the Arp2/3 complex which then nucleates new actin filaments at a 70 degree angle off of existing actin filaments [42,79].

When a cell receives growth factor signals, the Rho family small GTPases Rac, Rho and Cdc42 exchange their bound GDP for GTP, through the action of guanasine nu-cleotide exchange factors (GEFs) thereby moving into an active conformation [35]. Cdc42 and Rac are activated by the accumulation of phosphatidylinositol 3,4,5-trisphosphate

(PI(3,4,5)P3), which recrutis GEFs such as Sos1, Vav2 and Vav3, which in turn activate

Cdc42 and Rac [9, 93]. Activation of Rac is essential for the formation of lamellipo-dia [37,66], while Cdc42 activation favors the formation of filopolamellipo-dia [33,66]. The

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down-stream target of Rae are the SCAR/WAVE complexes [58], via the adaptor molecule Nck [23]. Cdc42 directly binds to neuronal WASP (N-WASP) [1, 39], although in vitro data suggests that a higher level of activation occurs through Nck as well [103].

WASPs and SCAR/WAVEs are fundamental actin cytoskeleton remodeling proteins that act to integrate upstream signaling events and translate them to downstream ef-fectors. The WASPs and WAVEs have in common a carboxy-terminus VCA region

(verprolin homology domain, connecting domain and acidic region) through which they bind Arp2/3 and G-actin [48,59, 78]. WASP is auto-inhibited, and activation relieves interactions between its GTPase binding domain and the carboxy terminal [44].

Bind-ing of both Cdc42 to the CRIB domain and (PI(4,5)P2) to the basic region enhance its

activity [92]. Once the auto-inhibition is relieved, WASP binds to Arp2/3. Dimerization enhances the activity of WASP, which then binds to Arp2/3 in a 1:1 complex [68]. The N-terminus of N-WASP can bind to F-actin, while the C-terminus can bind to G-actin, thus bringing Arp2/3, an actin monomer and an actin filament into close proximity [24]. SCAR/WAVEs are trans-inhibited, and it is through the action of Rac and Nck that this trans-inhibition is relieved [23]. Once the trans-inhibition is relived, SCAR/WAVEs can go on to bind to Arp2/3. This binding of the Arp2/3 complex by WASP or SCAR/WAVE promotes actin nucleation [54,55,92,120]. WAVE2 can also be recruited to the membrane

via binding to PI(3,4,5)P3 through its basic domain and this recruitment o the leading

edge is necessary for lamellipodia formation [67].

The Arp2/3 complex is composed of seven proteins- ARPC (Arp complex subunits)

1-5 and Arps (actin related proteins) 2 and 3. The complex localizes to the lamellipodium

close to the leading edge and other sites of actin polymerization [115]. Arp2 and Arp3 are 45% homologous to an actin monomer, and the Arp2/3 dimer is thought to mimic an actin dimer and promote nucleation of a daughter filament, with the complex remaining at the pointed end of the new filament [18]. Studies show that the CA domain of WASP and SCAR/WAVEs can bind actin, and it is hypothesized that it is this trimer of G-actin, Arp2 and Arp3 that form the nucleating site of a new actin filament [56]. However, this activity alone is not enough to account for the increase in actin polymerization, and further studies show that the VCA domain is also responsible for bringing Arp2 and

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Arp3 closer together [31,91], and it is this conformational change that contributes to the increased polymerization. Experiments done by Amann and Pollard show that Arp2/3 nucleates new filaments from existing ones [6, 7], and cryo-electron microscopy shows Arp2/3 binding to a mother actin filament and nucleating a daughter filament at the pointed end [109]. In vitro experiments have shown that polymerization occurs more efficiently when Arp2/3 is pre-incubated with existing filaments. These observations have led to the proposed dendritic nucleation model of Arp2/3 action [55], in which these daughter filaments branch off the sides of pre-existing filaments at an angle of

700 [64]. The Arp2/3 complex then remains at the site of the branch, with Arp2 and

Arp3 remaining as the first two subunits of the daughter filament [109].

Also contributing to actin polymerization at the leading edge is cofilin, which is acti-vated at the leading edge of the cell prior to Arp2/3 recruitment [21], and acts to generate new barbed ends through the severing of existing capped actin filaments [8,30]. A pool of

cofilin is held in an inactive state at the membrane through binding to PI(4,5)P2 [105,121],

and stimulation of the cell with a growth factor such as EGF triggers a transient release of this cofilin at the leading edge [17]. (It should be noted that cofilin has widely been observed to act as an F-actin depolymerizing and debranching factor, but evidence in-dicates that this function is distinct from its role as an F-actin severing protein.) Upon stimulation with EGF, phospholipase Cy (PLCy) is activated via phosphorylation by

EGFR [63]. PLCy can then hydrolyze PI(4,5)P2 into diacylglycerol (DAG) and inositol

trisphospate (IP 3) and this hydrolysis releases cofilin. Once released, the cofilin locally

binds to F-actin and severs filaments, releasing free barbed ends [105]. Actin polymer-ization occurs at the site of these free barbed ends, and Arp2/3 preferentially binds the ATP-cap of an actin filament, so the action of cofilin could create more Arp2/3 binding sites [21]. These free barbed ends are also available for capture by the Ena/VASP family of proteins, Mena, EVL and VASP, which act as anti-capping factors, promote the elon-gation of actin filaments, and bundle F-actin [11, 15,94]. Ena/VASP proteins also work to catalyze the transition of profilin-bound actin monomers from the cytosolic pool onto a growing actin filament [25]. The pathway leading to membrane protrusion is outlined in figure 1-2.

