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Effect of vicrostatin on integrin based signaling molecules in cancer

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Effect of vicrostatin on integrin based signaling molecules in cancer

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EFFECT OF VICROSTATIN ON INTEGRIN BASED SIGNALING MOLECULES IN
CANCER

BY
SHRENIK RAJESH SHAH

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

AUGUST 2013

Copyright 2013 SHRENIK RAJESH SHAH
i

DEDICATION

To my Family, who laughed, cried and supported me all the time

ii

ACKNOWLEDGEMENTS
I would like to thank the members of my laboratory including Dr. Francis
Markland, Dr. Steve Swenson, and Dr. Radu Minea, who thought that I could make a
difference to their lab and provided me with the opportunity. I would like to thank my
colleagues Joshua Chen, Steven Barnes and Adrian Gil for being always supportive and
helpful.
I would like to thank Dr Thomas Chen, for allowing me to use equipment’s in this
lab without which I could not have had completed my research. I also would like to thank
Brian Idoni for explaining me and familiarizing me with protocols for
immunoprecipitation using magnetic beads.
I extend my gratitude to my grandparents, my parents and all my family members
who have been always supportive and have constantly been a source of motivation for
me. I would like to thank all my friends Manasi, Asmiti, Yudi, Pranali, Rajesh, Nemi,
Ruchika, Ankit, Hiten, Rikin, Pankaj, Vandit present here as well as all those back in
India too, without whose help and support I would not have made it this far . Also I
would like to thank my committee members Dr Zolatn Tokes, Dr Joseph Hacia and Dr
Florence Hofman for guiding me in this process of writing and defending my thesis.
iii

TABLE OF CONTENTS

Dedication i
Acknowledgements ii
List of Figures v
List of Abbreviations vi
Abstract viii
Chapter 1 – Introduction
1.1 Cancer 1
1.2 Angiogenesis 1
1.3 Metastasis 4
1.4 Role of Integrins in Cancer 5

1.5 Integrin Structure 6
1.6 Integrin Activation & Functions 9
1.7 Important Intracellular Signaling molecules 12
1.7.1 Talin 12
1.7.2 Vinculin 13
1.7.3 Paxillin and α -Actinin 13
1.7.4 Integrin-Linked Kinase 14
1.7.5 Focal Adesion Kinase 15
1.8 Disintegrins 15
1.9 Disintegrin Contortrostatin(CN) and Vicrostatin (VCN) 17

iv

Chapter 2 – Materials And Methods
2.1 Cell culture and Antibodies 22
2.2 Expression and Purification of VCN 22
2.3 Cell Adhesion and Cell Spreading Assay 24
2.4 Lysate preparation, Immunoprecipitation and Western Blotting 24

2.5 Studying effect of VCN of FAK Phosphorylation 26
2.6 Statistical Analysis 27

Chapter 3 – Results
3.1 Inhibition of cell adhesion and cell spreading 28
3.2 Disruption of Talin Association to β1 on treatment of VCN 31
3.3 Disruption of key associations between focal adhesion proteins
in cells treated with VCN 33
3.4 Effect of VCN on Phosphorylation status of Focal Adhesion Kinase 36
(FAK and pFAK-
Y397
)

Chapter 4 – Discussion 39

Bibliography 45

v

LIST OF FIGURES
Fig. No. Figure Title Pg. No.
Fig 1.1 The Angiogenic Switch 3
Fig 1.2 Integrin Receptor Family 6
Fig 1.3 Primary Structure and Organization of Integrin 8
Fig 1.4 Stages of Integrin Activation 11
Fig 1.5 Actin – Integrin Linkage 14
Fig 1.6 Sequence Alignment of Contortrostatin and Vicrostatin 20
Fig 3.1.1 Effect of VCN on Adhesion and spreading of MDA-MB-231
cells after 1HOUR of incubation 29
Fig 3.1.2 Effect of VCN on Adhesion and spreading of MDA-MB-231
cells after 3HOUR of incubation 30
Fig 3.2 Effect of VCN on disrupting the interactions between
β1 and talin associations in MDA-MB-231 cells 31
Fig 3.3 Effect of VCN on disrupting associations between
key focal adhesion proteins in MDA-MB-231 cells 33

Fig 3.4 Effect of VCN on phosphorylation status of adhesion kinase
in MDA-MB-231 37

vi

ABBREVIATIONS

IC 50 50% Inhibitory Concentration

ADMIDAS Adjacent to MIDAS

α Alpha

RGD Arginine-glycine-aspartic acid

β Beta

BCA Bicinchoninic acid

CN Contortrostatin

DMEM Dulbecco’s modified Eagle’s medium

ECM Extracellular matrix

FBS Fetal bovine serum

FN Fibronectin

FA Focal adhesion

FAK Focal adhesion kinase

HPLC High pressure liquid chromatography

MIDAS Metal ion-dependent adhesion site

vii

PBS Phosphate buffered saline

PBST Phosphate buffered saline with 0.1% Tween

PSI Plexin-sempahorin-integrin

RIPA Radioimmunoprecipitation assay

SDS-PAGE Sodium dodecyl sulphate – Polyacrylamide gel electrophoresis

VCN Vicrostatin

viii

ABSTRACT

Cell adhesion molecules have been found to play a pivotal role in progression of
cancer. Integrins are one the molecules among these various cell adhesion molecules that
have been found to play an important role due their ability to bind to the extracellular
matrix. Integrins have been also found to be involved in mediating a large number of
signaling pathways. Disintegrins are small molecules which act as integrin antagonist and
can disrupt integrin function by directly binding them. However regarding how these
disintegrin molecules initiate signaling events once they bind to their ligand remains
unanswered. Recently vicrostatin (VCN), a recombinantly produced disintegrin, was
successfully produced in Markland laboratory. Based on the availability of this reagent
mechanistic studies can be initiated to determine the action of disintegrins upon binding
to the integrin ligand.
In this report, we show how VCN affects cell adhesion and cell spreading of the
metastatic breast cancer cell line MBA-MD-231. We also demonstrate effect of VCN on
one of the key molecule of integrin activation, Talin, and show how VCN binding to the
extracellular domain of β1 disrupts the binding of talin on the cytoplasmic domain.
Through a series of experiments involving immunoprecipitation and western blotting we
were able to show that VCN causes disruption of interactions between talin and the
cytoplasmic tails of β1. We also show how VCN disrupts associations within the key
focal adhesion (FA) proteins and how VCN affects the activation and phosphorylation
status of Focal Adhesion Kinase, a key kinase molecule involved in activation and
phosphorylation of a number of downstream signaling molecules. The data from all the

ix

events in this study shows VCN to produce a global inactivation signal for beta1
integrins. The findings from this study will help in increasing knowledge regarding
disintegrin signaling and their function.

1

CHAPTER 1
INTRODUCTION

1.1 Cancer
Malfunction of genes that regulate cell growth and cell division mainly lead to
cancer. A small fraction of cancers are attributed to genetics, in which damaged gene(s)
are present in somatic chromosomes, which are later transferred from one generation to
other. Various external factors such as radiation, chemicals, drugs and other mutagens
can act as carcinogens. The body has a number of repair mechanisms to correct the errors
in DNA(Collins, Jacks et al. 1997). However, multiple hits on the genome and
uncorrectable errors cause mutations in important cell cycle genes which may lead to
cancer. According to NCI & CDC data 1,660,290 new cancer cases and 580,350 cancer
deaths are estimated to take place in United States itself in the year of 2013(Siegel,
Naishadham et al. 2013).

