CHARACTERIZING ROLES OF ANNEXIN 1 IN BREAST CANCER DEVELOPMENT BY MASS SPECTROMETRY BASED QUANTITATIVE PROTEOMICS

CHARACTERIZING ROLES OF ANNEXIN-1 IN
BREAST CANCER DEVELOPMENT BY MASS
SPECTROMETRY-BASED QUANTITATIVE
PROTEOMICS

SWA LEE FOON HANNAH
B. Sc.(Merit), NUS

A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHYSIOLOGY
NATIONAL UNIVERSITY OF SINGAPORE
2013



Acknowledgements

This graduate journey is akin to a jigsaw puzzle. Besides the science involved,
completion of the full picture would not be possible without any of the
following ‘jigsaw pieces’. I would like to acknowledge and express my
gratitude to:
A/P Lina Lim, my supervisor, for her patience and guidance. I am also
thankful for the freedom, help and support she has graciously given me in this
journey.
Asst Prof Jayantha Gunaratne, my co-supervisor and mentor, for his grace,
guidance and support. His hard-working and passion in science greatly
motivates me.
Prof Walter Blackstock, my ex-boss of the lab. He was the one who cleared
the obstacles-filled path for me, making the commencement of this graduate
studies possible.

Past and present members of the Quantitative Proteomics Group (QPG),
especially Suat Peng and Sheena for their friendship, support and hearing ears
whenever I whine. Siok Ghee, for her expertise in mass spectrometry and
Rachel for her help in all the bioinformatics and visualization plots.
Past and present members of the Inflammation and Cancer Lab, especially
Durkesh, Sunitha and Suruchi for their friendship, for always being there for
me. This graduate fellowship we have shall be a beautiful memory.
Institute of Molecular and Cell Biology, A*STAR, for funding part of this
graduate programme.
My family members for their support, and especially my grandmother for her
constant love and concern for me.

Above all, I present my heartfelt thanksgiving and gratitude to God, the One
who loves me, never gives up on me and the One who holds the pieces of the
jigsaw puzzle together.

ii


Table of Contents
DECLARATION ................................................................................................. i 
Acknowledgements ............................................................................................. ii 
Summary………….. ......................................................................................... vii 
List of Tables ..................................................................................................... ix 
List of Figures ..................................................................................................... x 
List of Abbreviations ....................................................................................... xiii 
List of Symbols ................................................................................................ xvi 
Chapter 1: 

Introduction ................................................................................... 1 

1.1: 

Breast cancer ........................................................................................ 1 

1.2: 

Annexin 1 ............................................................................................. 2 

1.2.1:  Structure of ANXA1......................................................................... 2 
1.2.2:  Functions of ANXA1 ....................................................................... 3 
1.2.2.1: 

Anti-inflammatory role .......................................................... 3 

1.2.2.2: 

Regulator of Cellular Processes ............................................. 4 

1.2.3:  ANXA1 and Cancer.......................................................................... 6 
1.3: 

Models for breast cancer ...................................................................... 8 

1.3.1:  Cell line models for breast cancer .................................................... 8 
1.3.2:  Mouse models for breast cancer ....................................................... 9 
1.3.2.1: 

Xenograft models .................................................................. 9 

1.3.2.2: 

Genetically-modified models .............................................. 10 

1.3.3:  Mammary gland development ........................................................ 10 
1.3.4:  Hallmarks of cancer ........................................................................ 12 
1.4: 

Proteomics .......................................................................................... 15 

1.5: 

MS-based proteomics ......................................................................... 17 

1.6: 

Quantitative MS-based proteomics .................................................... 22 

1.6.1:  Label-free Quantification ............................................................... 25 
1.6.2:  Chemical labeling ........................................................................... 26 
1.6.3:  Metabolic Labelling ........................................................................ 32 
iii


1.6.3.1:  Stable isotope labelling by amino acids in cell culture
(SILAC)
. ...................................................................................... 32 
1.6.3.2: 
1.7: 

Super-SILAC ....................................................................... 35 

Aims of Study..................................................................................... 36 

Chapter 2: 

Methods and Materials ................................................................ 37 

2.1: 