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U

* EGF Arp213 EGFR F-acn coflin 0 Cdc42 Gactin + profdtn

o

Rac Prti SWASP V M. Scar/WAVE V pmffn

Figure 1-2: The process of membrane protrusion downstream of growth factor signaling. Stimulation with EGFR activates PL Cy, which cleaves PIP2, releasing cofilin, which sev-ers capped barbed ends. Cdc42 and Rac are activated concurrently, which leads to the activation of WASPs and Scar/WAVEs, resulting in activation of Arp2/3. Arp2/3 can bind to actin filaments and promote nucleation and branching. Mena binds to barbed ends and acts as an anti-capping factor as well as helping to recruit profilin- G-actin com-plexes, leading to growth of actin filaments and membrane protrusion. Figure adapted from [76,78].

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1.3

The Ena/VASP Family of Proteins: Actin

Cy-toskeletal Regulators

Mena, a member of the Ena/VASP (Enabled/vasodilator stimulated phosphoprotein) family of proteins, is the mammalian version of Drosophila enabled (ena), which was first described by Gertler, et al. in 1990 as a genetic suppressor of abl [28]. It was later found that the protein contains proline-rich SH3 motifs, and can bind both Abi and Src in

Drosophila, and that ena mutants have defects in the axonal architecture of the central

and peripheral nervous systems [27]. In 1996, the murine version of ena, mammalian ena, or Mena, was reported, along with EVL (Ena/VASP like protein) [29]. The Ena/VASP family of proteins are important in axon guidance, as mice homozygous null for Mena

display defects in nerve fiber tract formation [51]. Mena(-) VASPC-/-) double mutant

mice die perinatally and also show defects in the formation of fiber tracts in the central and peripheral nervous systems [49,57]. Ena/VASP proteins have a role in neuritogenesis and neural tube closure [49,51]. They localize to focal adhesions, the leading edge of migratory cells and the tips of filopodia in neuronal growth cones [29, 51, 89]. This localization implies an important function in the rearrangement of the actin cytoskeleton in response to external cues.

The members of the Ena/VASP family share a common structure of an N-terminal EVH1 (Ena/VASP homology 1) domain, a proline-rich domain, and an EVH2 domain. Mena also contains a 5-amino acid repeat with the consensus sequence LERER between the EVH1 and proline-rich domain. The EVH1 domain is involved in directing its sub-cellular localization via protein-protein interactions with a proline-rich motif, FPPPP

(FP4). Mena specifically has several alternative exons, which will be discussed in more

detail later. (Figure 1-3)

Mena can bind zyxin and vinculin, components of focal adhesions, as well as

Lamel-lipodin, a regulator of lamellipodial dynamics, all of which contain FP4 motifs [29,46].

Mena and VASP also bind to profilin [29,88]. This binding is mediated by the proline-rich domain, which can bind SH3- and WW-domain containing proteins as well [13]. The proline-rich domain has three distinct regions: a regulatory site, a recruiting site for

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+++ ;l

SH3, G-actin, ,

WW Domain Binding Binding

FP4 Binding ,' # Profiin Binding F-actin Coiled

Binding Coil

Figure 1-3: Mena, with the domains shown as well as the location of alternatively spliced

exons. The EVH1 domain binds FP4 motifs, the proline rich domain binds SH3 and

WW domains, as well as profilin, and the G- and F-actin binding motifs are in the EVH2 domain, as well as the coiled-coil motif, which mediates tetramerization The sites of alternative exons are shown.

binding profilin- G-actin, and a loading site for moving profilin- G-actin to the G-actin

binding site in the EVH2 domain [25]. The EVH2 domain mediates tetramerization and

also binds and bundles F-actin, in addition to binding G-actin [11,110]. Structural stud-ies suggest that profilin- G-actin first binds to the proline-rich domain of Ena/VASP, and then the proline-rich region directs the complex to the GAB domain within the EVH2 domain [25]. It is the EVH2-mediated interactions with actin filaments that tar-get Ena/VASP proteins to the leading edge of cells [53], and free barbed ends are required to target Ena/VASP proteins to the leading edge and filopodial tips [15]. Ena/VASP proteins are also subject to phosphorylation by the c-AMP and c-GMP serine/threonine protein kinases, PKA and PKG. VASP has three phosphorylation sites: Ser157, Ser239 and Thr278, while Mena has just the first two, and EVL has only the first one [45]. Studies show that phosphorylation of Mena at Ser236 is essential for its function, but not its sub-cellular localization

[53].

Work by Bear, et al. demonstrates that Ena/VASP proteins negatively regulate cell movement. When overexpressed, Mena and VASP cause decreased cell migration in a dose dependent manner. Sequestration of Mena or VASP (using a construct that directs

an EGFP tag fused to the EVH1 binding motif FP4 to the mitochondrial membrane

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significantly faster than control cells. Another construct that sequesters Ena/VASP

proteins away from focal adhesions was used, (FP44-cyto), and this displacement has no

effect on cell motility. A third construct, FP4-CAAX, localizes Ena/VASP proteins to

the leading edge. Upon localization to the leading edge, cell motility decreases. This was a surprising find, given previous research that showed that Ena/VASP proteins were required for actin tail formation and rocketing in Listeria monocytogenes [14].