1.2 Angiogenesis
Angiogenesis plays an important role in number of biological processes namely
growth and development, reproduction and wound repair; in these conditions it is a
regulated process turning ON and OFF according to physiological needs. Angiogenesis
also plays an important role in progression of many diseases such as diabetic retinopathy,
rheumatoid arthritis and cancer (Folkman 1990). Tumor growth and metastasis are
dependent on angiogenesis, and this was demonstrated through the initial experiments

2

performed by Greenblatt and Shubi, which showed that tumor angiogenesis was mediated
by diffusible factor(s) produced by the tumor cells(Greenblatt and Shubi 1968).
Tumor angiogenesis involves a large number of different steps starting right from
separation of endothelial cells from pericytes followed by basement membrane
degradation & migration across basement membranes, and finally eventually resulting in
the extension into the tumor body (Hanahan and Folkman 1996, Carmeliet 2000). Soluble
growth factors such as vascular endothelial growth factor (VEGF), angiopoietins,
transforming growth factors (TGF), platelet-derived growth factor (PLGF), tumor
necrosis factor alpha(TNF α), and fibroblast growth factor (FGF) are some of the
inducers/positive regulators of angiogenesis.(Goto, Goto et al. 1993, Thurston 2003,
Presta, Dell'Era et al. 2005). In addition to these soluble factors a number of membrane-
bound proteins are found to play an important role in angiogenesis(Folkman 2007).
Integrins, ephrins, and cadherins are membrane- bound proteins that affect many
functions involved in blood vessel assembly. Integrins α
2
β
1
, α
3
β
1
, α5β
1
, α6β
1
, α
IIb
β
1
, α
V
β
3
are among the few cell surface integrins are found to be important in angiogenesis
(Vihinen, Riikonen et al. 1996, Lee, Jin et al. 2013).

3

Figure 1.1:-The angiogenic switch is a discrete step in tumor development that can occur at
different stages in the tumor-progression pathway, depending on the nature of the tumor and its
microenvironment. Source:(Bergers and Benjamin 2003).

To sustain tumor growth beyond a particular size increased blood supply
becomes necessary; this is when angiogenic switch takes place which cause endothelial
cell to migrate toward the tumor site. Many factors are involved which include:-
1. Hypoxia is triggered in the area near the tumor cells due to lack to blood supply
by the normal blood system causes release of Hypoxia Induced Factor(HIF)
which in turn recruits pro-angiogenic factors (Maxwell, Pugh et al. 2001).

4

2. Tumors also release soluble growth factors themselves (e.g., VEGF, FGF, and
PlGF) which then induce angiogenesis (Carmeliet, Moons et al. 2001, Ferrara
2002).
3. Tumors have been shown to up regulate or/and over activate various signaling
receptors like Integrins, Eph receptors, VE-cadherin, insulin like growth factor- 1
receptor(Wang, Chen et al. 1998, Carmeliet, Lampugnani et al. 1999, Moser,
Schachtschneider et al. 2008).

A continuous supply of nutrients is needed by the tumor cells for its continued
growth and spread; it is this reason why tumors need to have a sufficient supply of blood
flow which provides it with required nutrients and oxygen. This in turn makes
angiogenesis a good candidate for anti-tumor therapies, as the blood supply is seen as a
good target at the tumor site. Observing and understanding the crucial role played by
angiogenesis in progression of cancer; proangiogenic factors such as VEGF, FGF and
PlGF are all potential targets for tumor therapy (Bergers and Benjamin 2003).

1.3 Metastasis
Tumor progression during metastasis is often described as a multistage process in
which malignant cells spread from the tumor of origin to colonize distant organs (Gupta
and Massagué 2006). Metastasis can be characterized as a multistage process that
requires cancer cells to escape from the primary tumor, the detached cancer cell must
then survive in the circulation, reach at the target organ (seeding), extravasate into the
parenchyma and show persistent growth (Chambers, Groom et al. 2002).

5

During metastasis a major phenomenon seen is EMT (epithelial–
mesenchymal transitions), under normal circumstances EMT plays a crucial role in
embryonic development, However it has been also found to be important in pathogenesis
of cancer(Baum, Settleman et al. 2008).The term EMT, refers to a complex cellular and
molecular programming by which the normal epithelial cells lose their differentiated
characteristics, which includes lack of motility, cell to cell attachment, and apical–basal
polarity, and acquire instead mesenchymal features, which include ability to move
(motility), invasiveness and a heightened ability to resist apoptosis(Thiery and Sleeman
2006, Hugo, Ackland et al. 2007). Invasion, metastatic dissemination and increased
resistance to therapeutics are some of the features imparted by EMT to cancer
cells(Polyak and Weinberg 2009)

1.4 Role of Integrins in cancer progression.
Integrins are a family of receptor which have been most studied, they play a
crucial role in majority of the biological processes, they have been identified to play key
roles in growth and development, immune response and defense, leukocyte trafficking,
blood flow and hemostasis, and cancer also they have been found to play important role
in many human diseases—genetic, autoimmune, and others.(Hynes 1987). These
molecules tend to have low binding affinity for their substrate (i.e ECM) however being
present in a large number on the cell surface they are able to produce a strong signal.
Integrins are considered to be bi-directional signaling machines, as they are able to
transduces signals in two directions, which means that it can make intracellular changes

6

according to the ligand it binds to which is called ―OUTSIDE TO IN‖ signaling and also
it can modify or alter its conformation or expression profile in accordance to the way cell
wants to interact with the ligand, this is considered to be ―INSIDE TO OUT‖
signaling(Hynes 2002)

Figure 1.2:- Integrin receptor family and their preferred ligands. Source:(Hynes 2002)

1.5 Integrin Structure
Integrins are transmembrane structures, present on the cell surface in large
number. Integrins are mainly glycoproteins with multiple transmembrane domains.
Integrins exist as two non-covalently bound α and β subunits, which pair to form
heterodimers. There are 18 α and 8 β known subunits which combine to form at least 24
distinct integrin heterodimers (Hynes 2002). Each subunit contains a proportionally large
extracellular domain, a transmembrane domain and a short cytoplasmic tail(Takada, Ye
et al. 2007).

7

The extracellular domain of integrins are generally large, ~80–150 kD structures,
the extracellular portion of α and β subunits are comprised of several sub-domains
organized into a globular N-terminal head domain standing on C-terminal legs that
connect to the transmembrane and cytoplasmic domains. The α subunit head consists of a
folded seven-bladed β propeller head domain, a thigh domain and two calf domains(calf-
1 and calf-2)(Xiong, Stehle et al. 2001). Half of integrin α subunits contain a domain that
is inserted between β-sheets 2 and 3 of the β-propeller domain known as I domain.
(Springer 1997).This I domain mainly acts as extracellular ligand binding site. Within
this I domain is an important ―metal-ion-dependent adhesive site‖ (MIDAS) which binds
to divalent cations and is important in protein ligand binding. Ligand binding to this site
cause displacement of the metal ion and changes the conformation of the integrin
molecule further leading to increased ligand affinity and integrin activation(Liddington
and Ginsberg 2002).
The β subunit is composed of 5 distinct components; the I-like domain, which is
structurally similar to the I-domain in α subunits, a hybrid domain, four EGF repeats, a
membrane proximal β tail (β TD) domain and a PSI (plexin/semaphorin/integrin) domain.
The β subunit plays important roles in ligand binding in α subunits which lack the I-
domain. In these integrin heterodimers, ligand, which is the RGD motif of the ECM,
protein bind to a small cleft in the head domain between α and β subunit interfaces. The
ligand on binding to the integrin mainly interacts with a metal-ion-occupied site
(MIDAS) located within the β subunit and the propeller domain of α subunit.