Isolation and culture of primary mammary gland epithelial cells ...... 37 

2.2: 

Adaptation of cell lines to SILAC media ........................................... 37 

2.3:  Filter-aided sample preparation (FASP) for SILAC incorporation
check ……………......................................................................................... 37 
2.4: 

Cell lysates preparation ...................................................................... 38 

2.5: 

Murine mammary tumors lysates preparation .................................... 39 

2.6: 

In-Gel Dehydration, Reduction, Alkyation and Tryptic Digestion .... 39 

2.7: 

Super-SILAC mix preparation ........................................................... 40 

2.8: 

Protein quantification ......................................................................... 40 

2.9: 

Western blot analysis ......................................................................... 40 

2.10:  RT-PCR analysis ................................................................................ 42 
2.11:  Heat treatment .................................................................................... 43 
2.12:  Comet assay........................................................................................ 44 
2.13:  Transfection experiments ................................................................... 44 
2.14:  Cell adhesion assay ............................................................................ 45 
2.15:  Hydrogen peroxide (H2O2) treatment and Cell proliferation (WST-1)
assay ….. ....................................................................................................... 45 
2.16:  Migration assay .................................................................................. 46 
2.17:  Wound healing assay.......................................................................... 46 
2.18:  Reactive oxygen species (ROS) assay ............................................... 46 
2.19:  Liquid Chromatography-Mass Spectrometry (LC-MS) ..................... 46 
2.20:  Identification and Quantification of Peptides and Proteins ................ 47 
2.21:  Bioinformatics Analysis ..................................................................... 47 
Chapter 3: 

Results ......................................................................................... 48 

3.1:  Proteome profiling of ANXA1+/- and ANXA1-/- normal mammary

gland cells...................................................................................................... 48 
3.1.1:  SILAC workflow for mass spectrometer analysis .......................... 48 
3.1.2:  MS data analysis ............................................................................. 52 
3.1.2.1:  Clustering proteins into up- and down-regulated with
ANXA1-/-………… .............................................................................. 55 
3.2: 

Analysis of down-regulated proteins with ANXA1-/- ........................ 57 

3.2.1:  Pathway analysis............................................................................. 57 
iv


3.2.1.1:  Identification of proteins in DNA-damage response
pathway…………………………….. ................................................... 57 
3.2.2:  Biochemical validation of selected down-regulated proteins ......... 59 
3.2.3:  Functional validation of pathway analysis ..................................... 60 
3.2.3.1:  Comet assay analyses of thermal-stressed mammary gland
cells……………. .................................................................................. 61 
3.2.3.2:  Reactive oxygen species (ROS) assay of mammary gland
cells……….. ......................................................................................... 63 
3.2.3.3:  Cell proliferation assays of oxidative-stressed mammary
gland cells……….. ............................................................................... 65 
3.2.3.4:  Over-expression of down-regulated protein reverses DNAdamage response in ANXA1-/- mammary gland cells ........................... 66 
3.3: 

Analysis of up-regulated proteins with ANXA1-/- ............................. 69 

3.3.1:  Pathway analysis............................................................................. 69 
3.3.1.1: 

Identification of proteins in cell adhesion/motility pathway69 

3.3.2:  Biochemical validation of selected up-regulated proteins.............. 72 
3.3.3:  Functional validation of pathway analysis ..................................... 72 
3.3.3.1: 

Migration assay of the mammary gland cells ...................... 73 

3.3.3.2: 

Wound healing assay of mammary gland cells ................... 74 

3.3.3.3:  Silencing of up-regulated protein reverses adhesive
phenotype in ANXA1-/- mammary gland cells.................................... 76 
3.4: 

Proteome profiling of ANXA1+/- and ANXA1-/- mammary tumors ... 82 

3.4.1:  Western blot analysis of the mammary tumors .............................. 82 
3.4.2:  Workflow for Super-SILAC mass spectrometry analysis .............. 83 
3.4.3:  Relative quantification of proteins ................................................. 88 
3.4.4:  Analysis of the Super-SILAC mix.................................................. 89 
3.5: 

