The 5' inositol phosphatase SHIP2 regulates EGFelicited protrusion in MTLn3 cells
by
MASSACHUSETTS INSTTUTE
OF TECHNOLOGY
JUN 0 2 2010
Georgiana L. Kuhlmann
Submitted to the Department of Biology
in partial fulfillment of the requirements for the degree of
LIBRARIES
ARCHIVES
Master of Science in Biology
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June :2010
@Massachusetts Institute of Technology 2010. All rights reserved.
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
'2
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, upregulated in metastastic cells, while an epithelial isoform, Menalla, is downregulated. SH2domain 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 dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) to phosphatidylinositol
3,4-bisphosphate (PI(3,4)P 2 ) as well as the dephosphorylation of phosphatidylinositol 4,5bisphosphate (PI(4,5)P 2). PI(3,4,5)P 3 and P][(4,5)P 2 are two major phosphoinositides at
the plasma membrane and regulate a variety of cellular functions, including receptor signaling, 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
4
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.
6
Contents
1
9
Introduction
. . . . . . . . . . . . . . . . . . . .
9
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
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
1.1
Metastatic Disease in Breast Cancer
1.2
The Motility Pathway
27
2 Methods
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 . . . . . . . . . .
3.2
SHIP2 Knockdown Increases Membrane Protrusion as Compared to Control Cells.............
3.3
3.4
. .... .... ..
. . . .
. . . . . ...
31
31
The Amount of Mena Localized to the Leading Edge Upon Stimulation
with EGF Increased in SHIP2 Knockdown Cells . . . . . . . . . . . . . .
33
Loss of SHIP2 Expression Results in a Reduction in Focal Adhesions
37
4 Discussion
41
8
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, 1015% have aggressive disease, and will develop distant metastases within 3 years, though
metastases can occur ten years or more after the initial diagnosis [113]. Attempts have
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 eightyfiver 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 epithelial cell undergoes multiple biochemical changes that allow it to assume a mesenchymal
phenotype. Hallmarks of a mesenchymal phenotype include increased migratory capabilities, decreased proliferation, increased invasiveness, resistance to apoptosis, and enhanced
ability to produce extracellular matrix components [43]. This process happens both during normal development as well as in metastasis initiation. In metastasis, miesenchymal
cells are capable of exiting the primary tumor and breaking through the basement membrane. 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 (CSF1), and the macrophage secretes epidermal growth factor (EGF), which is chemotactic for
Cancerous cell in situ
Normal Tissue
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
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, antiapoptotic 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 upregulated genes in the motility pathway are involved in the process of membrane protrusion, 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
a growing filament. Actin monomers are also bound to other proteins that make spontaneous 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 protein, 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 polymerization. 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 elusive. 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)P 2). 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 nucleotide 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)P 3), 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 filopodia [33,66]. The 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 effectors.
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]. Binding of both Cdc42 to the CRIB domain and (PI(4,5)P 2 ) 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)P 3 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 G-actin, and it is hypothesized that it is this trimer of Gactin, 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
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 activated 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)P 2 [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 indicates 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)P
2
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 polymerization 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 elongation 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.
U
* EGF
Arp213
EGFR
F-acn
coflin
0
Cdc42
o
Rac
SWASP
Gactin + profdtn
Prti
V
Scar/WAVEV
M.
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 severs 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 complexes, leading to growth of actin filaments and membrane protrusion. Figure adapted
from [76,78].
1.3
The Ena/VASP Family of Proteins: Actin Cytoskeletal 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 subcellular localization via protein-protein interactions with a proline-rich motif, FPPPP
(FP 4 ). 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 Lamellipodin, a regulator of lamellipodial dynamics, all of which contain FP 4 motifs [29,46].
Mena and VASP also bind to profilin [29,88]. This binding is mediated by the prolinerich 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
+++ ;l
SH3,
WW Domain Binding
FP4 Binding ,'
#
Profiin Binding
G-actin,
Binding
,
F-actin
Binding
Coiled
Coil
Figure 1-3: Mena, with the domains shown as well as the location of alternatively spliced
exons. The EVH1 domain binds FP 4 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 studies 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 target 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 FP 4 to the mitochondrial membrane
(FP 4-mito), localizing the proteins away from the leading edge) causes cells to move
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, FP 4-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 lamellipodium. This follow-up paper used the same targeting constructs to show that when
FP 4-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 FP 4-mito cells have
shorter and more branched actin filaments, while FP 4-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 concentrations 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
profilin- G-actin
EVH
Domain
Recruitng Prohine-rich
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 FP 4 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 anticapping 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.
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 +++. Mena 1 1a
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
the splice variant as MenaINV
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 protrusions. 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 metastases 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 membrane 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
(PLCy) and cofilin [63]. Both the extension of a lamellipodium and actin incorporation
5'-ptase
SH27
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 FP 4 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)P 2 , making PI(3,4,5)P 3. (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)P 2.
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
catalyzes the removal of a 5-phopshate from PI(3,4,5)P 3, producing PI(3,4)P 2 [41, 74].
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)P 2 from PI(3,4,5)P 3 . (Figure 1-6)
These 3'-phosphoinositides are key players in cell motility signaling. PI(4,5)P 2 accounts for only about 5% of the lipids in a cell membrane, and only 0.25% of inositolcontaining 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)P 3 accumulates 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)P 2 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].
A reduction in SHIP2 levels also alters the distribution of EGFR-containing vesicles and
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)P 3 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)P 3 levels both under resting and stimulated
conditions [122].
A second substrate for SHIP2 is PI(4,5)P 2 [102], which plays a key role in the dynamics 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 localized 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)P 2 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)P 2 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)P 2 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)P 2 via its plextrin homology (PH) domain. Lpd also contains six FP 4 motifs,
and was shown biochemically to bind Ena/VASP proteins. This interaction showed the
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 FP 4 -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.
Chapter 2
Methods
2.1
Cell Culture and Fluorescence-Activated Cell Sorting
MTLn3 cells were cultured in alpha-MEM (Gibco), with 5% fetal bovine serum, Lglutamine and antibiotics added. Fluorescence-activated cell sorting (FACS) was used
to sort cells for GFP positive population. The effectiveness of the knockdown was confirmed by Western blot. Lystates were prepared using an NP-40 buffer (130 mM NaCl,
.875% NP-40, 43.8 mM TRIS, 1 mM Na 3 VO 4 , 40 mM #-glycerophosphate, 50 mM NaF,
1:1000 leupeptin, 1:200 Pefabloc), separated by SDS-PAGE for 1.5 hours at 120V, transferred 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 Signaling]. 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
cassette is included in the vector to monitor for expression. The SHIP2 hairpin sequence
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 MgCl 2 , 120 mM
sucrose, 4% paraformaldehyde) after 60 seconds of stimulation. Cells were stained using
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 microscope 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.