8

The transmembrane (TM) domains of integrins are single spanning structures
comprised of ~25–29 amino acid residues that form α -helical coiled coils that either
homo- or heterodimerize. Structural information from the αIIbβ3 heterodimer, which was
recently elucidated, shows that αIIb TM domain is a 24 residue a-helix followed by a
backbone reversal that lacks a significant helix tilt while the β3 TM domain is a 30-
residue linear a helix that is somewhat longer than the width of a typical lipid bilayer,
implying that a pronounced helix tilt is present within the plasma membrane (Adair and
Yeager 2002, Lau, Partridge et al. 2008).

Figure 1.3:-Organization of the domains within the primary structure and the arrangement of the
domains with three dimensional crystal structure of integrin with an I domain added.
Source:(Shimaoka and Springer 2003)

9

Integrin cytoplasmic domains are generally short, largely unstructured and
comprised of 10–70 amino acid residues. β cytoplasmic tails are highly homologous,
while α subunit tails are highly divergent. The conserved GFFKR and HDR(R/K) E
sequences located in the membrane proximal regions of α and β subunits, respectively,
are proposed to form a salt bridge between arginine (R) from the a subunit and aspartic
acid (D) from the β subunit (Vinogradova, Haas et al. 2000, Adair and Yeager 2002).The
importance of the salt bridge is considered to be that it is responsible for maintaining
integrins in a low affinity, inactive state while disruption of the salt bridge is considered
to be responsible for integrin activation state (Hughes, O'Toole et al. 1995, Hughes, Diaz-
Gonzalez et al. 1996).

1.6 Integrin Activation and Function
Integrins can exist in one of the two states either open active state or closed bent
state. When in bent state the ligand cannot access ligand binding site because the ECM
molecules cannot interact with the integrin (Luo, Carman et al. 2007).Depending on the
cell type, the integrin present on the surface might be either basally active or basally
inactive; as in case of most adherent cells that are attached to a basement membrane the
integrins are found to be basally active, while platelets or leukocytes that freely circulate;
until activated to undergo platelet aggregation or mediate an inflammatory response have
their integrin inactive. Integrins have the capacity to shift between high and low affinity
conformations for ligand binding. In many cases integrins exists in low-affinity state

10

during which it has a bent confirmation and depends on intracellular signaling to become
activated.
Activation signals from within the cells causes the straightening of the
extracellular domains and stabilizes the extended active conformation. This is the Inside
– Out signaling which allows the extracellular domain to bind to the ligand(Ratnikov,
Partridge et al. 2005). In order to achieve high affinity state the trans-membrane domains
of the integrins are required to be separated, this disruption of the interaction between the
two subunits, causes conformational changes in both the extracellular or cytoplasmic
domains and subsequent integrin activation (Wegener and Campbell 2008). A large
number of cytoskeletal and signaling proteins bind to integrin cytoplasmic tails, however,
among those there are only two major binding proteins namely talin and the kindlins,
which both bind to β integrin cytoplasmic tails and have been demonstrated to be
important for separation of the trans membrane domains and subsequent integrin
activation (Calderwood, Fujioka et al. 2003).
Apart from the unexceptional Inside-Out signaling, integrins also show the
classical Outside-In signaling. This results due to ligand binding to the extracellular
domain. Integrins themselves lack intrinsic catalytic activity. These signals affect cellular
growth, differentiation and apoptosis and also cause formation of Focal Adhesion
complexes (FA), a large, dynamic multi-protein complex involving over 150 intracellular
proteins(Zaidel-Bar, Itzkovitz et al. 2007). The extracellular ligand binding causes the
extracellular domains to cluster while it also leads to assembly of actin filaments. Several
key FA proteins are involved in establishing and maintaining the integrin–cytoskeleton

11

linkage, these include integrin bound proteins that directly or indirectly bind the actin
cytoskeleton also there are present non-integrin-bound actin-binding proteins and other
various adaptor and signaling molecules.

Figure 1.4:-Various stages of integrin activation show the ―Inside-Out‖ signaling which helps
integrin to bind to the ligand and the ―Outside-In‖ signaling which allows transduction of signals
inside the cells. Source:(Pozzi 2010)

The basic primary function of integrin is attachment of the cells to the ECM; they
mainly act as a link between the ECM and the cell cytoskeleton. Integrins transduces
signals inside the cell to modify the cell cytoskeleton depending on the type of ECM the
cell attach to. Inside the cell, are present a large number of signaling molecule that are
attached to the cytoplasmic tail of the integrins which transduce the signal inside and
chance the cytoskeleton. These are namely talin, α-actinin, and filamin, the anchor

12

proteins, which interact with other proteins like vinculin which later go on to interact
with the actin cytoskeleton. To initiate the signaling cascade there are a large number of
kinases present that tend to phosphorylate their substrates and lead to signal propagation,
these kinases mainly involve Focal Adhesion Kinase (FAK) and Src family kinase, these
connections then further lead to final formation of Focal Adhesions complexes (FA).
Integrins are critical in cancer metastasis, angiogenesis and tissue invasion and
also they are found to be important cancer cell survival and proliferation (Hanahan and
Weinberg 2000). Apart from cancer, involvement of integrins has been documented in a
large number of diseases such as rheumatoid arthritis (RA), asthma and inflammatory
bowel disease and asthma, as well as cardiovascular diseases and thrombosis. Alteration
of expression of integrin or its functionality is the reason for causing these diseases.
Borradori et al showed that deletion of α6β4 integrin leads to Epidermolysis bullosa ,a
skin blistering disease(Borradori and Sonnenberg 1999) deletion of α7β1 causes
congenital muscular dystrophy, while due to quantitative or qualitative defects of αIIbβ3
on platelets which fail to aggregate such as in the case of patients with Glanzmann’s
Thrombasthenia(Vachon, Xu et al. 1997, Hodivala-Dilke, McHugh et al. 1999).

1.7 Important Intracellular Signaling Molecules
1.7.1 Talin
It is the first binding partner in the entire cascade of integrin-cytoskeleton
signaling. Talin plays an important role in both the signaling processes. During the
―Inside-Out‖ signaling the FREM (F for Band 4.1, E for Ezrin, R for Radixin, M for

13

Moesin) domain located in the head of the talin molecule is necessary while during the
―Outside- In‖ signaling both the head and the rod are required. Studies in Talin-1
deficient mice have shown that these deficient mice tend to die during gastrulation due to
a defect in cytoskeletal organization and cell migration (Monkley, Zhou et al. 2000).

1.7.2 Vinculin
Vinculin is the second important molecule in the signaling cascade; it is a non-
integrin binding protein. It binds directly to talin and actin, it binds talin at a large number
of sites and act as a cross linker that stabilizes talin-actin interactions. Once integrins
have bound to talin, vinculin is recruited to the nascent focal adhesion. Studies have
shown that vinculin knocked out fibroblasts make fewer and smaller FA that don’t
mature; which provides evidence regarding the importance of the role of vinculin in
reinforcing links between integrins and the actin cytoskeleton(Humphries, Wang et al.
2007).