In a subsequent study, Bear, et al. went on to show that Ena/VASP proteins regulate cell motility by controlling the geometry of the actin filament network in the lamel-lipodium. This follow-up paper used the same targeting constructs to show that when

FP4-mito is expressed and Ena/VASP proteins are sequestered away from the leading

edge lamellipodia protrude much more slowly but persist for longer periods of time. Cells in which Ena/VASP proteins are targeted to the membrane have increased protrusion

velocity. Examination of the underlying actin network shows that FP4-mito cells have

shorter and more branched actin filaments, while FP4-CAAX cells have longer and less

branched filaments. This study also showed that, in vitro, VASP can capture uncapped, but not capped, barbed ends. The Ena/VASP-dependent changes in the actin network of the lamellipodium are consistent with this observation. All of this evidence suggests that Ena/VASP proteins act in an anti-capping manner [15].

Further evidence for the anti-capping activity was provided in a study by Barzik et al., which showed that Ena/VASP proteins associate at or near actin filament barbed ends, promote polymerization, and restrict the access of capping proteins. In in vitro studies, VASP prevents capping proteins from binding to barbed ends. This effect could be explained by two hypotheses that are not mutually exclusive: VASP increases the rate of actin polymerization, but is not involved in actin nucleation and/or that VASP works as an anti-capping factor by antagonizing capping protein. VASP was shown to be capable of blocking the activity of several barbed end-binding proteins, suggesting that it can prevent capping by direct association with barbed ends. A decrease in G-actin con-centrations further suggests that VASP can block the binding of capping protein without interfering with filament elongation, although F-actin is also increased as compared to controls without VASP in the presence of capping protein. The EVH2 domain is sufficient

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profilin- G-actin EVH Recruitng Prohine-rich Domain Loading GAB FAR

Pro

Figure 1-4: Ena/VASP proteins as anti-capping factors. Tetramers of VASP bind to

actin filaments, and the EVH1 domain binds to FP4 motifs to help localize the protein

at the membrane. The mechanism through which profilin- G-actin is loaed and actin monomers are added onto a growing filament is shown. (Figure adapted from [13]).

for the anti-capping activity, and the G- and F-actin binding domains are necessary. In order for VASP to have anti-capping activity, it must form a tetramer. The binding of profilin to VASP enhanced actin polymerization and VASPs anti-capping activity [12,72].

A study using total internal reflection fluorescent (TIRF) microscopy showed the

anti-capping function of Ena/VASP proteins, as well. Ena/VASP proteins promote actin assembly by interacting directly with barbed ends, recruiting profilin-actin, and blocking capping. A Mena tetramer working as an anti-capping factor can be seen in figure 1-4.

Taken together, this work shows that Ena/VASP proteins capture free barbed ends and act as anti-capping factors to promote actin filament elongation. This role allows them to exert direct effects on cell motility and on the ability of cells to protrude. This in turn suggest a role for Ena/VASP proteins in metastatic cancer.

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1.4

Mena in Breast Cancer

Mena is expressed in several tumor types, including pancreatic, glioblastoma and breast

[38, 77, 112]. In the in vivo invasion assay utilized by the Condeelis lab, Mena was overexpressed four-fold in both xenograft and primary mammary carcinomas [111,112],

and it is in this cancer type that most of the research has been done. Overexpression of Mena seems to be an early event in breast cancer and higher levels of Mena expression have been shown to correlate with increased invasiveness [61], and a similar correlation has been shown for EVL [40].

In an attempt to discover prognostic markers for hematogenous dissemination, a test to define a tumor microenvironment of metastasis (TMEM) was devised. The idea of a TMEM was based around the observation that motile tumor cells about to intravasate are found in association with a perivascular macrophage. Therefore, a TMEM was defined as a tumor cell staining positively for Mena in contact with a macrophage and an endothelial cell. Tumor samples from patients with metastatic disease were matched to samples from patients without metastatic disease, and stained for Mena, macrophages and endothelial cells. The results showed no association between TMEM density and tumor size, grade or lymph node metastasis. However, for every 10-unit increase in TMEM density, the odds ratio for systemic metastasis was 1.9 [90]. This could be a possibly useful prognostic marker clinically for risk of metastasis.

When mammalian Ena was first reported, three alternatively spliced forms of it were

found, denoted

+,

++ and

+++

[29], and another exon was later found by Di Modugno,

et al., and named 1la [60]. Mena+ is preferentially expressed in the nervous system,

though no tissue-specific expression pattern has been found for ++ or +++. Mena11a

is expressed in normal epithelial cells and poorly invasive breast cancers that have an epithelial phenotype, but not in cancers that have a mesenchymal phenotype [60]. When invasive cells collected from the in vivo invasion assay were analyzed for the various

Mena isoforms, they found that the

+++

exon was up-regulated, while the 11a exon was

down-regulated [32]. In light of these findings, the +++ exon was renamed INV, and

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Philippar et al. studied the MenaINV isoform in more detail using MTLn3 cells. When MTLn3 tumor cells expressing either EGFP-Mena or EGFP-MenaINV are orthotopically injected into the rat mammary gland, Mena (or MenaINV) preferentially localized to the cytoplasm with enrichment at the leading edge of cells in 15-20% of motile cells. Within the primary tumor, Mena localizes to cell-cell junctions and rapidly forming pro-trusions. Expression of Mena increases the fraction of motile cells within the tumor, as compared to the control, and expression of MenaINV increased the percentage even further. Expression of MenaINV also significantly increases the number of lung metas-tases in animals injected with these cells as compared to Mena or GFP control cells. Measurements of primary tumors shows no significant difference in growth between cell types, therefore the phenotype is not due to a growth advantage. The expression of both