30
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 Protrusion 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
250
150
100
a-SHIP2
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 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 altered 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
2.15
-MTLn3 Wild Type
+SHIP2knockdown
LuciferaseControl
1.95
-MTLn3 Wild Type
+SHIP2Knockdown
LuciferaseControl
1.95
01.75-
'01.75i
1755
2
1.35
S1.35
.
-
-
0.95
0
50
150
-
-
--
-
100
-
-
250
300
200
Time (seconds)
350
0.95
-
-
400
1
450
0
500
50
100
150
200
250
300
350
400
450
500
Time(Seconds)
(b)
(a)
-MTLn3 WildType
+SHIP2Knockdown
Luciferase
Control
1.95
01
21.s
1.5
0
50
100
150
200
250
300
Time(Seconds)
350
400
450
500
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.
GFP
Mena
Actin
Merge
Actin
Merge
Luciferase
SHIP2 kd
0"
GFP
Mena
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.
GFP
Lpd
Actin
Merge
Actin
Merge
Luciferase
SHIP2kd
0"
GFP
Lpd
Luciferase
SHIP2 kd
60"
Figure 3-4: Localization of Lpd to the leading edge upon stimulation with EGF is unchanged 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.
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.)
Mena 60 seconds
Mena 0 seconds
140
140
-
120
Luciferase
2- 120
-Ship2
100-
_2 100
80.
80
C
C
-I
40
C
40
0
20
20
0
.
E
0
-1
2
0
z
E
-1
x (ptm)
x ( m)
Lamellipodin 0 seconds
140
40U
-
120
-
Luciferase
120
Ship2
100
1001
80-
CD
2
80
2
o40
60
2 0
60
4020
0L
-1
0
OW
2
1
0
0
1
x
00
u..
1
([Lm)
x([pm)
Actin 60 seconds
Actin 0 seconds
14
0
- Luciferase
120
-
--
0
Ship2
0
0
100
0
> 80
C
4o 0
C
Luciferase
-Ship2
-LLi
460
8
00
2
-1
x
([Lm)
1
0
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 indicated time points after 5 nM EGF stimulation. Error bars indicate SEM.
GFP
pan pY
Actin
Merge
Actn
Merge
Luciferase
SHIP2 kd
0"
GFP
pan pY
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.
40
Chapter 4
Discussion
SHIP2 is a 5'-phosphatase that can act on either PI(3,4,5)P 3 or PI(4,5)P 2 to dephosphorylate 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 communication). Dephosphorylation of PI(3,4,5)P 3 by SHIP2 produces PI(3,4)P 2 , to which Lamellipodin binds. Lamellipodin 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
significant difference in SHIP2 knockdown cells as compared to controls. We observed a
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)P
3
to produce PI(3,4)P 2 , and can also dephosphorylate
PI(4,5)P 2 [102]. The finding that SHIP2 can act on PI(4,5)P 2 was confirmed by Nakatsu,
et al. They also show that knocking down SHIP2 results in an increase in the PIP 2 /PIP
ratios by 40% as compared to control cells (F. Gertler, personal communication). Therefore loss of SHIP2 has a very significant effect on the balance of phosphoinositides in the
cell, resulting in an increase in PI(4,5)P 2 levels. Given their critical importance in signaling, 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)P 2 leads to the dissociation of Arp2/3 at the membrane [123]. This is due at least in part to the fact that
PI(4,5)P 2 can activate Cdc42 and can bind N-WASP, triggering a conformational change
that allows N-WASP to bind to and activate Arp2/3 [69]. It is possible that in SHIP2
knockdown cells the increase in PI(4,5)P 2 results in increased recruitment of Arp2/3 to
the leading edge, resulting in increased protrusion. PI(4,5)P 2 also facilitates actin filament elongation at the leading edge by preventing capping protein from capping filaments
as well as promoting the dissociation of profilin- G-actin, increasing the amount of Gactin available for incorporation into actin filaments [69,93]. Increased PI(4,5)P 2 levels
pre-stimulation could also cause a large pool of cofilin to be sequestered at the membrane,
which would lead to an increase in severing of capped filaments upon EGF stimulation.
These effects could also contribute to increased actin polymerization at the leading edge
that would result in increased membrane protrusion. Unpublished observations from the
Gertler lab show that in Rat2 cells, knockdown of SHIP2 results in increased filopodia
formation. This further supports a model in which PI(4,5)P 2 is up-regulated in SHIP2
knockdown cells, as the activation of Cdc42 and N-WASP promotes the formation of
filopodia. If in fact there is increased actin polymerization occurring at the leading edge
in SHIP2 knockdown cells, it is conceivable that the levels of Mena might be increased
at the edge at later time points post-stimulation. There could be due to a slower but
steadier recruitment of Mena to the leading edge, where it acts as an anti-capping factor
and allows more actin polymerization to occur.
Nakatsu et al. also demonstrated that while steady-state levels of PI(3,4,5)P 3 are not
increased in SHIP2 knockdown cells, they are increased upon acute serum stimulation.
PI(3,45)P 3 recruits WAVE2 to the membrane and this recruitment is required for the
formation of lamellipodia [67]. This could also contribute to the increase in membrane
protrusion in SHIP2 knockdown cells. There are other inositol 5'-phosphatases in the cell,
such as synaptojanin 1 and 2, OCRL (occulocerebrorenal syndrome of Lowe) and INPP5B
(inositol polyphosphate 5-phosphatase B), that could then dephosphorylate PI(3,4,5)P 3
to PI(3,4)P 2 , which could explain why no decrease in the amount of Lpd at the leading
edge is seen in SHIP2 knockdown cells.