1.7.3 Paxillin and α -Actinin
Paxallin is a structural protein which is usually localized at the leading g edge of
the cell and detected in the early adhesion stage. It creates a ground for large number of
simultaneous interactions which are further modified with the help of phosphorylation by
kinses. Paxallin is involved in binding talin to the α-integrin cytoplasmic talin, leading to
increase in the stability of integrin–talin–actin interaction (Alon, Feigelson et al. 2005).α
-Actinin can work as a binding partner of both talin and vinculin. α -Actinin can directly

14

bind β integrins and operates as a close partner of actin and has been shown to have an
essential role in adhesion strengthening.

Figure 1.5:-The figure shows actin integrin linkage it shows the linkage between the extracellular
matrix and the actin cytoskeleton and the components involved in successfully caring out this
interaction. Source:(Vicente-Manzanares, Choi et al. 2009)

1.7.4. Integrin-Linked Kinase
ILK is a multi-domain, scaffolding adaptor protein which links integrins to the
actin cytoskeleton. It is a direct integrin binding protein which further associates with
actin through its main binding partner, parvin. ILK also binds to the cytoskeleton through

15

its associations with paxillin, which also binds parvin and vinculin. ILK plays critical
roles in stabilizing the integrin–actin interaction which is shown by the fact that deletion
of ILK from the skeletal muscle results in detachment of basement membranes and
accumulation of extracellular matrix(Wang, Chang et al. 2008).

1.7.5 Focal Adhesion Kinase
Focal adhesion kinase (FAK) is a signaling protein that most likely interacts
indirectly with β integrins through its association with paxillin; it is a non receptor
tyrosine kinase signaling protein. FAK is required to stabilize the linkage to actin by
modulating the affinity of α-actinin to actin, which occurs via FAK-mediated
phosphorylation of α -actinin. FAK is although not required for nascent FA formation or
for the initial connection of integrins to the actin cytoskeleton. In addition to cytoskeletal
stabilization, FAK also plays an essential role in promoting FA turnover(Ilić, Furuta et al.
1995).

1.8 Disintegrins
Different snake venoms act differently on its prey, indicating that each venom is
unique with respect to its specificity and selectivity. Snake venom from snakes belonging
to Elapidae and Hydrophidae families are highly toxic and are involved in deaths due to
blocking of neuromuscular transmission, while venoms from snakes belonging to the
Viperidae family are mainly responsible for systemic and local hemorrhage, intravascular
clotting, edema and necrosis (Teng and Huang 1991).Snakes venom contains differing

16

amounts of various proteins in the venom , that play role in hemostasis and thrombosis.
These include aggregation inducers, fibrinogenolytic enzymes, prothrombin activating
inhibitors, platelet aggregation inhibitors, factor X activating inhibitors.
One of the family of proteins found in Viperidae venom are the disintegrins,
which are low molecular weight integrin antagonist. They are typically about 40-100
amino acids and found in low abundance in the snake venom. However interestingly they
are part of the venom but not toxic themselves, they are mainly present in the venom to
counteract the effect of integrins present on the blood platelets and inhibiting platelet
aggregation and as a result the venom can spread through the blood more quickly and
easily. The first disintegrin molecules purified from the snake venom include trigramin
and echistatin from Trimeresurusgramineus and Echiscarinatus venom respectively
(Teng and Huang 1991).A common feature observed among many disintegrins is the
presence of a specific Argenine-Glycine-Aspartic acid (RGD) which seems to be
imparting the biological activity to disintegrins against the integrins.
Structurally disintegrins are similar to other snake venom proteins in the fact that
they also have a large number of disulfide bonds. And peculiarly these molecules are
small in size. Depending on the number of disulfide bonds, number of subunits and the
polypeptide length the disintegrin molecules can be classified under different groups
namely dimeric group (~67 amino acids with 10 cysteines involved in 4 inter chain and 2
intrachain disulfide linkages), monomeric long group (~84 residues and 7 disulfide
bonds), monomeric medium group (~70 amino acids and 6 disulfide bonds), monomeric
short group (41-51 residues and 4 disulfide bonds) (McLane, Joerger et al. 2008). The

17

most important structural feature present in case of these molecules is the binding loop
which often contains an RGD motif which plays an important role in modulating the
binding of the disintegrin molecule to the integrin and inhibiting them. A slight
modification in the sequence can disrupt the loop and may cause inactivity or inability of
the molecule to bind to the integrin molecule.
Disintegrins have been characterized by many researchers as anti-adhesive
molecules since they bind to cells adhesive molecules, integrins, and disrupt their
function of attachment. As a result they have been seen as molecules with application as
cancer therapeutics as they can induce anti-migratory effects by blocking the integrins
present on cancer cells, which cells use for metastasis and attachment(Staniszewska,
Walsh et al. 2009). In case of snake bites the disintegrins act by mainly binding to the
αIIbβ3 integrins present on the platelet surface and as a result will inhibit platelet
aggregation. However, these disintegrins also binds to many other integrins like α5β1,
αvβ3, αvβ5 as well as αIIbβ3 which play an important role in cancer cell metastasis.
These integrins usually bind to the ECM proteins like fibronectin, vitronectin and
fibrinogen respectively, on which these cancer cells do migrate, indicating that these
molecules can be used as weapons against cancer.

1.9 Disintegrins Contortrostatin (CN) and Vicrostatin (VCN)
The Markland laboratory has a long history of studying proteins isolated from
snake venom. In early 1980’s Markland lab was working on isolation and
characterization of fibrinolytic enzymes from snake venom. In 1988, Markland lab was

18

successful in isolating and characterizing a novel venom protein fibrolase, a fibrinolytic
enzyme ,which is a direct fibrin acting molecule (Retzios and Markland 1988). Later a
modified version of fibrolase was created by Amgen and later it even passed Phase 1 and
2 clinical trials, however, unfortunately it failed to pass Phase III trials and it never made
it to the market. However, Markland lab continued exploring snake venoms for other
potential blockbuster drug molecules.
In 1994 Markland lab was successful in identifying a snake venom disintegrin
―Contortrostatin‖ (CN) which targeted integrins on cancer cells and inhibited
experimental metastasis. Contortrostatin was found in venom of Agkistrodoncontortrix
and was purified via a 3 step HPLC process. CN is a homodimer with a molecular weight
of 13,500 with each chain having an RGD motif and individual weight of 6750(Trikha,
Rote et al. 1994). CN binds to integrins of the β1, β3 and β5 subclasses, CN was found to
inhibit the process of invasion, tube formation and migration which are crucial steps of
angiogenesis and migration by up to 90 percent. CN was found not only to be an integrin
antagonist but was also found to cause disruption of variety of signaling pathways upon
ligation to integrins CN was shown to cause tyrosine phosphorylation of different
intracellular proteins also it was involved in causing severe disruption of actin
cytoskeleton and disassembly of focal adhesive structures (Trikha, De Clerck et al. 1994,
Schmitmeier, Markland et al. 2005). This lead to further progression of CN from in vitro
experiments to in vivo animal models where CN was found to reduce the growth rate in
an experimental breast cancer model and also reduce pulmonary metastasis, Further in
order to avoid issues related to efficient delivery and immunogenicity arising due to the

19

protein being venom based, a novel delivery strategy was devised, CN was stared to be
encapsulated in liposomes and these were used as vectors for drug delivery. It was found
that by doing this CN retained its full biological activity at the same time it did not elicit
any immune response and also it was seen that there was a significant improvement in the
half-life of CN (Swenson, Costa et al. 2005). Despite all the advantages the problem with
CN was its availability, limited quantities are present in snake venom which made it hard
to use CN as therapeutic agent.
Later in early 2000’s Dr. Minea in Dr. Markland’s lab designed and engineered a
novel recombinant disintegrin ―Vicrostatin‖ (VCN). The sequence of VCN was based on
CN, however both the molecules were different in terms of their structure and size; it was
found that VCN as a molecule was a monomer and not a homodimer as seen in case of
CN, the molecular weight of VCN is found to be 7146.0 daltons.VCN is generated
recombinantly by grafting C-terminal tail of viperid snake venom disintegrin echistatin to
the sequence of crotalid disintegrin contortrostatin (CN). It is believed that C-terminal of
snake venom disintegrins are important structural elements and are thought to be essential
for full disintegrin activity and it was this reason that in case of VCN the C-terminal tail
is grafted from other snake venom disintegrin echistatin (Minea, Helchowski et al. 2012).
VCN is produced in a particular strain of bacteria called Origami B (DE 3) Escherichia
coli. This strain has been uniquely designed to overcome the shortcomings of disulfide-
rich recombinant protein production.