Categorization of up- and down-regulated proteins with ANXA1-/- .. 91 

3.6:  Bioinformatics analyses of down- and up-regulated proteins with
ANXA1-/- ....................................................................................................... 92 
3.6.1:  Down-regulated proteins with ANXA1-/- ....................................... 92 

3.6.2:  Up-regulated proteins with ANXA1-/- ............................................ 93 
3.6.3:  Cancer-Associated Proteins (CAPs) and Cancer Driver Mutations
(CDMs) ...................................................................................................... 94 
3.6.3.1: 

Mapping up- and down-regulated CAPs to CDMs ............. 99 

3.6.3.2: 

Network with the up-regulated proteins ............................ 102 

3.6.3.3: 

Network with the down-regulated proteins ....................... 102 

3.7:  Comparison of normal mammary gland cells (SILAC-based) and
mammary tumors (Super-SILAC based)..................................................... 102 
3.7.1:  Up-regulated in both mammary gland cells and mammary tumors103 
v


3.7.2:  Down-regulated in both mammary gland cells and mammary
tumors ...................................................................................................... 104 
3.7.3:  Inverse correlation between up- and down-regulated proteins in
mammary gland cells and mammary tumors ........................................... 105 
3.7.4:  Up- or down-regulated proteins in mammary gland cells but no
change in mammary tumors .................................................................... 107 
3.7.5:  Up- or down-regulated protein in mammary tumors but not
detected in mammary gland cells. ........................................................... 108 
Chapter 4: 

4.1: 

Discussion ................................................................................. 110 

Characterization of ANXA1 from SILAC experiments ................... 110 

4.2:  Possible roles of ANXA1 in tumorigenesis revealed by Super-SILAC
experiments ................................................................................................. 118 
4.3:  Distinct differential regulation by ANXA1 between normal
mammary gland cells and mammary tumors .............................................. 125 
4.4: 

Use of SILAC, Super-SILAC and the mouse models ...................... 128 

4.5: 

Conclusion ........................................................................................ 129 

4.6: 

Future directions ............................................................................... 130 

Bibliography . ................................................................................................. 132 
Appendix

. .................................................................................................. 153 

Tables………….. ........................................................................................ 153 
Publications ................................................................................................. 190 
Supplementary CD .......................................................................................... 191 

vi


Summary…………..
Annexin-1 (ANXA1) has been reported to be involved in important physiopathological implications including cell proliferation, apoptosis, cancer and
metastasis. However, with controversies in ANXA1 expression in breast
carcinomas, its role in breast cancer initiation and progression remains unclear.
The study presented here seeks to characterize ANXA1 in initiation and
tumorigenesis of breast cancer by understanding the dysregulated pathways
involved. Through quantitative mass spectrometry-based proteomics, SILAC
(stable isotope labelling by amino acids in cell culture) and Super-SILAC
were applied to normal mammary gland cells and mammary tumors
respectively from ANXA1-heterozygous (ANXA1+/-) and null (ANXA1-/-)
mice to study their changes at the proteome level.
Quantitative comparison of the proteomes of normal mammary gland cells
using SILAC quantified over 4000 proteins with 214 up-regulated and 169
down-regulated in ANXA1-/-. Bioinformatics analysis of the up- and downregulated proteins revealed that ANXA1 is potentially implicated in DNAdamage response and cell adhesion/motility pathways. Relevant functional
assays showed accumulation of more DNA damage with slower recovery on
heat stress and an impaired oxidative damage response in ANXA1-/- cells in
comparison to ANXA1+/- cells. Over-expressing Yes-associated protein 1
(Yap1), the most down-regulated protein in DNA-damage response pathway
cluster, reversed the proliferative response in ANXA1-/- cells when insulted,
indicating the protective role ANXA1 plays via regulating a group of proteins
involved in the DNA-damage response pathway. Both migration and wound
healing assays showed that ANXA1+/- cells possess higher motility with better
wound closure capability than ANXA1-/-. Silencing of β-parvin in ANXA1-/-,
the protein with the highest fold change in the cell adhesion protein cluster
reversed its motility phenotype. This indicated that the pro-migratory role
ANXA1 plays via regulating a group of proteins that are involved in cell

adhesion/motility pathway.