An alteration in phosphoinositide levels has also been shown to have a direct effect
on clathrin-coated pit lifetime. Endocytosis through clathrin-coated pits is the major
pathway of EGFR internalization, and had been thought to attenuate EGFR signaling [108]. EGFR can also ben endcoytosed in a clathrin-independent manner [98]. In a
further study, Sigismund et al. propose that when EGFR is endocytosed in a clathrinindependent manner, it is targeted for degradation. Conversely, when EGFR is endocytosed in clathrin-coated pits, it is recycled back to the membrane. They showed that
clathrin-mediated internalization is essential for sustained EGFR signaling, but dispensable for degradation [97]. A shift in the balance between these two pathways could result
in significant alterations in EGFR signaling. Upon knockdown of SHIP2 in COS-7 cells,
clathrin-coated pit lifetime is decreased by 25% as compared to control cells (F. Gertler,
personal communication). This could lead to increased turnover of clathrin-coated pits
in SHIP2 knockdown cells, and this could shift the balance between recycling and degradation of EGFR in favor of recycling. A second possibility is that the pits are unable to
collect cargo, leaving EGF on the surface. Either mechanism would lead to more EGFR
on the cell surface and amplification of EGFR signaling in SHIP2 knockdown cells. This
hypothesis disagrees with previously published work, which showed that the loss of SHIP2
led to enhanced receptor degradation, increased EGFR ubiquitination and increased association of EGFR with c-Cbl, an E3-ubiquitin ligase both in HeLa cells [82,83] and breast
cancer cells [81]. However, in pulse-chase experiments done in breast cancer cells using
AlexaFluor- conjugated EGF, they see an increase in number of EGF- containing vesicles
in SHIP2- silenced cells. Thirty minutes after stimulation, control cells have fewer but
larger EGF- containing vesicles that localize at the perinuclear endosomal sorting area.
In SHIP2 knockdown cells there is a decrease in fluorescence signal, which is scattered as
fine puntae [81]. This could perhaps be due to an increase in receptor being recycled to
the membrane, rather than targeted for degradation, though further experiments would
be necessary to determine this.
The basic model is then one in which the loss of SHIP2 results in the balance of phosphoinositides in the cell, shifting it towards PI(4,5)P 2 . This in turn leads to a greater
activation of Cdc42, N-WASP and localization of Arp2/3 at the cell membrane, resulting in increased actin polymerization and greater membrane protrusion. An increase in
PI(4,5)P
2
could lead to more cofilin being sequestered at the membrane, and therefore
an increase in barbed end generation, which would also lead to increased actin polymerization. SHIP2 also negatively regulates lifetime of clathrin-coated pits, helping to
maintain a balance between degradation and recycling of EGFR. Loss of SHIP2 may
lead to an increase in receptor recycling to the surface, and therefore an amplification
of EGFR signaling. Further experiments must be done to determine if this model has
is valid. Crucial next steps would be to stain cells for Arp2/3, PI(3,4)P 2 , PI(4,5)P 2 and
PI(3,4,5)P
3
to determine if the balance is in fact shifted and if more Ap2/3 is recruited
to the leading edge. Stimulating cells and fixing them over several time points and then
staining for Mena would also be informative to determine if over time the amount of Mena
at the leading edge is increased in SHIP2 knockdown cells. Inhibiting coflin, Arp2/3 or
Ena/VASP and asking if knocking down SHIP2 still causes an increase in protrusion over
controls might help to delineate which step(s) are affected by SHIP2. It would also be of
interest to determine if in fact actin filaments grow longer and less branched in SHIP2
knockdown cells, perhaps through electron microscopy. Experiments are currently being
carried out by other members of the Gertler lab to study EGFR dynamics in MenaINV
cells. Similar experiments in SHIP2 knockdown cells would be informative, as well. Additionally, it would be informative to look at the change in cell motility upon SHIP2
knockdown. Lastly, it is interesting to consider hte apparent paradox that SHIP2 is
upregulated in tumors and yet seems to act as a negative regulator of protrusion. It is
possible that in vitro loss of SHIP2 leads to longer and less branched actin filaments,
which leads to an increase in protrusion but would not lead to an increase in motility.
Upregulation of SHIP2 may lead to more stable actin filaments which could, in vivo lead
to the stabilzation of invadopodium, similar to the effect observed by Philippar et al. in
MenaINV cells [76].
46
Bibliography
[1] N. Abdul-Manan, B. Aghazadeh, G. A. Liu, A. Majumdar, 0. Ouerfelli, K. A. Siminovitch, and M. K. Rosen. Structure of Cdc42 in complex with the GTPase-binding
domain of the "Wiskott-Aldrich syndrome" protein. Nature, 399(6734):379-83, May
1999.
[2] M. Abercrombie, J. E. Heaysman, and S. M. Pegrum. The locomotion of fibroblasts
in culture. I. Movements of the leading edge. Exp. Cell Res., 59(3):393-8, Mar 1970.
[3] M. Abercrombie, J. E. Heaysman, and S. M. Pegrum. The locomotion of fibroblasts
in culture. II. "Ruffling". Exp. Cell Res., 60(3):437-44, Jun 1970.
[4] M. D. Althuis, J. M. Dozier, W. F. Anderson, S. S. Devesa, and L. A. Brinton.
Global trends in breast cancer incidence and mortality 1973-1997. Int. J. Epidemiol,
34(2):405-12, Apr 2005.
[5] K. J. Amann and T. D. Pollard. Cellular regulation of actin network assembly.
Curr. Biol., 10(20):R728-30, Oct 2000.
[6] K. J. Amann and T. D. Pollard. The Arp2/3 complex nucleates actin filament
branches from the sides of pre-existing filaments. Nat. Cell Biol., 3(3):306-10, Mar
2001.
[7] K. J. Amann and T. D. Pollard. Direct real-time observation of actin filament
branching mediated by Arp2/3 complex using total internal reflection fluorescence
microscopy. Proc. Natl. Acad. Sci. USA, 98(26):15009-13, Dec 2001.
[8] E. Andrianantoandro and T. D. Pollard. Mechanism of actin filament turnover
by severing and nucleation at different concentrations of ADF/cofilin. Mol. Cell,
24(1):13-23, Oct 2006.
[9] K. Aoki, T. Nakamura, K. Fujikawa, and M. Matsuda. Local phosphatidylinositol
3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42
and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells. Mol.
Biol. Cell, 16(5):2207-17, May 2005.
[10] R. Arriagada, L.-E. Rutqvist, H. Johansson, A. Kramar, and S. Rotstein. Predicting
distant dissemination in patients with early breast cancer. Acta Oncol., 47(6):111321, Jan 2008.