20

Figure 1.6:-Sequence alignment of contortrostatin (CN), vicrostatin (VCN), and echistatin. The
figure also shows how the C-terminal of VCN has been generated by modification of CN and also
shows the Arg–Gly–Asp tripeptide motifs in bold and the graft in the structure of VCN has been
underlined. Source:(Minea, Helchowski et al. 2012)

To evaluate the activity of the recombinantly produced disintegrin, it was tested
for efficacy against platelet aggregation. Since being snake venom disintegrin in nature
they are presumed to bind to activated αIIbβ3 integrins on the platelets and inhibiting the
final step of blood coagulation and hence allowing the venom to spread through the
blood. It was found that VCN had full activity against activated αIIbβ3. VCN was found
to have the same IC
50
as that of CN in inhibiting the process of platelet aggregation
(~60nM)(Minea, Helchowski et al. 2010).

21

SPECIFIC AIMS:-
1) Integrins are needed by the cells to bind to the ECM matrix and for metastasis.
Here we test VCN, which is considered to be integrin inhibiting molecule, on breast
cancer cells MDA-MB-231 and observe its effect on cell attachment and spreading.
2) To study the effect of VCN on β1 integrin signaling and various components
such as Talin, Actin and Vincullin via Co-immunoprecipitation and Western Blotting.
3) To study the effect of VCN on FAK phosphorylation at Tyr397.

22

CHAPTER 2
MATERIALS AND METHODS
2.1 Cell Culture and Antibodies

MDA-MB-231 (ATTC, Manassas, VA) human breast cancer cells are grown in
Dulbecco’s modified Eagle’s medium (DMEM)(USC- cell culture core) containing 10%
fetal bovine serum (FBS)(Omega Scientific, membrane filtered), 100 U/ml penicillin, and
0.1 mg/ml of streptomycin, and incubated in a humidified atmosphere at 37
O
C and 5%
CO
2
in tissue culture treated flasks. The cells are grown up to 90% conflunecy before
being passed.
The antibodies for the assay were purchased from Santa Cruz Biotechnologies
(Talin: TA205, H-300 and C-20; β1: P5D2 and N-20; FAK: A-17; pFAK: Tyr 397-R;
Vinculin: G-11; Actin:I-19) The secondary antibodies labeled with Infrared Dyes were
purchased from Li-Cor Biosciences (Donkey anti-goat: 926-32214, Donkey anti- mouse:
926-32212, Donkey anti- rabbit: 926- 32213). The magnetic beads and the magnet used
for the Immunoprecipitation experiments were purchased from Life Technologies
(Protein A beads Dynabeads, 10007 D, and magnet: DynaMag-2 12321D). The bovine
fibronectin solution was purchased from PromoCell (Heidelberg, Germany).

2.2 Expression and Purification of VCN
Method for production and purification of recombinant VCN described in
previous publication by Minea et al. (Minea, Helchowski et al. 2012) was employed. We
used pET32a vector system along with BglII/NcoI restriction enzymes to insert the

23

VCN sequence downstream of Thioredoxin (Trx) and the whole construct was expressed
in Origami B (DE3) E. coli strain. The transformed cells are then grown in autoclaved
media, induced with IPTG and are then lysed in microfluidizer (Microfluidics M-110 L,
Microfluidics, Newton, MA).The lysates are then centrifuged at 40,000X g in order to
remove cellular debris and collect the soluble supernatant. The soluble lysate now
contains VCN-TRX fusion protein which was then cleared by incubating with
recombinant TEV protease overnight. The proteolyzed lysates are collected and then
passed through a 0.22mm filter and diluted 1:100 in deionizer water, then the lysate is
ultra-filtered twice by using two different molecular weight cartridges initially we pass
the lysate is passed through 50000 MW cut off cartridge (Biomax50, Millipore) and then
we re-concentrate against 5000MW cut off cartridge (Biomax5, Millipore); for this the
tangential flow ultra-filtration deice was used (Labscale TFF system, Millipore).
Further purification is carried out by Reverse phase HPLC using C-18 column
according to the method previously elucidate (Minea, Helchowski et al. 2010). The
filtered lysates are loaded on Vydac C-18 column (218TP54, Temecula, CA). Initially the
column is washed by using an aqueous solution containing 0.1% TFA at a rate of
5ml/min, after the washing we start the elution process in a linear gradient 0-100% for
about 150 minutes in which the mobile phase contains 80%acetonitrile and 0.1%TFA.
During the process we find the elution of VCN to take place at 35% acetonitrile. After
rigorous purification we went ahead for characterization of VCN. Sodium dodecyl-sulfate
polyacrylamide gel electrophoresis is used to characterize the purified VCN and we
found the molecular weight to be approximately 7000 daltons under reducing conditions.

24

In order to make sure that the protein purified is active we tested it against the activated
platelet integrin αIIbβ3, inhibiting the last step of aggregation of platelets.

2.3 Cell Adhesion and Spreading Assay
Twelve well plates are coated with fibronectin at a concentration of 10μg/cm
2
,
with appropriate amount of media containing matrix, the coated plates are then kept at
room temperature overnight for the ECM matrix to attach to the well surface .MDA-MB-
231 cells are grown in T-75 culture flasks till they achieve 90% confluency, they are then
detached by using 0.05% trypsin and are counted by mixing 100μl of cell sample with
100μl of 0.1% trypan blue and then loading 10-15μl onto haemocytometer. 2 X10
5
cells
are counted and the required amount of cell suspension was aliquoted in 1.7ml centrifuge
tubes, these cells are then transferred to a rotating device in an incubator at 37
O
C for 45-
60 mins to allow integrin re expression. The tubes are centrifuged 1000rpm for 2 minutes
and the media is removed from the tubes and replaced with serum-free media. The cells
are then treated with different concentration of VCN or are left untreated for 1 hour and
later are seeded on the wells coated with fibronectin and allowed to be incubated for 1- 3
hours in a humidified atmosphere at 37
O
C and 5% CO
2
.