Mena and MenaINV promotes macrophage-independent invasion in vitro. This decreased

dependence on macrophages indicates an increased sensitivity to growth factor signals, as the paracrine loop is no longer present. Addition of an EGFR inhibitor to the assay, decreases invasion to baseline levels, indicating that the small amount of EGF present in the culture media is enough to promote invasion. Cells expressing the Mena isoforms were analyzed for their responsiveness to EGF by looking at the fold change in mem-brane protrusion. MenaINV is sensitive to EGF stimulation down to 0.025 nM EGF, as compared to wild type MTLn3 cells, which respons maximally at 5 nM EGF, and stops responding at 0.5 nM EGF. These results were recapitulated in an in vivo invasion assay. When cells were stimulated with 0.5 nM EGF in vitro, the number of free barbed ends increases in cells expressing Mena or MenaINV. Actin polymerization at the leading edge is not due to global changes in EGFR signaling, but instead is cofilin dependent. This work shows that MenaINV works as a regulator of carcinoma cell invasion by potentiating the cells response to EGF and increasing cell motility [76].

MTLn3 cells respond to stimulation by EGF with a cessation of ruffling followed by an extension of a broad lamellipodium, with a maximal increase of membrane area at 5 nM

EGF. This membrane extension is a result of actin polymerization at the leading edge [95].

EGF-induced increases in barbed ends are due to the activation of phospholipase C'y

(23)

SH27

5'-ptase

Protine

Ri'ch,'

SAM

...FP4... FP4... FP4..

Mena

Figure 1-5: The domain structure of SHIP2. The SH2 (Src Homology 2) domain binds phosphotyrosines. The 5-phosphatase domain is the catalytically active region. The

proline rich domain contains FP4 motifs which are thought to interact with the EVH1

domain of Mena. The SAM domain mediates protein-protein interactions. at barbed ends play roles in invasion and metastasis in breast cancer [21,63].

1.5

SHIP2, an SH2-Domain Containing 5'-Phosphoinositide

Phosphatase

When activated by the binding of EGF, EGFR activates phosphatidylinsoitol-3-kinase

(P13K), which in turn phosphorylates PI(4,5)P2, making PI(3,4,5)P3. (P13K is an effector

of the EGFR pathway, and is often disregulated in cancer [106].) PI(3,4,5)P 3 is then

dephosphorylated by the src homology 2- containing inositol 5'-phosphatase 2 (SHIP2)

to form PI(3,4)P2. SHIP2, also known as inositol polyphosphate 5'-phosphatase-like

protein- 1, was first identified in 1997, based on homology to SHIPI, a phosphoinositol 5'-phosphatase discovered in 1996. It is highly homologous to SHIP1, however SHIP1 expression is restricted to hematopoietic cells, while SHIP2 is more ubiquitously expressed in tissues [741. SHIP2 contains a src homology 2 (SH2) domain at its N-terminus, a short proline-rich domain, a catalytic 5'-phosphatase domain, another proline rich domain containing an NPXY motif that can bind to phosphotyrosine binding (PTB) domains, and a sterile a motif (SAM) domain at its C-terminus [47]. (Figure 1-5)

As a regulator of P13K-mediated events downstream of growth factor signaling, SHIP2

(24)

Pl(4,5)P2 - --3

|

PI(3,4,5)P2

PIPl(3,4)P2

Lpd

Figure 1-6: The action of SHIP2 downstream of EGF signaling.

PTEN acts with SHIP2 to suppress the P13K pathway by catalyzing the formation of

PI(4,5)P2 from PI(3,4,5)P3. (Figure 1-6)

These 3'-phosphoinositides are key players in cell motility signaling. PI(4,5)P2

ac-counts for only about 5% of the lipids in a cell membrane, and only 0.25% of inositol-containing lipids are phosphorylated at the 3-position, which suggests that these lipids

serve regulatory functions in the cell [87,93]. It has been shown that PI(3,4,5)P3

accu-mulates at the front of chemotaxing cells, and helps to translate spatial information from gradients into directed movement [26]. In a study using SHIP1(-/-) neutrophils, Nishio

et al. showed that SHIPI and P13K were critical for the accumulation of PI(3,4,5)3 and

PI(3,4)P2 at the leading edge of chemotaxing neutrophils [65].

SHIP2 is tyrosine phosphorylated upon stimulation with growth factors and insulin,

and co-precipitates with EGFR and the adaptor protein Shc upon EGF stimulation

[34,75]. Phosphorylation at tyrosines 986, 987 and 1135 were thought to be necessary for

SHIP2 activation [86], but other studies show that this phosphorylation is not required for SHIP2s phosphatase activity [102]. In the absence of any stimuli, the SH2 domain and C-terminus of SHIP2 are inhibitory, keeping the basal activity level low [86]. SHIP2 localizes to focal adhesions and lamellipodia and interacts with the cytoskeleton proteins filamen and vinexin [22,73]. When overexpressed, an increase in adhesion is seen, whereas a decrease in adhesion and an inhibition of cell spreading is seen upon loss of SHIP2 [85].

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enhances receptor degradation [82]. Loss of SHIP2 increases ligand-induced receptor internalization of the ephrin receptor Eph2A [122]. A study done in cells derived from a

SHIP2 knockout mouse showed that when the cells are stimulated with serum, there is

an increase in PI(3,4,5)P3 levels. The baseline levels remain unchanged, and this effect is

not seen when cells are stimulated with EGF [16]. However, another study showed that

knocking down SHIP2 increased PI(3,4,5)P3 levels both under resting and stimulated

conditions [122].