[11] C. Bachmann, L. Fischer, U. Walter, and M. Reinhard. The EVH2 domain of the
vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding,
and actin bundle formation. J. Biol. Chem., 274(33):23549-57, Aug 1999.
[12] M. Barzik, T. I. Kotova, H. N. Higgs, L. Hazelwood, D. Hanein, F. B. Gertler, and
D. A. Schafer. Ena/VASP proteins enhance actin polymerization in the presence
of barbed end capping proteins. J. Biol. Chem., 280(31):28653-62, Aug 2005.
[13] J. E. Bear and F. B. Gertler. Ena/VASP: towards resolving a pointed controversy
at the barbed end. J. Cell Sci., 122(Pt 12):1947-53, Jun 2009.
[14] J. E. Bear, J. J. Loureiro, I. Libova, R. F.ssler, J. Wehland, and F. B. Gertler.
Negative regulation of fibroblast motility by Ena/VASP proteins. Cell, 101(7):71728, Jun 2000.
[15] J. E. Bear, T. M. Svitkina, M. Krause, D. A. Schafer, J. J. Loureiro, G. A. Strasser,
I. V. Maly, 0. Y. Chaga, J. A. Cooper, G. G. Borisy, and F. B. Gertler. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast
motility. Cell, 109(4):509-21, May 2002.
[16] D. Blero, J. Zhang, X. Pesesse, B. Payrastre, J. E. Dumont, S. Schurmans, and
C. Erneux. Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient
mouse embryonic fibroblasts. FEBS J., 272(10):2512-22, May 2005.
[17] A. Y. Chan, M. Bailly, N. Zebda, J. E. Segall, and J. S. Condeelis. Role of cofilin
in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion. J. Cell Biol., 148(3):531-42, Feb 2000.
[18] E. S. Chhabra and H. N. Higgs. The many faces of actin: matching assembly
factors with cellular structures. Nat. Cell Biol., 9(10):1110-21, Oct 2007.
[19] S. C16ment, U. Krause, F. Desmedt, J. F. Tanti, J. Behrends, X. Pesesse, T. Sasaki,
J. M. Penninger, M. Doherty, W. Malaisse, J. E. Dumont, Y. L. Marchand-Brustel,
C. Erneux, L. Hue, and S. Schurmans. The lipid phosphatase SHIP2 controls insulin
sensitivity. Nature, 409(6816):92-7, Jan 2001.
[20] S. S. Coughlin and D. U. Ekwueme. Breast cancer as a global health concern.
Cancer Epidemiol., 33(5):315-8, Nov 2009.
[21] V. Desmarais, F. Macaluso, J. S. Condeelis, and M. Bailly. Synergistic interaction
between the Arp2/3 complex and cofilin drives stimulated lamellipod extension. J.
Cell Sci., 117(Pt 16):3499-510, Jul 2004.
[22] J. M. Dyson, C. J. O'Malley, J. Becanovic, A. D. Munday, M. C. Berndt, I. D.
Coghill, H. H. Nandurkar, L. M. Ooms, and C. A. Mitchell. The SH2-containing
inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin. J. Cell Biol., 155(6):1065-79, Dec 2001.
[23] S. Eden, R. Rohatgi, A. V. Podtelejnikov, M. Mann, and M. W. Kirschner. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature,
418(6899):790-3, Aug 2002.
[24] C. Egile, T. P. Loisel, V. Laurent, R. Li, D. Pantaloni, P. J. Sansonetti, and M. F.
Carlier. Activation of the Cdc42 effector N-WASP by the Shigella flexneri IcsA
protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based
motility. J. Cell Biol., 146(6):1319-32, Sep 1999.
[25] F. Ferron, G. Rebowski, S. H. Lee, and R. Dominguez. Structural basis for the
recruitment of profilin-actin complexes during filament elongation by Ena/VASP.
EMBO J., 26(21):4597-606, Oct 2007.
[26] J. Franca-Koh, Y. Kamimura, and P. N. Devreotes. Leading-edge research: Ptdlns(3,4,5)P3 and directed migration. Nat. Cell Biol., 9(1):15-7, Jan 2007.
[27] F. B. Gertler, A. R. Comer, J. L. Juang, S. M. Ahern, M. J. Clark, E. C. Liebl,
and F. M. Hoffmann. enabled, a dosage-sensitive suppressor of mutations in the
drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding
properties. Genes Dev., 9(5):521-33, Mar 1995.
[28] F. B. Gertler, J. S. Doctor, and F. M. Hoffmann. Genetic suppression of mutations
in the Drosophila abl proto-oncogene homolog. Science, 248(4957):857-60, May
1990.
[29] F. B. Gertler, K. Niebuhr, M. Reinhard, J. Wehland, and P. Soriano. Mena, a relative of VASP and drosophila Enabled, is implicated in the control of microfilament
dynamics. Cell, 87(2):227-39, Oct 1996.
[30] M. Ghosh, X. Song, G. Mouneimne, M. Sidani, D. S. Lawrence, and J. S. Condeelis.
Cofilin promotes actin polymerization and defines the direction of cell motility.
Science, 304(5671):743-6, Apr 2004.
[31] E. D. Goley, S. E. Rodenbusch, A. C. Martin, and M. D. Welch. Critical conformational changes in the Arp2/3 complex are induced by nucleotide and nucleation
promoting factor. Mol. Cell, 16(2):269-79, Oct 2004.
[32] S. Goswami, U. Philippar, D. Sun, A. Patsialou, J. Avraham, W. Wang, F. D.
Modugno, P. Nistico, F. B. Gertler, and J. S. Condeelis. Identification of invasion
specific splice variants of the cytoskeletal protein Mena present in mammary tumor
cells during invasion in vivo. Clin. Exp. Metastasis, 26(2):153-9, Jan 2009.
[33] S. L. Gupton and F. B. Gertler. Filopodia: the fingers that do the walking. Sci.
STKE, 2007(400):re5, Sep 2007.
[34] T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker. Growth factors and insulin
stimulate tyrosine phosphorylation of the 51C/SHIP2 protein. J. Biol. Chem.,
273(29):18605-9, Jul 1998.
[35] A. Hall. The cytoskeleton and cancer. Cancer Metastasis Rev., 28(1-2):5-14, Jun
2009.