2.4 Lysate preparation, Immunoprecipitation and Western Blotting
MDA-MB-231 cells (3 x 10
6
) are either left untreated or treated with VCN (1 nM,
10 nM, 100 nM) for 30-min at 37 C. Fresh cell lysates are produced by aspirating the
media and washing the pellet with phosphate buffered saline (pH 7.4).The PBS with the

25

cells is collected in 1.7ml centrifuge tube and it is spun down at 1000rpm for 3 minutes to
pellet out the cells, the supernatant containing PBS mainly is discarded and freshly
prepared RIPA ( Santa Cruz Biotechnlogy, sc-24948) is added to the pellet, depending on
the size of the pellet. The lysate containing tubes are kept at 4
O
C or on ice and vortexed
after every 10 minutes up to 60 minutes along with passing them througha 30 gauge
syringe twice each time after vortexing during each of the 10 minutes incubation. The
lysates are then spun down at 12000 rpm for 20 minutes. The tubes are cooled and the
supernatant, considered to be protein lysate, is transferred to a fresh centrifuge tube and
saved for further analysis and protein estimation by BCA assay (BCA Protein Assay Kit,
Thermo Scientific, IL, 23225).
For the purpose of Immunoprecipitation we used magnetic beads (Dynabeads,
10007D, Life Technologies, CA). We used 20 μl of beads and separated from the solution
by placing on the magnet (DynaMag-2 12321D, Life Technologies,CA), the antibodies of
interest (Anti-Beta 1,Anti-Talin) are mixed with 200 μl of the antibody binding buffer
and added to the beads and mixed by gentle pipetting. The tubes are then incubated at
4
O
C for 1-2 hours on a rotating device for allowing the antibodies to bind to the beads.
Further the tubes are kept on the magnet and the beads are separated from the mixture,
they are washed twice with 200 μl of washing buffer and later re-suspended in 200 μl of
the washing buffer. Cell lysate (750 μg) area liquoted in separated PCR tubes, the tubes
are further kept for incubation at 70
O
C using PCR machine, the tubes are allowed to cool
and the lystaes are transferred to the beads. The beads are cleared of the washing buffer
by keeping them near the magnet and pulling out the excess buffer before mixing of the

26

lysates to the beads. The lysates with the beads are incubated for 2hours-overnight.After
incubation the beads are separated by using the magnet and the bead pellet is given three
washes using washing buffer and 20 μl of sample buffer is added and boiled for 5
minutes. The sample tubes are then kept over magnet so that the beads attach the sides of
the tube and the sample buffer containing the protein is pulled out and loaded in the wells
of the gel (8% Tris glycine pre-cast gels, NuSep # NG 21-008). The gel is run for 60-75
mins at 200V at 4
o
C. After completion of electrophoresis, proteins are transferred on
PVDF membrane (Western S PVDF membrane, Whatman, # 10413096) using wet
transfer method. After completion of the transfer the membranes are then blocked using
blocking buffer (Li-Cor Odyssey Blocking Buffer # 927-40000) at room temperature for
1 hour. The membranes are then sealed in plastic pouches along with 5ml of the primary
antibody of interest at a concentration of 1:1000 and kept on a rotating device for
overnight incubation. The membrane is then washed with Phosphate Buffered Saline
(PBS) for 15minutes 4 times. The membrane was then incubated in secondary antibodies
conjugated with infrared dye (IR) (Li-Cor IR secondary antibodies) for 90-120 minutes.
The membrane is removed from secondary antibody incubation and given 4 washes of 15
minutes each. The membrane is then developed on Li-cor Odyssey classic infrared
imaging system.

2.5 Studying the effect of VCN on FAK phosphorylation
In order to study the effect of VCN on FAK phosphorylation, the cells are serum
starved overnight the next day the cells are harvested with 0.05% Trypsin-EDTA.

27

Suspended cells (3X10
6
) cells are separated and replated on dishes coated with
Fibronectin (10μg/cm
2
) The dishes are allowed to be incubated for 3 hours at 37
O
C at 5%
CO
2
to allow the cells to attach to the ECM matrix and later the cells are further
incubated for 2 hours either untreated or with treatment of cells with varying
concentration of VCN (1nM, 10nM, 100nM). After treatment the media was aspirated
and the cells are washed with PBS and later the cells are scrapped using cell lifter
collected in 1,7ml centrifuge tubes and are then later lysed, the cell lysates are resolved
on 4-20% polyacrylamide gels. Proteins are transferred to PVDF membranes for 2 hours
4°C, blocked with blocking buffer and incubated with primary antibodies overnight and
probed with IR based secondary antibodies andthe membrane is then developed on Li-cor
Odyssey classic infrared imaging system.

2.6 Statistical Analysis
For each of the experiments above a student T-test was performed for statistical
analysis, generating a p-value. Error bars were included on the graphs to illustrate the
standard deviation between the multiple experiments.

28

CHAPTER 3
RESULTS
3.1 Inhibition of cell adhesion and cell spreading.
Cell adhesion and spreading is an important process in cancer metastasis. For a
cell to sucessfully metastasize it needs to move in the body and adhere to the new site,
where it it involved in making cell to ECM adhesions using adhesion molecules such as
integrins. Due to the crucial role played by integrins in the entire process we were very
much intrested in looking into the effect of VCN on the MDA-MB-231 cells in presence
of ECM matrix VCN.
Metastatic breast cancer cells MDA-MB-231 were grown as described . The cells
were treated with varying concentration of VCN or were left untreated and were plated
on ECM matrix and incubated for short (1 hr) and long(3 hrs) time period. After 1 hour of
incubation (Figure 3.1.1) one quarter of the population of the untreated cells were found
to have started attaching to the basement membrane and were seen to have more of
extended spread cell morphology. In case of 1nM concentration of VCN very few cells
were able to attach to the basement mebrane while the remaining were found to be
unattached. In case of cells with VCN at concnetrations of 10nM and 100nM almost no
cell adhesion was seen. After 3 hour of incubation (Figure 3.1.2) the majority of cells in
the untreated well were found to be adherent and were seen spreading . In the well with
1nM VCN also we observed a large number of cells adhering and beginning to spread. In

29

the 10nM VCN treatment well we observed a greater number of cells being adhered than
seen in 1 hour incubation, also certain amount of adhesion was also seen in cells plated
with 100nM after 3 hours of plating as compared to 1 hour of plating under the same
VCN concentration.

Figure 3.1.1:- Effect of VCN on Adhesion and spreading of MDA-MB-231 cells after 1HOUR
of incubation

Panel I shows untreated cells where we see a quarter of the population of the cells being attached.
Panel II shows cells treated with 1nM of VCN where we see very less cell attachment as
compared to the no treatment ones. Panel III shows cells treated with 10nM VCN where we see
almost no attachment and Panel IV shows cells treated with 100nM of VCN, where we see no
attachment

30

Figure 3.1.2:- Effect of VCN on Adhesion and spreading of MDA-MB-231 cells after 3 HOUR
of incubation

Panel I shows untreated cells where we observe maximum attachment, Panel II shows cells
treated with 1nM of VCN where we also observe considerable attachment as compared to the no
treatment Panel III shows cells treated with 10nM VCN were also we find moderate attachment
as compared to the no treatment and Panel IV shows cells treated with 100nM of VCN where we
observe least attachment as compared to the no treatment group.

31

3.2 Disruption of Talin Association to β1 on treatment of VCN.
The primary molecule in the entire process of signaling with integrins is talin.
Talin binds to cytoplasmic tails of integrin β1 and further acts as an anchor which allows
large number of different signaling molecules to attach and further transduce the
incoming signal. Here we examine whether treatment with various different
concentrations of VCN leads to decrease in the interaction between the β1 integrin and
talin molecule.
To carry this out, the beta1 integrin receptor was immunoprecipitated and further
probed it for association with talin. On immunoprecipitatingβ1 integrin from treated and
non-treated cells we find that VCN does infact reduce the amount of talin binding to the
β1 integrin in the treated cells as compared to non-treated cells and the reduction in
binding of talin happens is dose dependent with an increase in the concentration of VCN.