vii


After establishing roles of ANXA1 in DNA-damage response and cell
adhesion/motility that could contribute to breast cancer initiation, its role in
breast cancer progression was investigated. Super-SILAC analysis on
mammary tumors derived from PyMT+ANXA1+/- and PyMT+ANXA1-/- mice
quantified over 5000 proteins with 369 up- and 365 down-regulated proteins
observed in PyMT+ANXA1-/-. Bioinformatics analysis of up-regulated
proteins showed that ANXA1 may be involved in cell cycle regulation
pathways, whereas the analysis of down-regulated proteins showed its possible
roles in cystic fibrosis and cytoskeletal remodelling during tumorigenesis. As
these outcomes are different from pathway analyses performed for the
regulated protein clusters from the normal mammary gland cell experiments,
overlapping up- and down-regulated protein clusters between mammary cells
and tumors were closely compared. Interestingly, the non-overlapped up- and
down-regulated clusters were enriched in cell adhesion/migration and DNAdamage response pathway respectively, further indicating that ANXA1 plays a
different role in tumorigenesis. Out of the differentially-regulated proteins, 32
from the up-regulated and 25 from the down-regulated were identified to be
cancer-associated. Together with functional interaction network mapping,
ANXA1 is proposed to play a modulatory role in several pathways in
tumorigenesis. Altogether the study here suggests that ANXA1 plays different,
yet important roles in breast cancer initiation and progression.

viii


List of Tables

Table 1:

The status of ANXA1 in clinical cancer tissues

Table 2:

Antibodies used for western blotting

Table 3:

Primers used for RT-PCR analysis

Table 4:

Down-regulated proteins involved in DNA-damage response
pathway

Table 5:

Up-regulated proteins involved in cell adhesion and motility

Table 6:

Cell lines used in the Super-SILAC mix

Table 7:

Up-regulated proteins (from Super-SILAC experiments) known
to be CAPs.

Table 8:

Down-regulated proteins (from Super-SILAC experiments)
known to be CAPs.

Table 9:

Overlapping up-regulated proteins in SILAC and Super-SILAC
experiments.

Table 10:

Overlapping down-regulated proteins in SILAC and SuperSILAC experiments.

Table 11:

Proteins with inverse regulations between SILAC and SuperSILAC experiments.

ix


List of Figures

Figure 1:

A ribbon presentation of ANXA1 molecular structure

Figure 2:

The diverse biological functions of ANXA1

Figure 3:

Morphology of the murine mammary gland

Figure 4:

Overview of proteomics and its applications

Figure 5:

Typical workflow in top-down proteomics approach

Figure 6:

Typical workflow of shotgun proteomics

Figure 7:

Summary of quantitative MS-based proteomics approaches

Figure 8:

Chemical structure of the ICAT Reagent

Figure 9:

Workflow of ICAT methodology

Figure 10:

Components of the iTRAQ label

Figure 11:

Workflow of a typical isobaric labelling experiment

Figure 12:

Chemistry involved in reductive dimethyl labeling

Figure 13:

Typical workflow of a SILAC experiment

Figure 14:

Super-SILAC methodology

Figure 15:

Incorporation plot of heavy-labelled
mammary gland cells.

Figure 16:

Workflow of the SILAC experiment involving Fwd and Rev
experiments

Figure 17:

Correlation plots of all Fwd and Rev experiments against
protein fold changes

Figure 18:

Scatter and density plots of both Fwd and Rev experiments

Figure 19:

Biologically-mapped pathways of the down-regulated proteins

Figure 20:

Protein and mRNA levels of selected down-regulated proteins

Figure 21:

Graph of comets’ mean tail moments after heat treatment

x

ANXA1+/- normal


Figure 22:

Comet snapshots of control and heat-stressed mammary gland
cells

Figure 23:

FACS analysis of ROS assay

Figure 24:

Proliferation rate of oxidative-stressed mammary gland cells

Figure 25:

Western blot of over-expression of Yap-1 in ANXA1-/mammary gland cells

Figure 26:

FACS analysis of the Yap1-transfected ROS assay

Figure 27:

Comet snapshots of mock-treated and Yap1–transfected

Figure 28:

Proliferation of Yap1-transfected in control and oxidativestressed ANXA1-/-

Figure 29:

Biologically-mapped pathways of the up-regulated proteins

Figure 30:

Sub-folders of tissue remodeling/wound repair pathway

Figure 31:

Protein and mRNA levels of selected up-regulated proteins

Figure 32:
Figure 33:

Migratory capabilities of ANXA1+/- and ANXA1-/- mammary
gland cells.
Wound healing assay snapshots and analysis

Figure 34:

Silencing of -parvin in ANXA1-/- and its migratory capability

Figure 35:

Wound healing snapshots and analysis of -parvin-silenced in
ANXA1-/-

Figure 36:

Illustration of adhesion/migration assay setup

Figure 37:

Adhesion/migration assay of -parvin-silenced in ANXA1-/-.

Figure 38:

Status of ANXA1 level in the murine mammary tumors.

Figure 39:

Incorporation plots of murine mammary epithelial and tumor
cell lines.

Figure 40:

1:1 ratio mix of the murine mammary tumors with SuperSILAC mix

Figure 41:

Unimodal distributions for Super-SILAC experiments

Figure 42:

Density plot on the Super-SILAC experiments

Figure 43:

Biologically-mapped pathways by the down-regulated proteins
xi


Figure 44:

Biologically-mapped pathways by the up-regulated proteins

Figure 45:

Network mapping of up-regulated proteins with CDMs

Figure 46:

Network mapping of down-regulated proteins with CDMs

Figure 47:

GeneGo analysis of overlapped down-regulated proteins
between SILAC and Super-SILAC

Figure 48:

GeneGO analysis of oppositely-regulated proteins between
SILAC and Super-SILAC

Figure 49:

GeneGO analysis of up-/down-regulated proteins in SILAC but
unregulated in Super-SILAC

Figure 50:

GeneGo analysis of up-/down-regulated proteins in SuperSILAC but not detected in SILAC

Figure 51:

Overview of the differentially-regulated proteins between
SILAC and Super-SILAC

Figure 52:

Summary workflow of this study

xii


List of Abbreviations

ABC

Ammonium bicarbonate

ACN

Acetonitrile

ANXA1

Annexin-A1

ATP

Adenosine triphosphate

BAD

Bcl-2-associated death

CAMs

Cancer-associated mutations

CAPs

Cancer-associated proteins

CDMs

Cancer driver mutations

Cldn

Claudin

CM-H2-DCFDA

5-(and 6)-chloromethyl-2’,7’dichlorodihydrofluorescein diacetate

COL5A2

Collagen, type V, alpha 2

cPLA2

cytosolic phospholipase A2

Da

Daltons

DLBCL

B-cell lymphoma-diffuse large B-cell lymphoma

ECM

Extracellular matrix

EGFR

Epidermal growth factor receptor

EMT

Epithelial-mesenchymal transition

ER

Estrogen receptor

Erbb2ip

Erbb2 interacting protein

FAK

Focal adhesion kinase
xiii


FACS

Fluorescence-associated cell sorting

FASP

Filter-aided sample preparation

FBS

Fetal bovine serum

FT-MS

Fourier transform mass spectrometer

Fwd

Forward

H2O2

Hydrogen peroxide

HER2

Human epithelial growth factor receptor 2

iTRAQ

isobaric tagging for relative and absolute
quantification

IAA

Iodoacetamide

ICAT

Isotope-coded affinity tagging

K0R0

12

C614N2-lysine and 12C614N4-arginine

K8R10

13

C615N2-lysine and 13C615N4-arginine

m/z

mass-to-charge

MAPK/ERK

Mitogen-activated protein kinase extracellular
signal-regulated kinase

MMP14

Matrix metallopeptidase 14

MMTV

Mouse mammary tumor virus

MS

Mass spectrometry

nFPRs

n-formyl peptide receptors

NP40

Nonidet P-40

p53AIP

p53-regulated apoptosis-inducing protein 1

pSILAC

Pulse-SILAC

PAI1

Plasminogen activator inhibitor 1

xiv


PAIs

Protein abundance indices

PMF

Peptide mass fingerprinting

PMN

Polymorphonuclear

PR

Progesterone receptor

PS

Phosphotidylserine

PTK2

Protein tyrosine kinase

PyMT

Polyomavirus middle T antigen

Rev

Reverse

RB

Retinoblastoma protein

ROS

Reactive oxygen species

SILAC

Stable isotope labeling by amino acids in cell
culture

STAT

Signal transducer and activator of transcription

TGF

Transforming growth factor beta

TIMP2

Tissue inhibitor of metalloproteinase 2

TP53

Tumor protein 53

TJP1

Tight junction protein 1

WST-1

Water soluble tetrazolium

Yap1

Yes-associated protein 1

xv


List of Symbols

α

Greek alphabet alpha

β

Greek alphabet beta

μ

Prefix meaning 10-6

xvi


Part of this work was presented as a poster in
1) IMPAKT Breast Cancer Conference held in Brussels, Belgium from
3 – 5 May 2012
2) Frontiers in Cancer Science held in National University of Singapore,
Singapore from 5 – 8 November 2012
3) Yong Loo Lin School of Medicine 3rd Annual Graduate Scientific
Congress held in National University Health System, Singapore on 30
January 2013

This work was presented as a poster in Proteomics Forum held in Berlin,
Germany from 17 – 21 March 2013

xvii

Chapter 1: Introduction

1.1:

Breast cancer

Breast cancer is the second most common cancer worldwide. Besides being
the leading cause of cancer death in women, it has been reported that the
incidence of breast cancer is increasing and that the burden it poses globally
far exceeds other cancers (Jemal et al., 2010).
As breast cancer is known to be a clinically diversified and molecularly
heterogeneous disease, gene expression studies from a variety of human breast
tumors identifying four major molecular subtypes of breast cancer has been a
major breakthrough for targeted therapies and treatments. The molecular
subtypes are the luminal, human epithelial growth factor receptor 2 (HER2)enriched, the basal-like and the normal-like tumors (Perou et al., 2000). The
luminal subtype typically express the estrogen and/or progesterone hormone
receptors and are usually denoted as estrogen receptor (ER)-positive (Habashy
et al., 2012). Tamoxifen has been the gold standard in treating this type of
breast cancer whereby it acts as a competitive inhibitor to the estrogen which
is often responsible for the proliferation of the breast cells (Pearson et al.,
1982).
The HER2-enriched, or commonly known as the HER2-positive breast cancer
accounts for about 25% of all breast cancers. It is characterized by the overexpression of HER2 which results in the activation of the Ras/mitogenactivated protein kinase (MAPK) signalling pathway, increasing the
proliferation of the breast cells (Janes et al., 1994). Despite the recombinant
monoclonal antibody trastuzumab shown to obtain great success in curbing
HER2-positive breast cancer, there have been non-responsive cases as well
(Dean-Colomb and Esteva, 2008). This has thus also driven researches in
search of alternative therapies or treatments.
The basal-like subtype, more commonly presented as the triple-negative
phenotype clinically with the absence of ER, progesterone receptor (PR) and

HER2. This type of breast cancer is known to have the worst prognosis as
1


there is yet any successful targeted therapy, though chemotherapy is currently
the primary therapy (Irvin and Carey, 2008). This has thus driven many breast
cancer researches in an attempt to understand its biology for prevention of this
disease and improving the current therapies.

1.2:

Annexin 1

1.2.1: Structure of ANXA1
ANXA1 is one of the members of the annexin superfamily of 13 members
(A1-A13). All members of this superfamily have the same principal property
of binding to negatively-charged phospholipids in a calcium-dependent
manner. There are two main domains in ANXA1, which are also applicable to
members of the annexin superfamily: the NH2-terminal and the COOHterminal domains. It has been known that the NH2-terminal domain is the
regulatory domain with varying length and sequences among the annexins,
which specifies their functions and activities. The NH2-terminal domain of
ANXA1 comprises about 49 amino acid residues which contain sites for
phosphorylation and proteolysis (Raynal and Pollard, 1994), of which the first
10-14 of them are binding sites for the S100 calcium-binding family member
S100A11 (Mailliard et al., 1996; Seemann et al., 1996). This interaction
between S100A11 and ANXA1 in the presence of calcium confers ANXA1 a
totally different structure. In the absence of calcium, the NH2-terminal domain
of ANXA1 forms an amphipathic -helix which replaces a helix (helix D in
repeat III) in its core domain and causes unwinding of its core domain
(Rosengarth et al., 2001). However, this helix D is forced back to its original