[36] D. Hanahan and R. A. Weinberg. The hallmarks of cancer. Cell, 100(1):57-70, Jan
2000.
[37] S. J. Heasman and A. J. Ridley. Mammalian Rho GTPases: new insights into their
functions from in vivo studies. Nat. Rev. Mol. Cell Biol., 9(9):690-701, Sep 2008.
[38] M. Higashi, C. Ishikawa, J. Yu, A. Toyoda, H. Kawana, K. Kurokawa, M. Matsuda,
M. Kitagawa, and K. Harigaya. Human Mena associates with Rac1 small GTPase
in glioblastoma cell lines. PLoS One, 4(3):e4765, Jan 2009.
[39] H. N. Higgs and T. D. Pollard. Regulation of actin filament network formation
through ARP2/3 complex: activation by a diverse array of proteins. Annu. Rev.
Biochem., 70:649-76, Jan 2001.
[40] L.-D. Hu, H.-F. Zou, S.-X. Zhan, and K.-M. Cao. EVL (Ena/VASP-like) expression
is up-regulated in human breast cancer and its relative expression level is correlated
with clinical stages. Oncol. Rep., 19(4):1015-20, Apr 2008.
[41] M. Huber, C. D. Helgason,. J. E. Damen, M. Scheid, V. Duronio, L. Liu, M. D.
Ware, R. K. Humphries, and G. Krystal. The role of SHIP in growth factor induced
signalling. Prog. Biophys. Mol. Biol., 71(3-4):423-34, Jan 1999.
[42] R. H. Insall and L. M. Machesky. Actin dynamics at the leading edge: from simple
machinery to complex networks. Dev. Cell, 17(3):310-22, Sep 2009.
[43] R. Kalluri and R. A. Weinberg. The basics of epithelial-mesenchymal transition.
J. Clin. Invest., 119(6):1420-8, Jun 2009.
[44] A. S. Kim, L. T. Kakalis, N. Abdul-Manan, G. A. Liu, and M. K. Rosen. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein.
Nature, 404(6774):151-8, Mar 2000.
[45] M. Krause, E. W. Dent, J. E. Bear, J. J. Loureiro, and F. B. Gertler. Ena/VASP
proteins: regulators of the actin cytoskeleton and cell migration. Annu. Rev. Cell
Biol., 19:541-64, Jan 2003.
[46] M. Krause, J. D. Leslie, M. Stewart, E. M. Lafuente, F. Valderrama, R. Jagannathan, G. A. Strasser, D. A. Rubinson, H. Liu, M. Way, M. B. Yaffe, V. A.
Boussiotis, and F. B. Gertler. Lamellipodin, an Ena/VASP ligand, is implicated
in the regulation of lamellipodial dynamics. Dev. Cell, 7(4):571-83, Oct 2004.
[47] G. Krystal, J. E. Damen, C. D. Helgason, M. Huber, M. R. Hughes, J. Kalesnikoff,
V. Lam, P. Rosten, M. D. Ware, S. Yew, and R. K. Humphries. SHIPs ahoy. Int.
J. Biochem. Cell Biol., 31(10):1007-10, Oct 1999.
[48] S. Kurisu and T. Takenawa. The WASP and WAVE family proteins. Genome
Biol., 10(6):226, Jan 2009.
[49] A. V. Kwiatkowski, D. A. Rubinson, E. W. Dent, J. E. van Veen, J. D. Leslie,
J. Zhang, L. M. Mebane, U. Philippar, E. M. Pinheiro, A. A. Burds, R. T. Bronson,
S. Mori, R. Fassler, and F. B. Gertler. Ena/VASP is required for neuritogenesis in
the developing cortex. Neuron, 56(3):441-55, Nov 2007.
[50] J. V. Lacey, S. S. Devesa, and L. A. Brinton. Recent trends in breast cancer
incidence and mortality. Environ. Mol. Mutagen., 39(2-3):82-8, Jan 2002.
[51] L. M. Lanier, M. A. Gates, W. Witke, A. S. Menzies, A. M. Wehman, J. D. Macklis,
D. Kwiatkowski, P. Soriano, and F. B. Gertler. Mena is required for neurulation
and commissure formation. Neuron, 22(2):313-25, Feb 1999.
[52] D. A. Lauffenburger and A. R. Horwitz. Cell migration: a physically integrated
molecular process. Cell, 84(3):359-69, Feb 1996.
[53] J. J. Loureiro, D. A. Rubinson, J. E. Bear, G. A. Baltus, A. V. Kwiatkowski,
and F. B. Gertler. Critical roles of phosphorylation and actin binding motifs, but
not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein
(VASP) function during cell migration. Mol. Biol. Cell, 13(7):2533-46, Jul 2002.
[54] L. M. Machesky and R. H. Insall. Scar and the related Wiskott-Aldrich syndrome
protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr.
Biol., 8(25):1347-56, Jan 1998.
[55] L. M. Machesky, R. D. Mullins, H. N. Higgs, D. A. Kaiser, L. Blanchoin, R. C.
May, M. E. Hall, and T. D. Pollard. Scar, a WASp-related protein, activates
nucleation of actin filaments by the Arp2/3 complex. Proc. Natl. Acad. Sci. USA,
96(7):3739-44, Mar 1999.
[56] J. B. Marchand, D. A. Kaiser, T. D. Pollard, and H. N. Higgs. Interaction of
WASP/Scar proteins with actin and vertebrate Arp2/3 complex. Nat. Cell Biol.,
3(1):76-82, Jan 2001.
[57] A. S. Menzies, A. Aszodi, S. E. Williams, A. Pfeifer, A. M. Wehman, K. L. Goh,
C. A. Mason, R. Fassler, and F. B. Gertler. Mena and vasodilator-stimulated
phosphoprotein are required for multiple actin-dependent processes that shape the
vertebrate nervous system. J. Neurosci., 24(37):8029-38, Sep 2004.
[58] H. Miki, S. Suetsugu, and T. Takenawa. WAVE, a novel WASP-family protein
involved in actin reorganization induced by Rac. EMBO J., 17(23):6932-41, Dec
1998.
[59] H. Miki and T. Takenawa. Regulation of actin dynamics by WASP family proteins.