Figure 3.2:- Effect of VCN on disrupting the interactions between β1 and talin associations in
MDA-MB-231 cells.

Panel A: Western blot image showing IP of beta1 integrin with immuno-blot for talin head and
talin rod. The data shows significant decrease in the pull-down of Talin head at all the treatment
concentrations indicating severe disruption of binding between β1 and talin H region, while we
see significant decrease in the Talin rod only at 10nM and 100nM concentrations of VCN and not
at 1nM concentration.
A

32

Panel B: Densitometry plot for the western blots in panel A, in the plot for talin head we can see
considerable decrease in talin head pull down at all concentrations and at 100nM treatment we
can see only 10% pull down of talin head as compared to the control no treatment one. The error
bars indicate SD among the 3 measurements for each of the conditions. Significant differences
from the control are indicated as * P < 0.05

B

33

3.3 Disruption of key associations between focal adhesion proteins in cells treated with
VCN.
Vinculin is a key regulator in the formation of focal adhesion complex it binds to
the Rod region of talin and further cause’s attachment of talin to the actin cytoskeleton
hence it was important to know whether treating cells with VCN does have some effect
on vinculin binding to talin rod region. On treating cells with VCN we find that there is a
significant reduction in the association betweenβ1, Talin and Vinculin.
Observing this the next logical step was to see the associations between β1, Talin
and Actin and to observe this we pulled down β1 molecule and probed it for its
association with actin and as expected the amount of actin associated to the Rod region of
Talin was found to be dose dependently decreasing as the concentration of VCN
increases indicating that VCN does systematically disrupt association among the focal
adhesion complex proteins.

Figure 3.3:- Effect of VCN on disrupting associations between key focal adhesion proteins in
MDA-MB-231 cells

Panel A: Western blot image showing IP of beta1 integrin with immunoblot for Vinculin. The
data shows significant decrease in binding of vinculin at 10nM and 100nM concentration of VCN
as compared to control
A

34

Panel B: Densitometry plot for the western blots in panel A. The graph shows significant
disruption of vinculin binding at 10nM and 100nM concentration of VCN which is about 47%
and 18% respectively as compared to 0nM treatment. The error bars indicate SD among the 3
measurements for each of the conditions Significant differences from the control are indicated as
* P < 0.05

Panel C: Western blot image showing IP of beta1 integrin with immunoblot for Actin. The blot
shows a marginal decrease in actin binding at 1nM treatment however significant decrease in
actin is seen at treatments of 10nM and 100nM concentrations.
B

C

35

Panel D: Densitometry plot for the western blots in panel C. The graph shows a marginal
decrease in actin binding at 1 nM concentration which is about 78% as compared to the standard,
while at 10nM and 100nM the decrease in actin binding is significant, about 39% and 19 %
respectively as compared to 0nM treatment. The error bars indicate SD among the 3
measurements for each of the conditions. Significant differences from the control are indicated as
* P < 0.05

D

36

3.4 Effect of VCN on Phosphorylation status of Focal Adhesion Kinase (FAK and pFAK-
Y397
)
FAK is an important molecule in transducing external signals to the inside of the
cell. Under normal circumstances FAK acts as an adaptor protein which is involved in
binding of paxallin, vimentin and talin. Integrins are found to be involved in activation of
FAK. Under normal state the FAK proteins are auto inhibited mainly because of intra-
molecular interaction between its kinase domain and the amino terminal FREM domain,
this auto inhibitory interaction is disrupted by integrins causing transient dimerization of
the FAK molecules causing increased phosphorylation on Try397 and produces a high
affinity anchoring sites for binding of Src, leading to formation of stable FAK-Src
complex which is further involved in phosphorylation of many other substrates like CAS,
paxillin, and p190RhoGAP which have a central role in the reorganization of the actin
cytoskeleton and migration (Parsons 2003, Zhao and Guan 2009).
Since FAK plays an important role in phosphorylation of a large number of
downstream molecules and the activation of FAK is dependent largely on integrins, we
were interested to look into the effect of VCN on inhibiting FAK phosphorylation. On
treating the metastatic MDA-MB-231 cells bound to fibronectin with VCN for 3 hours
we found VCN to considerably affect the FAK phosphorylation at Try-397. At 10nM and
100 nM concentration of VCN we were able to see significant decrease in Try-397
phosphorylation levels at 10nM and 100 nM.

37

Figure 3.4:- Effect of VCN on Phosphorylation status of Focal Adhesion Kinase (FAK) in
MDA-MB-231 cells

Panel A: Western blot image showing levels of pFAK
Y397
from cells treated with VCN. 1nM
treatment shows an overall decrease in the phosphorylation level however the decrease in the
phosphorylation levels is significant in cells treated with 10nM and 100nM

A

38

Panel B: shows densitometry plot for panel A. The graph shows the significant extent of decrease
in the phosphorylation of FAK when the cells are treated with 10nM and 100nM of VCN which
is about34% and 13% respectively as compared to control 0nM treatment. The error bars indicate
SD among the 3 measurements for each of the conditions. Significant differences from the control
are indicated as * P < 0.05

B

39

CHAPTER 4
DISCUSSION

A major health problem in large parts of world including United States is Cancer,
statistics shows that 1 in 7 deaths in world and 1 in 4 deaths in United States is caused by
Cancer (Siegel, Naishadham et al. 2013). Current standard of care for cancer involves
used of drugs and compounds on the basis of their cytotoxicity, yet a majority of people
with advanced cancer fail to respond and do not survive. The predominant cause of
morbidity and mortality associated with cancer is the emergence of metastasis in distant
organs, due to which even by the time the primary cancer is detected and diagnosed;
metastasis might have already taken place to a distant site (Fidler 1985). Cell migration
has been be acknowledged to play a critical role in tumor invasion and cancer metastasis
(Hanahan and Weinberg 2000). This makes the migratory machinery employed by the
cell (cell adhesion molecules) to move an attractive set of targets which can be exploited
in order to win the war against cancer.
Among the various molecules and cellular machineries employed by the cell to
migrate are the immunoglobulin super family, integrins, cadherins and selectins. Integrins
are an important class of molecules and have often been targets of interest for treatment
of cancer due to their ability to promote intracellular signaling in cancer cells; notably
integrins have the capacity to conduct inside- out as well as outside- in signaling (Hynes
2002). Also integrins have been found to participate in a large number of different
intramolecular interactions such as crosstalk with growth factor cytokines and also

40

cooperation with oncogenes. In addition, integrins are involved in formation of focal
adhesion complexes which play an important role in cellular metastasis. (Trusolino,
Bertotti et al. 2001, Guo, Pylayeva et al. 2006).
MDA- MB-231 is a highly metastatic breast cancer cell line, which also expresses
β1 in huge amounts (Morini, Mottolese et al. 2000).Disintegrins are small low molecular
weight, integrin antagonist proteins found in snake venom. Vicrostatin is a recombinant
snake venom disintegrin containing the RGD binding domain. This report mainly shows
how VCN in involved in disruption of mechanical associations between key regulatory
focal adhesion proteins. In this report VCN is shown to affect cell adhesion and spreading
of the metastatic MDA-MB-231 cancer cell line.
To study the effect VCN on metastasis, both cell adhesion and cell migration were
investigated. Fibronectin coated plates were employed to see the effect on cells after
being treated with VCN. Fibronectin is one of the ECM matrix proteins used to mimic in
vivo tumor microenvironment in addition tumor cells preferentially spread more rapidly
on the matrix, On treating the cells with VCN and plating them on plates coated with an
ECM matrix protein it was seen that after 1 hour of incubation one quarter of the
untreated cells were found to have attached to the matrix while in case of treated cells
few cells were found to be attached. After longer incubation of 3 hours it was seen that a
majority of untreated cells were found to be attached while there was a concentration
dependent loss of attachment in cells treated with VCN with no attachment seen in case
of cells treated with high concentrations of VCN indicating that at longer time points a
greater effect was seen for VCN in disrupting cell adhesion.