A second substrate for SHIP2 is PI(4,5)P2 [102], which plays a key role in the

dynam-ics of clathrin-coated pits, as it binds all known endocytic clathrin adaptors and other endocytic factors including dynamin [69,123]. Recent work shows that SHIP2 is local-ized at clathrin-coated pits and is a component of early-stage pits, leaving just before fission (F. Gertler, personal communication). This localization is mediated by inter-sectin, which interacts with SHIP2 [119]. Upon knockdown of SHIP2, clathrin-coated pit lifetime decreases by 25%, suggesting that SHIP2 negatively regulates the maturation of clathrin-coated pits (F. Gertler, personal communication). This study also confirmed

the previous observation that PI(4,5)P2 is a substrate for SHIP2, and discovered that

the ration of PIP2/PIP in SHIP2 knockdown cells is approximately 40% higher than in control cells. They suggest as an explanation for faster growth of clathrin-coated pits

in SHIP2 knockdown cells that higher levels of PI(4,5)P2 promote faster recruitment of

endocytic clathrin adaptors and their accessory proteins. This work also shows that an

increase in the levels of PI(3,4,5)P 3 shortens the lifetime of clathrin-coated pits by 20%.

SHIP2 is relevant in human disease. Much work has been done on SHIP2 in insulin

signaling, as knockout mouse studies show it to have a crucial role in insulin sensitivity

[99]. SHIP2 is also involved in host cell colonization of pathogenic bacteria. Enteropathic E. coli (EPEC) form actin-rich pedestals when they adhere to epithelia cells. This

pedestal formation occurs via the recruitment of SHIP2, which engages Shc and creates

a PI(3,4)P2 enriched raft for the binding of Lamellipodin [101]. Lamellipodin (Lpd), a

protein involved in the regulation of lamellipodial dynamics, has been shown to bind to

PI(3,4)P2 via its plextrin homology (PH) domain. Lpd also contains six FP4 motifs,

(26)

first direct link between Ena/VASP proteins and phospholipid signaling molecules. Lpd localizes to the tips of filopodia and the leading edge of cells, similar to Ena/VASP proteins. Upon overexpression, lamellipod protrusion is more rapid and frequently turns into ruffles, similar to the phenotype seen by Bear et al. when Ena/VASP proteins

are overexpressed. If Ena/VASP proteins are sequestered using the FP4-mito construct,

the Lpd phenotype is suppressed. Upon knockdown of Lpd, lamellipodial protrusion is impaired, even more so that when Ena/VASP is sequestered away from the leading edge [463. Therefore Lamellipodin is crucial for lamellipodial dynamics and could function as a link between P13K signaling at the leading edge and effectors of the actin cytoskeleton. SHIP2 is also implicated in cancer, including breast cancer, with high expression of

SHIP2 in tumors correlating with decreased disease survival [84]. SHIP2 protein levels

are upregulated in several breast cancer cell lines [83]. Overexpression of SHIP2 in the human breast cancer cell line MDA-MB-231 enhances cell proliferation, while suppression of SHIP2 slows tumor growth and results in fewer lung metastases in nude mice. A further study shows that SHIP2 knockdown cells migrate significantly more slowly than control cells in wound healing assays [81]. Taken together, these results indicate an important role for SHIP2 in cancer. The fact that PTEN, a known tumor suppressor, acts on the same substrate as SHIP2 further supports a role for SHIP2 in cancer progression and metastasis.

Based on these observations, as well as the fact that SHIP2 catalyzes the production of Lpds binding partner and Lpd binds Ena/VASP proteins, we looked at the effects of knocking down SHIP2 in MTLn3 cells, a rat-derived metastatic breast carcinoma line. In these experiments we observed that decreased SHIP2 expression causes increased lamellipodial protrusion in response to EGF, and that reduction of SHIP2 does not change the amount of Mena or Lpd that translocate to the leading edge of cells as compared to control cells upon EGF stimulation.

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Chapter 2

Methods

2.1

Cell Culture and Fluorescence-Activated Cell

Sort-ing

MTLn3 cells were cultured in alpha-MEM (Gibco), with 5% fetal bovine serum, L-glutamine and antibiotics added. Fluorescence-activated cell sorting (FACS) was used to sort cells for GFP positive population. The effectiveness of the knockdown was con-firmed by Western blot. Lystates were prepared using an NP-40 buffer (130 mM NaCl,

.875% NP-40, 43.8 mM TRIS, 1 mM Na3VO4, 40 mM #-glycerophosphate, 50 mM NaF,

1:1000 leupeptin, 1:200 Pefabloc), separated by SDS-PAGE for 1.5 hours at 120V,

trans-ferred to an Immobilon membrane (Millipore) for 1.5 hours at 75V, 4C, and probed with anti-SHIP2 (1:1000) [a gift from Pietro de Camilli] and anti-GAPDH (1:1000) [Cell Sig-naling]. Species-specific secondary antibodies conjugated to horse radish peroxidase and Amersham ECL Plus detection reagents (GE Healthcare) were used to detect the signal.

2.2

Transient Transfections

The SHIP2 and luciferase hairpins were previously subcloned into the lentiviral vector

pLL3.7 that expresses shRNA under the mouse promoter U6. A CMV-EGFP reporter

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was made against rat SHIP2, and recognizes the sequence 5' - GGATTAGCATTGAT AAGGA -3' in exon 24. Transient transfections of SHIP2 and luciferase were done using

the Neon transfection system (Invitrogen). The optimized conditions for MTLn3 cells with the Neon system were 2 pulses of 1400 volts for 20 milliseconds. To do the Neon transfection system, cells were harvested and pelleted by spinning them down at 1000 rpm for 3 minutes. The pellet was then washed by resuspending in PBS, and then pelleted again. Cells were then resuspended in the Neon kit resuspension buffer, mixed with the appropriate plasmid, electroporated and plated in drug-free alpha-MEM.