J. Biochem., 134(3):309-13, Sep 2003.
[60] F. D. Modugno, L. DeMonte, M. Balsamo, G. Bronzi, M. R. Nicotra, M. Alessio,
E. Jager, J. S. Condeelis, A. Santoni, P. G. Natali, and P. Nistico. Molecular
cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth
factor increases their expression and stimulates hMena+11a phosphorylation in
breast cancer cell lines. Cancer Res., 67(6):2657-65, Mar 2007.
[61] F. D. Modugno, M. Mottolese, A. D. Benedetto, A. Conidi, F. Novelli, L. Perracchio, I. Venturo, C. Botti, E. Jager, A. Santoni, P. G. Natali, and P. Nistico.
The cytoskeleton regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions with high risk of transformation and human epidermal growth
factor receptor-2-positive/hormonal receptor-negative tumors. Clin. Cancer Res.,
12(5):1470-8, Mar 2006.
[62] A. Mogilner and G. Oster. Cell motility driven by actin polymerization. Biophys.
J., 71(6):3030-45, Dec 1996.
[63] G. Mouneimne, L. Soon, V. Desmarais, M. Sidani, X. Song, S.-C. Yip, M. Ghosh,
R. J. Eddy, J. M. Backer, and J. S. Condeelis. Phospholipase C and cofilin are
required for carcinoma cell directionality in response to EGF stimulation. J. Cell
Biol., 166(5):697-708, Aug 2004.
[64] R. D. Mullins, J. A. Heuser, and T. D. Pollard. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of
branching networks of filaments. Proc. Natl. Acad. Sci. USA, 95(11):6181-6, May
1998.
[65] M. Nishio, K. ichi Watanabe, J. Sasaki, C. Taya, S. Takasuga, R. Iizuka, T. Balla,
M. Yamazaki, H. Watanabe, R. Itoh, S. Kuroda, Y. Horie, I. Fdrster, T. W. Mak,
H. Yonekawa, J. M. Penninger, Y. Kanaho, A. Suzuki, and T. Sasaki. Control of
cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1. Nat. Cell
Biol., 9(1):36-44, Jan 2007.
[66] C. D. Nobes and A. Hall. Rho, Rac, and Cdc42 GTPases regulate the assembly
of multimolecular focal complexes associated with actin stress fibers, lamellipodia,
and filopodia. Cell, 81(1):53-62, Apr 1995.
[67] T. Oikawa, H. Yamaguchi, T. Itoh, M. Kato, T. Ijuin, D. Yamazaki, S. Suetsugu,
and T. Takenawa. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Nat. Cell Biol., 6(5):420-6, May 2004.
[68] S. B. Padrick, H.-C. Cheng, A. M. Ismail, S. C. Panchal, L. K. Doolittle, S. Kim,
B. M. Skehan, J. Umetani, C. A. Brautigam, J. M. Leong, and M. K. Rosen.
Hierarchical regulation of WASP/WAVE proteins. Mol. Cell, 32(3):426-38, Nov
2008.
[69] G. D. Paolo and P. V. D. Camilli. Phosphoinositides in cell regulation and membrane dynamics. Nature, 443(7112):651-7, Oct 2006.
[70] D. M. Parkin, F. Bray, and S. S. Devesa. Cancer burden in the year 2000. the
global picture. Eur. J. Cancer,37 Suppl 8:S4-66, Oct 2001.
[71) D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani. Global cancer statistics, 2002. CA
Cancer J. Clin., 55(2):74-108, Jan 2005.
[72] L. Pasic, T. I. Kotova, and D. A. Schafer. Ena/VASP proteins capture actin
filament barbed ends. J. Biol. Chem., 283(15):9814-9, Apr 2008.
[73] N. Paternotte, J. Zhang, I. Vandenbroere, K. Backers, D. Blero, N. Kioka, J.-M.
Vanderwinden, I. Pirson, and C. Erneux. SHIP2 interaction with the cytoskeletal
protein Vinexin. FEBS J., 272(23):6052-66, Dec 2005.
[74] X. Pesesse, S. Deleu, F. D. Smedt, L. Drayer, and C. Erneux. Identification
of a second SH2-domain-containing protein closely related to the phosphatidylinositol polyphosphate 5-phosphatase SHIP. Biochem. Biophys. Res. Commun.,
239(3):697-700, Oct 1997.
[75] X. Pesesse, V. Dewaste, F. D. Smedt, M. Laffargue, S. Giuriato, C. Moreau,
B. Payrastre, and C. Erneux. The Src homology 2 domain containing inositol
5-phosphatase SHIP2 is recruited to the epidermal growth factor (EGF) receptor
and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF-stimulated
COS-7 cells. J. Biol. Chem., 276(30):28348-55, Jul 2001.
[76] U. Philippar, E. T. Roussos, M. Oser, H. Yamaguchi, H.-D. Kim, S. Giampieri,
Y. Wang, S. Goswami, J. B. Wyckoff, D. A. Lauffenburger, E. Sahai, J. S. Condeelis,
and F. B. Gertler. A mena invasion isoform potentiates EGF-induced carcinoma
cell invasion and metastasis. Dev. Cell, 15(6):813-28, Dec 2008.
[77] M. S. Pino, M. Balsamo, F. D. Modugno, M. Mottolese, M. Alessio, E. Melucci,
M. Milella, D. J. McConkey, U. Philippar, F. B. Gertler, P. G. Natali, and P. Nistico. Human Mena+11a isoform serves as a marker of epithelial phenotype and
sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines. Clin. Cancer Res., 14(15):4943-50, Aug 2008.
[78] T. D. Pollard. Regulation of actin filament assembly by Arp2/3 complex and
formins. Annu. Rev. Biophys. Biomo.l Struct., 36:451-77, Jan 2007.
[79] T. D. Pollard, L. Blanchoin, and R. D. Mullins. Molecular mechanisms controlling
actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomo.l Struct.,
29:545-76, Jan 2000.
[80] T. D. Pollard and G. G. Borisy. Cellular motility driven by assembly and disassembly of actin filaments. Cell, 112(4):453-65, Feb 2003.
[81] N. K. Prasad. SHIP2 phosphoinositol phosphatase positively regulates EGFR-Akt
pathway, CXCR4 expression, and cell migration in MDA-MB-231 breast cancer
cells. Int. J. Oncol, 34(1):97-105, Jan 2009.