41

After demonstrating the ability of VCN to disrupt cell adhesion, the next logical
step was to examine the mechanism VCN uses to disrupts the cell adhesion to examine
this, co-immunoprecipitation was employed to study the intracellular binding partners of
integrins and to see whether treatment of cells with VCN have an effect on the
recruitment of these binding partners.
The primary molecule to be studied in the signaling pathway is talin; this is
because talin binds to the cytoplasmic domain of β1 which is an event that represents the
common final step in integrin activation. The data revels that that the exposure of MDA-
MB-231 cells to no treatment showed normal high levels of Talin-H and Talin-R regions
attached to the β1 integrin cytoplasmic tail. However when cells were exposed to VCN in
varying concentration i.e 1nM, 10nM and 100nM showed a dose dependent decrease in
the binding of both talin-H and talin-R to the beta1 cytoplasmic tail was observed.
Though we were able to precipitate the head and the rod region separately we were
unable to pull down the intact 270kD talin molecule. This can be attributed to the fact,
that β1 tails have a higher affinity for each of the talin individual sub-domains than for
the intact protein which is the reason why we tend to lose the intact protein in this
experiment. To explain these concentration dependent decreases in the talin regions
binding to β1 cytoplasmic tails results, it can be hypothesized that : (i) VCN tends to bind
the integrin molecule in the active conformation and causes it to remain active or tends to
freeze the molecule in active conformation, (ii) since integrins are bi-directional signaling
machines the binding of VCN to one of the integrins on the cell surface (outside→in

42

signaling) will cause the downstream propagation of signal which would cause the
neighboring unligated integrins to be inactivated (inside→out signaling)
Looking at the data from the disruption of binding of the talin to the cytoplasmic
tails of β1 it was logical to see the effect of VCN on binding of other intracellular binding
partner such as a linker molecule vinculin and also the effect of VCN on binding of the
final effector molecule actin. The data from the co–immunoprecipitation showed that
cells receiving dose dependent VCN treatment show a dose dependent decrease in the
binding the linker molecule vinculin and also showed similar results for decrease in the
levels of actin binding to talin. After looking into the data obtained from the
immunoprecipitation of the β1 subunits and looking at the decrease in the binding of the
intracellular molecules, the next step was to look at the phosphorylation status of one of
the important cellular kinase the focal adhesion kinase (FAK). FAK is said to have an
important role in cancer progression and metastasis, reports have shown that
mechanistically, talin up regulation leads to activation of FAK signaling and also leads to
excessive phosphorylation of FAK at Try-397 (Chang, Huang et al. 2005) .Under normal
circumstances the FAK molecule is auto inhibited due to intramolecular interaction,
however, integrins are involved in disrupting the auto inhibitory interaction and lead to
activation of FAK molecule and its phosphorylation at Try-397 (Zhao and Guan 2009).
Hence it would be interesting to see the effect of VCN on the ability to affect FAK
activation and FAK phosphorylation.
In order to study FAK phosphorylation, the cells were allowed to attach on to
fibronectin and then treated with VCN. The cells treated with VCN showed decreasing

43

concentration of pFAK
Y397
with increasing concentrations of VCN indicating that
inactivation/blocking of integrins by VCN further leads to a decrease in the
phosphorylation status of FAK and further confirming that integrins play an important
role in phosphorylation of FAK, which themselves are then further leads to
phosphorylation of a large number of intracellular signal transducing proteins.
From the data obtained in the studies presented here it can be determined that
VCN binding to the extracellular domain of integrins leads to displacement of talin from
the cytoplasmic tail region of the integrin molecules further causing inactivation and
alteration of the function of the integrin molecule. VCN is found elicit this effect via two
distinct routes from outside→in while at the same time inside→out generating a global
wave of integrin inactivation through the VCN exposed cell.
Cell adhesion molecules play a dynamic role in cancer disease progression and
integrins are one of the cell adhesion players that have a major role in cancer metastasis
and its progression. Due to an integral role played by integrins in cancer, they have
started to become a potential target for therapeutics. Active screening is being done to
find molecules with therapeutic potential as integrin antagonists for use in cancer therapy.
In this report we demonstrate and evaluate the potential of vicrostatin, a recombinantly
produced disintegrin molecule, to be used as an anti- cancer therapeutic agent. Here we
show how VCN is involved in mechanistically disrupting the signaling pathways
employed by integrins on the cancer cell surface and blocking adhesion and processes
integral to metastasis. Further studies are underway both in-vitro and in-vivo to elucidate

44

the mechanistic action of VCN as well as the therapeutic efficacy and clinical potential of
this novel drug molecule.

45

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Abstract (if available)

Abstract Cell adhesion molecules have been found to play a pivotal role in progression of cancer. Integrins are one the molecules among these various cell adhesion molecules that have been found to play an important role due their ability to bind to the extra cellular matrix. Integrins have been also found to be involved in mediating a large number of signaling pathways. Disintegrins are small molecules which act as integrin antagonist and can disrupt integrin function by directly binding them. However regarding how these disintegrin molecules initiate signaling events once they bind to their ligand remains unanswered. Recently vicrostatin (VCN), a recombinantly produced disintegrin, was successfully produced in Markland laboratory. Based on the availability of this reagent mechanistic studies can be initiated to determine the action of disintegrins upon binding to the integrin ligand. ❧ In this report, we show how VCN affects cell adhesion and cell spreading of the metastatic breast cancer cell line MBA-MD-231. We also demonstrate effect of VCN on one of the key molecule of integrin activation, Talin, and show how VCN binding to the extracellular domain of β1 disrupts the binding of talin on the cytoplasmic domain. Through a series of experiments involving immunoprecipitation and western blotting we were able to show that VCN causes disruption of interactions between talin and the cytoplasmic tails of β1. We also show how VCN disrupts associations within the key focal adhesion (FA) proteins and how VCN affects the activation and phosphorylation status of Focal Adhesion Kinase, a key kinase molecule involved in activation and phosphorylation of a number of downstream signaling molecules. The data from all the events in this study shows VCN to produce a global inactivation signal for beta1 integrins. The findings from this study will help in increasing knowledge regarding disintegrin signaling and their function.

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Asset Metadata

Creator Shah, Shrenik Rajesh (author)

Core Title Effect of vicrostatin on integrin based signaling molecules in cancer

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 08/05/2013

Defense Date 06/18/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag beta 1,cancer,disintegrins,integrins,OAI-PMH Harvest,signaling,vicrostatin

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Hacia, Joseph G. (committee member), Hofman, Florence M. (committee member), Markland, Francis S., Jr. (committee member)

Creator Email shreniks@usc.edu,shreniks30@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-315398

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Legacy Identifier etd-ShahShreni-1955.pdf

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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