2.3

Protrusion Assays

MTLn3 cells were plated on glass-bottom (MatTek) dishes pre-treated with IM HCl, washed with 70% ethanol followed by PBS, then coated with collagen at 100 pg/mL. Cells were plated at a density of 80,000 cells per dish and allowed to sit down overnight. Cells were starved in Leibovitzs L-15 media (Gibco) supplemented with 0.35% bovine serum albumin for 3-4 hours. Cells were then stimulated with 5 nM EGF by a bath application and imaged for 10 minutes with images taken every 10 seconds with a Hammamatsu CCD camera attached to a Nikon TE300 differential interference contrast (DIC) microscopy.

A 40X DIC oil-immersion objective was used. Time-lapse images were captured use

MetaMorph software (Molecular Devices, Downington, PA). Membrane protrusion was tracked and quantified ImageJ (National Institutes of Health). Area measurements for

each cell were standardized over the area of the corresponding cell at t = 0 and plotted

over time after EGF stimulation.

2.4

Immunofluorescence

Cells were plated onto glass coverslips coated with collagen at 100 pg/mL, starved in

L15 media, stimulated with 5 nM EGF or left unstimulated and then fixed using PHEM (60 mM PIPES pH 7, 60 mM HEPES pH7, 10 mM EGTA pH 8, 2mM MgCl2, 120 mM

(29)

a Mena monoclonal antibody (1:1000), an affinity purified Lamellipodin antibody (1:100) and AlexaFluor 647 phalloiden (1:250) [Molecular Probes]. Anti-EGFR pY1173 (1:100) [Epitomics] and a pan phospho-Tyrosine antibody(1:400) [Cell Signaling, no. 9411] were also used. Species-specific secondary antibodies conjugated to AlexaFluor594 were used at 1:250 to detect the primary antibodies. Cells were imaged using a DeltaVision micro-scope with an Olympus 60x/1.4NA Plan Apo oil-immersion objective (Applied Precision, Issaquah, WA). Exposure times ranged from 0.2 seconds to 1.25 seconds, and between

0.6 pm and 1.4 pm stacks were taken. Images were collected, deconvolved and projected

using softWoRX software (Applied Precision, Issaquah, WA). Line scans were done using ImageJ (National Institutes of Health) and data analysis was done using a MATLAB script.

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Chapter 3

Results

3.1

SHIP2 Is Knocked Down in GFP-Positive MTLn3

Cells

The vector pLL 3.7, a lentiviral vector containing an EGFP sequence for detection of expression, as well as an shRNA sequence against rat SHIP2, was used to decrease expression of SHIP2. Cells transfected with a luciferase shRNA were used as a control. Wild type MTLn3 cells, derived from a metastatic rat breast carcinoma, were transiently transfected and plated overnight before being sorted for GFP expression to obtain a pure population. After an additional 18 hours in culture, cells were lysed using an NP-40 buffer, and samples were separated using SDS-PAGE. A Western blot was performed using anti-SHIP2 and anti-GAPDH as a loading control. The results of the blot show that SHIP2 is effeciently knocked down in transfected cells (Figure 3-1)

3.2

SHIP2 Knockdown Increases Membrane

Protru-sion as Compared to Control Cells

The role of SHIP2 in actin cytoskeleton dynamics was assessed by membrane protrusion in MTLn3 cells in which SHIP2 expression was decreased. Cells transfected with a luciferase shRNA were used as a control. Wild type cells were analyzed as well. Cells

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250 a-SHIP2 150

100

soEF~h

37 ..O a-GAPDH

Figure 3-1: Successful knockdown of SHIP2 in shRNA transfected MTLn3 cells.

Cells were transfected with control or SHIP2 shRNA hairpins. Samples were blotted for SHIP2 and GAPDH as a loading control.

were directly plated after transfections on collagen coated MatTek dishes, and allowed to sit down overnight. Prior to stimulation cells were starved in serum-free media for four hours. Upon stimulation with 5 nM EGF, cells in which SHIP2 expression had been decreased showed a remarkable increase in membrane protrusion, measured as fold change, in comparison to wild type or luciferase control cells (Figure 3-2(a).) Cells in which SHIP2 expression was decreased had a lamellipod protrusion of an average of

1.7-fold(t0.08SEM) greater than their starting area after six minutes of stimulation

with 5 nM EGF, while wild type cells protruded 1.3-fold (±0.035SEM) and luciferase control cells protruded 1.2-fold (±0.024SEM) in comparison. (Cells were not sorted before performing these experiments, however only cells expressing GFP were quantified.)

A dose response experiment was then done to determine the degree of sensitivity to EGF stimulation. Cells in which SHIP2 expression had been decreased protruded in

response to significantly lower concentrations of EGF, continuing to protrude at 0.5 nM

EGF, and even at 0.1 nM EGF, as compared to control cells (Figures 3-2(b) 3-2(c).),

indicating that loss of SHIP2 sensitizes the cells to EGF by 20-fold. Cells in which SHIP2 expression was decreased had a lamellipod protrusion of 1.76-fold (t0.22SEM) greater than their starting area after six minutes of stimulation with 0.5 nM EGF, while wild type cells protruded 1.25-fold (±0.05SEM) and luciferase control cells protruded

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1.23-fold (±0.06SEM) in comparison. After stimulation with 0.1 nM EGF, cells in which

SHIP2 expression was decreased had lamellipod protrusions of an average of 1.35-fold

greater than their starting area after six minutes of stimulation, as compared to 1.23-fold

(±0.026SEM)

for wild type cells and 1.2-fold (±0.04SEM) for luciferase control cells.