position and structure upon calcium-dependent membrane binding, freeing the
NH2-terminal domain for interaction with S100A11 (Weng et al., 1993). This
thus highlights some interesting mechanistic involvement of the NH2-terminal
domain of ANXA1 (Gerke and Moss, 2002).

2


The NH2‐terminal ‐helix
Figure 1: A ribbon presentation of the COOH-terminal -helical molecular
structure of ANXA1 in the presence (left) and absence (right) of calcium.
(Reproduced/Adapted with permission from Rescher and Gerke, 2004 Journal
of Cell Science from Company of Biologists Ltd).

The COOH-terminal domain of ANXA1 is the conserved core domain that
contains four annexin repeats of 70 amino acid residues. These repeats are
packed into a -helical disk or the membrane binding module. This disk has
two sides, with the convex side having binding sites for calcium and facing the
membrane when ANXA1 binds to the phospholipids, and the concave side
facing away the membrane for accessibility of the NH2-terminal domain to
bind to cytoplasmic proteins (Weng et al., 1993).

1.2.2: Functions of ANXA1
1.2.2.1:

Anti-inflammatory role

ANXA1 has been known to play an anti-inflammatory role in the innate
immune

system

via

regulating

the

migratory

activities

of

the

polymorphonuclear (PMN) leucocytes (Perretti and D'Acquisto, 2009). These
cells, such as the neutrophils, monocytes and macrophages, are known to have
high levels of cytoplasmic ANXA1 during inactivated state (Morand et al.,
1995). Upon activation such as during inflammation where the immune cells
would migrate to the sites of inflammation, ANXA1 has been shown to
mobilize from the cytoplasm to the cell surface membrane and secreted
following PMN adhesion to the endothelium for extravasation (Perretti et al.,
3


1996). This results in the inhibition of the cells into the subendothelial matrix
tissue (Lim et al., 1998) and thus reduces the responsiveness of the immune
cells to inflammation by negatively regulating their migratory capability.
Though the exposure of PMN to glucocorticoids increases ANXA1 level and

its secretion, it has a different effect on the adaptive immune system.
In the adaptive immune system, the effect of glucocorticoids to curb
inflammation is also reported to be mediated by ANXA1. However, in
contrast to that of innate immunity, the immunosuppressive effect of
glucocorticoids on T-cells was brought about with a decrease in ANXA1 level
(D'Acquisto et al., 2008; D'Acquisto et al., 2007), resulting in the inhibition of
T-cell activation and thus, a reduction in adaptive immune response.
Phospholipase A2 (PLA2) is an important enzyme that is involved in the
production of arachidonic acid and eicosanoid, which are pro-inflammatory
and released during inflammation and fever. ANXA1 has also been shown to
interact with and inhibit cytosolic phospholipase A2 (cPLA2) activity,
preventing the phosphorylation and activation of PLA2 (Croxtall et al., 1995;
Kim et al., 1994a) and thus shown to be antipyretic (Davidson et al., 1991).

1.2.2.2:

Regulator of Cellular Processes

It has been reported that ANXA1 exerts both proliferative and antiproliferative effects in vitro. ANXA1 was shown to inhibit proliferation in
macrophages via the constitutive activation of the mitogen-activated protein
kinase extracellular signal-regulated kinase (MAPK/ERK) (Alldridge et al.,
1999). Whereas ANXA1 has been reported to play an inhibitory role in cell
growth and proliferation in lung cancer cells A549 (Croxtall et al., 1993a), it
has a proliferative effect on liver cancer cells with its increased expression (de
Coupade et al., 2000). Though the exact mechanisms of how ANXA1 exerts
its regulatory role in cell growth may not be fully elucidated, ANXA1 has
been reported to be a substrate for the epidermal growth factor receptor
tyrosine kinase (EGFR) (De et al., 1986), one of the important signalling
pathways involved in cellular proliferation and differentiation. Such would
result in an inhibition of the EGF-mediated proliferation (Croxtall et al., 1993b)