[82] N. K. Prasad and S. J. Decker. SH2-containing 5'-inositol phosphatase, SHIP2,
regulates cytoskeleton organization and ligand-dependent down-regulation of the
epidermal growth factor receptor. J. Biol. Chem., 280(13):13129-36, Apr 2005.
[83] N. K. Prasad, M. Tandon, S. Badve, P. W. Snyder, and H. Nakshatri. Phosphoinositol phosphatase SHIP2 promotes cancer development and metastasis coupled
with alterations in EGF receptor turnover. Carcinogenesis,29(1):25-34, Jan 2008.
[84] N. K. Prasad, M. Tandon, A. Handa, G. E. Moore, C. F. Babbs, P. W. Snyder, and
S. Bose. High expression of obesity-linked phosphatase SHIP2 in invasive breast
cancer correlates with reduced disease-free survival. Tumour Biol., 29(5):330-41,
Jan 2008.
[85] N. K. Prasad, R. S. Topping, and S. J. Decker. SH2-containing inositol 5'phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates
cellular adhesion and spreading. Mol. Cell Biol., 21(4):1416-28, Feb 2001.
[86] N. K. Prasad, M. E. Werner, and S. J. Decker. Specific tyrosine phosphorylations mediate signal-dependent stimulation of SHIP2 inositol phosphatase activity,
while the SH2 domain confers an inhibitory effect to maintain the basal activity.
Biochemistry, 48(27):6285-7, Jul 2009.
[87] L. E. Rameh and L. C. Cantley. The role of phosphoinositide 3-kinase lipid products
in cell function. J. Biol. Chem., 274(13):8347-50, Mar 1999.
[88] M. Reinhard, K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B. M. Jockusch,
and U. Walter. The proline-rich focal adhesion and microfilament protein VASP is
a ligand for profilins. EMBO J., 14(8):1583-9, Apr 1995.
[89] M. Reinhard, M. Halbriigge, U. Scheer, C. Wiegand, B. M. Jockusch, and U. Walter. The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel
protein associated with actin filaments and focal contacts. EMBO J., 11(6):206370, Jun 1992.
[90] B. D. Robinson, G. L. Sica, Y.-F. Liu, T. E. Rohan, F. B. Gertler, J. S. Condeelis,
and J. G. Jones. Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin.
Cancer Res., 15(7):2433-41, Apr 2009.
[91] A. A. Rodal, 0. Sokolova, D. B. Robins, K. M. Daugherty, S. Hippenmeyer, H. Riezman, N. Grigorieff, and B. L. Goode. Conformational changes in the Arp2/3 complex leading to actin nucleation. Nat. Struct. Mol. Biol., 12(1):26-31, Jan 2005.
[92] R. Rohatgi, L. Ma, H. Miki, M. Lopez, T. Kirchhausen, T. Takenawa, and M. W.
Kirschner. The interaction between N-WASP and the Arp2/3 complex links Cdc42dependent signals to actin assembly. Cell, 97(2):221-31, Apr 1999.
[93] J. Saarikangas, H. Zhao, and P. Lappalainen. Regulation of the actin cytoskeletonplasma membrane interplay by phosphoinositides. Physiol. Rev., 90(1):259-289,
Jan 2010.
[94] A. Schirenbeck, R. Arasada, T. Bretschneider, T. E. B. Stradal, M. Schleicher, and
J. Faix. The bundling activity of vasodilator-stimulated phosphoprotein is required
for filopodium formation. Proc. Natl. Acad. Sci. USA, 103(20):7694-9, May 2006.
[95] J. E. Segall, S. Tyerech, L. Boselli, S. Masseling, J. Helft, A. Y. Chan, J. G. Jones,
and J. S. Condeelis. EGF stimulates lamellipod extension in metastatic mammary
adenocarcinoma cells by an actin-dependent mechanism. Clin. Exp. Metastasis,
14(1):61-72, Jan 1996.
[96] S. Shapiro, E. A. Coleman, M. Broeders, M. Codd, H. de Koning, J. Fracheboud,
S. Moss, E. Paci, S. Stachenko, and R. Ballard-Barbash. Breast cancer screening
programmes in 22 countries: current policies, administration and guidelines. International Breast Cancer Screening Network (IBSN) and the European Network of
Pilot Projects for Breast Cancer Screening. Int. J. Epidemiol, 27(5):735-42, Oct
1998.
[97] S. Sigismund, E. Argenzio, D. Tosoni, E. Cavallaro, S. Polo, and P. P. D. Fiore.
Clathrin-mediated internalization is essential for sustained EGFR signaling but
dispensable for degradation. Dev. Cell, 15(2):209-19, Aug 2008.
[98] S. Sigismund, T. Woelk, C. Puri, E. Maspero, C. Tacchetti, P. Transidico, P. P. D.
Fiore, and S. Polo. Clathrin-independent endocytosis of ubiquitinated cargos. Proc.
Natl. Acad. Sci. USA, 102(8):2760-5, Feb 2005.
[99] M. W. Sleeman, K. E. Wortley, K.-M. V. Lai, L. C. Gowen, J. Kintner, W. 0.
Kline, K. Garcia, T. N. Stitt, G. D. Yancopoulos, S. J. Wiegand, and D. J. Glass.
Absence of the lipid phosphatase SHIP2 confers resistance to dietary obesity. Nat.
Med., 11(2):199-205, Feb 2005.
[100] J. V. Small, G. Isenberg, and J. E. Celis. Polarity of actin at the leading edge of
cultured cells. Nature, 272(5654):638-9, Apr 1978.
[101] K. Smith, D. Humphreys, P. J. Hume, and V. Koronakis. Enteropathogenic Escherichia coli recruits the cellular inositol phosphatase SHIP2 to regulate actinpedestal formation. Cell Host Microbe, 7(1):13-24, Jan 2010.
[102] V. Taylor, M. Wong, C. Brandts, L. Reilly, N. M. Dean, L. M. Cowsert, S. Moodie,
and D. Stokoe. 5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells. Mol. Cell Biol., 20(18):6860-71,
Sep 2000.
[103] N. Tomasevic, Z. Jia, A. Russell, T. Fujii, J. J. Hartman, S. Clancy, M. Wang,
C. Beraud, K. W. Wood, and R. Sakowicz. Differential regulation of WASP and
N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2. Biochemistry, 46(11):3494-502,
Mar 2007.
[104] M. J. van de Vijver, Y. D. He, L. J. van't Veer, H. Dai, A. A. M. Hart, D. W.