These results mimic the results seen by Philippar, et al. in the MenaINV cell lines [76].

3.3

The Amount of Mena Localized to the Leading

Edge Upon Stimulation with EGF Increased in

SHIP2 Knockdown Cells

We next asked if the localization of Mena or Lamellipodin to the leading edge was al-tered in MTLn3 cells in which SHIP2 expression had been decreased. Mena and Lpd translocate to the leading edge upon stimulation with growth factors [29,46. Cells were transiently transfected with SHIP2 or a luciferase control shRNA, plated directly onto collagen coated coverslips and allowed to sit down overnight. Sorting was not done prior to plating, however only cells expressing GFP were chosen for analysis. Prior to stimulation and fixing, cells were staved for four hours in serum-free meida. Cells were stimulated with 5 nM EGF and fixed after 1 minute. They were then stained for either Mena or Lamellipodin along with phalloidin conjugated to AlexaFluor 647, which binds to F-actin. Species specific secondary antibodies conjugated to AlexaFluor 594 were used for detection of primary antibodies. (Figures 3-3, 3-4.)

No obvious structural defects in the actin cytoskeleton were observed in cells in which SHIP2 expression had been decreased as compared to control cells. Line scans of cells at

60 seconds post stimulation to measure fluorescence intensity showed an increase in the

amount of Mena recruited to the leading edge in cells with reduced SHIP2 expression. Cells transfected with the SHIP2 shRNA also showed an increase in the level of Mena at the edge of cells prior to stimulation. The same observation was not true for Lamellipodin. There was no difference in the amount of Lpd at the edge of cells prior to stimulation, and the same amount of Lpd was recruited to the leading edge post-stimulation in cells

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-MTLn3 Wild Type +SHIP2 knockdown 1.95 Luciferase Control '01.75 i S1.35 0.95 - - - -- - - - -0 50 100 150 200 250 300 350 400 450 500 Time (seconds) (a) 2.15 -MTLn3 Wild Type +SHIP2 Knockdown 1.95 Luciferase Control 01.75-1755 2 1.35 . 1 0.95 0 50 100 150 200 250 300 350 400 450 500 Time (Seconds) (b) -MTLn3 Wild Type +SHIP2 Knockdown 1.95 Luciferase Control 01 21.s 1.5 0 50 100 150 200 250 300 350 400 450 500 Time (Seconds)

Figure 3-2: SHIP2 Knockdown Increases Membrane Protrusion in Response to EGF (a) Lamellipod protrusion after 5 nM EGF stimulation. Results represent 37 or more cells analyzed. Error bars indicate SEM.

(b) Lamellipod protrusion after 0.5 nM EGF stimulation. Results represent 6 or more

cells analyzed. Error bars indicate SEM.

(c) Lamellipod protrusion after 0.1 nM EGF stimulation. Results represent 12 or more cells analyzed. Error bars indicate SEM.

(35)

Mena

Actin

Luciferase

SHIP2 kd

0"

GFP

Mena

Actin

Merge

Lucierase

SHIP2 kd

60"

Figure 3-3: Localization of Mena to the leading edge upon stimulation with EGF is increased in SHIP2 knockdown Cells

Immunofluorescence of MTLn3 cells transfected with shRNA for SHIP2 or luciferase and stained for Mena and actin at the indicated time points.

Merge

(36)

Lpd

Actin

Luciferase

SHIP2kd

0"

GFP

Lpd

Actin

Merge

Luciferase

SHIP2 kd

60"

Figure 3-4: Localization of Lpd to the leading edge upon stimulation with EGF is un-changed in SHIP2 knockdown cells.

Immunofluorescence of MTLn3 cells transfected with shRNA for SHIP2 or luciferase and stained for Lpd and actin at the indicated time points.

(37)

with reduced SHIP2 as compared to control cells. (Figure 3-5.)

3.4

Loss of SHIP2 Expression Results in a Reduction

in Focal Adhesions

MTLn3 cells in which SHIP2 expression was reduced by transient transfection of an shRNA were plated on collage-coated coverslips and allowed to sit down overnight. They were then starved in serum-free media for 4 hours and stimulated with 5 nM EGF for 1 minute. A control luciferase shRNA was also used. Cells were then stained for pan phospho-tyrosine as a marker of focal adhesions. Although the staining was a bit dirty, focal adhesions could clearly be seen. Cells in which expression of SHIP2 was reduced appear to show a reduced focal adhesion signal, but further quantitative experiments will be needed to prove this. (Figure 3-6.)

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140 120 100-80. C C 40 C 20 0 . 0 E -1 Mena 60 seconds x (ptm) Lamellipodin 0 seconds 40U - Luciferase 120 - Ship2 100- 80-60 40-20 0 1 -1 0 1 x ([Lm) Actin 0 seconds 140 2- 120 _2 100 80 40 20 0 z 0 2 E -1 140 120 100 2 80 6 0 2 0 o40 OW 2 0 00 2 u.. 1 14 0 8 2 -LLi x ( m) x([pm) Actin 60 seconds -- Luciferase 0 -Ship2 0 0 0 0-0 -1 0 1 x ([Lm) x([ m)

Figure 3-5: More Mena is recruited to the leading edge of cells upon stimulation with

EGF.