4


ANXA1 is also a phosphorylation target for many signal transducing kinases
such as the platelet-derived growth receptor, hepatocyte growth factor receptor
(Skouteris and Schroder, 1996) and protein kinase C (Varticovski et al., 1988),
reinforcing its role in cellular proliferation.
ANXA1 has also been implicated to regulate apoptosis, being pro-apoptotic in
some cases but anti-apoptosis in other studies. Not much is known about such
contradiction in observations but there are some elucidations as to how
ANXA1 plays a role in apoptosis. For example, exogenously- administered
ANXA1 promoted apoptosis by inducing the dephosphorylation of Bcl-2associated death (BAD) protein which then translocate to the mitochondria in
neutrophils (Solito et al., 2003). This apoptotic effect is further reinforced with
the translocation of ANXA1 to the nucleus (Ishido, 2005) despite the
functional role of nuclear ANXA1 not known.
ANXA1 is also involved in apoptotic cells which relates to phagocytosis.
Exposure of phosphotidylserine (PS) on the outer leaflet of the plasma
membrane of apoptotic cells is one of the well-known signals for phagocytes
to phagocytosize. It has been shown that ANXA1 is able to be recruited to PSrich domains of apoptotic cell surfaces as a ‘eat-me’ signal for the phagocytes
(Arur et al., 2003). The use of siRNA-mediated silencing of ANXA1 or
antibodies against ANXA1 inhibited PS-mediated phagocytosis of apoptotic
cells (Fan et al., 2004). This further illustrates the involvement of ANXA1 in
apoptosis and PS-mediated phagocytosis.
All in all, ANXA1 plays regulatory role in the above-mentioned cellular
processes (Figure 2) and any dysregulation in cellular processes due to
ANXA1 may be informative in how ANXA1 may be involved in cancer
development.

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Figure 2: A summary diagram showing the functions of ANXA1. (Reproduced
Adapted with permission from Lim and Pervaiz, 2007 The FASEB Journal
from FASEB Office of Publications).

1.2.3: ANXA1 and Cancer
There is more and more evidence relating ANXA1 to cancer development in
recent years. Expressions of ANXA1 correlating with certain cancers have
been reported even though ANXA1 could be found over-expressed in some
cancers and loss in other cancers (Lim and Pervaiz, 2007). ANXA1 has been
shown to be lost in esophageal cancer (Hu et al., 2004), prostate cancer (Xin et
al., 2003), head and neck cancer (Garcia Pedrero et al., 2004) and overexpressed in hepatocarcinoma (de Coupade et al., 2000) as well as pancreatic
cancer (Bai et al., 2004). The differential expression of ANXA1 in different
types of cancer is also reflected by the variable effect of ANXA1 on the
proliferation of tumor-derived cell lines. The status of ANXA1 in certain
clinical cancer tissues has been tabulated in Table 1.

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Table 1: The status of ANXA1 in clinical cancer tissues. (Reproduced /
Adapted with permission from Lim and Pervaiz, 2007 The FASEB Journal
from FASEB Office of Publications).

ANXA1 has also been implicated in tumor growth. It has been reported that
ANXA1, being the agonist for formyl peptide receptor (FPR1), was released
by necrotic tumor cells. Its release allows its activation on the FPR1 expressed
on the live glioblastoma cells and promotes tumor growth and progression
(Yang et al., 2011). It has also been demonstrated that skin and lung tumors in
ANXA1-knockout mice had tumor growth retardation and were less

aggressive and less metastatic than the wild-type mice (Yi and Schnitzer,
2009). It has also been reported that ANXA1 was able to promote the
invasiveness of colorectal adenocarcinoma cells through its activation on the
FPR2 (Babbin et al., 2006).
The role of ANXA1 in breast cancer has been intriguing as there seems to
have conflicting reports on the status of ANXA1 in breast cancer. Loss of
ANXA1 was observed in ductal carcinomas while over-expression of ANXA1
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