Voskuil, G. J. Schreiber, J. L. Peterse, C. Roberts, M. J. Marton, M. Parrish,
D. Atsma, A. T. Witteveen, A. Glas, L. Delahaye, T. van der Velde, H. Bartelink,
S. Rodenhuis, E. T. Rutgers, S. H. Friend, and R. Bernards. A gene-expression
signature as a predictor of survival in breast cancer. N. Engl. J. Med., 347(25):19992009, Dec 2002.
[105] J. van Rheenen, X. Song, W. van Roosmalen, M. Cammer, X. Chen, V. Desmarais,
S.-C. Yip, J. M. Backer, R. J. Eddy, and J. S. Condeelis. EGF-induced PIP2
hydrolysis releases and activates cofilin locally in carcinoma cells. J. Cell Biol.,
179(6):1247-59, Dec 2007.
[106] B. Vanhaesebroeck, J. Guillermet-Guibert, M. Graupera, and B. Bilanges. The
emerging mechanisms of isoform-specific P13K signalling. Nat. Rev. Mol. Cell Biol.,
11(5):329-41, May 2010.
[107] L. J. van't Veer, H. Dai, M. J. van de Vijver, Y. D. He, A. A. M. Hart, M. Mao, H. L.
Peterse, K. van der Kooy, M. J. Marton, A. T. Witteveen, G. J. Schreiber, R. M.
Kerkhoven, C. Roberts, P. S. Linsley, R. Bernards, and S. H. Friend. Gene expression profiling predicts clinical outcome of breast cancer. Nature, 415(6871):530-6,
Jan 2002.
[108] A. V. Vieira, C. Lamaze, and S. L. Schmid. Control of EGF receptor signaling by
clathrin-mediated endocytosis. Science, 274(5295):2086-9, Dec 1996.
[109) N. Volkmann, K. J. Amann, S. Stoilova-McPhie, C. Egile, D. C. Winter, L. Hazelwood, J. E. Heuser, R. Li, T. D. Pollard, and D. Hanein. Structure of Arp2/3
complex in its activated state and in actin filament branch junctions. Science,
293(5539):2456-9, Sep 2001.
[110] B. Walders-Harbeck, S. Y. Khaitlina, H. Hinssen, B. M. Jockusch, and S. Illenberger. The vasodilator-stimulated phosphoprotein promotes actin polymerisation
through direct binding to monomeric actin. FEBS Lett., 529(2-3):275-80, Oct 2002.
[111] W. Wang, S. Goswami, K. Lapidus, A. L. Wells, J. B. Wyckoff, E. Sahai, R. H.
Singer, J. E. Segall, and J. S. Condeelis. Identification and testing of a gene
expression signature of invasive carcinoma cells within primary mammary tumors.
Cancer Res., 64(23):8585-94, Dec 2004.
[112] W. Wang, J. B. Wyckoff, S. Goswami, Y. Wang, M. Sidani, J. E. Segall, and
J. S. Condeelis. Coordinated regulation of pathways for enhanced cell motility
and chemotaxis is conserved in rat and mouse mammary tumors. Cancer Res.,
67(8):3505-11, Apr 2007.
[113] B. Weigelt, J. L. Peterse, and L. J. van't Veer. Breast cancer metastasis: markers
and models. Nat. Rev. Cancer,5(8):591-602, Aug 2005.
[114] H. K. Weir, M. J. Thun, B. F. Hankey, L. A. G. Ries, H. L. Howe, P. A. Wingo,
A. Jemal, E. Ward, R. N. Anderson, and B. K. Edwards. Annual report to the
nation on the status of cancer, 1975-2000, featuring the uses of surveillance data
for cancer prevention and control. J. Natl. Cancer Inst., 95(17):1276-99, Sep 2003.
[115] M. D. Welch, A. H. DePace, S. Verma, A. Iwamatsu, and T. J. Mitchison. The
human Arp2/3 complex is composed of evolutionarily conserved subunits and is
localized to cellular regions of dynamic actin filament assembly. J. Cell Biol.,
138(2):375-84, Jul 1997.
[116] J. B. Wyckoff, J. E. Segall, and J. S. Condeelis. The collection of the motile
population of cells from a living tumor. Cancer Res., 60(19):5401-4, Oct 2000.
[117] J. B. Wyckoff, W. Wang, E. Y. Lin, Y. Wang, F. Pixley, E. R. Stanley, T. Graf,
J. W. Pollard, J. E. Segall, and J. S. Condeelis. A paracrine loop between tumor
cells and macrophages is required for tumor cell migration in mammary tumors.
Cancer Res., 64(19):7022-9, Oct 2004.
[118] J. B. Wyckoff, Y. Wang, E. Y. Lin, J. feng Li, S. Goswami, E. R. Stanley, J. E.
Segall, J. W. Pollard, and J. S. Condeelis. Direct visualization of macrophageassisted tumor cell intravasation in mammary tumors. Cancer Res., 67(6):2649-56,
Mar 2007.
[119] J. Xie, I. Vandenbroere, and I. Pirson. SHIP2 associates with intersectin and
recruits it to the plasma membrane in response to EGF. FEBS Lett., 582(20):30117, Sep 2008.
[120] D. Yarar, W. To, A. Abo, and M. D. Welch. The Wiskott-Aldrich syndrome
protein directs actin-based motility by stimulating actin nucleation with the Arp2/3
complex. Curr. Biol., 9(10):555-8, May 1999.
[121] N. Yonezawa, Y. Homma, I. Yahara, H. Sakai, and E. Nishida. A short sequence
responsible for both phosphoinositide binding and actin binding activities of cofilin.
J. Biol. Chem., 266(26):17218-21, Sep 1991.
[122] G. Zhuang, S. Hunter, Y. Hwang, and J. Chen. Regulation of EphA2 receptor endocytosis by SHIP2 lipid phosphatase via phosphatidylinositol 3-kinase-dependent
Rac1 activation. J. Biol. Chem., 282(4):2683-94, Jan 2007.
[123] R. Zoncu, R. M. Perera, R. Sebastian, F. Nakatsu, H. Chen, T. Balla, G. Ayala,
D. Toomre, and P. V. D. Camilli. Loss of endocytic clathrin-coated pits upon
acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc. Natl. Acad. Sci.
USA, 104(10):3793-8, Mar 2007.