Representative line scans of the leading edge of MTLn3 cells transfected with either shRNA for SHIP2 or luciferase and stained for Mena or Lamellipodin and actin at indi-cated time points after 5 nM EGF stimulation. Error bars indicate SEM.

- Luciferase -Ship2

-I

1 CD 0L 120 100 > 80 460 C 4o 0 C - Lucif erase - Ship2 Mena 0 seconds 0

(39)

pan pY

Actin

Luciferase

SHIP2 kd

0"

GFP

pan pY

Actn

Merge

Luciferase

SHIP2 kd

60"

Figure 3-6: SHIP2 knockdown cells appear to have fewer focal adhesions.

Immunofluorescence of wild type cells transfected with shRNA for SHIP2 or luciferase control and stained for pan phospho-tyrosine as a marker of focal adhesions and actin at the indicated time points.

Merge

(40)
(41)

Chapter 4

Discussion

SHIP2 is a 5'-phosphatase that can act on either PI(3,4,5)P3 or PI(4,5)P2 to

dephospho-rylate the 5 position and has previously been implicated in a variety of human diseases, including diabetes and cancer. Loss of SHIP2 results in sensitivity to insulin [19], and overexpession of SHIP2 has been observed in human breast cancers [83]. SHIP2 has been shown to bind directly to EGFR [75], as well as be recruited to early-stage endocytic clathrin coated pits, via interaction with intersectin (F. Gertler, personal

communica-tion). Dephosphorylation of PI(3,4,5)P3 by SHIP2 produces PI(3,4)P2, to which

Lamel-lipodin binds. LamelLamel-lipodin can then bind Mena, an important regulator of the actin cytoskeleton [46].

Our results show that decreasing the expression of SHIP2 in MTLn3 cells results in increased membrane protrusion upon stimulation with 5 nM EGF. This increase in protrusion can be seen at 0.5 nM and even down to 0.1 nM EGF, suggesting that loss of SHIP2 sensitizes the cell to signaling by the EGFR pathway, and that SHIP2 normally has an inhibitory effect on EGFR signaling. We next looked to see if this effect could be due to an increased recruitment of Mena to the leading edge in SHIP2 knockdown cells as compared to controls. Upon stimulation with EGF, Mena is recruited to the leading edge of cells. There was a difference in the amount of Mena recruited in SHIP2 knockdown cells as compared to control cells. We then looked to see if less Lamellipodin

was recruited to the leading edge, due to a decrease in PI(3,4P)2, but did not see a

(42)

decrease in focal adhesions in SHIP2 knockdown cells as compared with controls using a pan phospho-tyrosine antibody as a marker of focal adhesions. The staining was not of high enough quality to quantify a loss in focal adhesions, and further experiments are need to obtain a quantitative result. However, a similar observation was made by Prasad et al, namely that knocking down SHIP2 in HeLa cells led to a decrease in cell adhesion [85]. A decrease in focal adhesions could result in an increase in free Mena, allowing it to accumulate at the edge, and we did see an increase in Mena at the edge of SHIP2 knockdown cells prior to stimulation. Increased Mena at the leading edge causes increased protrusion in fibroblasts, but these protrusions are unstable and are converted into ruffles, which are unproductive for motility [14, 15]. Lamellipodia with excess Ena/VASP have longer and less branched actin filaments, and is hypothesized that Ena/VASP proteins regulate cell motility by controlling the structure of the underlying actin filament network in lamellipodia [15]. Work by Prasad et al. shows that cells in which SHIP2 levels have been reduced via RNAi migrated significantly more slowly than control cells in would healing assays [81]. An increase in Mena at the leading edge of cells in which SHIP2 expression was decreased could therefore explain the increased membrane protrusion. Studying cell motility in cells with decreased SHIP2 would be an key next

step to determine if this increase in Mena at the leading edge decreases cell motility. It is possible that the increase in membrane protrusion could be due to a shift in the relative phosphoinositide levels in the cell. SHIP2 terminates P13K signaling

by dephosphorylating PI(3,4,5)P3 to produce PI(3,4)P2, and can also dephosphorylate

PI(4,5)P2 [102]. The finding that SHIP2 can act on PI(4,5)P2 was confirmed by Nakatsu,

et al. They also show that knocking down SHIP2 results in an increase in the PIP2/PIP

ratios by 40% as compared to control cells (F. Gertler, personal communication). There-fore loss of SHIP2 has a very significant effect on the balance of phosphoinositides in the cell, resulting in an increase in PI(4,5)P2 levels. Given their critical importance in

sig-naling, actin dynamics and other cellular processes, it is perhaps unsurprising that such a large fold change was observed in membrane protrusion upon loss of SHIP2 expression.

Zoncu et al. have previously shown that an acute loss of PI(4,5)P2 leads to the

Figure

Figure  1-1:  The  process  of  invasion  and  metastasis.  A  normal  cell  becomes  cancerous.
Figure  1-2:  The  process  of membrane  protrusion  downstream  of growth  factor  signaling.
Figure  1-3:  Mena,  with  the domains  shown  as well  as the  location  of alternatively  spliced exons
Figure  1-4:  Ena/VASP  proteins  as  anti-capping  factors.  Tetramers  of  VASP  bind  to actin  filaments,  and  the  EVH1  domain  binds  to  FP 4  motifs  to  help  localize  the protein at  the  membrane
+7

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